Application of flower-like gold nanoelectrodes in sandwich-type aptasensor for detecting Kana

Abstract

The present invention belongs to the field of antibiotic residue detection, and specifically relates to the application of flower-shaped gold nanoelectrodes in the detection of Kana in sandwich-type aptamer sensors. The present application stably prepares flower-shaped gold nanostructure electrodes with a large surface area by an electrodeposition method, so that the obtained flower-shaped gold nanostructures present a layered stacking and a flower-shaped dispersion to the surroundings, with an excellent specific surface area. And the EDS energy spectrum shows that the flower-shaped gold nanoparticles obtained by the preparation method of the present application have a very high purity. Therefore, when applied to aptamer sensors, their electrical properties and catalytic properties will show a significant improvement, thereby improving the sensitivity of the sensor. On the other hand, the flower-shaped gold nanoelectrode prepared by the present application can achieve a detection limit of 0.2nM for Kana in the sample to be tested by controlling the aptamer concentration, incubation time, etc. during the specific application process.

Description

Technical Field

[0001] The present invention belongs to the field of antibiotic residue detection, and particularly relates to the application of a flower-shaped gold nanoelectrode in the detection of Kana by a sandwich-type aptamer sensor. Background Art

[0002] Fluorescent aptamer sensors detect target molecules based on changes in fluorescence signals. They typically consist of a recognition unit, a signal transduction unit, and a signal output unit. The recognition unit often employs specific aptamers as molecular probes, selectively capturing the target through specific binding. The signal transduction unit connects the recognition unit and the signal output unit. Common signal transduction mechanisms include fluorescence resonance energy transfer (FRET), fluorescence quenching, and fluorescence enhancement. The signal output unit, the core component of the sensor, typically utilizes fluorescent materials such as fluorescent dyes or quantum dots (e.g., AuNPs). These materials produce a measurable change in fluorescence signal upon binding to the target molecule. The structure of a fluorescent sensor can be flexibly adjusted to enhance sensitivity and specificity, depending on the specific detection requirements.

[0003] Kanamycin (Kana), a widely used aminoglycoside antibiotic, is overused in animal husbandry, fisheries, and agriculture, leading to the entry of Kana residues into water bodies, posing a threat to aquatic life and human health. Therefore, accurate and sensitive detection of Kana is of great significance. Traditional methods such as liquid chromatography (LC), high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS), and capillary electrophoresis (CE) have the advantages of high sensitivity and good accuracy. However, due to factors such as long cycles, high costs, and the need for professional technicians to operate, the application of these methods is limited to a certain extent, and the detection limit is generally high. For example, the detection limit of an electrochemical aptamer sensor disclosed in prior art 1 (Qiu Y, Jiang L, Xiang L, et al. 2024. Anelectrochemical biosensor platform for kanamycin detection based on the target-induced spatial configuration of aptamer-complementary strand hybridization[J]. Monatshefte für Chemie - Chemical Monthly, 155: 467-475.) is 10nM; prior art 2 (Lu J, Xu X, Chen J, 2024. Polyoxometalate-based nanozyme with laccase-mimicking activity for kanamycin detection based on colorimetric assay[J]. Microchimica Acta, 191: 544.) discloses a colorimetric sensor with a detection limit of 6.28 nM; the prior art 3 (Liu Y, Guan B, Xu Z, et al. 2023. Afluorescent assay for sensitive detection of kanamycin by split aptamers and DNA-based copper / silver nanoclusters[J]. Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy, 286: 121953.) discloses a fluorescence sensor with a detection limit of 13.3nM; another fluorescence sensor disclosed in prior art 4 (Anbiaee G, Feizpour R, Khoshbin Z, et al. 2023. Asimple tag-free fluorometric aptasensing assay for sensitive detection of kanamycin[J]. Analytical Biochemistry, 672: 115183.) has a detection limit of 7.3nM; and a fluorescence-colorimetric dual-mode sensor disclosed in prior art 5 (Tian Y, Mou Y, Zhang W, et al. 2025. A fluorescence and colorimetric dual-mode aptasensor for kanamycin detection[J]. Biosensors and Bioelectronics, 268: 116911.) has a detection limit of 7.3nM. These prior art technologies all have detection limits exceeding 5nM, significantly limiting their applicability. Sandwich-type fluorescence sensors can effectively address these issues.

[0004] Sandwich-type fluorescence sensors are primarily based on a sandwich structure formed between target molecules and aptamers. In this structure, a gold nanoelectrode serves as the aptamer substrate. Its excellent surface area allows a large amount of split aptamer 1 (SPA1) to be immobilized on the electrode surface via Au-S bonds. When the target molecule, Kana, is present in the water sample, the free split aptamer 2 (SPA2) and SPA1 specifically recognize Kana, forming a ternary sandwich complex (SPA1-Kana-SPA2). This allows the ferrocenylnaphthyl imide (FND indicator) disclosed in prior art 6 (Deng YR, Li YF, Yang H, et al. 2024. Synthesis, DNA binding of bis-naphthyl ferrocenederivatives and the application as new electroactive indicators for DNA biosensor[J]. Journal of Inorganic Biochemistry, 257: 112615) to be effectively embedded within the sandwich, generating an electrochemical signal and enabling sensitive detection of Kana.

[0005] It can be seen that the sensitivity and accuracy of the above detection method are closely related to the amount of SPA1 immobilized on the gold nanoelectrode. The greater the amount of SPA1 immobilized, the greater the ability to form a ternary sandwich complex, which greatly improves the accuracy of Kana detection and correspondingly reduces the detection limit. Therefore, gold nanoparticles with large specific surface areas and their application in sandwich-type aptasensors for Kana detection are urgently needed in this field. Summary of the Invention

[0006] Based on the above reasons, the purpose of this application is to provide an application of flower-shaped gold nanoelectrodes in sandwich-type aptamer sensors to detect Kana. By using this application method to detect Kana in the sample to be tested, the detection limit of Kana can reach 0.2nM.

[0007] In order to achieve the above objectives, the technical solution of this application is:

[0008] The application of flower-shaped gold nanoelectrodes in the sandwich aptasensor for detecting Kana includes the following steps:

[0009] S1. Immerse the flower-shaped gold nanoelectrode in SPA1 buffer and react at room temperature to immobilize SPA1 on the flower-shaped gold nanoelectrode through Au-S bonds;

[0010] S2. Immerse the flower-shaped gold nanoelectrode immobilized with SPA1 in a mixed solution of SPA2 and the sample to be tested and incubate at room temperature to obtain a flower-shaped gold nanoelectrode with a SPA1-AuNFs-SPA2 sandwich structure;

[0011] S3. Place the flower-shaped gold nanoelectrode with SPA1-AuNFs-SPA2 sandwich structure in FND indicator and PBS buffer solution, enrich in the dark, and then wash, and then detect the Kana content in the sample by DPV method.

[0012] Furthermore, the preparation method of the flower-shaped gold nanoelectrode described in S1 is:

[0013] (1) The ITO electrode was alternately immersed in PAA and PEI solutions to form a PEI / PAA loaded film to obtain a modified ITO electrode;

[0014] (2) The modified ITO electrode was placed in a HAuCl4 precursor solution, and gold nanoparticles were deposited at a potential of -0.3 V using the chronoamperometry method to obtain a flower-shaped gold nanoelectrode.

[0015] Furthermore, in step (1), the concentration of the PEI solution is 0.25 mg / mL, and the concentration of the PAA solution is 1 mg / mL; and the number of alternating immersions is 3-5 times.

[0016] Furthermore, the concentration of the HAuCl4 precursor solution in step (2) is 1-3 mg / mL; and the deposition time of the chronoamperometry is 900-1200 s.

[0017] Furthermore, the concentration of the SPA1 solution in S1 is ≥0.3 μM.

[0018] Furthermore, the concentration of the SPA2 solution in S2 is ≥0.5 μM.

[0019] Furthermore, the incubation time in S2 is ≥120 min.

[0020] Furthermore, the SPA1 solution and SPA2 solution in S1 and S2 are obtained by diluting and preparing a DNA buffer solution, and the pH of the DNA buffer solution is 7-8.

[0021] Furthermore, the concentration of the FND indicator in S3 is 1-2.5 mM, and the enrichment time is ≥9 min.

[0022] Furthermore, the concentration of the SPA1 solution in S1 is 0.6 μM, the concentration of the SPA2 solution in S2 is 0.8 μM, the incubation time is 120 min, the pH of the PBS buffer in S3 is 7.6, the concentration of the FND indicator in S3 is 2 mM, and the enrichment time is 10 min; wherein, the SPA1 solution and SPA2 solution in S1 and S2 are diluted and configured with a DNA buffer, and the pH of the DNA buffer is 7.6.

[0023] Beneficial Effects: 1. This application provides a method for stably preparing an electrode with a large surface area and a flower-like gold nanostructure. The resulting flower-like gold nanostructure exhibits a layered stack and a flower-like dispersion, exhibiting an excellent specific surface area. EDS spectra indicate that the flower-like gold nanostructures obtained by this preparation method are of very high purity. Therefore, when applied to aptamer sensors, their electrical and catalytic properties are significantly improved, thereby enhancing the sensitivity of the sensor.

[0024] 2. The flower-shaped gold nanoelectrode prepared in this application can achieve a detection limit of 0.2 nM for Kana in the sample to be tested by controlling the aptamer concentration, incubation time, etc. during the specific application process. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The SEM images of gold nanoparticles at three deposition potentials in Experimental Example 1 are shown, where (A) is the SEM image of gold nanoparticles at a potential of -0.3 V; (B) is the SEM image of gold nanoparticles at a potential of -0.1 V; and (C) is the SEM image of gold nanoparticles at a potential of 0.3 V.

[0026] Figure 2The CV curves of gold nanoparticles at three deposition potentials in Experimental Example 1 and the linear equation diagram between the redox peak current and the square root of the scan rate, wherein (A) is the CV curve of gold nanoparticles at a potential of -0.1V; (B) is the CV curve of gold nanoparticles at a potential of 0.3V; (C) is the CV curve of gold nanoparticles at a potential of -0.3V; (D) is the linear equation diagram between the redox peak current of gold nanoparticles at a potential of -0.1V and the square root of the scan rate; (E) is the linear equation diagram between the redox peak current and the square root of the scan rate at a potential of 0.3V; (F) is the linear equation diagram between the redox peak current and the square root of the scan rate at a potential of -0.3V;

[0027] Figure 3 Optimization results of HAuCl4 solution concentration, including (A) 1 mg / mL (2000×); (B) 2 mg / mL (2000×); (C) 3 mg / mL (2000×); (D) 4 mg / mL (2000×);

[0028] Figure 4 Optimization results of the alternating immersion times of PAA and PEI solutions, where (A) polymer alternating loading 0 times (20000×); (B) polymer alternating loading 1 time (15000×); (C) polymer alternating loading 5 times (15000×); (D) polymer alternating loading 7 times (6000×);

[0029] Figure 5 Optimization results of deposition time, including (A) 180 s (10000×); (B) 300 s (10000×); (C) 600 s (10000×); (D) 900 s (10000×); (E) 1200 s (10000×); (F) 1500 s (10000×);

[0030] Figure 6 The structural characterization results of AuNFs, including (A) XPS full spectrum; (B) C 1s peak fitting diagram; (C) Au4f peak fitting diagram; (D) EDS point scan diagram; (E) EDS surface scan diagram; (F) EDS energy spectrum diagram;

[0031] Figure 7 Schematic diagram of the aptamer sensor based on flower-like gold nanoparticles;

[0032] Figure 8 is the ultraviolet spectrum;

[0033] Figure 9 This is the feasibility analysis result diagram;

[0034] Figure 10 The electrochemical characterization results are shown in Figure 2, where (A) is the CV characterization result diagram; (B) is the EIA characterization result diagram;

[0035] Figure 11 Optimize the results for SPA1 concentration;

[0036] Figure 12 Optimize the results for SPA2 concentration;

[0037] Figure 13 Results for DNA buffer pH optimization;

[0038] Figure 14 Optimize the enrichment time results for FND indicator;

[0039] Figure 15 Optimize the results for FND indicator concentration;

[0040] Figure 16 Optimize the results for Kana incubation time;

[0041] Figure 17 The results of the sensor linear range test are shown in Figure 2, where (A) is the DVP response in the presence of different concentrations of Kana; (B) is the linearity between the logarithm of Kana concentration and the DVP peak signal;

[0042] Figure 18 It is the sensor selectivity test result;

[0043] Figure 19 This is the sensor reproducibility test result;

[0044] Figure 20 This is the stability test result of the sensor. DETAILED DESCRIPTION

[0045] In order to more clearly illustrate the purpose, research methods and advantages of the present invention, the present invention is further described in detail through the following examples.

[0046] In this embodiment, all electrochemical experiments used a three-electrode system, wherein Ag / AgCl (saturated KCl) was the reference electrode, a platinum wire was the counter electrode, and an ITO electrode (tin-indium tungsten oxide electrode) was the working electrode.

[0047] Example 1 Selection and optimization of flower-shaped gold nanoelectrodes

[0048] This embodiment provides a method for preparing an optimal flower-shaped gold nanoelectrode: gold nanoelectrodes are deposited using a chronoamperometry method, with an ITO electrode as a carrier. The preparation method is as follows:

[0049] S1. Ultrasonic clean an ITO electrode (25 mm long, 8 mm wide, and 1.1 mm thick) in acetone, anhydrous ethanol, and ultrapure water, sequentially for 10 min each time. After ultrasonic treatment, dry the electrode with nitrogen. Then, alternately soak the electrode in 10 mL of 0.25 mg / mL PAA (polyacrylic acid) and 10 mL of 1 mg / mL PEI (polyethyleneimine) solutions five times for 10 min each, forming a PEI / PAA-loaded film on the electrode to obtain a modified ITO electrode.

[0050] S2. The modified ITO electrodes were placed in a 3 mg / mL HAuCl4 precursor solution and deposited for 1200 s at a potential of 0.3 V using the chronoamperometry method to construct a layer of gold nanostructure on the surface of the ITO electrode. The unreacted HAuCl4 precursor solution on the surface of the ITO electrode was rinsed with ultrapure water and dried at room temperature and pressure to obtain a flower-shaped gold nanoelectrode.

[0051] The above optimal embodiment is obtained through the following two experimental processes: selection of gold nanomorphology on the gold nanoelectrode and optimization of flower-shaped preparation conditions.

[0052] Experimental Example 1 Selection of gold nanomorphology on gold nanoelectrodes

[0053] The nucleation process of gold nanoparticles on the ITO electrode is mainly affected by the deposition potential. Therefore, in this experiment, the deposition potential was changed and the flower-shaped gold nanoparticles were screened by SEM images and the effective area of ​​the electrode.

[0054] Experimental method: The preparation method of flower-shaped gold nanoparticles is the same as that in Example 1, except that a waterproof sticker with a pore size of 5 mm is fixed on the surface of the modified ITO electrode described in S1, and deposition is performed at potentials of -0.3v, -0.1v, and 0.3v for different pore sizes to obtain flower-shaped gold nanoparticles.

[0055] Experimental results

[0056] 1. Morphology analysis: The flower-like gold nanoparticles grown on the ITO electrode obtained under three potentials were fixed on the sample stage. The morphology of the sample was observed under the conditions of a test voltage of 10.00 kV and a magnification of 1000×~40000×: the scanning results are as follows Figure 1 As shown, (A) (B) (C) are the scanning results at -0.3V, -0.1V, and 0.3V potentials respectively. It can be seen that when the deposition potential is -0.3 V ( Figure 1 (A) The gold nanoparticles deposited on the ITO electrode showed an irregular growth trend and branched growth, forming tree-like gold nanoparticles (AuNTs). When the potential rose to -0.1 V ( Figure 1(B) The gold nanoparticles deposited on the ITO surface become regular and orderly, forming spherical gold nanoparticles with a diameter of about 100 nm. This is because the reduction rate of gold ions is slow at this potential, so the nucleation process is dominant, which easily forms smaller nanoparticles, and the shape of the particles is generally regular. When the potential is further increased to 0.3 V ( Figure 1 (C) The gold nanoparticles deposited on the ITO electrode are not only evenly distributed but also exhibit a branching growth pattern, forming flower-like gold nanoparticles (AuNFs). The resulting AuNFs have a significantly increased diameter. This is likely because increasing the potential accelerates the reduction rate of gold ions, allowing nucleation and growth to proceed simultaneously. Although the number of gold nuclei formed is small, larger nanoparticles are more likely to form.

[0057] 2. Determination of effective electrode area: The ITO electrodes with flower-like gold nanoparticles grown at three potentials were placed in 5mM [Fe(CN)6] 3- / 4- CV scans were performed in a solution containing 0.1 M KCl at scan rates of 20 mV / s, 40 mV / s, 60 mV / s, 80 mV / s, 100 mV / s, and 200 mV / s (starting potential: 0.2 V, high potential: 0.7 V, low potential: -0.2 V). The effective area of ​​the ITO electrode was calculated from the relationship between the redox peak signal and the square root of the scan rate.

[0058] The test results are as follows Figure 2 As shown: CV scans at different rates were performed on ITO electrodes with gold nanoparticles (AuNTs, AuNPs, AuNFs) of different morphologies obtained under three potentials. As the scan rate increased from 20 mV / s to 200 mV / s, the redox peak current of CV also increased. Figure 2 (A) Figure 2 (B) Figure 2 (C) CV curves of AuNPs (-0.1 V), AuNFs (0.3 V), and AuNTs (-0.3 V) at different scan rates. When all are at the same scan rate, the redox peak current of AuNTs is the largest, followed by AuNFs, while the redox peak current of AuNPs is the smallest.

[0059] like Figure 2(D), (E), and (F) show the linear correlation equations between the redox peak current and the square root of the scan rate for AuNPs (-0.1 V), AuNFs (0.3 V), and AuNTs (-0.3 V), respectively. According to the Randles-Sevcik formula, the effective use area of ​​the ITO electrode deposited with AuNPs, AuNFs, and AuNTs was calculated, with the effective use area of ​​AuNPs being 13.88 cm 2 , AuNFs is 25.6 cm 2 , AuNTs are 31.44 cm 2 .

[0060]

[0061] in, ip Indicates peak current (A); A is the effective surface area of ​​the electrode (cm 2 ); D 0 is the diffusion coefficient of the reactant (1.0×10 -5 cm 2 / s); n is the number of electrons transferred during the electrode process (n=1); v is the scanning speed (V / s); C 0 is the concentration of the reactant (mol / cm 3 ).

[0062] However, in actual use, since AuNTs easily fall off from the surface of the ITO electrode, considering the reliability and stability of subsequent experimental data, AuNFs were used as the gold nanoparticles used in subsequent experiments, namely flower-shaped gold nanoparticles, and the deposition potential was determined to be 0.3V.

[0063] Experimental Example 2 Optimization of Preparation Conditions for Flower-Shaped Gold Nanoparticles

[0064] 1. Optimization of HAuCl4 solution concentration

[0065] Experimental method: The preparation method of flower-shaped gold nanoparticles was the same as that in Example 1, wherein the concentrations of HAuCl4 precursor solutions were set to 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, and 6 mg / mL, respectively.

[0066] Experimental results: The ITO electrode with gold nanoparticles prepared above was fixed on the sample stage. The morphology of the sample was observed under the conditions of a test voltage of 10.00 kV and a magnification of 1000×~40000×. The experimental results are as follows: Figure 3 shown.

[0067] Result analysis: HAuCl4 concentration plays a decisive role in regulating the morphology of AuNFs on the ITO electrode surface. Figure 3 It can be seen that when the concentration of HAuCl4 is 1 mg / mL, the AuNFs formed on the ITO electrode surface have a lower density and a smaller diameter ( Figure 3 (A)). With the increase of HAuCl4 concentration ( Figure 3 (B) 2 mg / mL, (C) 3 mg / mL, the AuNFs density increases while its diameter also gradually increases. It can be clearly observed that the deposited gold nanoparticles form more dendritic structures and the structure is more dense. When the concentration of HAuCl4 increases to 4 mg / mL ( Figure 3 (D) ), the AuNFs formed showed structural disorder and uneven morphology. Therefore, when the HAuCl4 concentration was between 1 and 3 mg / mL, dendritic structures were formed, with 3 mg / mL being the optimal concentration. This concentration was selected for subsequent experiments.

[0068] 2. Optimization of the number of alternating immersion times of PAA and PEI solutions

[0069] Experimental method: The preparation method of flower-shaped gold nanoparticles was the same as that in the embodiment, wherein the alternating immersion times in PAA and PEI solutions were set to 0, 1, 5, and 7 times, respectively, each time for 10 min.

[0070] Experimental results: The ITO electrode with gold nanoparticles prepared above was fixed on the sample stage. The morphology of the sample was observed under the conditions of a test voltage of 10.00 kV and a magnification of 1000×~40000×. The experimental results are as follows: Figure 4 shown.

[0071] Results: During electrodeposition, polymers can provide more effective nucleation sites for gold nanoparticles and offer a more precise growth path for the structured growth of gold nanoparticles. Therefore, multilayer polymers (PEI / PAA) can promote the formation of gold nanoparticles with high curvature and fine morphology. The number of polymer layers directly affects the shape of electrodeposited gold nanoparticles. Figure 4 It can be seen that when gold nanoparticles are grown on ITO without polymer loading by electrodeposition, the gold nanoparticles tend to grow in a spherical shape and grow more densely ( Figure 4 (A)). As the number of polymer loading layers increases, the gold nanoparticles gradually tend to grow in a flower-like manner. Figure 4 As shown in (B), when the polymer is loaded once, the gold nanoparticles become sparser and more branched structures are produced. When the polymer is loaded five times, the gold nanoparticles generated by electrodeposition have more branches and form flower-like structures, as shown in Figure 4(C) This may be because the polymer provides relatively fewer pores during the growth of gold nanoparticles, making the gold nanoparticles more sparse. However, because the polymer provides a more precise growth route and nucleation environment for gold nanoparticles, the gold nanoparticles tend to grow in a flower-like manner. As shown in 4 (D), when the number of polymer alternating loadings reaches 7 times, the growth of gold nanoparticles becomes chaotic. This may be because the gold nanoparticles have more branches, making the branches of gold nanoparticles more chaotic and aggregated during growth. In order to finally obtain structurally controllable AuNFs, a flower-like structure can be formed with a polymerization number of 3-5 times, of which 5 times is the best. Finally, the ITO electrode with 5 polymer alternating loadings was selected for subsequent experiments.

[0072] 3. Optimization of deposition time

[0073] Experimental method: The preparation method of flower-shaped gold nanoparticles was the same as that in Example 1, wherein the deposition time was set to 180s, 300s, 600s, 900s, 1200s, and 1500s, respectively.

[0074] Experimental results: The ITO electrode with gold nanoparticles prepared above was fixed on the sample stage. The morphology of the sample was observed under the conditions of a test voltage of 10.00 kV and a magnification of 1000×~40000×. The experimental results are as follows: Figure 5 shown.

[0075] Result analysis: The growth process of AuNPs on ITO electrode is mainly affected by the deposition time, such as Figure 5 As shown in the figure, when the deposition time is in the range of 180 s~1500 s, the density of AuNFs particles deposited on the ITO electrode hardly changes. This is because the generation of gold nuclei is instantaneous. Once a certain number of gold nuclei are formed, the subsequent electrochemical deposition will preferentially grow on the gold nuclei rather than on the bare electrode surface.

[0076] like Figure 5 As shown in (A), when the deposition time is 180 s, the gold nanoparticles deposited on the ITO electrode grow into a branched structure. Moreover, as the deposition time gradually increases to 300 s, 600 s, and 900 s, the size of the AuNFs particles increases with the increase of deposition time. Figure 5 As shown in (B), the size of AuNFs becomes larger when the deposition time is 300 s. When the deposition time is further increased to 600 s, AuNFs grow more branched structures, such as Figure 5 (C). When the deposition time continues to increase to 900 s, as shown in Figure 5 As shown in (D), the branched structure of AuNFs generated on the ITO electrode begins to show petal-like accumulation, indicating that the morphology of AuNFs has been initially formed. Figure 5 As shown in (E), when the deposition time reaches 1200 s, the petal-like structure of AuNFs generated on the ITO electrode begins to become more complex and an obvious layered structure appears. Figure 5 As shown in (F), when the deposition time is 1500 s, the generated AuNFs become disordered and uncontrollable. Therefore, when the deposition time is between 900-1200 s, the obtained flower-like AuNFs have a controllable morphology, and 1200 s is the optimal time. Therefore, the AuNFs deposition time is fixed at 1200 s for subsequent experiments.

[0077] Through Experimental Examples 1 and 2, the technical means for preparing flower-shaped gold nanoparticles with controllable morphology were clarified, which specifically include the following steps:

[0078] S1. Alternately soak the ITO electrode in PAA and PEI solutions for 3-5 times, each time for 10 min, to form a PEI / PAA loaded film to obtain a modified ITO electrode;

[0079] S2. Place the modified ITO electrode in a 1-3 mg / mL HAuCl4 precursor solution and use chronoamperometry to deposit gold nanoparticles at a potential of 0.3 V for 900-1200 seconds to obtain a flower-shaped gold nanoelectrode.

[0080] In some more preferred embodiments, the ITO electrode needs to be cleaned before the membrane is loaded, preferably by ultrasonic cleaning in acetone, anhydrous ethanol, and ultrapure water in sequence, with each cleaning lasting 10 minutes.

[0081] In some more preferred embodiments, the concentration of the PEI solution is 0.25 mg / mL, and the concentration of the PAA solution is 1 mg / mL.

[0082] In some more preferred embodiments, after obtaining the flower-shaped gold nanoparticles, washing and drying steps are further included.

[0083] Among them, the above-mentioned Example 1 is the optimal example of this application. In order to verify the feasibility of the preparation method of this application, the flower-shaped gold nanoparticles prepared in Example 1 are structurally characterized.

[0084] Experimental Example 3 Characterization of Flower-like Gold Nanostructures

[0085] Experimental method: The flower-shaped gold nanoparticles prepared in Example 1 were characterized by X-ray photoelectron spectroscopy (XPS). Specifically, the ITO electrode with the gold nanofilm deposited in Example 1 was adhered to the test platform, and an Al Kα X-ray with a power of 150.0 W was used as the excitation light source. The test was carried out in the instrument. The experimental results are shown in FIG. Figure 6 .

[0086] Experimental results and analysis: Figure 6 (A) The full XPS spectrum shows that the peaks at 285.32 eV and 84.15 eV correspond to the C 1s and Au 4f lines, respectively. The C 1s element likely originates from the ITO glass sheet, while the Au 4f line originates from the deposited gold nanoparticles.

[0087] Perform peak fitting on the elements, such as Figure 6 As shown in (B), the three peaks appearing in the C 1s peak fitting diagram are located at 284.80 eV, 286.14 eV and 288.91 eV, corresponding to the stretching vibrations of CC, COC and OC=C groups, respectively. Figure 6 (C) is the high-resolution XPS spectrum of Au 4f. After peak fitting, it was found that there were two peaks at 84.15 eV and 87.80 eV, corresponding to Au 4f 7 / 2 、Au 4f 5 / 2 The results show that zero-valent gold is generated on the surface of the ITO electrode, and the nucleation and growth of gold nanoparticles occur on the surface of the ITO electrode. It is proved that shape-controlled AuNFs are successfully deposited on the surface of the ITO electrode through electrochemical electrodeposition.

[0088] like Figure 6 (D) shows the EDS spectrum of AuNFs deposited on the electrode surface, which further determines the elemental composition and distribution on the electrode surface. Figure 6 As shown in (E), the Au elements on the entire electrode surface are evenly distributed in the flower-shaped area. The Au element content is the highest in the energy spectrum, and there are no other impurity peaks, which further demonstrates that the gold elements of the synthesized AuNFs are distributed in the flower-shaped area on the surface of the ITO electrode, and the AuNFs prepared by this method are of high purity. Figure 6 (F) shown.

[0089] This demonstrates that the embodiments of the present application can successfully and stably prepare flower-shaped gold nanostructures with large surface areas. The resulting flower-shaped gold nanostructures exhibit layered stacking and a flower-like dispersion, exhibiting excellent specific surface area. Furthermore, EDS spectra indicate that the flower-shaped gold nanostructures obtained using the present preparation method are of very high purity. Therefore, it is understood that when applied to aptamer sensors, their electrical and catalytic properties will be significantly improved, thereby enhancing the sensitivity of the sensor.

[0090] Example 2 Application of flower-shaped gold nanoelectrodes in the detection of Kana using a sandwich aptamer sensor

[0091] The flower-shaped gold nanoelectrode prepared by the method of Example 1 was used as a substrate to detect Kana in the sample to be tested in a sandwich aptamer sensor.

[0092] 1. Principle Analysis

[0093] like Figure 7 The schematic diagram shows the principle of the sandwich-type electrochemical aptamer sensor for detecting Lana based on flower-shaped gold nanoparticles in this application. As shown in the figure, the flower-shaped gold nanoparticles (AuNFs) described in this application are first deposited on an ITO electrode. The excellent specific surface area of ​​AuNFs is then utilized to immobilize a large amount of split aptamer 1 (SPA1) to the electrode surface via Au-S bonds. When the target Kana is present in the water sample to be tested, the free split aptamer 2 (SPA2) and SPA1 specifically recognize Kana and form a ternary sandwich complex (SPA1-Kana-SPA2). This allows the FND indicator to be effectively embedded within the complex, generating an electrochemical signal and enabling sensitive detection of Kana.

[0094] In the absence of Kana, SPA lacks a specific target for recognition and cannot form the SPA1-Kana-SPA2 structure, preventing the FND indicator from embedding into the sandwich structure. In this case, electrochemical measurements using differential pulse voltammetry (DPV) show no significant peak signal enhancement. However, due to the positive charge of the FND indicator, it can still bind to SPA through weak electrostatic adsorption, resulting in a very weak electrochemical background signal.

[0095] In the above process, the flower-shaped gold nanoelectrodes prepared by the present application have a large specific surface area, which makes the detection results more accurate and the Kana detection limit lower. The following specific test examples will demonstrate the effect of this technology.

[0096] In the following experimental examples, the oligonucleotides used (SPA1 and SPA2 sequences are as follows) were synthesized and modified by Sangon Biotech (Shanghai) Co., Ltd. and purified by HPLC:

[0097] SPA1: 5'-SH-(CH2)6-TGGGGGTTGAG 3';

[0098] SPA2: 5′-GCTAAGCCGA-3′.

[0099] In the following test examples, the solutions used and their preparation methods are as follows.

[0100] To prepare the DNA stock buffer solution: Accurately weigh 0.3028 g Tris, 0.073 g EDTA, and 1.46 g NaCl into a 250 mL beaker. Dissolve the solution in ultrapure water and adjust the pH to 7.6 with 0.5 M HCl. Transfer the solution to a 250 mL volumetric flask, bring to volume, shake well, and store in a refrigerator at 4°C.

[0101] Preparation of DNA buffer: Accurately weigh 0.3028 g Tris and 1.46 g NaCl into a 250 mL beaker. Dissolve in ultrapure water and adjust the pH to 7.6 with 0.5 M HCl. Transfer to a 250 mL volumetric flask, bring to volume, shake well, and store in a refrigerator at 4°C.

[0102] Preparation of SPA1 stock solution: Centrifuge a centrifuge tube containing 2.0 OD SPA1 dry powder at 4000 rpm for 1 min. Accurately pipette a certain volume of DNA stock buffer into the centrifuge tube to make the concentration of SPA1 stock solution 100 μM. Store at -20°C in the dark.

[0103] Preparation of SPA2 stock solution: Centrifuge a centrifuge tube containing 2.0 OD SPA2 dry powder at 4000 rpm for 1 min. Accurately pipette a certain volume of DNA stock solution buffer into the centrifuge tube to make the concentration of SPA2 stock solution 100 μM. Store at -20°C in the dark.

[0104] Preparation of 10 μM SPA1 stock solution: Pipette 100 μL of 100 μM SPA1 stock solution and 900 μL of DNA buffer into centrifuge tubes, shake thoroughly, and store in a 4°C refrigerator.

[0105] Preparation of 10 μM SPA2 stock solution: Pipette 100 μL of 100 μM SPA2 stock solution and 900 μL of DNA buffer into centrifuge tubes, shake thoroughly, and store in a 4°C refrigerator.

[0106] Preparation of 1 μM SPA1 stock solution: Pipette 100 μL of the prepared 10 μM SPA1 stock solution and 940 μL of DNA buffer into centrifuge tubes, shake thoroughly, and store in a 4°C refrigerator.

[0107] Preparation of 1 μM SPA2 stock solution: Pipette 100 μL of the prepared 10 μM SPA2 stock solution and 920 μL of DNA buffer into centrifuge tubes, shake thoroughly, and store in a 4°C refrigerator.

[0108] Preparation of 3 mg / mL HAuCl4 solution: Accurately weigh 0.3 g HAuCl4·3H2O and dissolve it in 100 mL ultrapure water. Then transfer it to a 100 mL volumetric flask, make up to volume, shake well, and store in a refrigerator at 4°C away from light.

[0109] Preparation of 10 mM TCEP solution ((Tris(2-carboxyethyl)phosphate)): Accurately weigh 0.0287 g of TCEP powder into a beaker, add 1 mL of ultrapure water, and shake thoroughly to prepare a 100 mM TCEP solution. Subsequently, accurately pipette 10 μL of the 100 mM TCEP solution into a glass bottle, add 90 μL of ultrapure water, and shake thoroughly to prepare a 10 mM TCEP solution. Store in a refrigerator at 4°C away from light.

[0110] To prepare a 10 mM MCH (methylcyclohexane) solution: Accurately pipette 7 μL of MCH into a 10 mL centrifuge tube, add ultrapure water to a total volume of 5 mL, and vortex thoroughly to mix to prepare a 10 mM MCH solution. Subsequently, accurately pipette 1 mL of 10 mM MCH into a centrifuge tube, add 9 mL of ultrapure water, and vortex thoroughly to prepare a 1 mM MCH solution. Store in a refrigerator at 4°C away from light.

[0111] 5 mM [Fe(CN)6] 3- / 4- Preparation of the solution (containing 0.1 M KCl): Accurately weigh 0.728 g KCl, 0.0823 g K3[Fe(CN)6], and 0.09363 g K4[Fe(CN)6]·3H2O, respectively. Add them to a 50 mL volumetric flask. Add ultrapure water to the volume, shake well, and store in a refrigerator at 4°C.

[0112] To prepare 0.2 M PBS buffer (pH = 7.6): Accurately weigh 17.907 g of Na₂HPO₄ and 7.805 g of NaH₂PO₄, respectively, and dissolve them in an appropriate amount of ultrapure water. Transfer the mixture to a 250 mL volumetric flask, bring to volume, and shake well to obtain 0.2 M Na₂HPO₄ and 0.2 M NaH₂PO₄ solutions. Adjust the pH of the 0.2 M Na₂HPO₄ solution to 7.6 using the 0.2 M NaH₂PO₄ solution. Shake thoroughly and store in a refrigerator at 4°C.

[0113] Preparation of 10 mM PBS buffer (pH = 7.6): Accurately measure 25 mL of the prepared 0.2 M PBS buffer solution (pH = 7.6) into a 500 mL volumetric flask, add ultrapure water to the volume, shake well, and store in a refrigerator at 4°C until use.

[0114] Preparation of 5 mM FND indicator: Accurately weigh 0.053 g of FND into a glass bottle and dissolve it with DMSO solution to 10 mL. Shake thoroughly and store in a refrigerator at 4°C until use.

[0115] Preparation of 1 mM Kana stock solution: Accurately weigh 0.05825 g of Kana and add it to a 100 mL volumetric flask. Add ultrapure water, shake thoroughly, and store in a refrigerator at 4°C until use.

[0116] Preparation of 1 μM Kana working solution: Accurately pipette 1 μL of 1 mM Kana solution into a 10 mL centrifuge tube, add 999 μL of ultrapure water, shake thoroughly, and store in a refrigerator at 4°C until use.

[0117] 2. Construction of aptasensors based on flower-like gold nanoelectrodes

[0118] This step constructs the optimal aptasensor for the flower-shaped gold nanoelectrode, specifically:

[0119] First, pretreat all nucleotides required for the experiment. To obtain linear chains, pipette 500 μL of SPA1 and 500 μL of SPA2 stock solutions into centrifuge tubes. These tubes are then placed in a water bath (95°C) for 5 minutes and immediately cooled in ice water at -4°C to form linear chains.

[0120] Next, 100 μL of a 10 mM TCEP solution was pipetted into the linearized SPA1, vortexed to mix, and placed in a -4°C refrigerator for 1 hour to inhibit disulfide bond formation. The prepared AuNFs electrode was immersed in this mixed solution (the SPA1 solution concentration was diluted to 0.6 μM using DNA buffer (pH = 7.6)). The reaction was allowed to proceed at room temperature and pressure for 12 hours, resulting in Au-S bond attachment to the electrode, thus obtaining ITO / SPA1. To prevent interference from nonspecific DNA adsorption on the electrode surface, the active sites on the electrode were blocked with a 1 mM MCH solution and incubated in the dark for 30 minutes, yielding ITO / SPA1 / MCH. After incubation, the electrode was immersed in a mixed solution of 0.8 μM SPA2 (diluted in DNA buffer (pH = 7.6)) and Kana, and incubated at room temperature for 2 hours, yielding ITO / [SPA1-Kana-SPA2] / MCH. The electrode was then placed in a PBS buffer solution (pH 7.6) containing 2 mM FND indicator and concentrated in the dark for 10 minutes. The unreacted FND indicator was then thoroughly washed away with PBS buffer (pH 7.6). The electrode was then rinsed with ultrapure water and dried with N₂ to obtain an AuNFs electrode embedded with the FND indicator, which was used in subsequent experiments.

[0121] Test Example 1 UV titration experiment

[0122] UV titration experiments can be used to study the interaction between small molecule structures and biomacromolecules. In this application, since the FND indicator contains a naphthalene dicarboxamide group in its structure, the FND indicator has a double or single absorption peak in the 300-400nm range of the visible spectrum. This experiment can prove whether FND interacts with SPA1-Knan-SPA2.

[0123] Experimental method: The experiment was divided into five groups, ae, each of which used 3 mL of 10 mM PBS buffer solution (pH = 7.6) as the base solution and 10 μL of 5 mM FND indicator. The mixture was mixed by pipetting and stabilized for 10 minutes, and then the SPA1-Kana-SPA2 mixture was added. Among them, the ae group added 0 μL (blank control group), 10 μL, 20 μL, 30 μL, and 40 μL of the SPA1-Kana-SPA2 mixture, respectively. The preparation method of the SPA1-Kana-SPA2 mixture was to pipette 50 μL of 1 μM SPA1, 50 μL of 1 μM SPA2, and 50 μL of 1 μM Kana into the pipette, and incubate at room temperature for 2 hours to form a SPA1-Kana-SPA2 sandwich structure. The ultraviolet absorption spectrum of the mixed solution in the wavelength range of 200~500 nm was measured by UV-visible spectrophotometer. The experimental results are shown in Figure 2. Figure 8 shown.

[0124] Experimental results and analysis: Figure 8 When FND is added alone (panel a), a pair of absorption peaks is clearly observed. However, when 10 µL of SPA1-Kana-SPA2 is added (panel b), the FND indicator's absorption peaks shift. This is because as the SPA1-Kana-SPA2 sandwich structure is gradually added to the PBS buffer containing the FND indicator, FND interacts with SPA1-Kana-SPA2, forming a complex that perturbs the FND indicator's electronic structure and electron transfer. The hypochromic effect observed in the UV-Vis absorption spectrum preliminarily indicates that the interaction between FND and the SPA1-Kana-SPA2 sandwich structure occurs via intercalation. This is because during intercalation, some of the π* orbitals in the small molecule are occupied by π electrons from the base pair, reducing the probability of spontaneous π-π* transitions within the small molecule. The degree of the hypochromic effect is related to the strength of the interaction between FND and the SPA1-Kana-SPA2 sandwich structure. The stronger the hypochromic effect, the stronger the insertion effect between FND and the SPA1-Kana-SPA2 sandwich structure. Figure 8 It can be seen that as the SPA1-Kana-SPA2 sandwich structure is gradually added, the hypochromic effect produced by the ultraviolet absorption spectrum becomes stronger.

[0125] Test Case 2 Feasibility Analysis

[0126] This test example was used to verify the feasibility of the electrochemical sensor constructed in Example 2-2, and a system containing different substances was analyzed by DPV.

[0127] Experimental method: The experiment was divided into groups ad. The electrochemical sensor constructed in Example 2-2 was immersed in a. 1 μM SPA1 for reaction at room temperature and pressure for 12 h, b. 1 μM SPA2 for incubation at room temperature and pressure for 2 h, c. 1 μM SPA1 for reaction at room temperature and pressure for 12 h and then placed in 1 μM SPA2 for incubation at room temperature and pressure for 2 h, d. 1 μM SPA1 for reaction at room temperature and pressure for 12 h and then placed in a mixed solution of 1 μM SPA2 and 1 μM Kana for incubation at room temperature and pressure for 2 h; then the electrode was placed in a PBS buffer solution (pH = 7.6) containing 2 mM FND indicator, enriched in the dark for 10 min, and then the unreacted FND indicator was thoroughly washed with PBS buffer (pH = 7.6), then washed with ultrapure water and blown dry with N2. Then, the DPV method was used for detection in 0.2 M blank PBS buffer solution (pH = 7.6): scanning potential: -0.2~0.7 V, scanning rate: 100 mV / s, pulse amplitude: 50 mV / s, pulse width: 0.05 s, and standing for 30 s. The experimental results are shown in Figure 2. Figure 9 shown.

[0128] Result analysis: Figure 9 As shown, after SPA1 was linked to the electrode via an Au-S bond, it was placed in a PBS buffer containing the FND indicator for DPV testing (panel a). The DPV signal generated was very weak. This is because, in the absence of the target, the SPA1-Kana-SPA2 sandwich structure was not formed, preventing the FND indicator from intercalating and enriching within it. However, the positive charge of FND electrostatically bound to SPA1, generating a weak DPV signal. Similarly, when the electrode was placed in a system containing only SPA2 and FND (panel b) or a system of SPA1, SPA2, and FND (panel c), the electrochemical signal was also very weak. When the target was added (panel d), SPA1 and SPA2 specifically recognized the target, forming a SPA1-Kana-SPA2 sandwich structure, which allowed the FND indicator to intercalate and enrich within it, resulting in a significant increase in the DPV electrochemical signal.

[0129] Experimental Example 3 Electrochemical Characterization

[0130] Experimental method: CV: The AuNFs electrode prepared in Example 2-2 was used as the working electrode, 5 mM [Fe(CN)6] 3- / 4- The solution was used as an indicator to characterize the charge change on the electrode surface during the electrode sensing interface modification process at a scan rate of 50 mV / s and a scanning potential of -0.2 V to 0.7 V.

[0131] EIS: The AuNFs electrode prepared in Example 2-2 was used as the working electrode, 5 mM [Fe(CN)6] 3- / 4- The solution is used as an indicator in the range of 0.1 to 10 6 The change of resistance during the modification of the electrode sensing interface was characterized at a scanning frequency of Hz.

[0132] Experimental results: Figure 10 As shown in Figure 2, the electrode sensing interface is characterized by CV and EIS, where CV is used to characterize the charge change on the electrode surface during the electrode modification process, and EIS is used to characterize the resistance change during the charge transfer process on the electrode surface. Figure 10 As shown in (A), when AuNFs are not deposited on the ITO electrode, the CV redox current signal is low; after the electrode is modified with polymer PEI / PAA, its surface electrochemical redox signal is improved to a certain extent. This is because the PEI loaded on the outermost layer has a negatively charged Fe[(CN)6] 3- / 4- Has good affinity. Since AuNFs have good conductivity, when AuNFs are further electrodeposited on the surface, the redox signal on the electrode surface increases significantly. When MCH and SPA1 are modified on the electrode, the redox signal decreases significantly. This is because both MCH and SPA1 carry a large amount of negative charge, which reacts with Fe[(CN)6] 3- / 4- Electrostatic repulsion exists between them. When Kana and SPA2 are added, the aptamer's specific targeting of the target forms a SPA1-Kana-SPA2 sandwich structure. SPA2's proximity to the electrode surface imparts a more negative charge, further reducing the redox potential. FND is an insulator, and its embedding into the SPA1-Kana-SPA2 sandwich further reduces the electrochemical redox signal.

[0133] like Figure 10(B) shows that under the same experimental conditions, EIS was used to characterize the changes in the surface impedance of the electrode during the modification process. When the bare electrode was modified with PEI / PAA and then AuNFs were deposited onto the electrode by electrodeposition, the resistance of the electrode gradually decreased. Subsequently, after the electrode was modified with MCH, MCH+SPA2+Kana, and MCH+SPA2+Kana+FND in sequence, the steric hindrance was greatly enhanced, resulting in R ct The above CV and EIS test results prove that the electrochemical aptasensor is successfully constructed.

[0134] Experimental Example 4 Optimization of reaction conditions

[0135] 1. Optimization of SPA1 concentration: SPA1 is the recognition element of the electrochemical aptasensor, and changes in its concentration will directly affect the performance of the sensor.

[0136] Experimental method: As shown in Example 2-2, the difference is that the concentrations of SPA1 solution are 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, and 0.8 μM, respectively. After the construction is completed, the DPV method is used for detection, the peak shape changes are observed, the oxidation peak current value is recorded, and the optimal concentration of SPA1 is analyzed; the experimental results are shown in FIG. Figure 11 shown.

[0137] Experimental results and analysis: Figure 11 As shown, the DPV peak signal gradually increases with increasing SPA1 concentration. This may be because as the SPA1 concentration increases, the amount of SPA1 attached to the AuNFs electrode via Au-S bonds also increases. This increases the number of SPA1-Kana-SPA2 sandwich structures that can be formed in the presence of fixed SPA2 and Kana concentrations, allowing more FND indicators to be embedded in these structures, resulting in a stronger electrochemical signal. When the SPA1 concentration reaches 0.6 μM, the DPV electrochemical signal gradually stabilizes. This may be because the amount of SPA1 attached to the AuNFs electrode reaches saturation at this point, causing the number of SPA1-Kana-SPA2 sandwich structures to gradually stabilize, and thus the amount of FND indicators that can be embedded in these structures to reach saturation. As can be seen, a DPV peak signal is obtained when the SPA1 concentration is ≥0.3 μM, with the optimal value being reached at 0.6 μM.

[0138] 2. Optimization of SPA2 concentration: SPA2 is an indispensable part of the electrochemical aptasensor system, and the concentration of SPA2 is directly related to the intensity of the generated electrochemical signal.

[0139] Experimental method: As shown in Example 2-2, the difference is that the concentrations of SPA2 solution are 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.79 μM, and 1 μM respectively. After the construction is completed, the DPV method is used for detection, the peak shape change is observed, and the oxidation peak current value is recorded; the experimental results are shown in FIG. Figure 12 shown.

[0140] Experimental results and analysis: Figure 12 As shown in the figure, with other conditions remaining unchanged, the DPV electrochemical signal value increases with increasing SPA2 concentration. This is because as the SPA2 concentration increases, the SPA1-Kana-SPA2 sandwich structure generated by the specific recognition of the target by SPA1 and SPA2 increases, allowing more FND indicators to be embedded in this structure, generating a larger electrochemical signal. When the SPA2 concentration increases further from 0.7 μM to 0.8 μM, the electrochemical signal increases slightly. This indicates that the generated SPA1-Kana-SPA2 sandwich structure has reached saturation at this point, and the amount of FND indicators that can be embedded in this structure has reached a maximum, resulting in the DPV electrochemical signal not continuing to increase with increasing SPA2 concentration. Therefore, it can be seen that the DPV peak signal is obtained when the SPA2 concentration is ≥0.5 μM, reaching an optimal value at 0.8 μM.

[0141] 3. Optimizing the pH of the DNA buffer solution: The pH of the PBS buffer solution directly affects the activity of SPA1 and SPA2, as well as their ability to specifically recognize their targets. Excessively acidic or alkaline pH in the DNA buffer solution will reduce the specific recognition ability of SPA1 and SPA2. Therefore, optimizing the pH of the DNA buffer solution is particularly important.

[0142] Experimental method: As shown in Example 2-2, the difference is that the pH of the DNA buffer is set to 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 respectively. After the construction is completed, the DPV method is used for detection to observe the changes in peak shape; the experimental results are shown in FIG. Figure 13 shown.

[0143] Experimental results and analysis: Figure 13 As shown, the electrochemical signal reaches its maximum value at a buffer pH of 7.6. This is likely because SPA1 and SPA2 achieve optimal target specificity and activity at a pH of 7.6, forming a larger SPA1-Kana-SPA2 sandwich structure and embedding more FND indicators. This indicates that good technical results are achieved within a pH range of 7-8, with the optimal value being 7.6.

[0144] 4. Optimization of FND indicator enrichment time: The enrichment time of the FND indicator is a key part of the sensor construction process. The length of the enrichment time will directly affect the amount of FND embedded in the SPA1-Kana-SPA2 sandwich structure. Therefore, in order to achieve better detection results, the enrichment time of the FND indicator needs to be optimized.

[0145] Experimental method: As shown in Example 2-2, the difference is that the electrode was placed in a PBS buffer solution (pH = 7.6) containing 2 mM FND indicator, and the light-proof enrichment time was 7 min, 8 min, 9 min, 10 min, 11 min, and 12 min respectively. After the construction was completed, the DPV method was used for detection, the peak shape change was observed, and the oxidation peak current value was recorded; the experimental results are shown in Figure 2-2. Figure 14 shown.

[0146] Experimental results and analysis: Figure 14 As shown in the figure, with other conditions remaining unchanged, the measured DPV signal value increases with increasing FND-embedded indicator enrichment time. This is likely due to the FND indicator gradually embedding into the SPA1-Kana-SPA2 sandwich structure, increasing the generated electrochemical signal value. When the incubation time gradually increases to 10 minutes, the generated DPV electrochemical signal begins to level off. This may be because the amount of FND indicator embedded in the sandwich structure reaches saturation. Therefore, an FND indicator enrichment time of 9 minutes or longer achieves optimal technical results, with 10 minutes being the optimal time.

[0147] 5. Optimizing FND Indicator Concentration: The concentration of the FND embedded indicator is crucial for electrochemical sensor performance. If the hybrid indicator concentration is too low, too little indicator will be embedded within the SPA1-Kana-SPA2 sandwich structure, making detection difficult and reducing the sensitivity of the electrochemical sensor. If the FND indicator concentration is too high, small molecules may aggregate, impairing indicator embedding.

[0148] Experimental method: As shown in Example 2-2, the difference is that the electrodes were placed in PBS buffer solution (pH = 7.6) containing 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, and 3.0 mM FND indicator, respectively, and the enrichment time was 10 min in the dark. After the construction was completed, the DPV method was used for detection, the peak shape change was observed, and the oxidation peak current value was recorded; the experimental results are shown in FIG. Figure 15 shown.

[0149] Experimental results and analysis: Figure 15As shown in the figure, as the concentration of the FND embedded indicator increases from 0.5 to 2 mM, the electrochemical peak signal measured by DPV gradually increases. When the concentration of the FND embedded indicator exceeds 2 mM, the change in peak current significantly decreases. This indicates that the electrochemical sensor performance is optimal when the FND indicator concentration is 2 mM. Therefore, a FND indicator concentration of 1-2.5 mM can achieve good results, with 2 mM being the optimal concentration.

[0150] 6. Kana incubation time optimization: The ternary sandwich complex formed in this study is based on the specific recognition of the target Kana by SPA1 and SPA2. Therefore, the incubation time of Kana will directly affect the number of SPA1-Kana-SPA2 ternary sandwich complexes formed, thereby affecting the performance of the sensor.

[0151] Experimental method: As shown in Example 2-2, the difference is that the electrode is immersed in a mixed solution of 0.8μM SPA2 (diluted with DNA buffer (pH=7.6)) and Kana, and the incubation time at room temperature is 30min, 60min, 90min, 120min, 150min, and 180min. After the construction is completed, the DPV method is used for detection, the peak shape change is observed, and the oxidation peak current value is recorded; the experimental results are shown in FIG. Figure 16 shown.

[0152] Experimental results and analysis: Figure 16 As shown in the figure, the measured electrochemical peak signal increases with increasing Kana incubation time. This is because as the incubation time increases, more ternary sandwich complexes are formed, allowing for the embedding of more FND indicators. After the incubation time reaches 120 minutes, the amplitude of the DPV peak signal decreases significantly and levels off. This is because the number of ternary sandwich complexes formed by SPA1, SPA2, and Kana has reached saturation at this point. Therefore, good technical results can be achieved with incubation times ≥120 minutes, with an incubation time of 120 minutes being optimal.

[0153] The above optimization experiments show that better measurement results can be achieved when the concentration of SPA1 solution is ≥0.3μM, the concentration of SPA2 solution is ≥0.5μM, the Kana incubation time is ≥120min, the pH of the DNA buffer of SPA1 solution and SPA2 solution is 7-8, the concentration of the FND indicator is 1-2.5mM, and the enrichment time is ≥9min. Among them, the best technical effect can be achieved when the concentration of SPA1 solution is 0.6μM, the concentration of SPA2 solution is 0.8μM, the Kana incubation time is 120min, the pH of the DNA buffer solution is 7.6, the concentration of the FND indicator is 2mM, and the enrichment time is 10min.

[0154] Test Example 5 Sensor Performance Investigation

[0155] 1. Linear range

[0156] Experimental method: The flower-shaped gold nanoelectrode aptasensor constructed in Example 2-2 was tested using the DPV method after construction, and the peak shape change was observed and the oxidation peak current value was recorded. The oxidation peak current value was linearly related to the Kana concentration, such as Figure 17 shown.

[0157] Experimental results and analysis: Figure 17 As shown in (A), as the concentration of Kana gradually increases, the measured DPV peak signal becomes larger and larger. This is because more and more SPA1-Kana-SPA2 ternary sandwich complexes are formed on the electrode, resulting in more and more FND indicators being embedded. Figure 17 (B) It can be seen that when the Kana concentration is in the range of 5 nM~2 μM, there is a good linear relationship between the peak signal of DPV and the logarithm of Kana concentration, and the linear equation is: y =-3.68144 x -5.31049 ( R 2 =0.997), where x represents the logarithm of Kana concentration, y Indicates the measured DPV peak signal. σ / k The detection limit (LOD) of the electrochemical aptasensor was calculated to be 0.2 nM.

[0158] The constructed electrochemical aptamer sensor was compared with other Kana detection methods in recent years. The results are shown in Table 1. In comparison, the electrochemical aptamer sensor proposed in this work has a lower detection limit, which may be due to the target-induced SPA binding strategy and the increase in sensor performance by AuNFs.

[0159] Table 1 Performance comparison of this application and other Kana detection methods

[0160] Detection method Linear range Detection limit References Electrochemical aptasensors 15.3 nM~0.24 mM 10 nM Prior Art 1 Colorimetric sensors 0.1 μM~100 μM 6.28 μM Prior Art 2 Fluorescence sensor 80 nM~10 μM 13.3 nM Prior Art 3 Fluorescence sensor 0.05 μM~20 μM 11.76 nM Prior Art 4 Fluorescence-colorimetric dual-mode sensor 50 nM~1 μM 7.3 nM Prior Art 5 Electrochemical sandwich aptasensor 5 nM~2 μM 0.2 nM This application

[0161] 2. Selectivity: Since the composition of the test solution is often complex during actual sample testing, to verify the sensor's ability to effectively identify Kana in practical applications, five aminoglycoside antibiotics with similar properties to Kana were evaluated: tobramycin, gentamicin, streptomycin, amikacin, and netilmicin. The target Kana concentration was 1 μM, and the concentrations of other interfering ions were all 10 μM.

[0162] Experimental method: As shown in Example 2-2, the difference is that the electrodes were immersed in 0.8μM SPA2 (diluted with DNA buffer (pH=7.6)) and a certain concentration of tobramycin (TOB), gentamicin (GEN), streptomycin (STR), amikacin (AMK), netilmicin (MET), and Kana mixed solution. After the construction was completed, the DPV method was used for detection, the peak shape change was observed, and the oxidation peak current value was recorded; the experimental results are shown in Figure 2-2. Figure 18 shown.

[0163] Experimental results and analysis: The test results are as follows: Figure 18 As shown, the electrochemical aptamer sensor exhibited a strong DPV response only when only Kana was present in the test water sample. This is because SPA1 and SPA2 specifically recognize Kana only in its presence, forming a ternary sandwich structure of SPA1-Kana-SPA2. This demonstrates that the electrochemical aptamer sensor constructed using this method has good selectivity for Kana and can be applied in real-world water testing.

[0164] 3. Reproducibility: Reproducibility is also an important factor in measuring sensor accuracy, so it needs to be examined. Six identical independent experimental groups were set up to test 1 μM Kana to evaluate the reproducibility of the electrochemical aptasensor.

[0165] Experimental method: As shown in Example 2-2, the difference is that 6 sets of electrodes were prepared separately, and the electrodes connected with SPA1 were immersed in a mixed solution of 0.8 μM SPA2 and 1 μM Kana and incubated at room temperature for 2 hours. After the incubation, the modified electrodes were stored in Tris-HCl buffer solution (pH = 7.6) and placed in a refrigerator at 4 ℃ for different days. After the construction was completed, the DPV method was used for detection, the peak shape changes were observed, and the oxidation peak current value was recorded; the experimental results are shown in Figure 2-2. Figure 19 shown.

[0166] Experimental results and analysis: Figure 19 As shown, the relative standard deviation (RSD) among six independent experimental groups was 2.6% (n=6), indicating that the constructed electrochemical aptasensor has good reproducibility.

[0167] 4. Stability

[0168] Experimental method: As in Example 2-2, the difference is that the electrode connected with SPA1 is immersed in a mixed solution of 0.8 μM SPA2 and 1 μM Kana and incubated at room temperature for 2 hours. After the incubation is completed, the modified electrode is stored in Tris-HCl buffer solution (pH = 7.6) and placed in a refrigerator at 4°C for different days. After the construction is completed, the DPV method is used for detection, the peak shape changes are observed, and the oxidation peak current value is recorded. The experimental results are shown in the figure. Figure 20 shown.

[0169] Experimental results and analysis: Figure 20 As shown in the figure, the electrochemical peak signal measured after the electrode was stored in the refrigerator for 5 days remained essentially unchanged. After 20 days of storage, the sensor's detection performance for Kana was still able to maintain over 85%, demonstrating the excellent stability of the constructed aptasensor.

[0170] Test Example 6 Actual Sample Testing

[0171] In order to demonstrate the feasibility and practicality of the constructed electrochemical aptasensor in actual water bodies, water samples from three different regions were spiked with the sensor and tested (the spiked concentrations were 10 nM, 100 nM, and 500 nM, respectively).

[0172] Experimental Method: Water samples for testing were collected from Shahe River, Qinglong Lake, and Dongfeng Canal in Chengdu, Sichuan Province. First, the water samples were spiked with 10 nM, 100 nM, and 500 nM, respectively. After spiking, the water samples were centrifuged at 8000 rpm for 10 minutes to remove larger impurities. The samples were then filtered using a 0.22 µm syringe filter to remove smaller impurities. Finally, Kana in the spiked samples was detected according to the electrochemical aptasensor detection procedures constructed in Example 2-2 (i.e., the electrode linked to SPA1 was immersed in a mixed solution of 0.8 μM SPA2 and the test sample and incubated at room temperature for 2 hours). The experimental results are shown in Table 2.

[0173] Experimental results and analysis: The electrochemical detection results were compared with the HPLC-MS detection results. The results are shown in Table 2. The recovery rate of the standard addition method experiment ranged from 86.4% to 116.8%, and the relative standard deviation (RSD) of the three parallel experiments ranged from 2.78% to 6.11%. The results detected in this work are similar to those obtained by high-performance liquid chromatography analysis, proving that the analytical method has high accuracy and precision, high reliability, and can be used for the determination of Kana in actual water samples.

[0174] Table 2 Actual water sample spiked test results

[0175]

[0176] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions that fall within the scope of protection of the present invention are within the scope of protection of the present invention. It should be noted that improvements and modifications that can be made by persons of ordinary skill in the art without departing from the principles of the present invention are also considered to be within the scope of protection of the present invention.

Claims

1. Application of flower-shaped gold nanoelectrodes in sandwich aptamer sensors for detecting kanamycin, characterized in that: The steps include: S1. Immerse the flower-shaped gold nanoelectrode AuNFs in SPA1 solution, and react at room temperature to fix SPA1 on the flower-shaped gold nanoelectrode through Au-S bonds; S2. Immerse the flower-shaped gold nanoelectrode immobilized with SPA1 in a mixed solution of SPA2 solution and the sample to be tested, and incubate at room temperature to obtain a flower-shaped gold nanoelectrode with a SPA1-AuNFs-SPA2 sandwich structure; S3. Place the flower-shaped gold nanoelectrode with the SPA1-AuNFs-SPA2 sandwich structure in a FND indicator and PBS buffer solution, protect from light, and then wash it. Then, detect the kanamycin content in the sample by the DPV method; Wherein, the nucleotide sequence of the SPA1 is 5'-SH-(CH2)6-TGGGGGTTGAG 3'; The nucleotide sequence of the SPA2 is 5'-GCTAAGCCGA-3'; The FND indicator contains a naphthalene dicarboxamide group; The preparation method of the flower-shaped gold nanoelectrode described in S1 is: The ITO electrode was alternately immersed in polyacrylic acid PAA and polyethyleneimine PEI solutions to form a PEI / PAA loaded film to obtain a modified ITO electrode; The modified ITO electrode was placed in a HAuCl4 precursor solution, and gold nanoparticles were deposited at a potential of -0.3 V using chronoamperometry to obtain flower-shaped gold nanoelectrodes. In step (1), the concentration of the PEI solution is 0.25 mg / mL, and the concentration of the PAA solution is 1 mg / mL; the number of alternating immersions is 3-5 times; The concentration of the HAuCl4 precursor solution in step (2) is 1-3 mg / mL; the deposition time of the chronoamperometry is 900-1200 s; The concentration of SPA1 solution described in S1 is ≥0.3 μM; The concentration of the SPA2 solution in S2 is ≥0.5 μM; The incubation time in S2 is ≥120 min; The SPA1 solution and SPA2 solution described in S1 and S2 are diluted and prepared with a DNA buffer, wherein the pH of the DNA buffer is 7-8; The concentration of the FND indicator in S3 is 1-2.5 mM, and the enrichment time is ≥9 min.

2. The application according to claim 1, characterized in that The concentration of the SPA1 solution in S1 is 0.6 μM, the concentration of the SPA2 solution in S2 is 0.8 μM, the incubation time is 120 min, the concentration of the FND indicator in S3 is 2 mM, and the enrichment time is 10 min; wherein, the SPA1 solution and SPA2 solution in S1 and S2 are diluted and prepared with a DNA buffer, and the pH of the DNA buffer is 7.6.

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