CN102286371A - Alternating current impedance type deoxyribonucleic acid (DNA) electrochemical sensor based on probe DNA control assembly interface - Google Patents

Abstract

The invention discloses an AC impedance type DNA electrochemical sensor based on probe DNA to control the assembly interface . At room temperature, the DNA and the gold electrode are modified to the surface of the gold electrode through the chemical bonding of the Au-S bond and the adsorption of the probe base and the gold, so that the capture probe DNA lies flat on the gold electrode surface to form a capture probe DNA. Assembly layer; on the surface of the capture probe DNA assembly layer, bovine serum albumin is used as a blocking agent and a protective agent. Using the change of impedance value before and after hybridization as an indicator signal, this method uses surface assembly chemistry technology to construct a "flat-lying" DNA probe recognition interface, and at the same time keeps the shape of the probe lying flat on the electrode surface without being affected by blank hybridization conditions. ; This method not only has the characteristics of high sensitivity and good specificity.

Description

AC Impedance DNA Electrochemical Sensor Based on Probe DNA Controlled Assembly Interface

technical field

The invention relates to a preparation method of an AC impedance type DNA electrochemical sensor based on a DNA-controlled assembly interface, and belongs to the technical field of biosensing.

Background technique

The AC impedance method is the process of using the change of the electrode surface resistance to reflect the change of the electrode surface morphology. The modified electrode is studied by electrochemical impedance spectroscopy, and the Randle circuit is used for simulation. The obtained Nyquist diagram includes two parts: semicircle and oblique line, of which the semicircle The part is the high-frequency region, which is controlled by the electron transfer process; the linear part is the low-frequency region, which represents the diffusion-controlled step. The change of the Nyquist diagram can reflect the magnitude of the electron transfer rate on the electrode surface, so as to understand the information about the dynamics of DNA hybridization and the structural properties of the electrode interface. This method reduces the non-specific adsorption caused by the addition of beacon substances in the enzymatic method, and does not require the use of hybridization indicators, so it has received more and more attention in the research of sensors.

There are two factors that affect the electron transfer resistance of the electrode surface. One is the charge effect, that is, the increase of the electronegative substance on the electrode surface causes the electronegative electrochemically active substance in the electrolyte solution [Fe(CN) ₆ ] ^3-/4- The repulsion of the electrode increases, the Ret value of the electrode surface increases; the other is the channel effect, that is, when the electrode surface assembly layer has pores, [Fe(CN) ₆ ] ^3-/4- in the electrolyte solution can easily pass through the pores The channel reaches the electrode surface, which accelerates the electron transfer rate and reduces the Ret value of the electrode surface.

The principle and detection process of the traditional AC impedance type DNA electrochemical sensor are: (1) The single-stranded DNA probe is fixed to the surface of the gold electrode by low-temperature self-assembly through the combination of thiol and gold. In addition to sulfhydryl groups, there are a small amount of DNA bases; (2) Mercaptohexanol (MCH) is used as a blocking agent for secondary assembly. While MCH blocks the exposed gold sites, its terminal sulfhydryl groups compete for DNA bases. The gold site makes the DNA chain detached from the electrode surface and is in a relatively upright state; (3) When the probe DNA assembly layer on the electrode surface hybridizes with the target DNA solution under hybridization conditions, the DNA chain on the electrode surface increases, phosphoric acid The number of negative charges generated by the skeleton increases, and the steric hindrance also increases, which has a greater hindrance to the electron exchange of negatively charged electrochemical probes such as [Fe(CN) ₆ ] ^3-/4- on the electrode surface, and the resistance Ret increase.

However, due to the high density of DNA probes on the electrode surface, the traditional AC impedance DNA electrochemical sensor has obvious steric hindrance and charge effects before DNA hybridization. When the concentration of the detected target DNA is low, the increased The steric hindrance and charge are very limited, and the AC impedance value of the hybridization reaction increases but does not change significantly, which makes the sensitivity of the method not high.

The present invention designs a new method, uses surface assembly chemistry technology to construct a "flat" DNA probe recognition interface, and detects target DNA by controlling the shape change of DNA before and after hybridization on the electrode surface, which greatly improves the sensitivity of the method.

Contents of the invention

The object of the present invention is to provide a kind of surface assembly chemistry technology to construct " flat " type DNA probe recognition interface and corresponding high-sensitivity AC impedance type DNA electrochemical sensor and its preparation method, its principle and detection process are shown in Figure 1 Show.

In order to achieve the above object, the present invention adopts the following technical scheme, a kind of AC impedance type DNA electrochemical sensor based on DNA control assembly interface described in the present invention, comprises electrode and capture probe DNA , and electrode adopts gold electrode, capture probe DNA The thiol-modified DNA is characterized in that the thiol-modified DNA is modified to the surface of the gold electrode at room temperature through the chemical bonding of the Au-S bond and the adsorption of the base part of the probe to the gold electrode to capture the probe DNA. Lay flat on the surface of the gold electrode to form a capture probe DNA assembly layer; bovine serum albumin is used as a blocking agent and a protective agent on the surface of the capture probe DNA assembly layer.

The preparation method of the AC impedance type DNA electrochemical sensor based on the DNA control assembly interface of the present invention comprises the following steps:

(1) Construction of "flat-lying" probe DNA recognition interface

According to the literature [1], thiol-modified DNA will interact with gold in addition to thiol, and the interaction between the base and gold is relatively weak, but the speed is fast. According to this principle, the sulfhydryl-modified single-stranded capture probe DNA was quickly assembled at room temperature, and it was modified to the surface of the gold electrode through the chemical bonding of the Au-S bond and the adsorption of the probe base part and gold. At this time, the capture probe DNA lies flat on the surface of the electrode to form a capture probe DNA assembly layer, and a large number of gold sites are occupied. The assembly electrode with the capture probe DNA assembly layer is placed in the ferricyanide solution, [Fe(CN) ₆ ] ^3-/4- is difficult to reach the gold surface of the assembly electrode through the DNA assembly layer of the capture probe, the electron transfer rate is slow, and the AC impedance value Ret is very large (Figure 2).

Literature [1]: Tonya M. Herne, and Michael J. Tarlov, Characterization of DNA Probes Immobilized on Gold Surfaces, J. Am. Chem. Soc., 1997, 119 (38), 8916-8920.

(2) Sealing and protection of the probe DNA recognition interface

Bovine serum albumin (BSA) is used as a blocking agent and a protective agent, on the one hand to block the exposed gold sites to reduce non-specific adsorption, and on the other hand to protect the probe DNA assembly layer from other factors (such as heating, pH, etc.) The impact of the electrode remains flat on the electrode surface.

(3) Hybridization and detection

After the probe DNA hybridizes with its complementary sequence, a rigid DNA double helix structure is formed. At this time, the DNA base is separated from the electrode surface, and the double helix structure stands upright on the electrode surface, exposing the space originally occupied by the DNA chain and forming ions. The channel that can enter and exit freely, the electron transfer rate increases significantly, and the Ret value of the electrode decreases significantly. Since the width of a DNA strand is much smaller relative to its length, even a tiny amount of probe DNA undergoes a conformational change due to hybridization, exposing a greater number of gold surface sites and generating electrochemically usable probes. The channel for ions to freely enter and exit improves the detection sensitivity. If the measured sequence cannot hybridize with the probe DNA, the assembly layer on the electrode surface has no morphological change, and the impedance value does not change significantly at this time. Therefore, the identification and detection of complementary and non-complementary DNA strands can be realized through the change of impedance value. Utilize the present invention to the detection result of DNA as shown in Fig. 3, Fig. 4.

From the results in Figure 3A, it can be seen that when the capture probe hybridizes to a completely complementary base sequence (curve a), the impedance value decreases significantly, indicating that the probe can hybridize well with its completely complementary base sequence to form a double-helix DNA. The double-stranded DNA stands upright on the surface of the electrode. In the measurement impedance solution, [Fe(CN) ₆ ] ^3-/4- quickly passes through the pores between the DNA layers to reach the electrode surface for redox reaction, electron transfer occurs, and the electrode surface impedance value decrease. When the capture probe interacts with a single-base mismatch sequence (curve b), although the electrode surface impedance is significantly reduced compared with the blank signal, the signal generated by the fully complementary sequence is significantly increased, which fully reflects the single-base mismatch sequence. The difference in hybridization efficiency between the matched sequence and the complete complementary sequence. The signal generated by the completely mismatched synthetic sequence (curve d) is very close to the blank signal (curve e), indicating that the constructed DNA electrochemical sensor has good sequence specificity. In addition, the influence of long-chain sequences on the determination results was investigated with salmon sperm DNA. The experimental results show that as long as there is no sequence correlation, the signal generated by long-chain DNA (curve c) has no significant difference from the blank value (curve e). This further demonstrates the good selectivity of the method.

From the results in Figure 4, it can be seen that the built AC impedance DNA electrochemical sensor is used to quantitatively detect specific gene sequences. Within a certain range, with the increase of the target DNA concentration, the impedance value gradually decreases. When the concentration of the complementary chain is in the range of 1.0×10 ^-13 to 1.0×10 ^-8 M, the impedance value has a linear relationship with the logarithm of the DNA concentration, and the detection limit is 3.0×10 ^-14 M.

The invention provides a specific detection method of an AC impedance type DNA electrochemical sensor based on the DNA-controlled assembly interface, which is characterized in that the concentration of the target DNA is within the range of 1.0×10 ^-13 to 1.0×10 ^-8 M, and the impedance value is the same as that of the DNA The concentration has a linear-logarithmic function relationship, and the detection limit is 3.0×10 ^-14 M.

The AC impedance type DNA electrochemical sensor based on the DNA controlled assembly interface of the present invention is used to identify single base mismatch sequences and complete complementary sequences.

The advantages of the present invention are: the single-stranded probe DNA modified by sulfhydryl group is quickly assembled with the gold electrode at room temperature, and it is modified to the gold electrode through the chemical bonding of the Au-S bond and the adsorption of the probe base part and gold. On the electrode surface, the capture probe DNA lies flat on the surface of the gold electrode to form a capture probe DNA assembly layer, and a large number of gold sites are occupied. The assembly electrode with the capture probe DNA assembly layer is placed on the ferricyanide pair In the solution, it is difficult for [Fe(CN) ₆ ] ^3-/4- to reach the gold surface of the assembled electrode through the capture probe DNA assembly layer, the electron transfer rate is slow, and the AC impedance value Ret is very large, see the probe DNA in Figure 2 AC impedance plot of the identification interface. There are bovine serum albumin (BSA) blocking agent and protective agent on the surface of the capture probe DNA assembly layer; after the capture probe DNA hybridizes with the target DNA of its complementary sequence, a rigid DNA double helix is formed upright on the surface of the gold electrode structure, the resistance in ferricyanide solution decreases. The above-mentioned impedance-type electrochemical DNA sensor based on surface assembly chemistry is highly sensitive for identifying complementary sequences, with a detection limit of 3.0×10 ^-14 M, and can quantitatively detect specific gene sequences for identifying single bases Mismatched sequences and perfectly complementary sequences.

Description of drawings

Fig. 1 is a schematic diagram of an AC impedance type DNA electrochemical sensor based on probe DNA controlled assembly interface.

Among them: the capture probe DNA in the figure is marked as 1, the target DNA is marked as 2, the bovine serum albumin (BSA) blocking agent and protective agent is marked as 3, and the electrode is marked as 4.

Figure 2 is the AC impedance diagram of the probe DNA recognition interface: (a) bare gold electrode (b) assembled at 25 °C for 2 h.

Figure 3A is the AC impedance diagram of probe DNA hybridized with 10 nM DNA of different sequences: complete complementary DNA (a), single base mismatch DNA (b), complete mismatch DNA (c), salmon sperm DNA (d ), blank signal (e).

Fig. 3B is a histogram of AC impedance values after the probe DNA hybridizes with DNA of different sequences.

Figure 4 A is the AC impedance diagram of probe DNA hybridized with different concentrations of target DNA; the concentrations from a to f are 10 nM, 1 nM, 100pM, 10 pM, 1 pM, 100 fM, respectively.

Figure 4B is a logarithmic linear relationship graph between impedance value and target DNA concentration (concentration range 10 nM~100 fM).

Detailed ways

As shown in Figure 1, the AC impedance type DNA electrochemical sensor based on the probe DNA control assembly interface of the present invention includes electrodes, capture probe DNA and bovine serum albumin , the electrode is preferably a gold electrode 4, and capture probe DNA 1 The thiol-modified single-stranded DNA is preferred, and the thiol-modified DNA will also interact with gold in addition to the thiol, and the interaction between the base and gold is relatively weak, but the speed is fast. The sulfhydryl-modified single-stranded probe DNA was quickly assembled with the gold electrode at room temperature, and modified to the surface of the gold electrode through the chemical bonding of the Au-S bond and the adsorption of the probe base part and gold. The probe DNA 1 lies flat on the surface of the gold electrode 4 to form a capture probe DNA assembly layer, a large number of gold sites are occupied, and the assembly electrode with the capture probe DNA assembly layer is placed in the ferricyanide solution, [ It is difficult for Fe(CN) ₆ ] ^3-/4- to reach the gold surface of the assembly electrode through the DNA assembly layer of the capture probe, the electron transfer rate is slow, and the AC impedance value Ret is very large, see the AC of the probe DNA recognition interface in Figure 2 Impedance diagram. There are bovine serum albumin (BSA) blocking agent and protective agent 3 on the surface of the DNA assembly layer of the capture probe; after the hybridization reaction of the capture probe DNA 1 and the target DNA 2 of its complementary sequence, a rigid vertical structure is formed on the surface of the gold electrode. DNA double helix structure, the resistance of ferricyanide solution is reduced.

Example 1:

The preparation steps of the AC impedance type DNA electrochemical sensor based on the probe DNA controlled assembly interface are as follows:

(1) The gold electrode was ultrasonically cleaned with Piranha solution (30% H ₂ O ₂ and 98% concentrated H ₂ SO ₄ at a volume ratio of 1:3) for 5 min, and cleaned twice with deionized water. 5 min each time, and then polished to a mirror surface with a mixture of 0.3 μm, 0.05 μm Al ₂ O ₃ and water, and then ultrasonically cleaned with ethanol and distilled water. Place the sonicated electrode in 0.5 M H ₂ SO ₄ for cyclic voltammetry scanning until it is stable, wash it with double distilled water, and dry it with N ₂ before use;

(2) Take 4 μl of probe DNA (oligonucleotides synthesized by Baosheng Bioengineering Co., Ltd.) solution, drop-coat it on the surface of the pretreated bare gold electrode, leave it at room temperature for 2 hours, wash it with PBS washing solution, double distilled water Rinse the surface of the electrode separately, dry it with nitrogen, soak the electrode in 300 μl of 0.5% BSA solution for 15 min, wash it with double distilled water, and dry it with nitrogen for later use.

Example 2:

The detection steps of the target DNA by the AC impedance type DNA electrochemical sensor based on the probe DNA controlled assembly interface are as follows:

(1) The capture probe immobilized on the surface of the electrode obtained in Example 1 and the target DNA (oligonucleotides synthesized by Baosheng Bioengineering Co., Ltd.) were hybridized in a hybridization buffer solution at 37 °C for 40 min in a water bath to form double-stranded DNA. Rinse the surface of the electrode with 10 mM PBS washing solution (pH 7.4) to remove unhybridized DNA strands, and then rinse with double distilled water before testing;

(2) Immerse the electrode prepared in step (1) in a solution of 4 mM K ₃ [Fe(CN) ₆ ]/K ₄ [Fe(CN) ₆ ] (1:1) containing 0.1 M KCl, the open circuit potential is the initial potential, the frequency range is 10 ⁵ Hz to 1.0 Hz, and the AC impedance curve is recorded. The AC impedance diagram is shown in curve a of Fig. 3A.

Example 3:

The preparation of AC impedance type DNA electrochemical sensor and the detection steps of PML/RARα fusion gene are as follows:

(1) The gold electrode was ultrasonically cleaned with Piranha solution (30% H ₂ O ₂ and 98% concentrated H ₂ SO ₄ at a volume ratio of 1:3) for 5 min, and cleaned twice with deionized water. 5 min each time, and then polished to a mirror surface with a mixture of 0.3 μm, 0.05 μm Al ₂ O ₃ and water, and then ultrasonically cleaned with ethanol and distilled water. Place the sonicated electrode in 0.5 M H ₂ SO ₄ for cyclic voltammetry scanning until it is stable, wash it with double distilled water, and dry it with N ₂ before use;

(2) The sequence of the DNA capture probe labeled with thiol at the 5' end is: 5'-SH-(CH ₂ ) ₆ -T ₁₀ CTTCA GAACT GCTGC TCTGG GTCTC AATGG-3', prepared into a 1 μM solution;

(3) Take 4 μl of the capture probe DNA solution in step (2), drop-coat it onto the surface of the bare gold electrode that has been pretreated in step (1), and leave it at room temperature for 2 hours, then wash it with PBS washing solution and double distilled water respectively. On the surface of the electrode, dry it with nitrogen, soak the electrode in 300 μl of 0.5% BSA solution for 15 minutes, wash it with double distilled water, and dry it with nitrogen for later use;

(4) Hybridize the capture probe immobilized on the surface of the electrode with the target DNA in a hybridization buffer solution at 37°C for 40 minutes to form double-stranded DNA, and rinse the surface of the electrode with 10 mM PBS washing solution (pH 7.4) to remove unhybridized DNA. The DNA strand was washed with double distilled water and then tested;

(5) Immerse the electrode in a solution of 4mM K ₃ [Fe(CN) ₆ ]/K ₄ [Fe(CN) ₆ ] (1:1) containing 0.1 M KCl, the open circuit potential is the initial potential, and the frequency range is 10 ⁵ Hz～1.0Hz, record the AC impedance curve. The AC impedance diagram is shown in Figure 4A, and the relationship between impedance and concentration is shown in Figure 4B.

From the experimental data of the above examples and accompanying drawings, it can be proved that the sulfhydryl-modified probe DNA of the present invention can be quickly assembled with the gold electrode at room temperature, through the chemical bonding of the Au-S bond and the base part of the probe with the gold electrode. The adsorption of gold modifies it to the surface of the gold electrode. At this time, the capture probe DNA 1 lies flat on the surface of the gold electrode 4 to form a capture probe DNA assembly layer, which is the "flat-lying" type probe DNA recognition interface described in the present invention. , and based on this, an AC impedance DNA electrochemical sensor with a DNA-controlled assembly interface was constructed.

Claims (5)

1. An AC impedance DNA electrochemical sensor based on the DNA-controlled assembly interface, including an electrode and a capture probe DNA , the electrode adopts a gold electrode (4), and the capture probe DNA (1) adopts sulfhydryl-modified DNA, which is characterized by The thiol-modified DNA is modified to the surface of the gold electrode through the chemical bonding of the Au-S bond and the adsorption of the probe base and the gold electrode at room temperature, so that the capture probe DNA (1) lies flat on the gold electrode ( 4) A capture probe DNA assembly layer is formed on the surface; on the surface of the capture probe DNA assembly layer, bovine serum albumin is used as a blocking agent and a protective agent (3). 2. the preparation method of the AC impedance type DNA electrochemical sensor based on DNA control assembly interface described in claim 1, comprises the steps: (1) Construction of "flat-laying" capture probe DNA recognition interface The sulfhydryl-modified single-stranded probe DNA is used as the capture probe DNA, which is modified to the surface of the gold electrode at room temperature through the chemical bonding of the Au-S bond and the adsorption of the probe base part and gold. The needle DNA lies flat on the electrode surface to form a capture probe DNA assembly layer; (2) Sealing and protection of the DNA recognition interface of the capture probe Bovine serum albumin (BSA) was used as a blocking agent and a protective agent, which was added on the surface of the DNA assembly layer of the capture probe and the surface of the gold electrode. 3. The specific detection method of the AC impedance type DNA electrochemical sensor based on the DNA control assembly interface according to claim 1, characterized in that 1) after the capture probe DNA hybridizes with the target DNA of its complementary sequence, the capture probe The base of DNA breaks away from the electrode surface to form a rigid DNA double helix structure. The DNA double helix structure stands upright on the electrode surface, exposing the space originally occupied by the DNA chain and forming a channel for ions to enter and exit freely; 2) when The target DNA of the measured sequence cannot hybridize with the capture probe DNA, so the capture probe DNA assembly layer on the electrode surface has no morphological change, and the impedance value does not change significantly at this time, realizing the recognition and detection of non-complementary DNA strands. 4. According to the specific detection method of the AC impedance type DNA electrochemical sensor based on the DNA control assembly interface according to claim 3, it is characterized in that the concentration of the target DNA is in the range of 1.0×10 ^-13 ~ 1.0×10 ^-8 M, and the impedance value It has a linear logarithmic function relationship with the concentration of DNA, and the detection limit is 3.0×10 ^-14 M. 5. The AC impedance DNA electrochemical sensor based on the DNA controlled assembly interface of claim 1 is used to identify single-base mismatch sequences and complete complementary sequences.

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