CN108680629B

CN108680629B - Preparation method of DNA sensing electrode based on anionic porphyrin-carbon nano tube

Info

Publication number: CN108680629B
Application number: CN201810258857.1A
Authority: CN
Inventors: 宁静恒; 刘龙; 王敏; 罗新
Original assignee: Changsha University of Science and Technology
Current assignee: Changsha University of Science and Technology
Priority date: 2018-03-27
Filing date: 2018-03-27
Publication date: 2020-08-28
Anticipated expiration: 2038-03-27
Also published as: CN108680629A

Abstract

The invention belongs to the technical field of electrochemistry and nano materials, and relates to a preparation method of a DNA sensing electrode. The modification steps are as follows: firstly, Carbon Nanotubes (CNTs) are dispersed in Tris-HCl buffer solution by ultrasonic wave, then ssDNA and ssDNA which is complementary and matched with the ssDNA are added (the ssDNA is hybridized into dsDNA), and finally, anionic porphyrin (TSPP) solution is added. And obtaining the TSPP/dsDNA/CNTs/GCE modified electrode by a dripping method. The electrode preparation method has simple process and short preparation time, and is an excellent detection electrode. The DNA biosensor electrode prepared by the invention combines the specificity of DNA complementary pairing and the high sensitivity of electrochemical detection, and is expected to provide a new way for early diagnosis of cancer cells.

Description

Preparation method of DNA sensing electrode based on anionic porphyrin-carbon nano tube

Technical Field

The invention belongs to the technical field of electrochemistry and nano materials, relates to the technical field of preparation of tetra-sulfophenyl porphyrin (TSPP) modified electrodes, and particularly relates to a preparation method of a DNA sensing electrode based on an anionic porphyrin-carbon nano tube and application of the DNA sensing electrode in ssDNA detection.

Background

The nano material technology is an emerging subject crossed by multiple subjects developed in the early 90 s of the 20 th century, and the development thereof opens up a new level for human to know and understand the world. Carbon Nanotubes (CNTs) are of great interest to scientists because of their unique physicochemical properties, such as conductor and semiconductor properties, extremely high mechanical strength, large specific surface area and aspect ratio, large number of catalytic sites, etc. At present, the mass preparation of high quality CNTs is becoming more and more mature, however CNTs themselves have disadvantages, such as insoluble and infusible, easy to entangle and agglomerate, and lack of surface functional groups, which makes it difficult to process and manipulate, and poor in dispersibility and stability, and due to the above problems, scientists have also made efforts to find out modifications which can improve the properties of CNTs, and then turned the eye light to porphyrin compounds, which are compounds with 4 pyrrole molecules pi-pi large conjugated rings, have rigid planar structures and high stability, and these characteristics make them have molecular recognition and abundant photoelectric properties. It is known that a large polar group such as a pyridyl group, a sulfonic group, an amino group, or a carboxyl group, and a metal ion have good water solubility, and therefore, a water-soluble porphyrin derivative having good water solubility can be obtained by combining porphyrin and these ionic groups. The composite synthesized by the water-soluble porphyrin and the carbon nano material by a chemical method combines the excellent properties of the two substances, overcomes the respective defects at the same time, and has wide application prospect in the fields of chemistry and biological detection.

The DNA modified electrode is used as an important means for DNA structural analysis and detection, is widely applied to electrochemical research of gene biosensors and interaction of DNA and drug molecules, and in the preparation engineering of the DNA biosensors, the immobilization of DNA on the surface of the electrode is the key point of research. In summary, the present invention ultrasonically disperses CNTs in Tris-HCl buffer solution by a very simple drop-coating method, adds ssDNA and its complementary paired ssDNA, and finally adds an anionic porphyrin (TSPP) solution. The DNA biosensor electrode prepared by the invention combines the specificity of DNA complementary pairing and the high sensitivity of electrochemical detection, and is expected to provide a new way for early diagnosis of cancer cells.

Disclosure of Invention

The invention aims to develop a preparation method of a DNA sensing electrode based on an anionic porphyrin-carbon nano tube, and provides a modified electrode with simple operation and excellent performance and a preparation method thereof. The invention can achieve the qualitative detection of DNA while preparing the modified electrode, and in order to realize the purpose, the invention adopts the following technical scheme:

a preparation method of a DNA sensing electrode based on anionic porphyrin-carbon nanotubes comprises the following steps:

(1) 0.3 μm and 0.05 μm α -Al for Glassy Carbon Electrode (GCE)₂O₃Polishing and cleaning, ultrasonic cleaning in deionized water, ethanol and deionized water, and air drying at room temperature.

(2) Ultrasonically dispersing carbon nanotubes in 0.05M Tris-HCl buffer solution (with the pH value of 8.00) containing 0.1M KCl to obtain CNTs dispersion liquid with the mass concentration of 2.5 × 10^-3g L^-1。

(3) Firstly, carbon nano tube CNTs are dispersed in Tris-HCl buffer solution in an ultrasonic mode, then ssDNA and ssDNA which is complementary and matched with the ssDNA are added, dsDNA is formed through hybridization, and finally, tetrasulfophenylporphyrin TSPP solution is added. And then, the buffer solution is dripped on the surface of the electrode by a dripping method to obtain the TSPP/dsDNA/CNTs/GCE modified electrode.

(4) And (3) establishing a three-electrode system by taking the modified electrode as a working electrode, a platinum wire as an auxiliary electrode and AgCl-Ag as a reference electrode, detecting on a CHI-660E electrochemical workstation, and acquiring an electric signal.

The material of the invention can be uniformly modified on the surface of the electrode, and the electrode modified by the nano material has good electrochemical performance and good electrochemical response to DNA.

Drawings

FIG. 1 is a cyclic voltammogram of electrochemical responses of different material modified electrodes (a GCE; b CNTs/GCE; cTSPP/GCE; d TSPP/CNTs/GCE).

FIG. 2 is a differential pulse voltammogram of the electrochemical response of modified electrodes to single-double stranded DNA (a GCE; bTSPP/ssDNA/CNTs/GCE; c TSPP/dsDNA/CNTs/GCE).

FIG. 3 is a differential pulse voltammogram of the electrochemical response of different modified electrodes to single-double stranded DNA (a GCE; bssDNA/CNTs/GCE; c dsDNA/CNTs/GCE; d TSPP/ssDNA/CNTs/GCE; eTSPP/dsDNA/CNTs/GCE).

FIG. 4 is a graph of the AC impedance of the electrochemical response of different modified electrodes to single-double stranded DNA (a GCE; bsDNA/CNTs/GCE; c dsDNA/CNTs/GCE; d TSPP/ssDNA/CNTs/GCE; eTSPP/dsDNA/CNTs/GCE).

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings.

Example 1 preparation method of the present invention for an anionic porphyrin-carbon nanotube based DNA sensing electrode

Mixing GCE with α -Al of 0.3 μm and 0.05 μm₂O₃Polishing to form a mirror surface, respectively ultrasonically cleaning by using ethanol and secondary distilled water in sequence, and airing at room temperature. Ultrasonic dispersing carbon nanotube in0.05M Tris-HCl pH 8.00 in 0.1M KCl buffer to give a CNTs dispersion at 2.5 × 10 mass concentration^-3g L^-1The ssDNA (2.0 × 10)^-6M) and their complementary counterparts ssDNA (2.0 × 10)^-7M) was added to the prepared CNTs dispersion followed by TSPP solution (2.5 × 10)^-7M), TSPP can adsorb on the surface of CNTs through pi-pi action, but cannot approach the CNTs due to the repulsion action of ssDNA; however, the dsDNA formed by hybridization reaction falls off from the surface of the carbon nano tube, and at the same time, TSPP is adsorbed on the surface of CNTs through pi-pi action, and finally, the modified electrode is obtained through a dropping coating method.

EXAMPLE 2 detection of electrochemical Properties of DNA biosensor electrode manufactured according to the present invention

(1) Electrochemical performance detection of different modified electrodes

Respectively taking GCE, CNTs/GCE, TSPP/CNTs/GCE modified electrodes as working electrodes, platinum wires as auxiliary electrodes and Ag-AgCl as reference electrodes to establish a three-electrode system, and performing electrochemical reaction on a CHI-660E working station at a potential range of-0.2-0.6V and at a potential range of 0.005M [ Fe (CN)₆]^3-/4-In a 0.10M KCl electrolyte solution, electrochemical performance characterization is carried out by adopting electrochemical Cyclic Voltammetry (CV), as shown in figure 1, the modified electrode (figure 1 curve d) has larger peak current and smaller potential difference compared with a single modified electrode (figures 1 curves a, b and c), which shows that the modified electrode has excellent conductivity, and the modified electrode prepared by the method is more beneficial to electron transfer on the surface of the electrode. The result of inspecting the electrochemical behavior of the composite membrane electrode by adopting a cyclic voltammetry shows that the modified electrode TSPP/CNTs/GCE obtained by the invention has good electrochemical response in an electrolyte solution.

(2) Detection of TSPP/CNTs/GCE modified electrode on DNA

The invention takes TSPP/ssDNA/CNTs/GCE and TSPP/dsDNA/CNTs/GCE as working electrodes, and differential pulse voltammetry scanning is carried out on Tris-HCl with 0.05M and pH 8.00 containing 0.1M KCl buffer solution to obtain a differential pulse voltammogram, as shown in figure 2. The peak current of the dsDNA modified electrode (curve c in FIG. 2) after complementary pairing can be obviously distinguished from the peak current of the modified electrode (curve b in FIG. 2) in the presence of ssDNA, which indicates that TSPP/dsDNA/CNTs/GCE can successfully detect complementary pairing base sequence DNA.

(3) Detection of DNA by different modified electrodes

Using GCE, ssDNA/CNTs/GCE, dsDNA/CNTs/GCE, TSPP/ssDNA/CNTs/GCE, TSPP/dsDNA/CNTs/GCE as working electrodes, and performing differential pulse voltammetry scanning on Tris-HCl containing 0.1M KCl buffer solution with the pH value of 8.00 at 0.05M to obtain a differential pulse voltammetry diagram, which is shown in FIG. 3. In the presence of TSPP, the peak current difference (curves d and e in FIG. 3) relative to TSPP/ssDNA/CNTs/GCE and TSPP/ssDNA/CNTs/GCE is significantly larger than that in the absence of TSPP (curves b and c in FIG. 3), which indicates that the qualitative detection of complementary paired DNA can be successfully achieved in the presence of TSPP and also indicates the importance of TSPP; also, 0.005M [ Fe (CN) ] in 0.05M Tris-HCl buffer (pH 7.40)₆]^3-/4-And 0.20M KCl buffer solution, the modified electrode is characterized by an alternating current impedance method to obtain an alternating current impedance diagram, as shown in FIG. 4, the conductivity is best in the presence of TSPP (FIG. 4, curves d and e), the electron transmission is facilitated, and complementary paired DNA exists, (FIG. 4, curve e) the impedance is minimum (consistent with a differential pulse voltammogram, the peak current is maximum), which indicates that the complementary paired DNA can be detected by the alternating current impedance method.

Claims

1. A preparation method of a DNA sensing electrode based on anionic porphyrin-carbon nano tubes is characterized by comprising the following steps:

firstly, ultrasonically dispersing carbon nano tube CNTs in a Tris-HCl buffer solution, then adding ssDNA and ssDNA complementary and matched with the ssDNA, hybridizing to obtain dsDNA, finally adding a tetrasulfophenylporphyrin TSPP solution, and then obtaining the TSPP/dsDNA/CNTs/GCE modified electrode by a dripping method.

2. The method for preparing the DNA sensing electrode based on the anionic porphyrin-carbon nano tube as claimed in claim 1, which is characterized by comprising the following steps:

(1) 0.3 μm and 0.05 μm α -Al for GCE₂O₃Polishing and cleaning, ultrasonic cleaning in ethanol and deionized water respectively, and air drying at room temperature;

(2) firstly, ultrasonically dispersing carbon nano tube CNTs in a Tris-HCl buffer solution, then adding ssDNA and ssDNA complementary and matched with the ssDNA, hybridizing to obtain dsDNA, finally adding a tetrasulfophenylporphyrin TSPP solution, and finally obtaining a TSPP/dsDNA/CNTs/GCE modified electrode by a dripping method;

(3) and establishing a three-electrode system by taking the modified electrode as a working electrode, jointly inserting the three-electrode system into an electrolyte solution, carrying out electrochemical detection on the three-electrode system, collecting an electric signal, carrying out data analysis and obtaining an analysis result.

3. The method for preparing DNA sensing electrode based on anionic porphyrin-carbon nanotube as claimed in claim 1, wherein the carbon nanotube is multi-walled carbon nanotube MWCNTs with length of 5-15 μm and diameter of 10-20nm, and mass concentration is 2.5 × 10^-3g L^-1The molarity of the tetrasulfophenylporphyrin TSPP was 2.5 × 10^-7M。

4. The method of claim 1, wherein ssDNA concentration is 2.0 × 10^-6M, complementary paired ssDNA concentration 2.0 × 10^-7M。

5. The method of claim 2, wherein the buffer solution is Tris-HCl buffer solution with concentration of 0.05M and pH 8.00, and contains 0.1M KCl.

6. The method for preparing the DNA sensing electrode based on the anionic porphyrin-carbon nano tube as claimed in claim 2, wherein the adopted three-electrode system is as follows: the TSPP/dsDNA/CNTs/GCE modified electrode is used as a working electrode, a platinum wire is used as an auxiliary electrode, and AgCl-Ag is used as a reference electrode.

7. The method for preparing a DNA sensing electrode based on anionic porphyrin-carbon nanotubes as claimed in claim 2, wherein the electrochemical detection method is cyclic voltammetry, differential pulse voltammetry or alternating current impedance method.

8. The method for preparing DNA sensing electrode based on anionic porphyrin-carbon nanotube as claimed in claim 7, wherein the electrolyte solution of the three-electrode system is Tris-HCl buffer solution with concentration of 0.05M and pH of 7.40, and contains 0.005M [ Fe (CN) ]according to cyclic voltammetry and AC impedance method₆]^3-/4-And 0.20M KCl, and the buffer solution of the three-electrode system is Tris-HCl buffer solution with the concentration of 0.05M and the pH value of 7.40 and contains 0.1M KCl by differential pulse voltammetry.