CN104267088A - An electrochemical biosensor for detecting glutathione and a preparing method thereof - Google Patents

Abstract

The invention relates to an electrochemical biosensor for detecting glutathione and a preparation method thereof. The sensor is a three-electrode system sensor, wherein the counter electrode is a platinum electrode, the reference electrode is a saturated calomel electrode, and the working electrode is a gold electrode. strand DNA. The invention uses the high-affinity combination of glutathione and copper ions to inhibit the synthesis of copper nano-clusters on the electrode surface, and realizes the indirect detection of glutathione through the electrochemical quantitative characterization of copper nano-clusters. The linear range for detecting glutathione of the present invention is 1-1000nM, and the detection limit is about 0.42nM. The invention also has the advantages of simple operation, low cost, convenient use, high selectivity, etc., and thus has great potential application value in the fields of biochemical research, clinical analysis and the like.

Description

Electrochemical biosensor for detecting glutathione and preparation method thereof

technical field

The invention relates to a novel electrochemical biosensor and a preparation method thereof, in particular to an electrochemical biosensor for detecting glutathione and a preparation method thereof.

Background of the invention

Glutathione is the most abundant small molecular weight non-protein sulfhydryl compound in cells, which is formed by condensation of glutamic acid, cysteine and glycine through peptide bonds. Glutathione is a regulator of sulfhydryl groups and sulfides in the body, and plays an important role in maintaining the reduction state of protein sulfhydryl groups and enzyme activity, anti-oxidation, and maintaining the balance of the redox environment in biological organisms. Several studies in recent years have shown that changes in the concentration of glutathione in the body are related to the occurrence and development of many diseases such as Alzheimer's disease, Parkinson's syndrome, diabetes, and atherosclerosis; and the sensitive detection of glutathione can Provide a lot of important information for the clinical diagnosis and treatment of these diseases. Today's techniques for detecting glutathione mainly include high-performance liquid chromatography, ultraviolet-calorie detection, capillary electrophoresis, mass spectrometry, and fluorescence spectrometry. These methods have their own advantages in detection sensitivity, selectivity, detection time and stability, but they also have disadvantages such as cumbersome operation and expensive equipment. Therefore, it is very urgent to develop a simple and sensitive new method for glutathione detection.

Electrochemical biosensors are a type of biosensors that use electrodes as signal converters to measure potential or current. They are mainly composed of molecular recognition and information conversion components. The electrochemical system realizes the input or output of electrical energy by means of electrodes, so as to obtain the electrical signal of the modified substances on the electrode surface. A three-electrode system is commonly used in electrochemical research, including a working electrode, an auxiliary electrode (also called a counter electrode), and a reference electrode. Current flows through the working electrode and the auxiliary electrode, and the potential measured by the working electrode is relative to the reference electrode. As a kind of analysis and detection method, electrochemical method has the advantages of low equipment, high sensitivity, simplicity and speed.

Contents of the invention

One of the objectives of the present invention is to provide an electrochemical biosensor for detecting glutathione, which realizes the indirect detection of glutathione through the quantitative analysis of copper nanoclusters on the electrode surface.

The second object of the present invention is to provide a preparation method of the sensor.

In order to achieve the above object, the present invention adopts the following mechanism: design a DNA single strand P1, its 3' end contains a sulfhydryl group, which can be self-assembled on the surface of the gold electrode through the effect of a gold-sulfhydryl covalent bond; DNA single-strand P2 that is completely complementary to the base, and the two hybridize to form a double-strand, which becomes a template for the synthesis of copper nanoclusters on the electrode surface. In the presence of a reducing agent, the divalent copper ions in the solution are reduced to monovalent cuprous ions, which undergo a disproportionation reaction and transform into divalent copper ions and zero-valent copper atoms, and the zero-valent copper atoms can be fixed on the electrode surface The major groove position of double-stranded DNA is enriched and copper nanoclusters are finally formed. The synthesized copper nanoclusters were oxidized and dissolved with hydrochloric acid, and detected by electrochemical techniques, which can realize the quantitative characterization of the copper nanoclusters on the electrode surface. On the other hand, glutathione can specifically bind copper ions in solution, thereby inhibiting the reduction process of copper ions, which eventually leads to a decrease in the number of copper nanoclusters synthesized on the electrode surface. The electrochemical signal obtained when the copper nanoclusters are quantitatively characterized by electrochemical techniques also decreases accordingly. By analyzing how much the electrochemical signal changes, we can calculate the concentration of glutathione.

According to above-mentioned mechanism, the present invention adopts following technical scheme:

An electrochemical biosensor for detecting glutathione is a three-electrode system sensor, wherein the counter electrode is a platinum electrode, the reference electrode is a saturated calomel electrode, and the working electrode is a gold electrode, which is characterized in that on the gold electrode It is modified with a DNA double-strand that can be used as a template for the synthesis of copper nano-clusters, and the double-strand is complementary.

The above DNA double strands are formed by the hybridization of P1 and P2 strands, wherein the sequence of the P1 strand is: 5'-TACTCATACGCTCATACGTTCATCACGACTAAAAA-C ₆ -SH-3', and the sequence of the P2 strand is: 5'- AGTCGTGATGAACGTATGAGCGTATGAGTA-3'.

The design principle of the above sequence is that the P1-P2 hybrid chain can maintain a stable double-strand structure on the electrode surface during the experiment, and the length of the double-strand is not less than 30 bases (the ratio of T-A base pairs is greater than 50%). Ensure the efficiency of copper nanocluster synthesis. Once the DNA sequence is designed, its preparation or chemical synthesis will be completed by a professional nucleic acid synthesis company.

A preparation method according to the above-mentioned biosensor for detecting glutathione is characterized in that the working electrode of the sensor is prepared, and the specific steps are:

a) Place the treated gold electrode in 0.5 M H ₂ SO ₄ , perform cyclic voltammetry scan in the voltage range of 0-1.6 V, and set the scan rate to 100 mV/s until it reaches a stable value; dry it for use;

b) Submerge the gold electrode obtained in step a in the DNA immobilization buffer solution, let it stand at room temperature for 15-18 hours, then immerse it in 1 mM mercaptohexanol aqueous solution and react in the dark for 0.5-2.0 hours, rinse with ultrapure water, Blow dry to obtain the gold electrode modified by the first strand; the DNA immobilization buffer solution contains the first strand with a concentration of 1 μM, Tris-HCl of 10 mM, EDTA of 1 mM, TCEP of 10 mM and 0.1 M NaCl, the pH of the solution is 7.4;

c) Submerge the gold electrode modified by the first strand obtained in step b in the DNA hybridization buffer, let stand at 4??C for 1~2 hours, rinse with ultrapure water, and dry to obtain a complete double strand DNA-modified gold electrode; the DNA hybridization buffer solution is 10 mM phosphate buffer at pH 7.4, which contains the second strand at a concentration of 1 μM and 1 M NaCl.

The specific steps of the treatment method of the above-mentioned gold electrode are: drop 20 μL of piranha solution on the surface of the gold electrode to be treated, that is, the volume ratio of concentrated sulfuric acid:hydrogen peroxide is 3:1, react for 2 minutes, and use ultrapure water Rinse it clean and dry it with nitrogen; after polishing the gold electrode on 5000-grit sandpaper for 2 minutes, polish it to the mirror surface successively on the silk of the mortar containing alumina with a particle size of 1 μm, 0.3 μm, and 0.05 μm, and then wash it in ethanol, Sonicate in ultrapure water for 2 minutes sequentially to remove impurities.

A detection method for glutathione, using the above-mentioned biosensor for glutathione, is characterized in that the specific steps of the method are:

a. Weigh an appropriate amount of ascorbic acid powder, dilute to 10 mM with ultrapure water, mix 40 μL with 320 μL reaction buffer, and then add 40 μL of preparation containing 10 μM copper ions and different concentrations of glutathione to the system. The mixed solution was tested and reacted at room temperature for 20 minutes; the reaction buffer solution contained 20 mM MOPS, 300 mM NaCl and 2 mM MgCl ₂ , and the pH value of the solution was 7.5.

b. Put the working electrode gold electrode in the biosensor into the mixed solution obtained in step a immediately, and react at room temperature for 30 minutes. After rinsing with ultrapure water and blowing dry with nitrogen, the working electrode was immersed in 200 μL of 0.1 M hydrochloric acid solution to dissolve the copper nanoclusters synthesized on the electrode surface. After 2 hours of reaction, the above solution was mixed with 4.8 mL of 0.5 M NaAc-HAc buffer, and used as the electrolyte solution for electrochemical detection. The specific steps of electrochemical detection are as follows: first deposit at -1.2 V for 8 minutes, and then use differential pulse voltammetry (DPV) to scan to obtain corresponding electrochemical data; the specific parameters of DPV experiment are: initial potential 0.1 V, end potential 0.35 V, pulse amplitude 50 mV, pulse period 200 m.

The present invention constructs an electrochemical biosensor for detecting glutathione, utilizes the characteristics of strong affinity between glutathione and copper ions, and combines the inhibitory effect of glutathione on the synthesis of copper nanoclusters with the sensitive and convenient electrochemical detection Combining the advantages of the present invention, the rapid and sensitive detection of glutathione has been carried out through the quantitative characterization of the copper nanoclusters synthesized on the electrode surface, which has broad application prospects.

Description of drawings

Figure 1 shows the DPV quantitative characterization results of copper nanoclusters synthesized on the electrode surface when different concentrations of copper ions exist in the solution. Curves from a to f are 0 M, 1 nM, 10 nM, 100 nM, 1 μM and 10 μM, respectively.

Figure 2 is the DPV spectrum obtained in the detection of 1 μM glutathione and the control experiment. (a) Control group, the system does not contain glutathione; (b) Experimental group, the system contains 1 μM glutathione.

Figure 3 is the DPV spectrum obtained when detecting different concentrations of glutathione (from a to g are 0 nM, 1 nM, 5 nM, 10 nM, 100 nM, 1 μM and 10 μM, respectively).

Figure 4 shows the relationship between the peak current value of DPV and the concentration of glutathione. The insert figure shows the linear relationship between the peak current value of DPV and the logarithmic value of glutathione concentration in the range of 1-1000 nM.

Figure 5 is the DPV spectrum obtained when detecting 1 μM glutathione and 10 μM interfering amino acids.

Specific implementation method

Example 1: Preparation of Gold Electrode Modified by Double-Stranded DNA

Drop 20 μL of piranha solution (concentrated sulfuric acid: hydrogen peroxide = 3:1) on the surface of the gold electrode to be treated for 2 minutes, rinse with ultrapure water, and blow dry with nitrogen. After the gold electrode was polished on 5000-grit sandpaper for 5 minutes, it was sequentially polished to a mirror surface on the silk containing alumina (particle size: 1 μm, 0.3 μm, 0.05 μm), and then ultrasonicated in ethanol and ultrapure water. 2 minutes to remove impurities. Place the treated gold electrode in 0.5 M H ₂ SO ₄ for cyclic voltammetry scanning. Set the scanning voltage range from 0 to 1.6 V, the scanning speed at 100 mV/s, set 30 cycles until the cyclic voltammetry curve is stable, and dry it with nitrogen to obtain a bare gold electrode with a clean surface, which can be used for the modification of thiol DNA. Immerse the gold electrode in a DNA immobilization buffer solution (10 mM Tris-HCl, 1 mM EDTA, 10 mM TCEP, and 0.1 M NaCl, pH 7.4) containing 1 μM P1 chain, and let it stand at room temperature for 15 to 18 hours to form a good Self-assembled monolayers of P1 chains without non-specific adsorption. Rinse the gold electrode with ultrapure water and dry it with nitrogen, then immerse the gold electrode in the hybridization buffer (10 mM pH 7.4 phosphate buffer containing 1 M NaCl) containing 1 μM P2 chain, 4?? After standing for 1 to 2 hours, rinse with ultrapure water and blow dry with nitrogen to obtain a complete double-stranded DNA modified gold electrode.

Example 2: Copper nanocluster synthesis and electrochemical quantitative characterization

Mix 40 μL of 10 mM ascorbic acid solution with 320 μL of reaction buffer (20 mM MOPS, 300 mM NaCl and 2 mM MgCl ₂ , pH 7.5), and then add 40 μL of copper ion solutions of different concentrations to the system, statically Leave for 20 minutes. Put the double-stranded DNA modified gold electrode into the above mixed solution, and react at room temperature for 30 minutes. After the reaction, the electrode was rinsed with ultrapure water and dried with nitrogen to remove excess copper ions adsorbed on the surface of the electrode. Subsequently, the electrode was immersed in 200 μL of 0.1 M hydrochloric acid solution to dissolve the copper nanoclusters synthesized on the electrode surface. After 2 hours, the above solution was mixed with 4.8 mL of 0.5 M NaAc-HAc buffer and used as the electrolyte solution for electrochemical detection.

Related nucleic acid sequences are as follows:

The sequence of the P1 chain is: 5'-TACTCATACGCTCATACGTTCATCACGACTAAAAA-C ₆ -SH-3'.

The sequence of the P2 chain is: 5'- AGTCGTGATGAACGTATGAGCGTATGAGTA-3'.

The specific steps of electrochemical detection: first deposit at -1.2 V for 8 minutes, and then DPV scan to obtain corresponding electrochemical data; the specific parameters of DPV experiment are: initial potential 0.1 V, end potential 0.35 V, pulse amplitude 50 mV, pulse period 200 ms.

As shown in the curve a of Figure 1, when there is no copper ion in the reaction solution, there is no obvious electrochemical response peak in the final DPV spectrum, which is due to the fact that copper nanoclusters are not formed on the electrode surface at this time. When copper ions exist in the reaction solution, the DPV spectrum obtains an obvious characteristic redox peak near 0.22 V, and the absolute value of the peak current increases with the increase of copper ion concentration (curves b to f). The above results indicate that the quantitative characterization of Cu nanoclusters synthesized on the electrode surface can be achieved through a series of steps including hydrochloric acid dissolution, electrodeposition, and DPV scanning.

Example 3: Detection of different concentrations of glutathione

Mix 40 μL of 10 mM ascorbic acid solution with 320 μL of reaction buffer (20 mM MOPS, 300 mM NaCl and 2 mM MgCl ₂ , pH 7.5), and then add 40 μL of 10 μM copper ions and different concentrations to the system Glutathione (0 nM, 1 nM, 5 nM, 10 nM, 100 nM, 1 μM and 10 μM) mixed solution to be tested, reacted at room temperature for 20 minutes. Subsequently, the double-stranded DNA-modified gold electrode was put into the above mixed solution, and electrochemical detection was performed after reacting at room temperature for 30 minutes. The specific detection steps were the same as in Example 2.

Curve a in Figure 2 shows the DPV spectrum obtained by scanning the double-stranded DNA-modified gold electrode in a reaction system containing only ascorbic acid and copper ions for a period of time and after a certain treatment. It can be seen that the double-stranded DNA molecules immobilized on the electrode surface can serve as templates for the synthesis of copper nanoclusters, and the generation of copper nanoparticles on the electrode surface will lead to obvious electrochemical responses in the DPV spectrum. However, when 1 μM glutathione exists in the system, the resulting DPV response is significantly weakened (Fig. 2 curve b), which is due to the fact that glutathione can combine with copper ions in the solution, thereby inhibiting the formation of copper nanoclusters. formed on the electrode surface.

As shown in Figure 3, as the concentration of glutathione increases, the electrochemical response in the DPV spectrum gradually decreases, which indicates that as the concentration of glutathione increases, more and more copper ions are combined with glutathione , leading to fewer and fewer copper nanoclusters formed on the electrode surface.

Figure 4 was obtained by plotting the peak current value of DPV and the concentration of glutathione. In addition, the insert of Figure 4 shows that when the concentration of glutathione varies between 1 and 1000 nM, the logarithm of glutathione concentration and the peak current value of DPV show a good linear relationship, which can be used as the Basis for quantitative testing. The lowest detection limit of glutathione was about 0.42 nM.

Example 4: Research on the Specificity of Bioelectrochemical Sensors

In order to verify the specificity of this bioelectrochemical sensor, we use other amino acids (alanine, phenylalanine, glutamic acid, etc.) . As shown in Figure 5, when the solution contained 1 μM glutathione, the absolute value of the DPV peak current obtained was the smallest, about 0.16 μA, while the absolute value of the DPV peak current was larger when the solution contained other interfering amino acids, compared with the blank control The time is similar, indicating that the bioelectrochemical sensor has high selectivity and specificity for glutathione.

The above results show that the biosensor has good selectivity and specificity for glutathione, simple design idea, low cost, convenient experimental operation, and sensitive detection results. It has great potential in the fields of biochemical research and clinical analysis. Value. No patent has been found that is about detecting GSH with a sensor prepared by a double-strand modified gold electrode. the

<120> Electrochemical biosensor for detecting glutathione and its preparation method

<160> 2

<210> 1

<211> 35

<212>DNA

<213> Artificial sequence

<400> 1

5'-TAACTC ATACG CTCAT ACGTT CATCA CGACT AAAAA-C ₆ -SH-3'

the

<210> 2

<211> 30

<212>DNA

<213> Artificial sequence

<400> 2

5'- AGTCG TGATG AACGT ATGAG CGTAT GAGTA-3'

Claims (5)

1. An electrochemical biosensor for detecting glutathione is a three-electrode system sensor, wherein the counter electrode is a platinum electrode, the reference electrode is a saturated calomel electrode, and the working electrode is a gold electrode, which is characterized in that the gold electrode The electrode is decorated with DNA double strands that can be used as templates for the synthesis of copper nanoclusters, and the double strands have a complementary structure. 2. The electrochemical biosensor for detecting glutathione according to claim 1, characterized in that said DNA double strand is formed by hybridization of P1 and P2 strands, wherein the sequence of P1 strand is: 5'-TACTCATACGCTCATACGTTCATCACGACTAAAAA-C ₆ -SH-3', the sequence of the P2 chain is: 5'- AGTCGTGATGAACGTATGAGCGTATGAGTA-3'. 3. a preparation method of a biosensor for detecting glutathione according to claim 1, characterized in that the working electrode of the sensor is prepared, and the concrete steps are: a) Place the treated gold electrode in 0.5 M H ₂ SO ₄ , perform cyclic voltammetry scan in the voltage range of 0-1.6 V, and set the scan rate to 100 mV/s until it reaches a stable value; dry it for use; b) Submerge the gold electrode obtained in step a in the DNA immobilization buffer solution, let it stand at room temperature for 15-18 hours, then immerse it in 1 mM mercaptohexanol aqueous solution and react in the dark for 0.5-2.0 hours, rinse with ultrapure water, Blow dry to obtain the gold electrode modified by the first strand; the DNA immobilization buffer solution contains the first strand with a concentration of 1 μM, Tris-HCl of 10 mM, EDTA of 1 mM, TCEP of 10 mM and 0.1 M NaCl, the pH of the solution is 7.4; c) Submerge the gold electrode modified by the first strand obtained in step b in the DNA hybridization buffer, let stand at 4??C for 1~2 hours, rinse with ultrapure water, and dry to obtain a complete double strand DNA-modified gold electrode; the DNA hybridization buffer solution is 10 mM phosphate buffer at pH 7.4, which contains the second strand at a concentration of 1 μM and 1 M NaCl. 4. method according to claim 3, it is characterized in that the specific steps of the processing method of described gold electrode are: drop 20 μ L piranha solution on the gold electrode surface to be treated, i.e. concentrated sulfuric acid: hydrogen peroxide The volume ratio was 3:1, reacted for 2 minutes, rinsed with ultrapure water, and blown dry with nitrogen; after polishing the gold electrode on 5000-grit sandpaper for 2 minutes, the gold electrodes with particle sizes of 1 μm, 0.3 μm, and 0.05 μm were respectively The silk of the aluminum mortar was polished to a mirror surface in sequence, and then ultrasonicated in ethanol and ultrapure water for 2 minutes to remove impurities. 5. a detection method of glutathione, adopts the biosensor of glutathione according to claim 1, is characterized in that the concrete steps of this method are: a) Weigh an appropriate amount of ascorbic acid powder, dilute to 10 mM with ultrapure water, mix 40 μL with 320 μL reaction buffer, and then add 40 μL of preparation containing 10 μM copper ions and different concentrations of glutathione to the system. The mixed solution was tested and reacted at room temperature for 20 minutes; the reaction buffer solution contained 20 mM MOPS, 300 mM NaCl and 2 mM MgCl ₂ , and the pH value of the solution was 7.5. b) Put the working electrode gold electrode in the biosensor into the mixed solution obtained in step a immediately, and react at room temperature for 30 minutes. After rinsing with ultrapure water and blowing dry with nitrogen, the working electrode was immersed in 200 μL of 0.1 M hydrochloric acid solution to dissolve the copper nanoclusters synthesized on the electrode surface. After 2 hours of reaction, the above solution was mixed with 4.8 mL of 0.5 M NaAc-HAc buffer, and used as the electrolyte solution for electrochemical detection. The specific steps of electrochemical detection are as follows: first deposit at -1.2 V for 8 minutes, and then scan by differential pulse voltammetry to obtain corresponding electrochemical data; the specific parameters of DPV experiment are: initial potential 0.1 V, termination potential 0.35 V, pulse Amplitude 50 mV, pulse period 200 ms.