A layered double hydroxide-based sensor, and a preparation method and application thereof

Abstract

The application relates to a layered double metal hydroxide-based sensor and a preparation method and application thereof, and belongs to the technical field of sensors. The preparation method comprises the following steps: S1, sequentially dropping gold nanoparticle solution, ferrocene@magnesium-aluminum layered double hydroxide solution and gold nanoparticle solution on a glassy carbon electrode, and drying to obtain sample A; S2, dropping thiolated SH-cDNA probe solution with a concentration of 0.5 muM-1.2 muM on the sample A, and incubating to obtain sample B; the thiolated SH-cDNA probe solution is obtained by diluting PBS buffer solution with a pH of 6.5-8; S3, incubating the sample B with mercaptoethanol solution to obtain sample C; S4, incubating the sample C with bladder cancer marker solution for 25 min-50 min to obtain sample D; and S5, dropping thiolated MB-cDNA probe@gold nanoparticle solution on the sample D, and incubating for 80 min-90 min to obtain the sensor. The sensor is used for sensitive, accurate and specific detection of bladder cancer markers.

Description

Technical Field

[0001] This invention belongs to the field of sensor technology, and particularly relates to a sensor based on layered bimetallic hydroxides, its preparation method and application. Background Technology

[0002] Bladder cancer is the tenth most frequently diagnosed cancer worldwide, accounting for nearly 170,000 deaths annually. It has the highest incidence rate in men, ranking fourth among male malignant tumors. Bladder cancer seriously threatens human health, making early prevention and treatment crucial for improving patient survival rates. The occurrence and progression of bladder cancer are often accompanied by changes in tumor marker concentrations. Tumor markers are substances present in blood, urine, or tissues, produced and changing during tumor development and progression. Their types and concentrations can indicate different stages of tumor development and progression; moreover, the measurement of tumor marker levels is of great significance for early screening, diagnosis, and prognosis of tumors. However, in the early stages of cancer, tumor marker concentrations are very low; therefore, trace detection of tumor markers is of great significance for early cancer prevention and treatment.

[0003] Electrochemical sensors are widely used due to their advantages such as simple operation, high sensitivity, and fast detection speed. However, single-signal sensing strategies are susceptible to environmental changes, sensor concentration, and instrument efficiency, which limits the applicability of single-signal electrochemical sensors. Ratiometric methods have been widely applied in various analytical techniques (such as fluorescence, electrochemistry, and chemiluminescence). Compared with traditional single-signal electrochemical biosensors, ratiometric electrochemical DNA sensors use the ratio of two signals as the final output signal, effectively avoiding background interference, possessing better built-in calibration capabilities, and improving the accuracy, repeatability, and anti-interference ability of sensor detection and analysis.

[0004] In the construction of ratiometric electrochemical DNA sensors, selecting materials with good conductivity, biocompatibility, and large specific surface area is crucial for improving sensor performance. Traditional materials suffer from drawbacks such as poor conductivity, high cost, low mechanical properties, poor chemical stability, and structural vulnerability to water, oxygen, or solvents in the environment, leading to collapse. Compared to thermally stable materials like zeolites, most traditional materials decompose under high-temperature conditions. These shortcomings limit their practical application in certain fields.

[0005] Layered bimetallic hydroxides (LBHs) are anionic clay mineral materials with typical layered structure characteristics. They have a large specific surface area, allowing for the binding of more probe molecules. Their synthesis is simple, composition is easily modulated, and their structure (number of layers, interlayer spacing, etc.) is easily tailored. Furthermore, they are readily combinable with other materials to achieve functionalization, leading to their wide application in energy conversion and electrochemical energy storage, such as supercapacitors, secondary batteries, and electrocatalysis. However, as electrode materials, LBHs still suffer from insufficient conductivity. Combining them with materials of high conductivity is a common method to improve their conductivity. However, their application in ratio electrochemical DNA sensors still faces drawbacks such as expensive equipment, complex operation, and inability to achieve micro-level detection and bedside monitoring. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the problems of relatively cumbersome sample pretreatment, high cost of detection instruments, and the need for skilled operators in the prior art, which makes it difficult to achieve real-time, rapid and on-site detection.

[0007] To address the aforementioned technical problems, this invention provides a sensor based on layered bimetallic hydroxides, its preparation method, and its application.

[0008] The first objective of this invention is to provide a method for fabricating a sensor based on layered bimetallic hydroxides, comprising the following steps:

[0009] S1. Gold nanoparticle solution, ferrocene@magnesium aluminum layered double hydroxide solution and gold nanoparticle solution were sequentially added to a glassy carbon electrode and dried to obtain sample A;

[0010] S2. Add a 0.5 μM-1.2 μM thiolized SH-cDNA probe solution to sample A described in S1, and incubate to obtain sample B; the thiolized SH-cDNA probe solution is obtained by diluting with PBS buffer at pH 6.5-8;

[0011] S3. Incubate sample B described in S2 with mercaptoethanol solution to obtain sample C;

[0012] S4. Incubate sample C described in S3 with bladder cancer marker solution for 25 min-50 min to obtain sample D;

[0013] S5. Add thiolized MB-cDNA probe@gold nanoparticle solution to sample D as described in S4 and incubate for 80-90 minutes to obtain the sensor based on layered bimetallic hydroxide.

[0014] In one embodiment of the present invention, in S1, the preparation of the ferrocene@magnesium-aluminum layered double hydroxide specifically includes the following steps: dissolving ferrocene in an alkaline solution, adding aluminum salt solution and magnesium salt solution dropwise under stirring conditions, and reacting to obtain the ferrocene@magnesium-aluminum layered double hydroxide.

[0015] In one embodiment of the present invention, the alkaline solution is selected from one or more of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, and urea solution.

[0016] In one embodiment of the present invention, the molar ratio of the metals in the ferrocene, aluminum salt and magnesium salt is 1.5-3.0:127:317.

[0017] Furthermore, the aluminum salt is aluminum nitrate; the magnesium salt is selected from magnesium nitrate.

[0018] In one embodiment of the invention, the pH of the reaction is 9-10.

[0019] In one embodiment of the present invention, in S2, the concentration of the thiolated SH-cDNA probe solution is related to I. MB / I Fc The ratio has a significant impact on the electrochemical signal; as the concentration increases, I... MB / I Fc The ratio increases, and after reaching a certain concentration, I MB / I Fc The ratio decreased. Thiolized SH-cDNA probe solutions with concentrations of 0.5 μM–1.2 μM yielded stronger electrochemical signals.

[0020] In one embodiment of the invention, in S2, the thiolized SH-cDNA probe solution is obtained by diluting with PBS buffer at pH 6.5-8. The pH of the PBS buffer affects I... MB / I Fc The intensity of the electrochemical signal of the ratio has a significant impact. Excessive acidity or alkalinity has a significant impact on the physicochemical properties of thiolated SH-cDNA and miR-21, thereby impairing the complementary binding of SH-cDNA and miR-21.

[0021] In one embodiment of the present invention, in S2, the thiolated SH-cDNA probe is obtained by reacting SH-cDNA and tris(2-carboxyethyl)phosphine at a molar ratio of 1:1000.

[0022] In one embodiment of the present invention, in S4, the bladder cancer marker is selected from one or more of miR-21, miR-100, miR-143, miR-221 and miR-183.

[0023] In one embodiment of the present invention, in S5, the thiolized MB-cDNA probe@gold nanoparticles is prepared by grafting the thiolized MB-cDNA probe onto the surface of gold nanoparticles; the thiolized MB-cDNA probe is prepared by reducing MB-cDNA with dithiothreitol to convert the disulfide bond into a hydrosulfonyl group.

[0024] A second objective of this invention is to provide a sensor based on layered bimetallic hydroxides prepared by the method described above.

[0025] A third objective of this invention is to provide an application of the aforementioned layered bimetallic hydroxide-based sensor in the detection of bladder cancer biomarkers.

[0026] The technical solution of the present invention has the following advantages compared with the prior art:

[0027] (1) The sensor described in this invention uses MgAl-LDH because it has typical layered structure characteristics, large specific surface area, can bind more probe molecules, has a simple synthesis method, is easy to modulate composition, and its structure (number of layers, interlayer spacing, etc.) is easy to tailor. Fc is embedded in MgAl-LDH, which can be directly modified on the electrode or used as one of the signal substances of the ratio electrochemical DNA sensor. AuNPs are widely used in the construction of electrochemical DNA sensors due to their good biocompatibility and catalytic performance. The introduction of AuNPs effectively improves the electron transfer rate of the electrode and improves the performance of the sensor. The use of AuNPs can also significantly improve the biocompatibility and stability of electrochemical biosensors. At the same time, it can be loaded on layered bimetallic hydroxide nanomaterials to improve the conductivity of the material, and can also capture thiolated SH-cDNA through Au-S bonds, making it firmly fixed on the electrode surface. MB-cDNA@AuNPs is selected as the signal amplification element and is fixed on the DNA sensor as another signal substance of the ratio electrochemical DNA sensor.

[0028] (2) The sensor described in this invention fully utilizes the multi-metal center, porous structure, high biocompatibility, non-toxicity, and large surface area of ​​Fc@MgAl-LDH to increase the number of SH-cDNA capture probes and load more probe molecules; it also fully utilizes the good conductivity and biocompatibility of gold nanoparticles (AuNPs) to achieve better complementary advantages of different materials, thereby improving the performance of the ratio electrochemical DNA sensor to realize the detection of bladder cancer markers.

[0029] (3) The sensor described in this invention uses AuNPs, which are widely used in the construction of electrochemical DNA sensors due to their good biocompatibility and catalytic performance. The introduction of AuNPs effectively improves the electron transfer rate of the electrode and enhances the performance of the sensor. The use of AuNPs can also significantly improve the biocompatibility and stability of electrochemical biosensors, and at the same time, it can be loaded onto layered bimetallic hydroxide nanomaterials to improve the conductivity of the materials.

[0030] (4) The sensor described in this invention is used for sensitive, accurate and specific detection of bladder cancer markers; it can provide ideas for the development of portable detection devices suitable for home and bedside use, and has good theoretical and practical application significance. Attached Figure Description

[0031] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0032] Figure 1 This is a flowchart illustrating the assembly process of a sensor based on layered bimetallic hydroxides according to Embodiment 1 of the present invention.

[0033] Figure 2 The images show the characterization of Fc@MgAl-LDH and AuNPs in Test Example 1 of this invention; where A is the SEM image of Fc@MgAl-LDH, B is the SEM image of AuNPs, and C is the HTEM image of AuNPs.

[0034] Figure 3 The Fourier transform infrared (FTIR) spectrum of Test Example 2 of the present invention;

[0035] Figure 4 The X-ray diffraction (XRD) pattern of Test Example 3 of the present invention;

[0036] Figure 5 The image shows the X-ray photoelectron spectroscopy (XPS) spectrum of Test Example 4 of the present invention; where A is the Fc@MgAl-LDH spectrum, B is the Al 2p spectrum, C is the O 1s spectrum, D is the Fe 2p spectrum, and E is the Mg 1s spectrum.

[0037] Figure 6 Zeta potential and UV-Vis spectroscopic characterization of MB-cDNA@AuNPs in Test Example 5 of this invention; wherein, A is the zeta potential test diagram and B is the UV-Vis spectrophotometry.

[0038] Figure 7 The above are electrochemical characterization diagrams of test example 6 of the present invention; where A is a CV diagram and B is an EIS diagram.

[0039] Figure 8 The image shows the DPV test results of the sensor in Test Example 7 of this invention when detecting different concentrations of miR-21; where A is the DPV curve and B is the standard curve.

[0040] Figure 9 This is a stability test diagram of the sensor in Test Example 8 of the present invention;

[0041] Figure 10 This is a selective test diagram of the sensor in Test Example 9 of the present invention. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0043] In this invention, unless otherwise stated, the glassy carbon electrode (GCE) needs to be pretreated before modification, that is, polished on a cloth with an alumina slurry (0.3 μm then 0.05 μm), thoroughly cleaned with ethanol and water in an ultrasonic bath, and then naturally dried to obtain a new GCE surface.

[0044] Example 1

[0045] Reference Figure 1 As shown, the sensor based on layered bimetallic hydroxide and its preparation method of the present invention specifically include the following steps:

[0046] Materials involved in this embodiment:

[0047] (1) Synthesis of ferrocene-intercalated layered double hydroxide (Fc@MgAl-LDH): It was prepared by hydrothermal method. 0.2724 g of Fe(C5H5)2(Fc) was weighed and dissolved in 30 mL of 0.5 M NaOH solution. Under magnetic stirring, 15 mL of solution containing 0.127 M Al(NO3)3·9H2O and 0.317 M Mg(NO3)2·6H2O was slowly added dropwise to the above solution. The pH of the resulting mixed solution was adjusted to 9 with NaOH. The obtained yellow precipitate was left to stand in the dark for 24 h. The product was collected, washed with deionized water, centrifuged, and dried under vacuum at 50 °C to obtain ferrocene@MgAl-LDH.

[0048] (2) Synthesis of gold nanoparticles (AuNPs): 18 mg of chloroauric acid was dissolved in 100 mL of boiling aqueous solution and stirred continuously. Then, under stirring and heating, 3 mL of 1% sodium citrate solution was slowly added dropwise to the above solution. Under continuous stirring, the solution changed from light yellow to purple. The reaction mixture was naturally cooled to room temperature and stored at 4 °C in the dark to obtain gold nanoparticles (AuNPs).

[0049] (3) Thiolization of SH-cDNA probe: Mix 5 μL of 10 μM SH-cDNA with 5 μL of 10 mM tris(2-carboxyethyl)phosphine (TCEP) for 1 h to reduce disulfide bonds, and dilute the SH-cDNA concentration to 1 μM with 0.01 M pH 7 PBS buffer.

[0050] (4) Thiolization of MB-cDNA probe: 3.5 mg of dithiothreitol (DDT) was dissolved in 300 μL of 0.1 M Tris-HCl buffer to form a reducing agent. This was then added to the MB-cDNA solution and reacted at room temperature in the dark for 1 h. After the reaction was complete, the reduced MB-cDNA was regenerated by alcohol precipitation. 50 μL of 3MC2H3O2Na was added to the mixture. After thorough mixing, 1.5 mL of C2H5OH was added, and the mixture was kept at -20 °C for 20 min. Finally, the mixture was centrifuged and washed with 75% ethanol. The precipitate obtained was the thiolated MB-cDNA probe.

[0051] (5) Synthesis of the thiolized MB-cDNA probe@gold nanoparticle (MB-cDNA@AuNPs) signal amplification element: 2 mL of AuNPs were transferred to a centrifuge tube, and then 164 μL of 50 μM thiolized MB-cDNA was added. After stirring continuously for 16 h, the mixture was transferred to 0.1 M NaCl solution (dissolved in PBS) and incubated for 24 h. Then, the mixture was centrifuged and washed with the above 0.1 M NaCl solution, and the precipitate obtained was the thiolized MB-cDNA probe@gold nanoparticle (MB-cDNA@AuNPs). Finally, the MB-cDNA@AuNPs composite material was dissolved in 0.75 mL of Tris-EDTA buffer and stored at 4 °C for later use.

[0052] Sensor assembly:

[0053] S1. Add 2.5 μL AuNPs to a glassy carbon electrode and allow it to dry naturally. Then add 2.5 μL Fc@MgAl-LDH (1 mg / mL) to its surface. Repeat the addition of 2.5 μL AuNPs once. After drying at room temperature, AuNPs / Fc@MgAl-LDH / AuNPs nanocomposite film is formed, resulting in (AuNPs / Fc@MgAl-LDH / AuNPs) / GCE.

[0054] S2. Add 10 μL of thiolized SH-cDNA probe (1 μM) to (AuNPs / Fc@MgAl-LDH / AuNPs) / GCE and incubate at 4°C for 12 h to completely transplant SH-cDNA onto AuNPs; then rinse the electrode with PBS to remove unbound or weakly bound thiolized SH-cDNA from the AuNPs / Fc@MgAl-LDH / AuNPs nanocomposite membrane to obtain SH-cDNA / (AuNPs / Fc@MgAl-LDH / AuNPs) / GCE.

[0055] S3. To cover non-specific sites on the sensing interface, SH-cDNA / (AuNPs / Fc@MgAl-LDH / AuNPs) / GCE was incubated with 5 μL of mercaptoethanol (2 mM MCH, containing 2 mM MTCEP) for 30 min, followed by rinsing with PBS to remove excess MCH from the electrode surface, yielding MCH / SH-cDNA /

[0056] (AuNPs / Fc@MgAl-LDH / AuNPs) / GCE.

[0057] S4. Modify the electrode MCH / SH-cDNA / (AuNPs / Fc@MgAl-LDH / AuNPs) / GCE as a probe for capturing miR-21. Incubate MCH / SH-cDNA / (AuNPs / Fc@MgAl-LDH / AuNPs) / GCE with miR-21 solution (10 μL, in series of concentrations) for 40 min. After complete capture, wash the electrode with PBS to remove unbound miR-21 on the sensing interface, resulting in miR-21 / MCH / SH-cDNA / (AuNPs / Fc@MgAl-LDH / AuNPs) / GCE.

[0058] S5. Add 10 μL of MB-cDNA@AuNPs to miR-21 / MCH / SH-cDNA / (AuNPs / Fc@MgAl-LDH / AuNPs) / GCE, incubate at 37°C for 90 min, and wash with PBS to remove excess MB-cDNA@AuNPs from the electrode surface to obtain a layered double metal hydroxide-based sensor (MB-cDNA@AuNPs) / miR-21 / MCH / SH-cDNA / (AuNPs / Fc@MgAl-LDH / AuNPs) / GCE.

[0059] Test Example 1

[0060] Based on Example 1, Fc@MgAl-LDH and AuNPs were characterized using scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HTEM), and the results are as follows: Figure 2 As shown. From Figure 2 As can be seen from A, Fc@MgAl-LDH has a layered structure with an average particle size of approximately 100 nm; from Figure 2 B-2C shows that the HTEM and SEM results of AuNPs are consistent, indicating a spherical shape, which is typical of gold nanoparticles.

[0061] Test Example 2

[0062] Based on Example 1, Fc@MgAl-LDH was characterized by Fourier transform infrared spectroscopy (FTIR), and the results are as follows: Figure 3 As shown. From Figure 3 It can be seen that for Fc, due to the tensile vibrations of C-OH, C=C, and C=O, it reaches a point at approximately 1286 cm⁻¹. -1 1473cm -1 and 1655cm -1 An absorption peak is generated at the position of . In the infrared spectrum of MgAl-LDH, due to the bending and stretching vibrations of -OH, it has an absorption peak at approximately 1635 cm⁻¹. -1 and 3466cm -1 An absorption peak is generated at the position, and approximately at 1381 cm⁻¹. -1 The absorption peak at this position is a characteristic vibration of nitrate. Furthermore, for Fc@MgAl-LDH, due to the presence of Fc, there is an absorption peak at approximately 1357 cm⁻¹. -1 1472cm -1 and 1529cm -1 An absorption peak is generated at the position; and due to the presence of MgAl-LDH, an absorption peak is generated at approximately 1388 cm⁻¹. -1 and 3467cm -1 The presence of an absorption peak at the position indicates that Fc@MgAl-LDH has been successfully synthesized.

[0063] Test Example 3

[0064] Based on Example 1, X-ray diffraction was performed on Fc@MgAl-LDH, and the results are as follows: Figure 4 As shown. From Figure 4 It can be seen that the peaks of MgAl-LDH appear at (003), (006), (012), (015), (018), (110), and (113), while the peaks of Fc appear at (012), (110), and (022). Fc@MgAl-LDH shows diffractions of (003), (006), (012), (015), (110) and (012), (110), representing MgAl-LDH and Fc, respectively, indicating that Fc@MgAl-LDH was successfully synthesized.

[0065] Test Example 4

[0066] Based on Example 1, Fc@MgAl-LDH was analyzed using X-ray photoelectron spectroscopy (XPS), and the results are as follows: Figure 5 As shown. From Figure 5 It can be seen that Fc@MgAl-LDH contains a relatively large number of elements, namely Mg (magnesium), Al (aluminum), Fe (iron), C (carbon), O (oxygen), and N (nitrogen). Figure 5 A); Al 2p( Figure 5 B) and Mg 1s( Figure 5 The binding energies of E) at 73.84 eV and 1303.63 eV indicate the presence of Al in the hydroxide layer. 3+ and Mg 2+ O1s ( Figure 5 The binding energy spectrum of C) may be due to the O atoms in -CO, -C=O, -NO, and -OH. XPS analysis of Fc@MgAl-LDH shows Fe 2p3 / 2 and Fe 2p1 / 2 ( Figure 5 The peak value of D) indicates that Fc is integrated into the hydroxide layer.

[0067] Test Example 5

[0068] Based on Example 1, MB-cDNA@AuNPs were characterized by zeta potential, and the results are as follows: Figure 6 As shown in A. From Figure 6 As can be seen from Figure A, the potential of AuNPs was -31.8 mV, while after incubation with thiolized MB-cDNA, this value increased to -26.8 mV. This indicates that the thiolized MB-cDNA was successfully grafted onto AuNPs, and since MB carries a positive charge, this leads to a forward shift in the overall potential of the composite material.

[0069] Based on Example 1, MB-cDNA@AuNPs were characterized by ultraviolet-visible spectrophotometry (UV-Vis), and the results are as follows: Figure 6 As shown in B. From Figure 6 As shown in Figure B, curve a displays the characteristic absorption of AuNPs at 520 nm; curve b is the absorption spectrum of the MB-cDNA solution, with two absorption peaks at 258 nm and 662 nm; and curve c shows three absorption peaks at 258 nm, 520 nm, and 662 nm. These results indicate that MB-cDNA has been successfully grafted onto the surface of AuNPs, forming MB-cDNA@AuNPs.

[0070] Test Example 6

[0071] [Fe(CN)6] [3- / 4-]Redox coupling pairs are an effective tool for evaluating the microstructure of electrode surfaces. Therefore, CV and EIS were used to characterize the assembly process of the layered bimetallic hydroxide-based sensor of Example 1.

[0072] (1)CV is an important electrochemical technique that can be used to effectively characterize the assembly process of DNA sensors. Figure 7 A). After modifying the (AuNPs / Fc@MgAl-LDH / AuNPs) membrane on the bare GCE (curve b), the peak current intensity decreased significantly, indicating that the AuNPs / Fc@MgAl-LDH / AuNPs membrane had been immobilized on the GCE surface. The transfer of thiolated SH-cDNA on the (AuNPs / Fc@MgAl-LDH / AuNPs) / GCE resulted in a decrease in redox peak current (curve c) and an increase in interpeak separation. This is due to the negatively charged phosphate backbone and negatively charged [Fe(CN)6] in the thiolated SH-cDNA. [3- / 4-] The repulsion between them was observed; when MCH self-assembled onto SH-cDNA / (AuNPs / Fc@MgAl-LDH / AuNPs) / GCE to block the remaining active sites, a further decrease in the redox peak current was observed (curve d). Furthermore, after immobilization of thiolized SH-cDNA (curve c) and subsequent hybridization with miR-21 (curve e), the current continued to decrease due to spatial blockage of DNA molecules and negative charge density near the electrode surface, indicating successful immobilization of thiolized SH-cDNA and miR-21. Finally, after incubation of miR-21 / MCH / SH-cDNA / (AuNPs / Fc@MgAl-LDH / AuNPs) / GCE with MB-cDNA@AuNPs solution, a further decrease in the peak current (curve f) indicated that the MB-cDNA@AuNPs signal amplification composite material had successfully bound to miR-21 on the electrode surface.

[0073] (2) EIS is another technique for characterizing the DNA sensor assembly process. Figure 7 B). In the Nyquist plot, the semicircle diameter at high frequencies represents the electron transport resistance (Ret), and the linear portion at low frequencies represents the diffusion process. The impedance changes on the electrode surface during the layer-by-layer modification process can be observed using electrochemical impedance spectroscopy (EIS). The Nyquist plots of EIS at 0.1 M KCl and 5.0 mM [Fe(CN)6]... [3- / 4-]Bare GCE exhibits the largest Ret value (curve a). When AuNPs / Fc@MgAl-LDH / AuNPs are deposited on the surface of GCE, an increase in the size of the semicircle can be observed (curve b). After assembling thiolized SH-cDNA on (AuNPs / Fc@MgAl-LDH / AuNPs) / GCE, the semicircle diameter increases significantly (curve c), because the presence of thiolized SH-cDNA blocks interfacial electron transfer, and the assembly of MCH also leads to a significant increase in Ret (curve d). Then, after treatment with miR-21, the significant increase in Ret in curve e demonstrates the successful hybridization of thiolized SH-cDNA and miR-21. Subsequently, the addition of MB-cDNA@AuNPs resulted in a significant increase in the semicircle diameter in curve f as electron transport increased. With the successive layer-by-layer modification of MCH, miR-21, and MB-cDNA@AuNPs, the impedance value increases, proving that each layer of material was successfully modified on the electrode.

[0074] CV and EIS results show that the sensor based on layered bimetallic hydroxide was successfully assembled.

[0075] Test Example 7

[0076] The peak current response of the sensor based on layered bimetallic hydroxide prepared in Example 1 to different concentrations of miRNA-21 was detected, and the results are as follows: Figure 8 As shown. From Figure 8 It can be seen that as the concentration of miRNA-21 gradually increases, the MB signal increases, the Fc signal decreases, and the peak current blank value minus the peak current miRNA-21 value (ΔI=I0-I1) is directly proportional to the logarithm of the miRNA-21 concentration, with a linear range of 1.0×10⁻⁶. -15 -1.0×10 -10 mol / L (from a to f, each is 1×10⁻⁶) -10 mol / L, 1×10 -11 mol / L, 1×10 -12 mol / L, 1×10 -13 mol / L, 1×10 -14 mol / L, 1×10 -15 (mol / L); the standard curve is y = 0.0919x + 1.7006, and the coefficient of determination Rm is 1. 2 =0.9967, the limit of detection (LOD) is 0.757×10 -15 The electrochemical signal ratio of the sensor is linearly related to the logarithmic concentration of miR-21, and the prepared sensor shows good quantitative detection performance for miRNA-21.

[0077] Test Example 8

[0078] To test the stability and repeatability of the layered bimetallic hydroxide-based sensor prepared in Example 1, five identical sensors were prepared and electrochemical signals were detected over 14 days. The results are as follows: Figure 9 As shown. From Figure 9 It can be seen that the electrical signal value of the sensor did not change significantly within two weeks, with a relative standard deviation (RSD) of 3.8%. This indicates that the constructed sensor has good stability.

[0079] Furthermore, repeatability measurements were performed on the same seven modified electrodes. The results showed that the electrical signal values ​​of the seven sensors did not change significantly during repeated measurements, with an RSD of 0.45%. This indicates that the constructed electrochemical sensor has good repeatability and can easily detect different targets by replacing matching markers.

[0080] Test Example 9

[0081] To assess the anti-interference capability of the layered double hydroxide-based sensor prepared in Example 1, single-stranded nucleic acid molecules with different sequences were selected as interfering substances. For miRNA-21, nucleic acid molecules with single-base mismatch, double-base mismatch, and completely mismatched bases were designed. Simultaneously, miRNA-122, a nucleic acid molecule similar to miRNA-21, was also selected as an interfering target. miRNA-21 (1.0 × 10⁻⁶) was prepared... -9 mol / L) and interfering substances (1.0×10 -8 A mixed solution of (mol / L) was added dropwise to the sensor, and the electrochemical signal value was measured. The results are as follows: Figure 10 As shown. From Figure 10 It can be seen that the electrochemical sensor does not show a significant change in response to the presence or absence of interfering substances with miRNA-21, and the constructed electrochemical sensor has good selectivity.

[0082] Test Case 10

[0083] The linear range and detection limit of the sensor based on layered bimetallic hydroxide prepared in Example 1 were compared with those of different types of existing sensors for detecting miR-21, as shown in Table 1.

[0084] Table 1 Comparison of different miRNA detection methods

[0085]

[0086] As shown in Table 1, the layered bimetallic hydroxide-based sensor of Example 1 exhibits a lower limit of detection (LOD) and a wider linear range. This is because the Fc@MgAl-LDH nanocomposite provides a favorable layered environment for the immobilization of thiolated SH-cDNA, thus allowing for the adsorption of more analytes and improving sensor sensitivity. The addition of AuNPs not only enhances the conductivity of the nanocomposite but also amplifies the electrochemical signal. Furthermore, compared to other methods for detecting miRNA-21, the layered bimetallic hydroxide-based sensor demonstrates superior analytical performance, providing theoretical support and design ideas for large-sample bladder cancer screening and the development of novel, portable miR-21 biomarker detection devices.

[0087] In summary, this invention fully utilizes the advantages of Fc@MgAl-LDH to construct a sensor with high sensitivity, good accuracy, and strong anti-interference ability. It provides ideas for the development of detection devices for RNA tumor markers with high sensitivity, wide linear range, good stability, and strong anti-interference ability. It has good academic prospects and certain application value, and can provide ideas for the development of portable detection devices suitable for home and bedside use.

[0088] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for fabricating a sensor based on layered bimetallic hydroxides, characterized in that, Includes the following steps, S1. Gold nanoparticle solution, ferrocene@magnesium-aluminum layered double hydroxide solution and gold nanoparticle solution are added dropwise to a glassy carbon electrode in sequence, and dried to obtain sample A; The preparation of the ferrocene@magnesium-aluminum layered double hydroxide specifically includes the following steps: ferrocene is dissolved in alkaline solution, aluminum salt solution and magnesium salt solution are added dropwise under stirring conditions, and the reaction is carried out to obtain the ferrocene@magnesium-aluminum layered double hydroxide. S2. Add a 0.5 μM-1.2 μM thiolized SH-cDNA probe solution to sample A described in S1, and incubate to obtain sample B; the thiolized SH-cDNA probe solution is obtained by diluting with PBS buffer at pH 6.5-8; S3. Incubate sample B described in S2 with mercaptoethanol solution to obtain sample C; S4. Incubate sample C described in S3 with bladder cancer marker solution for 25 min-50 min to obtain sample D; S5. Add thiolized MB-cDNA probe@gold nanoparticle solution to sample D as described in S4 and incubate for 80-90 minutes to obtain the sensor based on layered bimetallic hydroxide.

2. The method for preparing a sensor based on layered bimetallic hydroxides according to claim 1, characterized in that, The alkaline solution is selected from one or more of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, and urea solution.

3. The method for preparing a sensor based on layered bimetallic hydroxides according to claim 1, characterized in that, The molar ratio of the metals in the ferrocene, aluminum salt, and magnesium salt is 1.5-3.0:127:

317.

4. The method for preparing a sensor based on layered bimetallic hydroxides according to claim 1, characterized in that, The pH of the reaction is 9-10.

5. The method for fabricating a sensor based on layered bimetallic hydroxides according to claim 1, characterized in that, In S2, the thiolized SH-cDNA probe is obtained by reacting SH-cDNA and tris(2-carboxyethyl)phosphine at a molar ratio of 1:1000.

6. The method for preparing a sensor based on layered bimetallic hydroxides according to claim 1, characterized in that, In S4, the bladder cancer marker is selected from one or more of miR-21, miR-100, miR-143, miR-221, and miR-183.

7. The method for preparing a sensor based on layered bimetallic hydroxides according to claim 1, characterized in that, In S5, the thiolized MB-cDNA probe@gold nanoparticles is prepared by grafting the thiolized MB-cDNA probe onto the surface of gold nanoparticles; the thiolized MB-cDNA probe is prepared by reducing MB-cDNA with dithiothreitol to convert the disulfide bond into a hydrosulfonyl group.

8. A sensor based on layered bimetallic hydroxide prepared by the method of any one of claims 1-7.

9. The use of the layered bimetallic hydroxide-based sensor of claim 8 in the detection of bladder cancer biomarkers for non-diagnostic and / or therapeutic purposes.

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