Preparation method and application of a DNA tetrahedron-based multivalent aptamer modified electrode

Abstract

The application discloses a preparation method and application of a multivalent aptamer modified electrode based on DNA tetrahedron. In the application, a co-blocked nucleic acid aptamer for recognizing antibiotics is assembled on the edge of a DNA tetrahedron structure through Hoogsteen hydrogen bond to construct a multivalent aptamer recognition interface. The antibiotics can be combined with the aptamer to cause the conformational change of the nucleic acid chain, inhibit the electron transmission efficiency of the interface, and thus cause the change of the peak current intensity, so that the quantitative detection of the antibiotics can be realized. In addition, the triple-stranded structure for fixing the nucleic acid aptamer has unique pH response characteristics. After the recognition interface constructed is combined with a target antibiotic, the co-blocked aptamer combined with the antibiotic can be dissociated from the DNA tetrahedron support by adjusting the environmental pH value, so that the regeneration of the modified electrode interface can be realized. The multivalent aptamer modified electrode prepared by the application can be regenerated and reused, the recognition efficiency is improved, the detection sensitivity is improved, and the detection of different targets can be realized.

Description

Technical Field

[0001] This invention relates to a method for preparing and applying a multivalent aptamer-modified electrode based on DNA tetrahedrons, belonging to the field of biosensing technology. Background Technology

[0002] Electrochemical biosensors based on nucleic acid aptamers have attracted widespread attention due to their high sensitivity and fast response speed. However, the nucleic acids directly immobilized on the electrode interface often exhibit conformations unfavorable to recognition, such as lying down, interstrand entanglement, and stacking, making uniform, controllable, and directional immobilization of nucleic acid aptamers on the electrode interface a challenge. Frame nucleic acids are a class of monodisperse DNA nanostructures with atomic-level precision and tunable size. Among them, the DNA tetrahedral structure formed by annealing four nucleic acid chains is widely used to modify the spacing between nucleic acid probes on the electrode interface, significantly improving its recognition kinetics and thermodynamics. However, its interface recognition characteristics depend on the sequence design of the frame nucleic acid chains, leading to the following limitations: 1. Embedding the recognition sequence into the DNA tetrahedral framework increases the cost of electrode modification; 2. The constructed recognition interface can only target a single target; to extend to other objects, re-modification is required, increasing the variability in electrode preparation; 3. The capture efficiency and detection sensitivity of single-vertex recognition domain probes for targets remain limited. Summary of the Invention

[0003] The purpose of this invention is to address technical issues such as how to construct multiple recognition domains to improve target capture efficiency and detection sensitivity, and how to achieve reusability and regeneration of modified electrode interfaces. This invention aims to provide a method for preparing a multivalent aptamer-modified electrode based on DNA tetrahedrons. In this invention, a co-block nucleic acid aptamer probe for antibiotic recognition is formed into a triplet via Hoogsteen hydrogen bonds and assembled onto the edges of the DNA tetrahedral framework nucleic acid, constructing a multivalent aptamer recognition interface. After the antibiotic binds to the aptamer, it causes a conformational change in the nucleic acid chain, inhibiting the interface electron transport efficiency, thereby resulting in a change in peak current intensity. Quantitative detection of antibiotics can then be achieved through electrochemical detection.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is to provide a method for preparing a multivalent aptamer-modified electrode based on a DNA tetrahedron, comprising the following steps:

[0005] Step 1): Pre-treat the gold electrode;

[0006] Step 2): The prepared DNA tetrahedron (1 μM) was co-incubated with the pretreated gold electrode (12-16 h), and then co-incubated with (2 mM) MCH solution (1 h) to obtain the DNA tetrahedron modified gold electrode Au-TDN;

[0007] Step 3): The DNA tetrahedral modified gold electrode Au-TDN obtained in Step 2) is incubated with the antibiotic-recognizing co-block aptamer recognition probe TE. A triple helix structure of DNA is formed through Hoogsteen hydrogen bonds, allowing the co-block aptamer recognition probe TE to assemble onto at least one of the three edges of the DNA tetrahedral structure, thus obtaining the multivalent aptamer-modified electrode Au-TDN-TE. The co-block aptamer recognition probe TE consists of three parts: from the 5' end to the 3' end, it is the target aptamer sequence recognition region, the polythymine spacer region, and the thymine-rich anchoring region. By changing the sequence of the aptamer recognition region, binding to different targets can be achieved, thereby realizing the detection of different targets.

[0008] Preferably, the pretreatment in step 1) specifically includes: polishing, ultrasonic treatment, cleaning and drying the gold electrode in sequence.

[0009] Preferably, the ultrasonic treatment includes: placing the electrodes in ethanol and Milli-Q water for ultrasonic treatment, respectively.

[0010] Preferably, the cleaning process includes: sequentially cleaning the electrode by CV scanning in NaOH solution and H2SO4 solution, and then rinsing the gold electrode surface with Milli-Q water.

[0011] Preferably, the drying is performed by blowing with nitrogen gas.

[0012] Preferably, in step 2), the four single-stranded nucleic acid molecules used to prepare the DNA tetrahedron are selected from single-stranded nucleic acid molecules with sequences as shown in SEQ ID NO: 1 to 8.

[0013] Preferably, the DNA tetrahedron is assembled from single strands of nucleic acid with sequences shown in SEQ ID NO: 1 to 4; or from single strands of nucleic acid with sequences shown in SEQ ID NO: 5, SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 7; or from single strands of nucleic acid with sequences shown in SEQ ID NO: 5, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 8.

[0014] Preferably, the sequence of the co-block aptamer recognition probe TE in step 3) is as shown in SEQ ID NO: 9 or SEQ ID NO: 10.

[0015] The present invention also provides the application of the multivalent aptamer-modified electrode prepared by the above preparation method in the detection of antibiotics.

[0016] Preferably, the method for detecting antibiotics includes: incubating the prepared Au-TDN-TE with the sample to be tested, and measuring the change in current intensity by electrochemical means to detect antibiotics in the sample to be tested; the principle of the detection is that the antibiotics in the sample to be tested bind to the target recognition sequence in Au-TDN-TE, causing a change in the conformation of the nucleic acid chain, inhibiting the interface electron transport efficiency, thereby causing a change in the peak current intensity.

[0017] Preferably, the detection includes quantitative detection and qualitative detection, wherein the quantitative detection method includes: mixing antibiotic solutions of different concentrations, recording the current intensity using differential pulse voltammetry in the range of 0.6 to -0.2V, establishing a linear relationship between different concentrations of antibiotics and the change in current intensity, i.e., establishing a standard curve; by detecting the change in current intensity of the sample to be tested after incubation with Au-TDN-TE, substituting it into the standard curve, the concentration of antibiotics in the sample to be tested is obtained, thereby achieving quantitative detection of antibiotics.

[0018] This invention first constructs three independent DNA tetrahedra by adjusting partial sequences, supporting the assembly of different numbers of co-cutting aptamer recognition probes. Each co-cutting aptamer recognition probe consists of three parts: from the 5' end to the 3' end, they are the aptamer sequence recognition region, the polythymine spacer region, and the thymine-rich anchoring region. Changing the aptamer sequence in the recognition region can extend to the detection of other targets; the polythymine sequence is used for the spacer recognition region and the anchoring region sequence; the thymine-rich anchoring region sequence, under neutral pH conditions, can form a triplet helical structure through Hoogsteen hydrogen bonds, assembling the co-cutting probe onto the edges of the DNA tetrahedral structure. By changing the number of assembly sites, up to three probes can be assembled simultaneously. Due to the unique pH dependence of triple-stranded DNA, adjusting the environmental pH to alkaline can dissociate the antibiotic-bound co-cutting aptamer from the DNA tetrahedral scaffold, achieving regeneration of the modified electrode interface. Figure 1 As shown.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1. The DNA triple-strand structure used to immobilize co-block nucleic acid aptamers in this invention has unique pH response characteristics; after the constructed recognition interface binds to the target antibiotic, the antibiotic-bound co-block aptamer recognition probe can be dissociated from the DNA tetrahedral scaffold by adjusting the environmental pH value, thereby realizing the regeneration of the modified electrode interface and improving the reproducibility of the modified electrode.

[0021] 2. The DNA tetrahedral-based nucleic acid scaffold in this invention not only adjusts the modification density of nucleic acid aptamers at the electrode interface, but also improves recognition efficiency and detection sensitivity through the anchoring of multiple recognition domains; in addition, by changing the recognition domain sequence in the co-modified aptamer, it can also be used for the detection of other targets. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the principle of the present invention.

[0023] Figure 2 Cyclic voltammetric scans of bare gold electrode, TDN-modified gold electrode, TDN-TE, and TDN-TE bound to kanamycin.

[0024] Figure 3 The graph shows the linear relationship between the signal differences obtained using three-edge (3TE'), two-edge (2TE'), one-edge (1TE') and single-stranded aptamers and different concentrations of kanamycin.

[0025] Figure 4 The binding kinetics of kanamycin with different concentrations were measured using three-sided (A) and single-chain (B) aptamers.

[0026] Figure 5 This is a linear relationship diagram when the recognition region sequence is replaced with the ampicillin aptamer sequence.

[0027] Figure 6 A schematic diagram illustrating how pH adjustment enables multiple regeneration of the modified electrode interface. Detailed Implementation

[0028] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0029] Example 1

[0030] Preparation of three independent DNA tetrahedra:

[0031] TA, TB, TC, and TD can be assembled to form a recognition probe containing three co-intercalated aptamer anchoring regions; TA-1, TB, TC-1, and TD-1 form a recognition probe containing one co-intercalated aptamer anchoring region; and TA-1, TB, TC, and TD-2 form a recognition probe containing two co-intercalated aptamer anchoring regions (sequences are shown in Table 1).

[0032] DNA tetrahedral assembly sequences (e.g., TA, TB, TC, TD) containing different numbers of co-block aptamer anchoring region recognition probes were mixed in equimolar ratios, and then 30 mM of TCEP (tris(2-carboxyethyl)phosphine) was added to bring the final DNA concentration to 1 μM. The mixture was then subjected to slow annealing using a PCR instrument and cooled to 4°C for storage.

[0033] Example 2

[0034] A method for preparing a multivalent aptamer-modified electrode based on DNA tetrahedrons:

[0035] 1) Pretreatment of gold electrodes (polishing - ultrasonic treatment - cleaning - drying)

[0036] The gold electrodes were polished for 3 minutes each in 0.3 and 0.05 μm alumina solutions. Next, the electrodes were ultrasonically treated for 3 minutes each in ethanol and Milli-Q water. The electrodes were then cleaned by CV scanning in 0.5 M NaOH and 0.5 M H₂SO₄. Finally, the gold electrode surface was rinsed with Milli-Q water and dried with nitrogen for later use.

[0037] 2) Preparation of DNA tetrahedrons

[0038] The preparation method is the same as in Example 1.

[0039] 3) Multivalent aptamer-modified electrodes based on DNA tetrahedrons

[0040] The prepared DNA tetrahedrons (1 μM) were incubated overnight at 4 °C with the polished electrode. The modified gold electrode was then immersed in 2 mM MCH (6-mercaptohexanol) for 1 hour, followed by incubation at 25 °C for 90 minutes with TE solution. Finally, the modified gold electrode was incubated in target solutions of different concentrations for 40 minutes.

[0041] Table 1 Nucleic Acid Sequences

[0042]

[0043] The tetrahedral DNA sequence can form a triple-stranded DNA structure with the probe TE (TE*) via Hoogsteen hydrogen bonds of "T·A·T" and "C·G·C". The co-modular aptamer recognition probe TE (TE*) consists of three parts: from the 5' end to the 3' end, it comprises the kanamycin aptamer sequence recognition region, the polythymine spacer region, and the thymine-rich anchoring region. Changing the aptamer sequence in the recognition region can extend the detection to other targets; the polythymine sequence is used for the spacer recognition region and the anchoring region sequence; the thymine-rich anchoring region sequence, under neutral pH conditions, can form a triplet helical structure via Hoogsteen hydrogen bonds, assembling the co-modular probe onto the edges of the DNA tetrahedral structure. By changing the number of assembly sites, up to three probes can be assembled simultaneously.

[0044] Example 3

[0045] Regeneration and repeated measurement of the identification interface: After the measurement was completed, the electrode was incubated with the dissociation buffer solution for 90 min, then incubated with TE solution at 25 °C for 90 min, then incubated with the target solution for 40 min, and finally electrochemical measurement was performed.

[0046] Example 4

[0047] Construction and measurement of universal identification interfaces:

[0048] The prepared DNA tetrahedron (1 μM) was incubated overnight at 4 °C with a polished gold electrode. The modified gold electrode was then immersed in 2 mM MCH (6-mercaptohexanol) for 1 hour, followed by incubation at 25 °C for 90 minutes with TE* (a co-block aptamer containing an ampicillin aptamer). Finally, the modified gold electrode was incubated in ampicillin solutions of different concentrations for 40 minutes, and electrochemical measurements were performed.

[0049] Figure 2 The images show cyclic voltammetry scans of (a) bare gold electrode, (b) TDN-modified gold electrode, (c) TDN-TE, and (d) TDN-TE bound to kanamycin. The current intensity gradually decreases with DNA modification, which to some extent indicates the successful preparation of TDN-TE.

[0050] Figure 3 The results showed that, within a certain concentration range of kanamycin solution, the signal differences obtained from the detection of three-edge, two-edge, one-edge, and single-stranded aptamers were all linearly correlated with the kanamycin concentration. Furthermore, the highest detection limit obtained by this method was two orders of magnitude higher than the lowest detection limit, thus improving the recognition performance.

[0051] Figure 4 The results showed that when using three-edged and single-chain aptamers to detect different concentrations of kanamycin, the results were as follows: (using three-edged aptamers...) Figure 4The k' value of A is higher than that when using a single chain ( Figure 4 B) is about 4 times higher, indicating that it binds to kanamycin at a faster rate.

[0052] Figure 5 The results showed that when the ampicillin aptamer sequence was changed (TE*), the current response signal still had a good linear relationship with the concentration of ampicillin solution, indicating that the proposed recognition strategy can be extended to other targets.

[0053] Figure 6 The results show that the recognition interface can still be reconstructed after five elutions and reassemblies, indicating that the proposed construction strategy can achieve the regeneration of the modified electrode interface and improve the reproducibility of the modified electrode.

[0054] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a multivalent aptamer-modified electrode based on DNA tetrahedrons, characterized in that, Includes the following steps: Step 1): Pre-treat the gold electrode; Step 2): The prepared DNA tetrahedrons were co-incubated with the pretreated gold electrode, and then co-incubated with MCH solution to obtain the DNA tetrahedron modified gold electrode Au-TDN; Step 3): The DNA tetrahedral modified gold electrode Au-TDN obtained in Step 2) is incubated with the antibiotic-recognizing co-block aptamer recognition probe TE. A triple helix structure of DNA is formed through Hoogsteen hydrogen bonds, allowing the co-block aptamer recognition probe TE to assemble onto at least one of the three edges of the DNA tetrahedral structure, thus obtaining the multivalent aptamer-modified electrode Au-TDN-TE. The co-block aptamer recognition probe TE consists of three parts: from the 5' end to the 3' end, it is the target aptamer sequence recognition region, the polythymine spacer region, and the thymine-rich anchoring region. Binding to different targets can be achieved by changing the sequence of the aptamer recognition region.

2. The preparation method according to claim 1, characterized in that, The pretreatment in step 1) specifically includes: polishing, ultrasonic treatment, cleaning and drying the gold electrode in sequence.

3. The preparation method according to claim 2, characterized in that, The ultrasonic treatment includes: placing the electrodes in ethanol and Milli-Q water for ultrasonic treatment, respectively. And / or, the cleaning includes: sequentially cleaning the electrode using CV scanning in NaOH solution and H2SO4 solution, followed by rinsing the gold electrode surface with Milli-Q water; And / or, the drying is performed by blowing with nitrogen gas.

4. The preparation method according to claim 1, characterized in that, In step 2), the four single-stranded nucleic acid molecules used to prepare the DNA tetrahedron are selected from single-stranded nucleic acid molecules with sequences as shown in SEQ ID NO: 1 to 8.

5. The preparation method according to claim 4, characterized in that, The DNA tetrahedron is formed by assembling single strands of nucleic acid with sequences shown in SEQ ID NO: 1 to 4; or by assembling single strands of nucleic acid with sequences shown in SEQ ID NO: 5, SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 7; or by assembling single strands of nucleic acid with sequences shown in SEQ ID NO: 5, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:

8.

6. The preparation method according to claim 1, characterized in that, The sequence of the co-block aptamer recognition probe TE in step 3) is shown in SEQ ID NO: 9 or SEQ ID NO:

10.

7. The application of the multivalent aptamer-modified electrode prepared by the preparation method according to any one of claims 1 to 6 in the detection of antibiotics.

8. The application according to claim 7, characterized in that, The method for detecting antibiotics includes: incubating the prepared Au-TDN-TE with the sample to be tested, and measuring the change in current intensity by electrochemical measurement to detect antibiotics in the sample to be tested; the principle of the detection is that the antibiotics in the sample to be tested bind to the target recognition sequence in Au-TDN-TE, causing a change in the conformation of the nucleic acid chain, inhibiting the interface electron transport efficiency, thereby causing a change in the peak current intensity.

9. The application according to claim 8, characterized in that, The detection includes quantitative and qualitative detection. The quantitative detection method includes: mixing antibiotic solutions of different concentrations, recording the current intensity using differential pulse voltammetry in the range of 0.6 to -0.2V, establishing a linear relationship between different concentrations of antibiotics and the change in current intensity, i.e., establishing a standard curve; by detecting the change in current intensity of the sample to be tested after incubation with Au-TDN-TE, substituting it into the standard curve, the concentration of antibiotics in the sample to be tested is obtained, thus achieving quantitative detection of antibiotics.

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