CN110779967B - An electrochemical detection method of NF-κB based on traditional glassy carbon electrode - Google Patents

Abstract

The invention belongs to the technical field of analytical chemistry, and relates to an electrochemical detection method for NF-κB based on a traditional glassy carbon electrode. The present invention mainly utilizes the principle that peptide nucleic acid (PNA) can competitively bind complementary ssDNA in dsDNA containing NF-κB binding sequence to form a stable PNA-DNA hybrid chain, and the existence of NF-κB can inhibit the generation of PNA-DNA hybrid chain . In the experiment, the surface of the activated electrode is first plated with gold and covalently modified with PNA. The PNA competitively binds the complementary ssDNA in the dsDNA containing the NF-κB binding sequence to form a stable PNA-DNA hybrid chain on the electrode surface. The presence of NF-κB will interact with the dsDNA. Binds and inhibits the formation of PNA-DNA hybrid chains, and the difference in the content of NF-κB will lead to different amounts of PNA-DNA hybrid chains. MBs specifically chimeric in the PNA-DNA hybrid chains are selected as electrical signal molecules, and the differential pulse voltage is used. The peak current value of MB at different concentrations of NF-κB was measured by the amperometric method, the relationship between the concentration of NF-κB and the peak current value was drawn to obtain a linear equation, and the content of NF-κB in the sample to be tested was calculated by detecting the electrical signal. This method has high sensitivity and provides a new idea for the detection of NF-κB.

Description

An electrochemical detection method of NF-κB based on traditional glassy carbon electrode

technical field

The invention relates to an electrochemical detection method of NF-κB based on a traditional glassy carbon electrode, belonging to the field of analytical chemistry.

Background technique

The nuclear transcription factor NF-κB is a transcription factor belonging to the zinc finger structure family, which can bind to the promoter region of the B cell immunoglobulin κ light chain and regulate the expression of genes in this region. NF-κB is widely present in various cell types of mammals. Different types of studies have found that the expression of NF-κB is significantly different between healthy and pathological states. It plays an important indicator role in the occurrence of inflammatory response, tumor development and other disease processes, and is of great significance for disease development, early diagnosis or prevention. Therefore, it is very necessary to establish a sensitive, rapid and convenient method to detect the level of NF-κB.

Nowadays, the detection methods of NF-κB include electrophoretic mobility shift assay, western blotting and so on. However, these methods often require specific antibodies, cumbersome labeling or special instrumentation. It is well known that the labeling process is often time-consuming and expensive, and may even lead to denaturation of biomolecules. Electrochemical detection technology has the advantages of simple equipment, low price, high sensitivity, simple and fast, and can realize label-free detection. Among them, glassy carbon electrodes have the advantages of high chemical stability, small thermal expansion coefficient, good air tightness, and can be made into cylindrical and disc electrode shapes. They have been widely used in electrochemical experiments. In recent years, they have been successfully applied. For qualitative or quantitative detection of biological macromolecules.

SUMMARY OF THE INVENTION

The purpose of the present invention is to take advantage of the electrochemical detection technology to establish a simple, label-free, low-cost and high-sensitivity NF-κB detection method.

The technical solution of the present invention: an electrochemical detection method of NF-κB based on a traditional glassy carbon electrode, which utilizes the combination of PNA and DNA with high affinity and specificity, and can competitively bind the complementary ssDNA in dsDNA to form a stable PNA- DNA hybrid strands; MBs can be chimeric in hybrid duplexes and act as redox indicators to amplify electrical signal responses; DNAs containing NF-κB binding sequences were designed, and when NF-κB protein exists, NF-κB protein and dsDNA target The binding energy can inhibit the generation of PNA-DNA hybrid chains, the interaction between the PNA single chain modified on the electrode surface and MB is not strong, and the signal is weak; the difference in NF-κB content leads to different degrees of signal response, the standard curve is measured, and the statistical The linear equation of NF-κB detection was obtained by chemical analysis and its content was calculated. The method has good repeatability and high sensitivity, and can be applied to the detection of NF-κB.

The method includes the following steps: pretreatment of glassy carbon electrode, gold particle modified glassy carbon electrode, PNA modified gold-coated glassy carbon electrode, co-incubation of sample and modified electrode, co-incubation of MB and modified electrode, and electrochemical detection of NF-κB.

(1) Pretreatment of glassy carbon electrodes

The glassy carbon electrode was pretreated by the classical pretreatment method. The specific steps are as follows: polishing the disc glassy carbon electrode with a diameter of 3 mm with 1 and 0.3 μm dioxide trioxide powder respectively, and then ultrasonicating with alcohol and ultrapure water for 5 min respectively. Afterwards, the treated electrode was placed in 0.5MH ₂ SO ₄ , and cyclic voltammetry was performed in the voltage range of 0V-1.5V, and the scan speed parameter was set to 0.1V/s, and it was dried at room temperature until it reached stability.

(2) Gold particle modified glassy carbon electrode

The glassy carbon electrode pretreated by the classical method in the above (1) was immersed in 5 mL of 2 mM chloroauric acid solution (0.5 MH ₂ SO ₄ dissolved) for electrodeposition for 750 s, and the deposition potential was -200 mV.

(3) PNA modified gold-coated glassy carbon electrode

Add 0.5 μM 5.0 μL PNA (0.1 M PBS, pH=7.4) dropwise to the surface of the gold-coated glassy carbon electrode in (2) above, incubate at 37° C. for 1.5 h, and rinse with distilled water. Then, the PNA-modified electrode was placed in 100 μL of 1.0 mM MCH solution for 30 min to remove nonspecific adsorption.

The sequence of the above PNA is: 5'-Cys-ATG-GTC-GGG-ACT-TTC-CCT-3'.

(4) Co-incubation of samples with modified electrodes

Pretreatment of the sample to be tested: take 12.5μL of 0.06μM ssDNA1 (0.1M PBS, 0.25M NaCl, pH=7.4) and 12.5μL of 0.06μM ssDNA2 (0.1M PBS, 0.25M NaCl, pH=7.4), at 90°C for 5 min → 70℃10min→50℃10min→30℃10min→10℃25min hybridization to obtain dsDNA. It was then mixed with 25 μL solutions of different concentrations (0→0.1 ng/mL) NF-κB p50 (50 mM HEPES, 1 mM TCEP, 50 mM NaCl, pH=7.4), and incubated at 37° C. for 0.5 h. The electrode treated in (3) above was placed in a mixed solution of dsDNA and protein, and incubated at 37° C. for 2.0 h.

The sequence of the above ssDNA1 is: 5'-ATG-GTC-GGG-ACT-TTC-CCT-3';

The sequence of the above ssDNA2 is: 5'-AGG-GAA-AGT-CCC-GAC-CAT-3'

(5) Co-incubation of MB with modified electrode

The electrode treated in (4) above was placed in 100 μL of MB solution (20 mM Tris-HCl, pH=7.4) with a concentration of 20 mM, protected from light, and incubated at room temperature for 2 h.

(6) Electrochemical detection of NF-κB

The electrode processed in the above (5) was placed in a solution of 5mL 20mM Tris-HCl with a pH of 7.4, and electrochemical quantitative analysis was carried out. The electrochemical workstation (CHI 660E) used in this detection was a saturated calomel electrode as a reference electrode. , the platinum electrode is the counter electrode. The scanning method used was differential pulse voltammetry, with parameter settings: initial potential -0.6V, termination potential 0.3V, potential increment 0.004V, amplitude 0.05V, pulse width 0.05s, and pulse period 0.5s. When the content of NF-κB is high, the less hybrid chains of PNA-DNA are formed, and the less the amount of the electrical signal molecule MB is adsorbed, so the obtained electrochemical signal is also reduced. Taking the electrochemical signal of MB as the ordinate and the concentration of NF-κB as the abscissa, a standard curve was drawn, and the sensitive detection of NF-κB could be realized by calculating the concentration of NF-κB.

Beneficial effects of the invention: the method is simple, rapid, and highly sensitive, and successfully realizes the label-free detection of NF-κB. It simplifies the experimental steps and avoids the labeling process, which is of great significance to the monitoring and treatment of various diseases, including cancer.

Description of drawings

Figure 1: Schematic diagram of the detection of NF-κB.

Figure 2: Standard curve of the relationship between the electrochemical signal value and the concentration of NF-κB at different concentrations of NF-κB.

Detailed ways

Example 1. Determination of Electrochemical Signal Value-Concentration Standard Curve of NF-κB Standard Solution

25 μL of different concentrations of NF-κB standard solutions were incubated with DNA according to the above steps, and then modified on the electrode, and the electrochemical signal was measured after being treated with the electrical signal molecule MB. Figure 2 shows the relationship between the electrochemical signal value (ip) and the concentration of NF-κB. In the range of 0.02-0.1ng/mL of NF-κB, there is a linear relationship between ip and the concentration. The linear regression equation is: y=- 55.05x+7.101, R ² =0.995, where y is the peak current ip (μA) of DPV, and x is the concentration of NF-κB (ng/mL).

Claims (6)

1. A NF-kB electrochemical detection method based on a traditional glassy carbon electrode is characterized in that three probes which take a p50 structural domain in NF-kB as a target are designed: PNA, ssDNA1, and ssDNA 2;

PNA：5′-Cys-ATGGTCGGGACTTTCCCT-3′

ssDNA1：5′-ATGGTC-GGGACT-TTCCCT-3′

ssDNA2：5′-AGGGAAAGTCCCGACCAT-3′

the double-stranded DNA formed by s sDNA1 and ssDNA2 in a complementary mode contains NF-kB binding sequences, ssDNA2 can be competitively bound by peptide nucleic acid PNA to form a stable PNA-ssDNA2 hybrid chain, the principle that the existence of NF-kB can inhibit the generation of PNA-ssDNA2 hybrid chain is utilized, the PNA is firstly plated with gold and covalently modified on the surface of treated traditional glassy carbon, the PNA competitively binds complementary ssDNA2 in dsDNA containing the NF-kB binding sequences to form a stable PNA-DNA hybrid chain on the surface of an electrode, the existence of NF-kB can be combined with the dsDNA to inhibit the formation of the PNA-DNA hybrid chain, the difference of the content of NF-kB can cause the formation of the PNA-DNA hybrid chain to be different, MB specifically embedded in the PNA-DNA hybrid chain is selected as an electric signal molecule, and the peak current value of MB at different concentrations of the NF-kB is measured by a differential pulse voltammetry method, and drawing a relation curve of the NF-kB concentration and the peak current value to obtain a linear equation, and calculating the NF-kB content in the sample to be detected by detecting the electric signal.

2. The method of claim 1, wherein ssDNA2 is designed to be competitively bound by peptide nucleic acid PNA to form stable PNA-ssDNA2 hybrid chain, and the principle that the presence of NF- κ B inhibits the formation of PNA-ssDNA2 hybrid chain is utilized.

3. The method of claim 1, wherein the experiment is performed by first plating gold on the surface of treated conventional glassy carbon and covalently modifying PNA, and the modification process comprises pretreating the glassy carbon electrode by a classical method, and soaking the electrode in 5mL of 0.5M H₂SO₄Electrodepositing at-200 mV in a 2mM dissolved chloroauric acid solution for 750s, washing by double distilled water, dropwise adding 5.0 mu L of a solution containing 0.5 mu M PNA, 0.1M PBS and having a pH value of 7.4 on the surface of the electrode, incubating at 37 ℃ for 1.5h, washing by distilled water, soaking in a 1mM mercaptohexanol solution for 0.5h, and washing by distilled water to obtain the PNA stably modified gold-plated glassy carbon electrode surface.

4. The method of claim 1, wherein PNA competitively binds complementary ssDNA2 in dsDNA containing NF-. kappa.B binding sequence to form stable PNA-ssDNA2 hybrid chains on the surface of the electrode, NF-. kappa.B is present to bind to dsDNA and inhibit the formation of PNA-DNA hybrid chains and the difference in NF-. kappa.B content results in the formation of PNA-DNA hybrid chains in different amounts, 0.25M NaCl, 0.1M PBS is used in the experiment, 0.06. mu.M ssDNA1 dissolved in a solution of pH 7.4 and 12.5. mu.L each of 0.06. mu.M ssDNA2 are mixed together and hybridized with dsDNA obtained by hybridization at 90 ℃ 5min → 70 ℃ 10min → 50 ℃ 10 → min → 30 ℃ 10min → 10 ℃ 25min, then mixed with 25. mu.L standard solution of 1mM TCEP, 50mM NaCl, 50mM HEPES, and NF-. kappa.4 dissolved in a solution of pH 7.4, after incubation at 37 ℃ for 0.5h, the glassy carbon electrode of claim 2 was soaked in the solution and incubated at 37 ℃ for 2.0 h.

5. The NF-kB electrochemical detection method based on the traditional glassy carbon electrode as claimed in claim 1, wherein MB specifically embedded in PNA-DNA hybrid chain is selected as an electric signal molecule, peak current value of MB at different concentrations of NF-kB is measured by differential pulse voltammetry, the electrode in claim 4 is soaked in 100 μ L of solution containing 20mM MB and 20mM Tris-HCl and having pH value of 7.4, incubated for 2h at room temperature in a dark place, washed by double distilled water, and then placed in 5mL of solution containing 20mM Tris-HCl and having pH value of 7.4 for differential pulse voltammetry analysis, and parameters are set as follows: initial potential-0.6V, final potential 0.3V, potential increment 0.004V, amplitude 0.05V, pulse width 0.05s and pulse period 0.5 s.

6. The NF- κ B electrochemical detection method according to claim 1, wherein a linear equation is obtained by plotting the relationship between NF- κ B concentration and peak current value: -55.05x +7.101, R²And (5) when the concentration is 0.995, y is the peak current ip mu A of the DPV, x is the concentration ng/mL of the NF-kappa B, and the content of the NF-kappa B in the sample to be detected is calculated by detecting an electric signal.

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