G4-DNA tetrahedral probe electrode, its preparation method, application and application method

Abstract

This invention discloses a G4-DNA tetrahedral probe electrode, its preparation method, applications, and application methods. This invention utilizes four DNA strands to self-assemble into a DNA tetrahedral structure based on Watson-Crick hybridization. The DNA tetrahedron and G4 strand can be connected to the electrode based on the interaction between thiol groups and the gold electrode, and complementary base pairing. The addition of the DNA tetrahedron, through the construction of a reasonable probe density, further improves the detection sensitivity. This invention utilizes square wave voltammetry for electrochemical detection of malachite green. The content of malachite green can be determined based on the strength of the current signal of its characteristic peak. Rapid detection of malachite green can be achieved in a short time, with a minimum detection concentration of 3.65 μg / kg, showing potential for on-site detection. Six repeated tests using this electrode yielded almost identical detection signals, indicating that the electrode has good reusability and stability.

Description

Technical Field

[0001] This invention belongs to the field of rapid detection technology of malachite green and is an electrochemical detection method. Specifically, it relates to a G4-DNA tetrahedral probe electrode, its preparation method, application and application method. Background Technology

[0002] Aquatic products provide humans with high-quality protein and are among the fastest-growing food production sectors. Currently, aquatic product production meets consumer demand, but food quality and safety issues are increasing. Aquatic products suffering from certain diseases require veterinary drug treatment to maintain yield and economic value, but excessive drug residues reduce the quality and safety of these products. Malachite Green (MG) is a triphenylmethane industrial dye. In the 1930s, it was discovered that malachite green could kill bacteria, fungi, and parasites on the surface of fish, especially effective against saprolegniasis. Many countries widely use malachite green as an insecticide and fungicide in aquaculture to prevent and treat various saprolegniasis, gill rot, and ichthyophthiriasis in aquatic animals. Since the 1990s, researchers both domestically and internationally have discovered that the functional group of malachite green, triphenylmethane, has carcinogenic, teratogenic, and mutagenic (three-fold toxicity) side effects.

[0003] However, due to its effectiveness and low cost in preventing and treating fish diseases, and the current lack of effective substitutes, the illegal use of malachite green by some producers and operators driven by profit in the farming, transportation, temporary holding, and sales of aquatic products remains a prominent issue. Furthermore, coupled with the influence of industrial wastewater pollution and residual fishpond conditions, reports of malachite green residues in aquatic products still occur frequently. Therefore, rapid and sensitive detection of malachite green in water is one of the effective means to monitor and ensure the quality and safety of fresh aquatic products and food.

[0004] Currently, common detection methods for malachite green in China generally involve large-scale instruments and equipment, including high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), enzyme-linked immunosorbent assay (ELISA), and colloidal gold immunochromatography. These methods offer advantages such as high sensitivity and accuracy, but chromatographic and mass spectrometric methods require complex sample pretreatment. Separation by chromatography followed by detection is time-consuming and cumbersome. With the deepening of nanomaterial research, nano-assembly systems and artificially synthesized nanostructures are attracting increasing attention. This means that nanomaterial research can now design, assemble, and create new systems according to people's wishes, and purposefully imbue these systems with desired properties. This technological leap provides a solid foundation for the application of nanomaterials. Electrochemical sensors achieve sensing by converting the chemical signal of the analyte into an electrical signal. Compared with other sensing signals, electrochemical signals are not affected by sample turbidity or background fluorescence, and electrochemical sensors are easily integrated with nanotechnology to achieve miniaturization and the construction of portable devices. The literature (Analytica Chimica Acta, 2020, 1099, 39-45) describes electrodes modified with cysteine-coated black scales and gold nanoparticles, using RNA aptamers modified with thiol groups and methylene blue, respectively, as recognition probes for malachite green. When malachite green is added, it causes a structural change in the aptamer, transforming it into a hairpin structure, which draws methylene blue closer to the electrode, generating an electrical signal change and thus achieving sensitive detection of malachite green. While this method is sensitive, it requires the use of multiple nanomaterials such as gold nanoparticles and cysteine-coated black scales. Furthermore, RNA aptamers are easily degraded, unstable, difficult to label, and expensive, increasing the cost of electrode construction, and the probe stability is insufficient. Summary of the Invention

[0005] To address the shortcomings of the aforementioned technologies, this invention proposes a G4-DNA tetrahedral probe electrode, its preparation method, application, and application method. Utilizing the DNA G-quadruplex (G4) capable of binding malachite green as a molecular probe, and employing DNA self-assembly tetrahedral passivation of the electrode and optimized probe density, malachite green is efficiently captured and enriched. The electrochemical signal of malachite green is then acquired using square wave voltammetry (SWV), achieving sensitive and rapid detection of malachite green.

[0006] This invention specifically utilizes a G4 chain with good affinity for malachite green, effectively enriching malachite green onto the surface of a gold electrode and generating a significant electrochemical signal. Four DNA strands are used to self-assemble into a DNA tetrahedron structure based on Watson-Crick hybridization, a simple and high-yield synthesis method. The DNA tetrahedron is attached to the electrode via the interaction of thiol groups with the gold electrode, and then the G4 chain is attached to the DNA tetrahedron based on base complementarity. The addition of the DNA tetrahedron, through the construction of a reasonable probe density, further improves the detection sensitivity. This invention uses square wave voltammetry for electrochemical detection of malachite green. The strength of the current signal from its characteristic peak can determine the content of malachite green, enabling rapid detection of malachite green within a short time, with a minimum detection concentration of 3.65 μg / kg, showing potential for on-site detection. The detection signal of this electrode remained almost unchanged after six repeated tests, indicating good reusability and stability.

[0007] The technical solution adopted in this invention is as follows:

[0008] I. A G4-DNA tetrahedral probe

[0009] The G4-DNA tetrahedral probe is composed of DNA tetrahedra bound to G4 strands.

[0010] The nucleotide sequence of the G4 chain is SEQ ID No. 5.

[0011] The DNA tetrahedron is self-assembled from four DNA strands based on Watson-Crick hybridization. The four DNA strands include DNA strand A, DNA strand B, DNA strand C, and DNA strand D. The nucleotide sequence of DNA strand A is SEQ ID No. 1; the nucleotide sequence of DNA strand B is SEQ ID No. 2; the nucleotide sequence of DNA strand C is SEQ ID No. 3; and the nucleotide sequence of DNA strand D is SEQ ID No. 4.

[0012] II. A G4-DNA tetrahedral probe electrode

[0013] The G4-DNA tetrahedral probe electrode is obtained by binding the bottom face of the DNA tetrahedron in the G4-DNA tetrahedral probe to the electrode.

[0014] The electrode is a gold electrode.

[0015] III. A method for preparing a G4-DNA tetrahedral probe electrode

[0016] The preparation method includes the following steps:

[0017] 1) Dissolve the four DNA strands separately with TE buffer to obtain four DNA solutions;

[0018] 2) Add TM buffer, TCEP solution and equal volumes of the four DNA solutions to a centrifuge tube, shake thoroughly to mix, heat in a metal bath, then remove and place on ice to cool rapidly to obtain a DNA tetrahedral probe working solution with a concentration of 1 μM.

[0019] 3) Drop the DNA tetrahedral probe working solution onto the electrode surface and cover the droplet with an object to prevent it from drying out. Incubate at room temperature and then blow dry the electrode.

[0020] 4) Drop 6-mercaptohexanol onto the electrode surface that has been dried in 3) and incubate to seal the electrode surface. Then rinse with PBS buffer and dry.

[0021] 5) After dropping the G4 probe solution onto the dried electrode surface from step 4), incubate it to allow the G4 probe to successfully connect to the DNA tetrahedron on the electrode surface, thus obtaining the G4-DNA tetrahedron probe electrode.

[0022] In step 3), the electrode is a cleaned gold electrode. The specific cleaning steps are as follows:

[0023] 1) The gold electrode was polished sequentially with 0.3 and 0.05 μm alumina, and then the polished gold electrode was subjected to ultrasonic treatment in ultrapure water, ethanol and ultrapure water in sequence to obtain the gold electrode after initial cleaning.

[0024] 2) Electrochemically clean the gold electrode after the initial cleaning in H2SO4 using cyclic voltammetry until a repeating CV curve is obtained, thus obtaining a cleaned gold electrode.

[0025] IV. Application of a G4-DNA tetrahedral probe electrode

[0026] The G4-DNA tetrahedral probe electrode is used to detect malachite green.

[0027] V. An application method for a G4-DNA tetrahedral probe electrode

[0028] The application method includes the following steps:

[0029] First, the G4-DNA tetrahedral probe electrode is inserted into a solution containing malachite green. The malachite green is enriched onto the surface of the G4-DNA tetrahedral probe electrode by stirring. Then, the malachite green is detected by square wave voltammetry, and the concentration of malachite green is obtained based on the signal intensity of the characteristic peak.

[0030] In the detection of malachite green using the square wave voltammetry method, the potential range is set to 0.2-0.8V.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] 1. This invention utilizes DNA tetrahedral nanostructures to control the density and spatial orientation of G4 probes on the electrode surface, thereby enhancing detection sensitivity;

[0033] 2. The modified electrode assembled by the present invention is regenerable and can be reused after electrochemical elution following testing;

[0034] 3. When the method constructed in this invention is combined with screen-printed electrodes and a handheld electrochemical workstation, a portable sensor can be built for on-site detection. Attached Figure Description

[0035] Figure 1 A schematic diagram of an electrode modified with DNA tetrahedrons and G4 strands for rapid detection of malachite green.

[0036] Figure 2 This is a diagram used to verify the tetrahedral assembly of DNA by polyacrylamide gel electrophoresis.

[0037] Figure 3 This is a characterization diagram of the electrode modification process.

[0038] Figure 4 The electrochemical response signals of bare gold electrode (Au), electrode directly modified with G4 chain (Au-G4), and electrode simultaneously modified with DNA tetrahedron and G4 (Au-TDN-G4) to the same concentration of malachite green are compared.

[0039] Figure 5 To optimize enrichment time.

[0040] Figure 6 To rapidly detect malachite green using DNA tetrahedral and G4-modified electrodes, the electrochemical response and linear regression equation were determined. Figure 6 (A) represents the malachite green concentration gradually increasing from 0 to 1×10⁻⁶. -4 Electrochemical response diagram of the modified electrode at mol / L; Figure 6 (B) shows the relationship between the current signal at 0.5V and the logarithmic value of malachite green concentration for the DNA tetrahedral and G4 modified electrodes.

[0041] Figure 7 Electrodes modified with DNA tetrahedrons and G4 exhibit good specificity for the detection of malachite green.

[0042] Figure 8 Electrodes modified with DNA tetrahedrons and G4 are regenerable and exhibit good detection stability.

[0043] Figure 9Performance of DNA tetrahedral and G4 modified electrodes for detecting malachite green in a temporary holding tank.

[0044] Figure 10 This is a schematic diagram of the planar structure of a G4-DNA tetrahedral probe. Detailed Implementation

[0045] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0046] G4-DNA tetrahedral probes are composed of DNA tetrahedra bound to G4 strands, and their structure is as follows: Figure 1 and Figure 10 As shown. Each single strand folds to form a triangle, constituting one face of a tetrahedron. Each edge of the tetrahedron consists of two DNA single strands, and the two DNA strands on each edge of the tetrahedron structure are complementary. One edge of the DNA tetrahedron is not completely complementary, consisting of the double-stranded portion of the edge formed by the other single-stranded DNA and a single-stranded DNA. This single-stranded DNA can bind to the G4 strand to form a G4-DNA tetrahedron probe. That is, each DNA strand forms three edges of each face, and the two nucleotide sequences corresponding to the common edges of adjacent faces are complementary, so that the four DNA strands form the four faces of the DNA tetrahedron. Among them, DNA strand B forms the base of the DNA tetrahedron, and the other three DNA strands form the three lateral faces of the DNA tetrahedron. The 3' end of DNA strand A extends from the top of the DNA tetrahedron and binds to the 3' end of the G4 strand, thereby forming a G4-DNA tetrahedron probe.

[0047] The nucleotide sequence of the selected G4 chain is SEQ ID No. 5.

[0048] A DNA tetrahedron is formed by the self-assembly of four DNA strands based on Watson-Crick hybridization. The four DNA strands are DNA strand A, DNA strand B, DNA strand C, and DNA strand D. The nucleotide sequence of DNA strand A is SEQ ID No. 1; the nucleotide sequence of DNA strand B is SEQ ID No. 2; the nucleotide sequence of DNA strand C is SEQ ID No. 3; and the nucleotide sequence of DNA strand D is SEQ ID No. 4. The selected G4 strand has a strong affinity for malachite green, and the addition of the DNA tetrahedron, through the construction of a reasonable probe density, further improves the detection sensitivity.

[0049] The G4-DNA tetrahedral probe electrode is obtained by binding the base face of the DNA tetrahedron in the G4-DNA tetrahedral probe to the electrode. The electrode is a gold electrode.

[0050] A method for preparing a G4-DNA tetrahedral probe electrode, the method comprising the following steps:

[0051] 1) Dissolve the four DNA strands separately in TE buffer to obtain four DNA solutions;

[0052] 2) Add TM buffer, TCEP solution and equal volumes of the four DNA solutions to a centrifuge tube, then shake thoroughly to mix and heat in a metal bath. After heating, remove the tube and place it on ice to cool rapidly to obtain a DNA tetrahedral probe working solution with a concentration of 1 μM.

[0053] 3) Drop the DNA tetrahedral probe working solution onto the electrode surface and cover the droplet with an object to prevent it from drying out. Incubate at room temperature and then blow dry the electrode.

[0054] 4) Drop 6-mercaptohexanol onto the electrode surface that has been dried in 3) and incubate to seal the electrode surface and prevent non-specific adsorption. Then rinse with PBS buffer and dry.

[0055] 5) After dropping the G4 probe solution onto the dried electrode surface from step 4), incubate it to allow the G4 probe to successfully connect to the DNA tetrahedron on the electrode surface, thus obtaining the G4-DNA tetrahedron probe electrode.

[0056] The assembly process of DNA tetrahedrons is as follows:

[0057] 1) First, weigh 17.20 mg of TCEP, add 2 mL of ultrapure water, and shake thoroughly to obtain a TCEP solution with a concentration of 30 mM.

[0058] Then, the buffer solution was prepared by weighing 242.80 mg of Tris and 74.50 mg of EDTA, dissolving them thoroughly in water, adjusting the pH to 8.0, and then bringing the volume to 200 mL using a volumetric flask to obtain 10 times the TE buffer (100 mM Tris + 10 mM EDTA, pH = 8.0).

[0059] Weigh 0.48g of Tris and 2.03g of MgCl2, dissolve them thoroughly in water, adjust the pH to 8.0, and then bring the volume to 200mL using a volumetric flask to obtain a 1-fold TM buffer (20mM Tris + 50mM MgCl2, pH = 8.0).

[0060] 2) Dissolve Tetra A, B, C and D strands separately in 1×TE buffer, dilute appropriately and determine the concentration to accurately prepare a DNA solution with a concentration of 50 μM;

[0061] 3) Add 41 μL of 1×TM buffer and 5 μL of 30 mM TCEP solution to a 600 μL centrifuge tube. Take 1 μL of each DNA strand (Tetra A, B, C, D) and add it to the centrifuge tube. Shake well to mix. Then heat the reaction solution in a 95°C metal bath for 2 min. After removing it, place it on ice and cool it rapidly for 1 min. The 1 μM DNA tetrahedral probe working solution can be successfully assembled and stored at 4°C for later use.

[0062] like Figure 2 As shown, a tetrahedral probe composed of four DNA strands was validated and analyzed using 10% polyacrylamide gel electrophoresis (PAGE). The formation of the tetrahedral probe product was verified by combining these four DNA strands in different ways. The results showed that the bands of two DNA strands migrated significantly slower than those of one DNA strand, the bands of three DNA strands migrated significantly slower than those of two DNA strands, and the bands with all four DNA strands present were the slowest. This is because the DNA tetrahedron has a complex three-dimensional structure, and the resulting tetrahedral probe has a much larger relative molecular mass than single-stranded DNA, thus reducing its migration rate in the gel. The significant comparison of migration distances between the bands indicates successful assembly of the DNA tetrahedron.

[0063] The electrode modification process is as follows:

[0064] 1) Polish the gold electrode with a diameter of 2 mm with 0.3 and 0.05 μm aluminum oxide respectively, and then sonicate it in ultrapure water, ethanol and ultrapure water for 1 min respectively.

[0065] 2) Electrochemically clean the electrode in 0.5M H2SO4 using cyclic voltammetry (CV) (scan rate: 100mV / s; measurement range: -0.3-1.55V) until a repeatable CV curve is obtained.

[0066] 3) Take 4 μL of the synthesized DNA tetrahedral probe working solution and drop it onto the surface of the gold electrode. Then cover the electrode with a plastic cap to prevent the solution from drying out. Incubate overnight at room temperature and then dry the electrode with N2.

[0067] 4) Take 4 μL of 3 mM 6-mercaptohexanol (MCH) and drop it onto the electrode surface. Incubate for 1 h to seal the electrode surface and prevent non-specific adsorption. Rinse with PBS buffer (0.1 M Na2HPO4 + 0.1 M NaH2PO4 + 0.1 M KCl, pH = 7.0) and dry with N2.

[0068] 5) Drop a 1 μM G4 probe prepared with 1×TM buffer onto the electrode and incubate at room temperature for 1 h to allow the G4 probe to successfully connect to the electrode surface. Finally, use the assembled electrode for the detection of malachite green.

[0069] like Figure 3 As shown in (A), electrochemical impedance spectroscopy (EIS) was used to monitor the resistance changes during electrode fabrication to characterize the electrode assembly process. The figure shows that the resistance of the bare gold electrode is very small, almost a straight line. After the DNA tetrahedra are assembled, the resistance increases significantly. With further modification by the blocking agent and G4 probe, the resistance increases further, indicating that the modified DNA self-assembly probe has been successfully assembled onto the electrode surface. The increase in resistance is caused by the passivation of the electrode surface by the tetrahedra, the blocking of MCH hindering electron transfer between the electrode surface and the solution, and the electrostatic repulsion between nucleic acid chains, indicating successful electrode construction. Figure 3 As shown in (B), biotin was modified onto G4, and horseradish peroxidase modified with streptavidin was used to verify the reliability of the electrode construction method. The results showed that horseradish peroxidase played a catalytic role, and the electrical signal was significantly enhanced, indicating that the electrode construction method designed in this invention is feasible and efficient.

[0070] The G4-DNA tetrahedral probe electrode is used to detect malachite green.

[0071] A method for applying a G4-DNA tetrahedral probe electrode, the method comprising the following steps:

[0072] First, the G4-DNA tetrahedral probe electrode is inserted into a solution containing malachite green. Malachite green is enriched onto the surface of the G4-DNA tetrahedral probe electrode by stirring, with an optimal enrichment time of 7 minutes. Then, square wave voltammetry is used to detect the malachite green, and its concentration is determined based on the signal intensity of the characteristic peak in the generated electrochemical signal.

[0073] The square wave voltammetry method was used to detect malachite green with a potential range of 0.2-0.8V, a potential increment of 0.004V, an amplitude of 0.025V, a frequency of 10Hz, and a settling time of 10s.

[0074] Specifically:

[0075] The gold electrode (Au-TDN-G4) assembled with tetrahedrons and G4 was immersed in a solution containing 1×10 -4The electrode was enriched by stirring in an electrolyte solution containing 1 mol / L MG for 0, 1, 3, 5, 7, and 9 min, respectively. Then, a cyclic voltammetry (SWV) test was performed with a potential range of 0.2–0.8 V, a potential increment of 0.004 V, an amplitude of 0.025 V, a frequency of 10 Hz, and a settling time of 10 s. After each test, the electrode was rinsed with ultrapure water and then placed in PBS electrolyte solution for 10 cycles of cyclic voltammetry at a potential range of 0.2–0.8 V and a scan rate of 50 mV / s to elute the MG from the electrode surface and regenerate the probe on the electrode surface. This allowed for the reuse of the constructed electrode and probe to detect sample solutions containing different concentrations of malachite green.

[0076] The electrochemical signal of malachite green was tested using an electrochemical workstation.

[0077] Comparative experiment on the detection effects of DNA tetrahedral structure presence and absence

[0078] Two types of electrodes were constructed: 1. G4 strand directly immobilized on the electrode surface. The G4 strand directly modified onto the electrode was Probe6-SH-2, with the sequence 5'-AATGGGTGGGATGGGTGGGAGT-C6-SH-3'. 2. G4 strand immobilized on the electrode surface using DNA tetrahedra. The same concentration of malachite green (1×10⁻⁶) was then used for detection. -4 mol / L, and compare their signal intensity.

[0079] like Figure 4 As shown, the current value measured after enriching the bare gold electrode in MG solution for 7 minutes was very small, indicating that the bare gold electrode without DNA probes had a very weak response to MG. After modifying the gold electrode with G4 chains that can recognize and bind malachite green, MG was enriched on the electrode surface due to the good affinity between G4 chains and MG. The oxidation of MG then generated a strong electrochemical signal. Modifying the electrode with G4 using DNA tetrahedra, due to the three-dimensional structure of the DNA tetrahedra, uniformly dispersed the density of the G4 probes and maintained their upright orientation, avoiding entanglement between G4 probes and non-specific adsorption to the electrode surface. Furthermore, the G4 probes formed G4 chain structures after adsorbing malachite green, further enhancing the performance of MG capture and enrichment, resulting in a significant enhancement of the electrochemical signal of MG on the electrode surface. Therefore, Au-TDN-G4 has a better ability to detect MG.

[0080] Figure 5The SWV curves of MG were measured at different enrichment times. As the enrichment time increased, the current at 0.5V gradually increased, because more MG was enriched on the electrode surface with increasing time. After 7 minutes of enrichment, the current signal tended to stabilize, indicating that at 7 minutes, the modified electrode at 1×10⁻⁶ MW⁻¹... -4 The adsorption capacity on the mol / L electrode surface had reached saturation. Therefore, the enrichment time for subsequent experiments was selected as 7 min.

[0081] Response analysis of DNA tetrahedral and G4 modified electrodes to different concentrations of malachite green

[0082] 1) Immerse the prepared DNA tetrahedron and G4-modified electrode in an immersion solution with a concentration of 1×10⁻⁶. -8 5×10 -7 1×10 -6 2×10 -6 5×10 -6 1×10 -5 5×10 -5 and 1×10 -4 SWV was detected in 1 mol / L MG electrolyte solution.

[0083] 2) Before measurement, enrichment was performed for 7 minutes. The potential range during measurement was 0.2-0.8V, the potential increment was 0.004V, the amplitude was 0.025V, the frequency was 10Hz, and the settling time was 10s. From low concentration to high concentration, each concentration was measured 3 times. After each measurement, the electrode was rinsed with ultrapure water and electrochemically eluted using cyclic voltammetry.

[0084] 3) The concentration of malachite green in the electrolyte solution is calculated based on the pre-plotted linear regression curve and the current signal of the reaction mixture at around 0.5V.

[0085] The pre-plotted linear regression curve is obtained using the following method:

[0086] The current signal of the modified electrode at around 0.5V is positively correlated with the logarithm of the malachite green concentration (Log(C / μg / kg)). The modified electrode was subjected to SWV testing in electrolyte solutions containing different concentrations of malachite green. The current signal at 0.5V was collected and fitted with the logarithm of the malachite green concentration to obtain a linear regression curve.

[0087] like Figure 6 As shown in (A), as the final concentration of malachite green increases from 0 to 1×10 -4 mol / L (from low to high, 0, 1×10 mol / L) -8 5×10 -7 1×10 -6 2×10-6 5×10 -6 1×10 -5 5×10 -5 and 1×10 -4 (mol / L), the current signal at 0.5V of the modified electrode gradually increases.

[0088] like Figure 6 As shown in (B), the current signal at 0.5V for the DNA tetrahedral and G4-modified electrodes is 1×10⁻⁶. -8 Up to 1×10 -4 The relationship between the mol / L concentration and the current change was linear. The linear regression equation obtained by fitting the logarithm of the malachite green concentration to the current change was y = 6.45 × 10⁻⁶. -7 lgC + 4.81 × 10 -6 Linear correlation coefficient R 2 =0.987.

[0089] The results showed that the linear detection range of malachite green was 1×10⁻⁶. -8 Up to 1×10 -4 mol / L, minimum detectable concentration is 1×10 -8 mol / L.

[0090] Changes in electrochemical signals: When malachite green is present in the electrolyte solution, a significant signal peak appears at around 0.5V, and this signal peak gradually increases with the increase of malachite green concentration, indicating that malachite green can be quantitatively analyzed by changes in current signals.

[0091] Specificity detection assay of malachite green using DNA tetrahedral and G4 modified electrodes

[0092] S1: Prepare an electrolyte solution with a concentration of 1×10⁻⁶. -3 Mol / L of leucomalle green, tetracycline, crystal violet, oxytetracycline, methylene blue, and doxycycline.

[0093] S2: Immerse the prepared Au-TDN-G4 electrode in the above solutions, stir and enrich for 7 min, then perform SWV testing. Compare the test results with those obtained at 1×10⁻⁶. -4 The electrochemical signals in mol / L malachite green were compared to determine the specificity of the electrochemical sensor for MG detection.

[0094] The results are as follows Figure 7 As shown, the concentrations of several other antibiotics were 10 times that of MG. By comparing the results with those tested in MG solution, the results showed that the electrode's response to MG was significantly higher than that to the other substances, indicating that the electrode has excellent selectivity.

[0095] Electrode regenerability test

[0096] S1: Place the electrode at 1×10 -4 The electrode was subjected to six consecutive measurements in mol / L malachite green. After each measurement, the electrode was electrochemically cleaned and regenerated using cyclic voltammetry (0.2–0.8 V).

[0097] The results are as follows Figure 8 As shown, the electrochemical signals obtained from the six measurements were almost identical, indicating that the assembled modified electrode has regenerative capability and good stability for the detection of malachite green.

[0098] Response analysis of DNA tetrahedral and G4 modified electrodes to different concentrations of malachite green in a holding tank.

[0099] S1: Weigh out appropriate amounts of Na2HPO4, NaH2PO4 and KCl, dissolve them in water from the temporary holding pond obtained from the outdoor fish pond, and prepare an electrolyte solution with pH=7.0 of 0.2 mol / L.

[0100] S2: Centrifuge the prepared solution at 3000 r / min for 3 min in a low-speed centrifuge to remove suspended impurities in the sample solution, and then filter it with a 0.22 μm filter membrane;

[0101] S3: Prepare 5×10⁻⁶ phosphate buffer solutions using 0.2 mol / L sample phosphate buffer. -8 mol / L, 1×10 -7 mol / L, 5×10 -7 mol / L, 1×10 -6 mol / L, 5×10 -6 mol / L and 1×10 -5 mol / L MG solution;

[0102] S4: The prepared Au-TDN-G4 electrode was subjected to SWV testing in solutions of different concentrations. Before measurement, enrichment was performed for 7 min, with a potential range of 0.2-0.8V, a potential increment of 0.004V, an amplitude of 0.025V, a frequency of 10Hz, and a settling time of 10s.

[0103] S5: After electrochemical elution using cyclic voltammetry, perform three parallel measurements of the sample solution containing the same concentration of malachite green, following the steps in S4 above.

[0104] The results are as follows Figure 9 As shown, in the holding tank water, the current signal gradually increased with the increase of malachite green concentration, and at 5×10 -8 Up to 1×10 -5 It exhibits a linear relationship within the range of mol / L, with the lowest detectable concentration being 5 × 10⁻⁶.-8 mol.

[0105] The gene sequences involved in this invention are as follows:

[0106] SEQ ID No.1;

[0107] Name: DNA strand A

[0108] Source: synthetic construct

[0109] Type: other DNA

[0110] 5'-ACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGC CGCCATAGTA-TTTTTTTTTT-3'

[0111] SEQ ID No.2;

[0112] Name: DNA strand B

[0113] Source: synthetic construct

[0114] Type: other DNA

[0115] 5'-HS-C6-TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATG CGAGGGTCCAATAC-3'

[0116] SEQ ID No. 3;

[0117] Name: DNA strand C

[0118] Source: synthetic construct

[0119] Type: other DNA

[0120] 5'-HS-C6-TCAACTGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCTCTTC-3'

[0121] SEQ ID No.4;

[0122] Name: DNA strand D

[0123] Source: synthetic construct

[0124] Type: other DNA

[0125] 5'-HS-C6-TTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTAT TGGACCCTCGCAT-3'

[0126] SEQ ID No. 5;

[0127] Name: G4 Chain

[0128] Source: synthetic construct

[0129] Type: other DNA

[0130] 5'-AATGGGTGGGATGGGTGGGAGT-C6-SH-3'

[0131] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for preparing a G4-DNA tetrahedral probe electrode, characterized in that, The preparation method includes the following steps: 1) Dissolve the four DNA strands separately in TE buffer to obtain four DNA solutions; the four DNA strands include DNA strand A, DNA strand B, DNA strand C and DNA strand D, the nucleotide sequence of DNA strand A is SEQ ID No. 1; the nucleotide sequence of DNA strand B is SEQ ID No. 2; the nucleotide sequence of DNA strand C is SEQ ID No. 3; the nucleotide sequence of DNA strand D is SEQ ID No. 4; 2) Add TM buffer, TCEP solution and equal volumes of the four DNA solutions to a centrifuge tube, shake thoroughly to mix, heat in a metal bath, then remove and place on ice to cool rapidly to obtain a 1 μM DNA tetrahedral probe working solution. 3) Drop the DNA tetrahedral probe working solution onto the electrode surface and cover the droplet with an object to prevent it from drying out. Incubate at room temperature and then blow dry the electrode. 4) Drop 6-mercaptohexanol onto the electrode surface that has been dried in 3) and incubate to seal the electrode surface. Then rinse with PBS buffer and dry. 5) After dropping the G4 probe solution onto the dried electrode surface from step 4), incubate it to allow the G4 probe to successfully connect to the DNA tetrahedron on the electrode surface, thus obtaining the G4-DNA tetrahedron probe electrode; the nucleotide sequence of the G4 chain contained in the G4 probe solution is SEQ ID No.

5.

2. The method for preparing a G4-DNA tetrahedral probe electrode according to claim 1, characterized in that, In step 3), the electrode is a cleaned gold electrode. The specific cleaning steps are as follows: 1) The gold electrode was polished sequentially with 0.3 μm and 0.05 μm aluminum oxide, and then the polished gold electrode was subjected to ultrasonic treatment in ultrapure water, ethanol and ultrapure water in sequence to obtain the gold electrode after initial cleaning. 2) Electrochemically clean the gold electrode after the initial cleaning in H2SO4 using cyclic voltammetry until a repeating CV curve is obtained, thus obtaining a cleaned gold electrode.

3. A G4-DNA tetrahedral probe electrode, characterized in that, The G4-DNA tetrahedral probe electrode was prepared using the method described in claim 1.

4. The application of the G4-DNA tetrahedral probe electrode according to claim 3, characterized in that, The G4-DNA tetrahedral probe electrode is used to detect malachite green.

5. The application of the G4-DNA tetrahedral probe electrode according to claim 3 or 4, characterized in that, Includes the following steps: First, the G4-DNA tetrahedral probe electrode is inserted into a solution containing malachite green. The malachite green is enriched onto the surface of the G4-DNA tetrahedral probe electrode by stirring. Then, the malachite green is detected by square wave voltammetry, and the concentration of malachite green is obtained based on the signal intensity of the characteristic peak.

6. The application of the G4-DNA tetrahedral probe electrode according to claim 5, characterized in that, In the detection of malachite green using the square wave voltammetry method, the potential range is set to 0.2-0.8V.

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