CN101464424B - Test method and system for researching DNA molecule conductivity - Google Patents

Abstract

The invention proposes a testing method and system for studying the conductivity of DNA molecules. The system is composed of a signal generator, a digital oscilloscope, an ultra-micro-integrated double-ring carbon electrode, a micro-current amplifier, and a computer combination. An electrostatic field is formed through an ultra-micro-integrated double-ring carbon electrode to induce DNA molecules to be polarized along the vector direction of the field strength. The two ends with positive and negative charges are formed, and they are arranged in parallel between the inner and outer ring carbon electrodes. The input signal is sent to the ultra-micro-integrated double-ring carbon electrode to obtain an output signal, which is amplified by a micro-current amplifier, collected by a digital oscilloscope, and A/D After the conversion, it is sent to the computer, and the relationship between the input signal and the output signal is analyzed to study the conductivity of DNA molecules. The system and method have a slight force without damaging the DNA molecule, know the overall conductivity, monitor the electron transfer process in real time, track the fast electron transfer behavior, avoid the influence of the joint quality, and are easy to renew the surface and simple in the instrument.

Description

A test method and test system for studying the conductivity of DNA molecules

technical field

The invention relates to the technical fields of electroanalytical chemistry, molecular electronic devices and biosensors, in particular to a system and method for studying the conductivity of DNA molecules.

Background technique

In recent years, research on the conductivity of deoxyribonucleic acid (DNA) molecules has attracted a lot of attention. Because of its double helix structure, the bases pair with each other and form large conjugated π bonds between the entire molecular chain. When the DNA molecule is subjected to Electrons are generated when ionizing radiation or ultraviolet light is irradiated, and the electrons move along the DNA molecular chain before being captured, causing DNA damage, which in turn interferes with the process of DNA replication or protein production, resulting in abnormalities or death of cells. Recent research results show ( http://www.its.caltech.edu/~jkbgrp/ ) that certain proteins can send charges along the long DNA chain, and there is another corresponding protein in the distance that can detect this charge, But when the above-mentioned abnormality occurs on the DNA, due to the destruction of the original stack state, the overlap of electron orbits is reduced, and the conductivity becomes poor, so that the second protein cannot receive the test charge, and the repair system will start. Therefore, the study of the conductivity of DNA molecules can deepen the understanding of DNA damage caused by light-induced charge transfer, cell mutation caused by charge transfer, and DNA repair. It has broad application prospects in tumor therapy and gene therapy. In addition, the simplicity of bases, the constancy and specificity of complementarity, the diversity of genetic information, the specificity of conformation and topological targeting in the process of DNA replication are all the designs required by nanotechnology. Principle; and DNA has stable physical and chemical properties and unique linear structure, such as the diameter of DNA is only 2nm, and its length spans microscopic and macroscopic. These properties make DNA an ideal material for making nanowires and nanodevices. Therefore, the research on the conductivity of DNA molecules occupies an extremely important position not only in the field of life sciences but also in the field of nanomaterials.

At present, the methods for studying the conductivity of DNA molecules mainly include: spectroscopy, biochemistry, electrochemical methods, electron microscopy and atomic force microscopy. Spectroscopy method (J.Am.Chem.Soc., 2000, 122: 5893; Biochemistry, 2000, 39: 6190; J.Am.Chem.Soc., 2000, 122: 11545) covalently bonds photooxidants in On the DNA chain, light-induced electron transport triggers base oxidation, causing DNA damage to study DNA conductivity. Conductivity of non-DNA molecules as a whole; biochemical method (J.Am.Chem.Soc., 1995, 117:6406; Biochem.Biophys.Res.Commun., 1992, 188:1) inserts a compound into DNA At one end of the helix, guanine is oxidized under light induction, and the DNA is broken at the position of oxidized guanine by treatment with piperidine, and the DNA fragment obtained is observed by radioactivity to study its conduction behavior. The main method of this method is The disadvantage is that this biochemical technique can only observe the final reaction result caused by electron transfer, and cannot monitor the electron transfer process in real time, and cannot study the overall conductivity of DNA molecules; electrochemical methods (Curr.Opin.Chem.Biol., 2001, 5: 209) to study the conductivity of DNA by detecting the electrochemical behavior of electroactive substances on DNA modified electrodes, that is, to select electroactive substances that can specifically bind to DNA molecules as electrochemical probes to adjust the base sequence control Its insertion position on the DNA chain can be used to study the distance between the electroactive substance and the electrode, the base accumulation and the sequence's influence on the charge transfer. The disadvantage of this method is that the electrochemical technique used For cyclic voltammetry, the information that can be provided is limited, and the scanning speed that can be used is relatively small. Even if the fastest known ultrafast voltammetry is used, it is only about 1MHz, and it is impossible to track the fast electron transfer inside the DNA molecule. Electron microscopy (Nature, 1999,398:407) and atomic force microscopy (Chinese patent application number: 200610147822.8), connect the two ends of the metal electrode with a piece of DNA, and directly measure its current-potential relationship. This method is straightforward. But its disadvantage is: since only one DNA is connected to the metal electrode, the quality of the joint between them seriously affects the measurement results. If the connection between the DNA and the electrode is not good, it is possible that the DNA itself is conductive, but the measured The result of insulation, on the contrary, if the electrode substrate used for measurement has a short circuit, it is also possible that the DNA itself is insulated, but the result of conductivity is measured. All in all, the stability and reliability of the result are not good.

Contents of the invention

The primary technical problem to be solved by the present invention is to provide a test method for studying the conductivity of DNA molecules in view of the existing background technology. It does not damage the DNA molecules, and what is known is the overall conductivity of the DNA molecules; the electron transfer process can be monitored in real time; The excitation signals are diverse and fast, which can provide rich information and track the fast electron transfer inside the DNA molecule; the number of contact molecules is relatively large, so as to avoid the impact of poor contact quality on the results; the surface is easy to renew, and the front end of the electrode is cut off to form a new section. Yes; the equipment used is simple.

Another technical problem to be solved by the present invention is to provide a related test system for studying the conductivity of DNA molecules in view of the existing background technology.

The technical solution adopted by the present invention to solve the above-mentioned primary technical problems is: a test method for studying the conductivity of DNA molecules, which is characterized in that an electrode whose inner and outer layers are conductive layers is used, so that an electric field is established between the inner and outer conductive layers of the electrode. Under the induction of an electric field, the DNA molecules freely dispersed in the solution are polarized along the vector direction of the field strength to form positive and negative charged ends, forming an oriented parallel arrangement of the DNA molecules between the inner and outer conductive layers of the electrode. One conductive layer inputs electrical signals, and the other conductive layer outputs electrical signals. After amplification and A/D conversion, the relationship between input electrical signals and output electrical signals is analyzed by computer, so as to study the conductivity of DNA molecules.

The technical scheme adopted by the present invention to solve the above-mentioned another technical problem is: a test system for studying the conductivity of DNA molecules, which is characterized in that the inner and outer layers are electrodes with conductive layers, one conductive layer of the electrode is connected to the signal generator, and the other The conductive layer is connected to the amplifier, so that an electric field is established between the inner and outer conductive layers of the electrode. Under the induction of the electric field, the DNA molecules freely dispersed in the solution are polarized along the vector direction of the field strength to form positive and negative ends. Form the directional parallel arrangement of DNA molecules between the inner and outer conductive layers of the electrode. The signal output of the amplifier passes through the A/D converter and is connected to the computer for analysis and processing. According to the relationship between the input electrical signal and the output electrical signal, the conduction of DNA molecules is studied. sex.

As an improvement, the electrode adopts a double-ring electrode, the inner and outer conductive layers of which are distributed between the inner surface and the outer surface of the ring-shaped electrode substrate, presenting a ring shape, forming an inner ring electrode and an outer ring electrode, which is convenient for the substrate. Forming on top.

As an improvement, the manufacturing method of the double-ring electrode includes the following steps:

(1) Take a glass capillary substrate and wrap its outer wall with tape for a certain length, such as 1/3 of the length;

(2) By heating and cracking organic gas such as alkane with a low boiling point, inner and outer carbon layers are formed on the inner and outer walls of the glass capillary substrate, and the inner carbon layer becomes the outer conductive layer and the inner conductive layer respectively, and the inner conductive layer acts as the inner carbon electrode. Lead out from the wire through the conductive glue, and fix it with epoxy resin as a connection end; remove the tape wrapped on the outer wall, and the remaining 2/3 length of the outer conductive layer acts as the outer ring carbon electrode, lead out from the wire through the conductive glue, and fix it with epoxy resin as another connection end;

(3) impregnating and covering the above-mentioned structure with an inner insulating layer and an outer insulating layer;

(4) The front end of the glass capillary substrate coated with carbon layer and insulating layer is cut vertically, and the neat section exposed is the ultramicro integrated double-ring carbon electrode.

Preferably, the carbon layer coating adopts the method of heating and cracking CH ₄ , and its thickness is adjusted by changing the time of heating and cracking, and is controlled between 5-10 nm, which is convenient for operation and operation at low temperature.

Preferably, the impregnation coating of the inner insulating layer and the outer insulating layer is to immerse the above-mentioned structure in insulating varnish, take it out after 5-15 minutes, and then dry it at 80-100°C to form the inner insulating layer and the outer insulating layer. The insulating layer is very convenient and easy to make in this way, and the cost is also very low.

Preferably, the inner ring electrode is connected to the input electrical signal, and the outer ring electrode is connected to the output electrical signal, which reduces measurement errors and is more convenient for testing.

Finally, the output signal generated by the signal generator is divided into two paths, one path is directly connected to a digital oscilloscope to be collected, and the other path enters from one end of the DNA molecule through the inner ring carbon electrode of the ultramicro-integrated double-ring carbon electrode, passes through the entire DNA molecule, Then the output signal is obtained through the outer ring carbon electrode. The output signal is amplified by the micro-current amplifier and then connected to a digital oscilloscope to be collected. Finally, the analog signal collected by the digital oscilloscope is converted into a digital signal by A/D and sent to a computer for analysis and processing. Record. This facilitates the real-time observation and comparison of various input signals and output signals.

Compared with the prior art, the present invention has the advantages of:

(1) In the present invention, on the inner ring carbon electrode and the outer ring carbon electrode of the ultramicro-integrated bicyclic carbon electrode, appropriate positive and negative DC potentials are respectively applied to form an electrostatic field, and under the induction of the electrostatic field, the The DNA molecule is polarized along the vector direction of the field strength to form positive and negative ends. The negative end is placed on the positively charged inner ring carbon electrode, and the positive end is placed on the negatively charged outer ring. On the carbon electrode, an oriented parallel arrangement of DNA molecules is formed between the inner ring carbon electrode and the outer ring carbon electrode. The force is mainly electrostatic attraction, which is slight and does not damage DNA molecules;

(2) In the present invention, one end of the DNA molecule is placed on the inner ring carbon electrode, and the other end is placed on the outer ring carbon electrode. The input signal enters from one end of the DNA molecule through the inner ring carbon electrode, passes through the entire DNA molecule, and then passes through the outer ring carbon electrode. The electrode gets the output signal, so what is known is the conductivity of the DNA molecule as a whole rather than the DNA fragment;

(3) In the present invention, after the input signal generated by the signal generator is sent to the ultramicro integrated double-ring carbon electrode, the output signal can be obtained immediately, and the relationship between the input signal and the output signal can be analyzed to study the conductivity of DNA molecules and realize real-time Monitoring the electron transfer process;

(4) The present invention can use multiple waveforms of the signal generator to conduct research as the excitation signal, and the information is rich and diverse;

(5) The present invention can use high-frequency input signals to track the fast electron transfer behavior inside the DNA molecule;

(6) In the present invention, there are more DNA molecules aligned in parallel between the inner ring carbon electrode and the outer ring carbon electrode, and the number of contact molecules is relatively large, so as to avoid the influence of poor contact quality on the measurement results;

(7) What the present invention knows is the conductivity of more DNA molecules arranged in parallel rather than the conductivity of a DNA molecule. The number of DNA molecules can be determined according to the size of the DNA molecule itself and the inner ring carbon electrode (2), the distance between the DNA molecules Estimate the mutual exclusion between them;

(8) It is very easy to renew the surface of the supermicro-integrated double-ring carbon electrode in the present invention, just cut off the front end vertically to expose the fresh surface, the operation is simple, and the stability is good;

(9) The present invention is used for studying the conductivity of DNA molecules, and the required instruments are simple and cheap, and only need an arbitrary waveform generator (or function generator), digital oscilloscope, microcurrent amplifier, and computer to build a system, and the total price is only About 1/30~1/50 of the price of STM or CF-AFM, and the research system does not require operating costs;

(10) The manufacturing process of the ultramicro-integrated double-ring carbon electrode in the present invention is simple, practical, easy to operate, easy to control the manufacturing conditions, and low in cost. It can be manufactured in general chemical laboratories and has good popularization and application value.

The system and method for studying the conductivity of DNA molecules provided by the present invention form an electrostatic field through an ultramicro-integrated double-ring carbon electrode, and induce DNA molecules freely dispersed in a solution to be polarized along the vector direction of the field strength to form positively and negatively charged The two ends of the two ends are arranged in parallel between the inner ring carbon electrode and the outer ring carbon electrode to study the conductivity of the DNA molecule. The force is slight without damaging the DNA molecule. Knowing the overall conductivity of the DNA molecule and monitoring the electron transfer process in real time can be used. Track the fast electron transfer behavior inside the DNA molecule, avoid the impact of poor contact quality on the results, the surface is easy to renew, and the instruments used are simple, which can be used to study the conductivity of DNA molecules.

Description of drawings

Figure 1 is a schematic diagram of the system for studying the conductivity of DNA molecules;

Fig. 2a, Fig. 2b are respectively the structural sectional view (M) and the cross-sectional view (N) of the ultramicro-integrated bicyclic carbon electrode described in the present invention;

Fig. 3 is a schematic diagram of the directional parallel arrangement of DNA molecules on the ultramicro-integrated double-ring carbon electrode;

Fig. 4 is the current-potential (I～V) curve of natural calf thymus deoxyribonucleic acid (CTDNA) on the ultramicro-integrated double-ring carbon electrode.

Its figure 1:

1. Glass capillary substrate, 2. Inner ring carbon electrode, 3. Outer ring carbon electrode, 4. Conductive adhesive, 5. Wire, 6. Epoxy resin, 7. Conductive adhesive, 8. Wire, 9. Epoxy resin , 10, inner insulating layer, 11, outer insulating layer; M, longitudinal section, N, cross section.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

Example

In this embodiment, a Tektronix AFG3021B arbitrary waveform generator, a Tektronix 2012B digital oscilloscope, a TJ-110 microcurrent amplifier, and a P4 computer are selected to build a research system, as shown in Figure 1. The inner and outer layers are double-ring electrodes with conductive layers. One conductive layer is connected to the digital signal generator, and the other conductive layer is connected to the micro-current amplifier. The output of the amplifier and the other output of the digital signal generator are respectively connected to the input terminals of each channel of the digital oscilloscope. The analog signal collected by the digital oscilloscope is then passed through The A/D converter converts it into a digital signal and connects it to the computer via the RS-232 interface.

Its working principle is as follows: the arbitrary waveform generator generates an input signal, one way is directly sent to the first channel of the digital oscilloscope to be collected, and the other way is sent to the ultra-micro integrated double-ring carbon electrode to obtain an output signal, which is passed through the micro-current amplifier After being amplified, it is sent to the second channel of the digital oscilloscope to be collected, and finally the analog signal collected by the digital oscilloscope is A/D converted into a digital signal, and sent to the computer through the RS-232 interface for recording and processing, and to analyze the relationship between the input signal and the output signal. The relationship between DNA molecules can be studied.

The ultra-micro-integrated double-ring carbon electrode is made of glass capillary as the substrate, ultrasonically cleaned with ethanol and double distilled water, carbon is used as the electrode material, silver conductive glue is used for the conductive glue, and insulating varnish is used for the insulating layer. The specific production method is: cut off a glass capillary with a length of 3-4 cm, an inner diameter of 4 μm, an outer diameter of 20 μm, and a wall thickness of 8 μm, ultrasonically clean it with ethanol and double distilled water and dry it completely, then wrap about 1/3 of the length of the outer wall with adhesive tape, and use CH ₄ continuously passes through the inside and outside of the capillary, and the resistance wire is heated for 30 minutes, so that the carbon produced by the cracking of CH ₄ adheres to the inner and outer walls of the capillary; the carbon layer on the inner wall acts as the inner ring carbon electrode 2, which is led out by the wire 5 through the conductive glue 4, and epoxy resin 6 Fixed to become the connection end of the input electrical signal of the double-ring electrode; remove the tape wrapped on the outer wall, and the remaining outer carbon layer of about 2/3 length acts as the outer ring carbon electrode 3, which is led out by the wire 8 through the conductive glue 7, and epoxy resin 9. Fix it to become the connection terminal of the output electrical signal of the double-ring electrode; immerse the front end of the above structure in the insulating varnish, take it out after about 5-10 minutes, and then dry it at 80-100°C to form the inner insulating layer 10, the outer insulating layer Layer 11: Use a special ceramic quartz cutting blade to cut off the front end vertically to expose a fresh surface, and obtain an ultra-micro-integrated double-ring carbon electrode, as shown in Figure 2.

In order to maintain the physiological pH of DNA and avoid DNA melting or precipitation, 50mmol/LNaCl+5mmol/LTris-HCl (THB) with a pH of 7.1 and low ionic strength was selected as the electrolyte solution to fully swell and disperse the DNA in it. Soak the above-mentioned ultramicro-integrated double-ring carbon electrode made in the above-mentioned bottom solution containing 0.1mg/mL natural calf thymus deoxyribonucleic acid (CTDNA), maintain the DC potential of the inner ring carbon electrode as +1.8V, and the outer ring carbon electrode The DC potential of the carbon electrode is -1.8V. After 30 minutes, take it out, wash it gently in ultrapure water, dry it, and install it according to attached figure 1. The arbitrary waveform generator emits a linear scanning potential of -0.5V～+0.5V, The speed is 1V/s, the input signal and the output signal are input to the oscilloscope as the X axis and the Y axis respectively, and the current-potential (I～V) curve is recorded, as shown in Figure 4, which shows that under the experimental conditions, CTDNA behaves as a resistance Properties of Molecular Electronic Devices. According to the slope of the I-V curve, the resistance at this time is about 10 ⁹ Ω. Considering the diameter of the DNA molecule itself is 2nm and the repulsion between each other, the influence range is estimated to be about 10nm. The circumference of the inner ring carbon electrode in this embodiment It is about 10 μm, so it can be estimated that there are about 1000 CTDNA molecules arranged in parallel between the inner ring carbon electrode and the outer ring carbon electrode, so it is estimated that the resistance of CTDNA under this experimental condition is about 10 ¹² Ω

Claims (13)

1. A test method for studying the conductivity of DNA molecules, characterized in that the inner and outer layers are used as the electrode of the conductive layer, and its electric field is established between the inner and outer conductive layers of the electrode, so that under the induction of the electric field, the DNA freely dispersed in the solution Molecules are polarized along the vector direction of the field strength to form positive and negative ends, forming an oriented parallel arrangement of DNA molecules between the inner and outer conductive layers of the electrode. One conductive layer of the electrode inputs electrical signals, and the other conductive layer The output electrical signal is amplified and A/D converted, and the relationship between the input electrical signal and the output electrical signal is analyzed by a computer, so as to study the conductivity of DNA molecules. 2. The test method according to claim 1, wherein said electrode adopts a double-ring electrode, and its inner and outer conductive layers are distributed between the inner surface and the outer surface of the ring-shaped electrode base material, presenting a ring shape, forming an inner ring electrode. ring electrode and outer ring electrode. 3. test method according to claim 2, is characterized in that the manufacture method of described double ring electrode comprises the steps: (1) Take a glass capillary substrate (1), and wrap its outer wall with adhesive tape for a certain length; (2) By heating and cracking the organic matter gas, inner and outer carbon layers are formed on the inner and outer walls of the glass capillary substrate (1). 2), lead out from the wire (5) through the conductive glue (4), and fix it with epoxy resin (6); remove the tape wrapped on the outer wall, and the remaining length of the outer conductive layer acts as the outer ring carbon electrode (3), through the conductive glue ( 7) Lead out from the wire (8) and fix with epoxy resin (9); (3) impregnating and covering the above-mentioned structure with an inner insulating layer (10) and an outer insulating layer (11); (4) The front end of the glass capillary substrate coated with carbon layer and insulating layer (1) is cut vertically, and the neat section exposed is the ultramicro integrated double-ring carbon electrode. 4. The test method according to claim 3, characterized in that the carbon layer coating adopts the method of heating and cracking CH4, and its thickness is adjusted by changing the time of heating and cracking, and is controlled between 5-10 nm. 5. The test method according to claim 4, characterized in that the impregnation coating of the inner insulating layer (10) and the outer insulating layer (11) is to immerse the above-mentioned structure in insulating varnish, and after 5 to 15 minutes, Take it out, and then dry it at 80-100° C. to form an inner insulating layer (10) and an outer insulating layer (11). 6. The testing method according to claim 5, characterized in that the inner ring electrode is connected to the input electrical signal, and the outer ring electrode is connected to the output electrical signal. 7. A test system for studying the conductivity of DNA molecules is characterized in that the inner and outer layers of the electrode are electrodes with conductive layers, one conductive layer of the electrode is connected to the signal generator, and the other conductive layer is connected to the amplifier, so that the inner and outer conductive layers of the electrode are connected to the amplifier. An electric field is established, and under the induction of the electric field, the DNA molecules freely dispersed in the solution are polarized along the vector direction of the field strength to form positive and negative charged ends, forming the orientation of the DNA molecules between the inner and outer conductive layers of the electrode Arranged in parallel, the signal output of the amplifier passes through the A/D converter and is connected to the computer for analysis and processing. According to the relationship between the input electrical signal and the output electrical signal, the conductivity of DNA molecules is studied. 8. The test system according to claim 7, characterized in that the electrode adopts a double-ring electrode, and its inner and outer conductive layers are distributed between the inner surface and the outer surface of the ring-shaped electrode base material, presenting a ring shape, forming an inner ring electrode and a ring electrode. outer ring electrode. 9. The test system according to claim 8, characterized in that the structure of the double ring electrode is: (1) The center is a glass capillary substrate (1); (2) The inner and outer carbon layers are formed on the inner and outer walls of the glass capillary substrate (1) by heating and cracking the organic matter gas. The outer and inner carbon layers become the outer conductive layer and the inner conductive layer respectively, and the inner conductive layer acts as the inner ring carbon electrode ( 2), lead out from the wire (5) through the conductive glue (4), and fix it on the upper end of the base material with epoxy resin (6); the outer conductive layer acts as the outer ring carbon electrode (3), and the wire (8) lead out, be fixed on the outer surface of base material with epoxy resin (9); (3) impregnating and coating the above structure with an inner insulating layer (10) and an outer insulating layer (11); (4) The front end of the glass capillary substrate coated with carbon layer and insulating layer (1) is cut vertically, and the neat section exposed is the ultramicro integrated double-ring carbon electrode. 10. The test system according to claim 9, characterized in that the carbon layer coating adopts the method of heating and cracking CH ₄ , and its thickness is adjusted by changing the time of heating and cracking, and is controlled between 5-10 nm. 11. The test system according to claim 10, characterized in that the impregnation coating of the inner insulating layer (10) and the outer insulating layer (11) is to immerse the above-mentioned structure in insulating varnish, and after 5 to 15 minutes, Take it out, and then dry it at 80-100° C. to form an inner insulating layer (10) and an outer insulating layer (11). 12. The test system according to claim 11, characterized in that the inner ring electrodes are connected to input electrical signals, and the outer ring electrodes are connected to output electrical signals. 13. The test system according to claim 12, characterized in that the output signal generated by the signal generator is divided into two paths, one path is directly connected to a digital oscilloscope to be collected, and the other path passes through the inner ring of the ultramicro integrated double-ring carbon electrode The carbon electrode (2) enters from one end of the DNA molecule, passes through the entire DNA molecule, and then passes through the outer ring carbon electrode (3) to obtain an output signal, which is amplified by a micro-current amplifier and then connected to a digital oscilloscope to be collected. The analog signal is converted into a digital signal by A/D and sent to a computer for analysis, processing and recording.

Images