Application of flower-like gold nanoparticles in detection of Kana by electrochemical-colorimetric dual-mode sandwich type aptamer sensor

Abstract

The invention relates to the field of antibiotic residue detection, in particular to application of flower-like gold nanoparticles in Kana detection by an electrochemical-colorimetric dual-mode sandwich type aptamer sensor. The flower-like gold nanostructure with the large surface area is stably prepared, the obtained flower-like gold nanostructure presents layered stacking and flower-like dispersion towards the periphery and has the excellent specific surface area, when the flower-like gold nanostructure is applied to an aptamer sensor, the electrical performance and the catalytic performance of the flower-like gold nanostructure can be remarkably improved, and the flower-like gold nanostructure can be applied to the aptamer sensor. The sensitivity of the sensor is improved; the gold nanoelectrode prepared by the invention is combined with optimized parameters (such as SPA1, SPA2 concentration and the like) in the application process, so that the detection limit of Kana in a to-be-detected sample can reach 0.074 nM.

Description

TECHNICAL FIELD

[0001] The application relates to the field of antibiotic residue detection, and particularly relates to application of a flower-shaped gold nanometer in an electrochemical-colorimetric dual-mode sandwich type aptamer sensor for detecting Kana. BACKGROUND

[0002] A fluorescent aptamer sensor is a sensor that detects target molecules through fluorescence signal changes, and is generally composed of a recognition unit, a signal transduction unit and a signal output unit to form a functional system. The recognition unit usually adopts a specific aptamer as a molecular probe to achieve selective capture of target molecules through specific binding. The signal transduction unit is responsible for connecting the recognition unit and the signal output unit, and common signal transduction mechanisms include fluorescence resonance energy transfer (FRET), fluorescence quenching and fluorescence enhancement. The signal output unit, as the core part of the sensor, usually adopts fluorescent materials such as fluorescent dyes or quantum dots (such as AuNPs), which will produce measurable fluorescence signal changes after being combined with target molecules. According to different detection requirements, the composition of the fluorescent sensor can be flexibly adjusted to improve its sensitivity and specificity.

[0003] Kana is a widely used aminoglycoside antibiotic. Its overuse in the fields of animal husbandry, fishery and agriculture leads to Kana residues entering water bodies, thereby causing harm to aquatic organisms and human health. Therefore, it is of great significance to accurately and sensitively detect Kana. 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 the long cycle, high cost and the need for professional technical personnel to operate, the application of these methods is limited to a certain extent. Aptamer sensors based on colorimetric method and electrochemical technology have attracted widespread attention from researchers due to their faster detection speed and simpler operation. However, due to the complexity of environmental samples, these aptamer sensors based on a single method often have poor accuracy in the detection process. In order to solve the limitations of single sensing method, dual-mode sensors combining two complementary technologies have attracted widespread attention due to their high accuracy and excellent anti-interference ability.

[0004] A sandwich type fluorescent sensor is mainly based on the sandwich structure formed between aptamers and target molecules. In this structure, gold nanometer electrode is used as the substrate of aptamer, and its excellent specific surface characteristics and high conductivity make gold nanometer material the most popular nanometer material in the field of electrochemical aptamer sensors. Therefore, a technology for rapidly detecting Kana by using gold nanometer is urgently needed in the field. SUMMARY

[0005] Based on the above reasons, the purpose of the present application is to provide an application of flower-shaped gold nanometer electrode in sandwich type aptamer sensor detection of Kana, which specifically comprises the following steps:

[0006] S1. Respectively mix the thiolated SPA1 solution and SPA2 solution with the flower-shaped gold nanometer solution to obtain the SPA1-AuNFs probe solution and the SPA2-AuNFs probe solution linked by Au-S bond;

[0007] S2. Add the sample to be measured to the mixed solution of the SPA1-AuNFs probe solution and the SPA2-AuNFs probe solution for incubation, add the H2O2 solution and the TMB solution after the incubation is completed, mix and incubate for the second time, then measure the absorbance of the solution by using the ultraviolet-visible spectrophotometer and determine the electrochemical signal value by using the chronoamperometry, and obtain the concentration of Kana in the sample to be measured.

[0008] Further, the flower-shaped gold nanometer solution is prepared by the seed growth method.

[0009] Further, the seed growth method specifically comprises:

[0010] (1) Mix and stir the HAuCl4 solution with ultrapure water, heat to vigorous boiling, quickly add the sodium citrate solution, mix and stir until the solution color changes from blue to purple and finally gradually changes to red, then continue to heat to obtain the colloidal gold seed solution;

[0011] (2) Drop the AuNPs solution into ultrapure water, then drop the colloidal gold seed solution and the sodium citrate solution, mix and stir to heat to 50℃, then add the hydroquinone solution while cooling, stir until the color changes from black gray to blue, continue to stir and cool, centrifuge to obtain the flower-shaped gold nanometer.

[0012] Preferably, the concentration of the HAuCl4 solution, the sodium citrate solution and the AuNPs solution is 1%, w / v.

[0013] Preferably, the concentration of the SPA1 solution and the SPA2 solution in S1 is greater than or equal to 1 μM.

[0014] Preferably, the concentration of the H2O2 solution in S2 is 6%-12%.

[0015] Preferably, the concentration of the TMB solution in S2 is ≥6 mM.

[0016] Preferably, the second incubation temperature in S2 is 20-50℃.

[0017] Preferably, the second incubation time in S2 is 100-150 min.

[0018] Preferably, the concentration of the SPA1 solution in S1 is 1.2 μM, the concentration of the SPA2 solution is 1 μM, the concentration of the H2O2 solution in S2 is 8%, the concentration of the TMB solution is 8 mM, the secondary incubation temperature is 40°C, and the secondary incubation time is 120 min.

[0019] Beneficial effects: The application provides a stable method for preparing flower-shaped gold nanostructures with a large surface area, and the obtained flower-shaped gold nanostructures present a layered stack and a flower-shaped dispersion around the periphery, have an excellent specific surface area, and when applied to an aptamer sensor, the electrical performance and catalytic performance of the flower-shaped gold nanostructures are significantly improved, thereby improving the sensitivity of the sensor; the gold nano electrode prepared by the application and the optimized parameters (such as the concentrations of SPA1 and SPA2) in the application process can make the detection limit of Kana in the sample to be measured reach 0.074 nM. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Flow chart for preparing AuNFs;

[0021] Figure 2 Characterization analysis results of the flower-shaped gold nanostructures, wherein (A) is the ultraviolet-visible spectroscopy of AuNPs and AuNFs; (B) is the TEM image of AuNPs and (C) is the TEM image of AuNFs; (D) is the DLS particle size analysis diagram of AuNFs, (E) is the EDS full spectrum diagram, (F) is the gold element EDS area scan diagram, (G) is the XPS full spectrum diagram, (H) is the Au 4f peak fitting diagram, and (I) is the C 1s peak fitting diagram;

[0022] Figure 3 Principle diagram of the sensor for detecting the application;

[0023] Figure 4 Feasibility analysis results of the dual-mode sensing system, wherein (A) is the ultraviolet-visible spectroscopy and (B) is the chronoamperometry method;

[0024] Figure 5 Peroxidase-like activity analysis results, wherein (a) is SPA-AuNFs+H2O2+TMB; (b) is AuNFs+H2O2+TMB; (c) is SPA1+H2O2+TMB; (d) is SPA2+H2O2+TMB; (e) is AuNF+TMB; and (f) is H2O2+TMB;

[0025] Figure 6 Steady-state kinetics analysis results, wherein (A) is the Michaelis-Menten curve and (B) is the Lineweaver-Burk curve;

[0026] Figure 7Results of different kinds of active oxygen scavengers, where (a) Blank; (b) 5 mM Tryptophan; (c) 5 mM 1,4-Benzoquinone; (d) 5 mM tert-Butanol;

[0027] Figure 8 Results of different concentrations of active oxygen scavengers, where (a) Blank; (b) 0.5 mM tert-Butanol; (c) 1 mM tert-Butanol; (d) 2 mM tert-Butanol; (e) 3 mM tert-Butanol; (f) 4 mM tert-Butanol; (g) 5 mM tert-Butanol;

[0028] Figure 9 Results of optimization of SPA1 concentration;

[0029] Figure 10 Results of optimization of SPA2 concentration;

[0030] Figure 11 Results of optimization of H2O2 concentration;

[0031] Figure 12 Results of optimization of TMB concentration;

[0032] Figure 13 Results of optimization of secondary incubation time;

[0033] Figure 14 Results of optimization of secondary incubation time;

[0034] Figure 15 Results of linear range, where (A) visualization of different concentrations of Kana; (B) UV-Vis spectrogram of different concentrations of Kana; (C) linear relationship between different concentrations of Kana and absorbance at 652 nm; (D) chronoamperometric response of different concentrations of Kana; (E) linear relationship between different concentrations of Kana and chronoamperometric intensity;

[0035] Figure 16 Results of sensor selectivity;

[0036] Figure 17 Results of sensor reproducibility, where (A) reproducibility of sensor AuNFs catalytic performance and (B) reproducibility of sensor detection performance;

[0037] Figure 18 Results of sensor stability. DETAILED DESCRIPTION

[0038] In order to clarify the purpose, research method and advantages of the present application, the present application is further described in detail through the following examples.

[0039] In this embodiment, all electrochemical experiments use a three-electrode system, in which Ag / AgCl (saturated KCl) is the reference electrode, platinum wire is the counter electrode, and ITO electrode is the working electrode.

[0040] Example 1 Selection and optimization of flower-shaped gold nanoelectrode

[0041] This example provides a preparation method of the flower-shaped gold nanoelectrode of the present application, as shown in Figure 1 (AuNFs preparation flow) Before the experiment, all glassware and stirrers involved in the experimental process need to be immersed in aqua regia for more than 24 hours. When used, the above glassware is cleaned several times with ultrapure water.

[0042] As shown in Figure 1 (A), first, a colloidal gold seed solution with a particle size of 20 nm was synthesized by citrate hydrothermal reduction: 1 mL of 1% (w / v) HAuCl4 was first added to a beaker containing 99 mL of ultrapure water, and a magnetic stirrer was used to heat the mixture to 100°C. After observing that the solution in the beaker began to boil vigorously, 2 mL of 1% (w / v) sodium citrate solution was quickly added under vigorous stirring for two minutes of mixing and observation of the color change in the beaker. When the solution color changed from blue to purple and finally gradually changed to red, the timer continued to heat for 10 min on the magnetic stirrer. After the solution was completely cooled to room temperature, a colloidal gold seed solution with a particle size of 20 nm (AuNPs) was obtained and stored in a refrigerator at 4°C for standby use.

[0043] As shown in Figure 2 (B), using the seed growth method strategy, AuNFs with multiple branches were synthesized. AuNFs with an average particle size of 73 nm were prepared using sodium citrate as a reducing agent. 150 μL of AuNPs solution was slowly added to a beaker containing 25 mL of ultrapure water under slow stirring, followed by slowly adding 180 μL of 1% (w / v) HAuCl4 and 660 μL of 1% (w / v) sodium citrate solution and timing for 2 min of mixing. After timing for 2 min, the solution was heated to 50°C with a magnetic stirrer, then the heat source was removed and the stirring speed was increased, while 1 mL of 30 mM hydroquinone solution was added dropwise to the mixed solution, and the color change of the solution was observed. When the solution color changed from black gray to blue, the stirring was continued for 30 min. After the solution was cooled to room temperature and centrifuged at 9000 x g for 10 min, the obtained AuNFs were redissolved in 25 mL of ultrapure water and stored in a refrigerator at 4°C for standby use.

[0044] Characterization of flower-like gold nanomaterials of test example 1

[0045] The flower-like gold nanomaterials prepared in example 1 were characterized as shown in Figure 2 (A). The synthesized gold nanomaterials were tested in the range of 400 nm to 800 nm using a UV-Vis spectrophotometer. The results showed that the gold seeds synthesized by citrate reduction had a maximum absorption peak at 520 nm, which was consistent with the theoretical absorption wavelength of AuNPs. After the synthesis of AuNFs by the seed-mediated growth method, it was observed through the UV-Vis spectrum that the UV spectrum of AuNFs showed a clear red shift, which was probably due to the successful transformation of gold nanomaterials from small-sized AuNPs to large-sized AuNFs with flower-like wrinkles. In order to further verify this conclusion, the synthesized gold seeds and AuNFs were characterized by TEM, respectively. As shown in Figure 2 (B), the gold seeds obtained by citrate reduction showed uniform spherical shape, which was the basis for the formation of AuNFs. Subsequently, the synthesized AuNFs were characterized and found to show a morphology of thick in the center and thin at the edges, i.e. a core and surface flower-like wrinkle morphology, which proved that AuNFs were generated from AuNPs, as shown in Figure 2 (C). As shown in Figure 2 (D), the particle size of the synthesized AuNFs was analyzed by dynamic light scattering (DLS), which showed that the particle size of the synthesized AuNFs was about 73.50.48 nm.

[0046] In order to further determine the elemental composition and distribution of the synthesized AuNFs, EDS mapping of the synthesized AuNFs was performed, as shown in Figure 2 (E). The EDS full spectrum of AuNFs showed the presence of C, Au and O elements. Due to the use of carbon film for testing, the peak value of C element was relatively high. The Au element was uniformly distributed in the flower-like region, indicating that the method could successfully synthesize AuNFs, as shown in Figure 2 (F). Subsequently, the AuNFs were characterized by X-ray photoelectron spectroscopy (XPS). As shown in Figure 2 (G), the XPS full spectrum of AuNFs showed that the peaks at 285.45 eV and 84.27 eV corresponded to the spectral lines of C1s and Au 4f, respectively. Subsequently, the peaks of each element were fitted. As shown in Figure 2 (H), the three peaks in the C 1s fitting diagram appeared at 284.80 eV, 286.48 eV and 288.02 eV, respectively, corresponding to the stretching vibration of C-C, C-O-C and O-C=C groups. As shown in Figure 2Au 4f peaks were fitted, two peaks appeared at 83.3eV and 86.97eV, which corresponded to Au 4f 7 / 2 , Au 4f 5 / 2 . It showed the generation of zero-valent gold.

[0047] The above analysis can see that the flower-shaped gold nano of the present application is successfully prepared in example 1.

[0048] Example 2 Application of flower-shaped gold nano in electrochemical-colorimetric dual-mode sandwich type aptamer sensor for detecting Kana.

[0049] The flower-shaped gold nano obtained by the preparation method of example 1 is used as a substrate to construct an electrochemical-colorimetric dual-mode sandwich type aptamer sensor, which is used for detecting Kana in a sample to be tested.

[0050] 1. Principle analysis

[0051] As Figure 3 shown, the principle diagram of the electrochemical-colorimetric dual-mode sandwich type aptamer sensor based on flower-shaped gold nano of the present application for detecting Lana, from the figure, it can be seen that: the sensor uses the peroxidase-like activity of AuNFs to catalyze H2O2 to oxidize colorless TMB into blue oxTMB, and through ultraviolet-visible absorption spectrum, a strong characteristic peak can be observed at 652nm. Compared with AuNPs, AuNFs have a larger specific surface area, which not only increases the catalytic activity, but also provides more linking sites for the aptamer. After linking SPA1 and SPA2 on the surface of AuNFs through Au-S bond, a large number of negative charges are provided on the surface of AuNFs, thereby increasing the affinity of AuNFs for TMB, so that the peroxidase activity of AuNFs is enhanced. As Figure 3 shown, when Kana is added, due to the specific recognition of SPA to the target, a sandwich structure of SPA1-Kana-SPA2 is formed. This causes AuNFs to be close to each other and aggregate, so that some of the catalytic sites are lost, which greatly inhibits the ability of AuNFs to catalyze H2O2 to oxidize TMB, resulting in a decrease in colorimetric signal and electrochemical signal.

[0052] The technical effects will be proved by specific test examples below.

[0053] In the following test examples, the oligonucleotides (SPA1, SPA2 sequences are as follows) used are synthesized and modified by Shenguo Biotechnology (Shanghai) Co., Ltd., and purified by HPLC:

[0054] SPA1: 5'-SH-(CH2)6-TGGGGGTTGAG-3'

[0055] SPA2: 5'-GCTAAGCCGA-(CH2)6-SH-3'.

[0056] The solutions used in the following test examples and their preparation methods are as follows.

[0057] 5mM HEPES buffer solution (DNA diluent): 0.11915g of HEPES was accurately weighed into a 100mL beaker, and then an appropriate amount of ultrapure water was added to dissolve it. The pH was then adjusted to 7.6 with a 0.5M HC1 solution, and then transferred to a 250mL volumetric flask for constant volume, shaken well, and stored in a 4°C refrigerator.

[0058] 500mM HEPES buffer solution: 1.1915g of HEPES was accurately weighed into a 10mL beaker, and then an appropriate amount of ultrapure water was added to dissolve it. The pH was then adjusted to 7.6 with a 0.5M HC1 solution, and then transferred to a 10mL volumetric flask for constant volume, shaken well, and stored in a 4°C refrigerator.

[0059] Preparation of SPA1 stock solution: the centrifuge tube containing 2.0OD of SPA1 dry powder was centrifuged at 4000rpm for 1min, and a certain volume of DNA diluent was accurately transferred to the centrifuge tube to make the concentration of SPA1 stock solution 100μM, and stored in a dark place at -20°C.

[0060] Preparation of SPA2 stock solution: the centrifuge tube containing 2.0OD of SPA2 dry powder was centrifuged at 4000rpm for 1min, and a certain volume of DNA diluent was accurately transferred to the centrifuge tube to make the concentration of SPA2 stock solution 100μM, and stored in a dark place at -20°C.

[0061] Preparation of 1μM SPA1 working solution: 10μL of prepared 100μM SPA1 stock solution and 990μL of DNA diluent were transferred to a centrifuge tube, shaken well, and stored in a 4°C refrigerator.

[0062] Preparation of 1μM SPA2 working solution: 10μL of prepared 100μM SPA2 stock solution and 990μL of DNA diluent were transferred to a centrifuge tube, shaken well, and stored in a 4°C refrigerator.

[0063] Preparation of 1% HAuCl4: 1g of HAuCl4·3H2O was accurately weighed and dissolved in 100mL of ultrapure water, and then transferred to a 100mL volumetric flask for constant volume, shaken well, and stored in a 4°C refrigerator in the dark.

[0064] Preparation of 1% sodium citrate: accurately weigh 1 g of sodium citrate into a 100 mL beaker, add an appropriate amount of ultrapure water to dissolve, then transfer to a 100 mL volumetric flask, dilute to volume, shake well, and store in a 4°C refrigerator.

[0065] 30 mM hydroquinone: accurately weigh 0.33 g of hydroquinone into a 100 mL beaker, then add an appropriate amount of ultrapure water to dissolve, then transfer to a 100 mL volumetric flask, dilute to volume, shake well, and store in a 4°C refrigerator.

[0066] Preparation of 10 mM TCEP solution: accurately weigh 0.0287 g of TCEP powder into a beaker, add 1 mL of ultrapure water, shake well to prepare a 100 mM TCEP solution. Then accurately transfer 10 μL of 100 mM TCEP solution into a glass bottle, accurately add 90 μL of ultrapure water, shake well to prepare a 10 mM TCEP solution and store it in a 4°C refrigerator in the dark.

[0067] Preparation of 10 mM TMB: accurately weigh 0.024 g of TMB into a 10 mL centrifuge tube, and dissolve with DMSO solution to 10 mL, shake well, and store in a 4°C refrigerator in the dark for future use.

[0068] Preparation of 0.1 M PBS buffer (pH = 7.6): accurately weigh 17.907 g of Na2HPO4 and 7.805 g of NaH2PO4, respectively, and dissolve with an appropriate amount of ultrapure water, transfer to a 250 mL volumetric flask, dilute to volume, to obtain a 0.2 M Na2HPO4 solution and a 0.2 M NaH2PO4 solution. Then, adjust the 0.2 M Na2HPO4 solution to pH = 7.6 with 0.2 M NaH2PO4 solution, shake well, and store in a 4°C refrigerator.

[0069] Preparation of 1 mM Kana stock solution: accurately weigh 0.05825 g of Kana, add it to a 100 mL volumetric flask, add ultrapure water, shake well, and store in a 4°C refrigerator for future use.

[0070] Preparation of 1 μM Kana working solution: accurately transfer 1 μL of 1 mM Kana solution into a 10 mL centrifuge tube, add 999 μL of ultrapure water, shake well, and store in a 4°C refrigerator for future use.

[0071] 2. Construction of aptamer sensor based on flower-shaped gold nanoparticles: this step constructs the optimal aptamer sensor for flower-shaped gold nanoparticle electrodes, which are:

[0072] Firstly, all the nucleotides required for the experiment were pretreated, in order to obtain straight chains, 1 mL of SPA1 working solution and 1 mL of SPA2 working solution were respectively taken to the centrifuge tube. Then, after being placed in a water bath (95℃) for 5 min, they were immediately cooled in ice water at -4℃ to form straight chains. Then, 100 μL of 10 mM TCEP was respectively taken to the centrifuge tube containing 1 mL of 1.2 μM SPA1 and 1 mL of 1 μM SPA2, and incubated at room temperature for 30 min to inhibit the formation of disulfide bonds.

[0073] The thiolated SPA1 and SPA2 were linked to AuNFs through Au-S bonds by low-pH assisted method to form SPA1 / SPA2-AuNFs probes.

[0074] Construction of electrochemical-colorimetric sandwich type aptamer sensor: 100 μL of SPA1-AuNFs probe and 100 μL of SPA2-AuNFs probe were respectively taken to the centrifuge tube containing 100 μL of 1 μM Kana, and after shaking and mixing, they were incubated at room temperature for 2 h. After the incubation was completed, 200 μL of the solution was taken to the centrifuge tube. Then, 100 μL of H2O2 (8%) and 20 μL of TMB (8 mM) were added, and after fully mixing, they were incubated at room temperature for 50 min, and then the absorbance of the solution was measured by ultraviolet-visible spectrophotometer and electrochemical experiments were carried out.

[0075] Test Example 1 Analysis of experimental feasibility

[0076] Experimental method: The experiment was divided into four groups a-d, wherein group a was as shown in Example 2-2, group b did not contain 100 μL of 1 μM Kana, group c replaced 100 μL of SPA1-AuNFs probe and 100 μL of SPA2-AuNFs probe with 100 μL of AuNFs probe and did not contain 100 μL of 1 μM Kana, and group d replaced 100 μL of SPA1-AuNFs probe and 100 μL of SPA2-AuNFs probe with 100 μL of AuNPs probe and did not contain 100 μL of 1 μM Kana.

[0077] The aptamer sensors of each group constructed above were respectively photographed by a camera to visualize the photos, and the absorbance of the solution (the maximum absorption wavelength of oxTMB was at 652 nm) was measured by ultraviolet-visible spectrophotometer; and the electrochemical signal of oxTMB was measured by chronoamperometry in the electrochemical workstation, the voltage was set to 100 mV, and the amperometric response time was 60 s, and the experimental results were as shown in Figure 4

[0078] Experimental results and analysis: as shown in Figure 4 ​(A) shows the analysis of the ability of AuNPs and AuNFs to catalyze the oxidation of TMB by H2O2 under the same conditions (curve c, curve d). The results show that the peak signal of the UV-Vis spectrum after adding AuNFs is significantly higher than that of AuNPs. This may be because AuNFs have a larger specific surface area than spherical AuNPs, which can provide more catalytically active sites and thus have stronger catalytic ability. When SPA1 and SPA2 are connected to the surface of AuNFs by Au-S bonds (curve a), the peak signal of the UV-Vis absorption spectrum at 652 nm is significantly increased, because when the aptamer is connected to the surface of AuNFs by Au-S bonds, a large amount of negative charge is brought to the surface of AuNFs, which enhances the affinity of AuNFs for TMB, making its ability to catalyze the oxidation of TMB by H2O2 improved.

[0079] When Kana is added, SPA1 and SPA2 connected to the surface of gold nanometer can specifically recognize Kana, forming a SPA1-Kana-SPA2 ternary sandwich complex, causing AuNFs to aggregate, resulting in a decrease in the measured absorbance signal (curve b). This may be because when AuNFs aggregate, their surface active sites are inhibited to some extent.

[0080] When TMB loses two electrons and is oxidized to oxTMB, chronoamperometry can be used to measure the electrochemical signal of oxTMB. As shown in Figure 4 (B) shows that when the catalytic ability of AuNFs is stronger, the number of TMB converted to oxTMB is also greater, i.e. the number of electron transfer is greater, and the chronoamperometry signal we measured is also greater. When Kana is added, the SPA1-Kana-SPA2 structure is formed, causing AuNFs to aggregate, and its peroxidase enzyme activity is reduced, resulting in a decrease in the production of oxTMB under the same conditions, and the electrochemical signal measured by chronoamperometry is reduced.

[0081] Test Example 2 Analysis of Class 2 Peroxidase Activity

[0082] Experimental method: The experiment is divided into groups a-f, among which group a is: 100 μL of SPA1-AuNFs probe and 100 μL of SPA2-AuNFs probe are taken into a centrifuge tube and shaken to mix. Then, 100 μL of H2O2 (8%) and 20 μL of TMB (8 mM) are added and mixed thoroughly. After incubation at room temperature for 50 min, a visual photograph is taken with a camera, and the absorbance of the solution (the maximum absorption wavelength of oxTMB is at 652 nm) is measured with a UV-Vis spectrophotometer.

[0083] In group b, 100 μL of SPA1-AuNFs probe and 100 μL of SPA2-AuNFs probe are replaced by 100 μL of AuNFs.

[0084] Group c replaced 100 μL SPA1-AuNFs probe and 100 μL SPA2-AuNFs probe with 100 μL thiolated SPA1 (1 μM).

[0085] Group d replaced 100 μL SPA1-AuNFs probe and 100 μL SPA2-AuNFs probe with 100 μL thiolated SPA2 (1 μM);

[0086] Group e replaced 100 μL SPA1-AuNFs probe and 100 μL SPA2-AuNFs probe with 100 μL AuNFs without H2O2;

[0087] Group f did not contain 100 μL SPA1-AuNFs probe and 100 μL SPA2-AuNFs probe.

[0088] The experimental results are shown in Figure 5 When AuNFs were added into the system containing H2O2 and TMB, the color of the solution changed from colorless to blue, and a characteristic absorption peak at 652 nm appeared in the UV-Vis spectrum (curve b), because the peroxidase-like activity of AuNFs could catalyze H2O2 to oxidize TMB to oxTMB. In addition, when the AuNFs linked with SPA were added into the centrifuge tube containing H2O2 and TMB (curve a), it could be clearly seen that the absorption peak at 652 nm was significantly higher than curve b, indicating that the negative charge carried by SPA improved the affinity of AuNFs for TMB. When SPA1 and SPA2 were added into the centrifuge tube containing H2O2 and TMB, no absorption peak at 652 nm was measured by UV-Vis spectrophotometer (curve c, curve d), which indicated that SPA1 and SPA2 themselves did not have peroxidase activity and could not catalyze H2O2 to oxidize TMB. Similarly, when AuNFs and H2O2 were added into the centrifuge tube containing TMB (curve e, curve f), no UV absorption peak at 652 nm was measured, because AuNFs did not have the ability to directly oxidize TMB, and H2O2 also could not directly oxidize colorless TMB to blue oxTMB in the absence of AuNFs. In summary, when SPA was linked to the surface of AuNFs through Au-S bond, the peroxidase activity of AuNFs could be further improved, and the detection performance of the dual-mode sensor could be improved.

[0089] Test Example 3 Steady-state Kinetics Experiment

[0090] Experimental method: 100 μL of SPA1-AuNFs probe and 100 μL of SPA2-AuNFs probe were taken respectively into a centrifuge tube and shaken to mix. Then, 100 μL of fixed concentration of H2O2(8%) was added, and the change in absorbance at 652 nm was recorded by measuring the change in absorbance at 652 nm in an ultraviolet-visible spectrophotometer with an enzyme kinetics module by changing the concentration of TMB. The Michaelis constant (K m ) and the maximum reaction rate (V max ) were calculated using the Michaelis equation (4-1) and the double-reciprocal equation (4-2).

[0091]

[0092] where V represents the initial speed of the enzymatic reaction, and [S] represents the substrate concentration.

[0093] Experimental results: As shown in Figure 6 , the catalytic kinetics of SPA-AuNFs on TMB conforms to the Michaelis-Menten equation curve of typical enzymatic reactions, as shown in Figure 6 (A). After transformation, the Lineweaver-Burk double-reciprocal curve of TMB can be obtained, as shown in Figure 6 (B). The K m value of SPA-AuNFs is 0.26 mM, which is much lower than the K m value of HRP (K m = 3.7 mM), indicating that SPA-AuNFs has a strong affinity for TMB. As shown in Table 1, the V max of AuNFs is less than that of HRP when TMB is used as a substrate, which is consistent with the observed results during the experiment, because the rate of HRP as a natural enzyme in the catalytic reaction is usually higher than that of gold nanoparticles.

[0094] Table 1 Comparison of kinetic parameters of SPA-AuNFs with other oxidase enzymes

[0095]

[0096] where reference 1 is Gao L, Zhuang J, Nie L, et al. 2007. Intrinsic peroxidase-like activity of ferromagnetic nanoparticles [J]. Nature Nanotechnology, 2: 577-583.

[0097] Reference 2 is Jiang X, Xu W, Chen X, et al. 2019. Colorimetric assay of Hg 2+ based on the inhibition of peroxidase mimetic activity of gold nanoclusters induced by Hg 2+ [J]. Analytical Methods, 11: 2179-2182.

[0098] Reference 3 is Jiang C, Zhang C, Song J, et al. 2021. Cytidine-gold nanoclusters as peroxidase mimetic for colorimetric detection of glutathione (GSH), glutathione disulfide (GSSG) and glutathione reductase (GR) [J]. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 250: 119316.

[0099] Reference 4 is Qi Y, Li B, Song D, et al. 2023. Ultrafast colorimetric detection of Cr(VI) based on competition of 8-HQ to Cr(VI) and TMB oxides using GO / AuNPs nanocomposites as peroxidase mimic [J]. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 297: 122722.

[0100] Test Example 4 Radical Scavenging Experiment

[0101] 1. Influence of Different Kinds of ROS Scavengers

[0102] Experimental Methods: The experiment was divided into groups a and b. Group a consisted of: 100 μL of SPA1-AuNFs probe and 100 μL of SPA2-AuNFs probe were placed in centrifuge tubes and vortexed to mix. Then, 100 μL of H2O2 (8%) and 20 μL of oxTMB (8 mM) were added, mixed thoroughly, and incubated at room temperature for 50 min. Visual images were then taken using a camera, and the absorbance of the solution was measured using a UV-Vis spectrophotometer (the maximum absorption wavelength of oxTMB is 652 nm).

[0103] Group b is the same as in Example 2-2, except that Kana is replaced with 20 μL of 5 mM tryptophan;

[0104] Group c is the same as in Example 2-2, except that Kana is replaced with 1,4-Benzoquinone;

[0105] Group d is the same as in Example 2-2, except that Kana is replaced with tert-Butanol.

[0106] Experimental results are as follows Figure 7 As shown.

[0107] 2. Effects of different concentrations of ROS scavengers

[0108] For example, in group d of 1, the concentration of tert-butanol is set as follows: group a 0 mM, group b 0.5 mM, group c 1 mM, group d 2 mM, group e 3 mM, group f 4 mM, and group g 5 mM.

[0109] Experimental results are as follows Figure 8 As shown.

[0110] Experimental results and analysis: such as Figure 7 As shown, the catalytic process of peroxidase generally involves the generation of reactive oxygen species (ROS). In this experiment, different types of ROS scavengers were added to determine the ROS that might participate in the reaction. Tert-Butanol was used as a scavenger. OH scavengers; tryptophan as... 1 O2 scavengers; while 1,4-benzoquinone acts as... O2 - The cleaner, from Figure 7 It can be seen that when the same concentration of the three ROS scavengers is added, the absorbance at 652 nm in the UV-Vis spectrophotometer decreases, indicating that all three ROS can be generated during this catalytic process. The decrease in absorbance is particularly significant when 5 mM tert-Butanol is added, which may be due to the peroxidase catalysis process... OH was dominant.

[0111] To further explain The role of OH in the peroxidase catalysis process was investigated by measuring the absorbance at 652 nm by varying the concentration of tert-Butanol. For example... Figure 8 As shown, the absorbance at 652 nm on the UV-Vis spectrophotometer gradually decreases with increasing tert-Butanol concentration. This further demonstrates the effectiveness of AuNFs in catalyzing the oxidation of TMB by H₂O₂. OH is a type of ROS that plays a major role.

[0112] Experiment Example 5: Condition Optimization Experiment

[0113] 1. Optimization of SPA1 at different concentrations: As a key component of this dual-mode sensor for identifying target objects, the concentration of SPA1 directly determines the performance of the sensor.

[0114] Experimental method: As shown in Example 2-2, the difference is that the concentrations of SPA1 solution were 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1 μM, 1.2 μM, 1.4 μM, and 1.6 μM, respectively. The experimental results are as follows: Figure 9 As shown.

[0115] Experimental results and analysis: such as Figure 9 As shown, when the concentration of SPA1 gradually increased from 0.2 μM to 1.2 μM, the absorption peak signal and electrochemical signal of the blank group at 652 nm gradually increased. Simultaneously, the solution color gradually changed from light blue to dark blue. This indicates that, within the low concentration range, SPA1 helps to improve the catalytic activity of AuNFs. This is because, with the increase of SPA1 concentration, the negative charge it provides increases the affinity of AuNFs for TMB.

[0116] The absorption peak signal value of the signal group first increased and then decreased. This may be because when the SPA1 concentration is 0.2 μM and 0.4 μM, the catalytic ability inhibited by the aggregation of AuNFs is insufficient to offset the catalytic activity brought by SPA1 to AuNFs. As the SPA1 concentration continues to increase from 0.4 μM to 1.2 μM, more and more SPA1-Kana-SPA2 ternary sandwich structures are formed, leading to more AuNFs aggregation and a decrease in their catalytic activity. When the SPA1 concentration reaches 1.2 μM, the difference between the blank group and the signal group is the largest (the area shown in the red dashed box), indicating that the sensor performance reaches its optimal level when the SPA1 concentration is 1.2 μM. Therefore, 1.2 μM was chosen as the SPA1 concentration for subsequent experiments, and it can be seen that good technical effects can be achieved at concentrations above 1 μM.

[0117] 2. Optimization of SPA2 concentration

[0118] Experimental method: as shown in Example 2-2, the difference is that the concentration of SPA2 solution is 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1 μM, 1.2 μM, 1.4 μM, 1.6 μM, respectively, and the experimental results are shown in Figure 10 .

[0119] Experimental results and analysis: as shown in Figure 10 , with the gradual increase of SPA2 concentration from 0.2 μM to 1 μM, the signal difference between the signal group and the blank group gradually increases, and when the concentration of SPA2 reaches 1 μM, the difference gradually tends to be stable. This may be because, at this time, the number of SPA2 connected to AuNFs through Au-S bond reaches saturation. So that the number of negative charges provided by SPA2 reaches the highest, so that the signal of the blank group no longer rises. And when Kana is added (signal group), the number of SPA1-Kana-SPA2 ternary sandwich structure formed also reaches the highest, so that the catalytic activity of AuNFs is inhibited to the greatest extent. Therefore, 1 μM is selected as the concentration of SPA2 for subsequent experiments.

[0120] 3. Optimization of different concentrations of H2O2.

[0121] Experimental method: as shown in Example 2-2, the difference is that the concentration of H2O2 is replaced by 0 (blank control), 1%, 2%, 4%, 6%, 8%, 10%, 12%, respectively, and the experimental results are shown in Figure 11 .

[0122] Experimental results and analysis: as shown in Figure 11 , with the increase of H2O2 concentration in the range of 1% to 8%, the signal of the blank group and the signal group gradually increases, indicating that when the concentration of H2O2 is in this range, it can effectively promote the catalysis of AuNFs to oxidize TMB to form blue oxTMB, thereby causing the increase of absorbance and electrochemical signal. When the concentration of H2O2 increases to 8% (red dotted line box area), the absorbance signal difference between the blank group and the signal group reaches the maximum, at the same time, the electrochemical signal also reaches the maximum. It is indicated that when the concentration of H2O2 is 8%, the detection signal of the sensor for the target is the most sensitive.

[0123] When the concentration of H2O2 continues to increase, the difference between the blank group and the signal group decreases, and when the concentration of H2O2 reaches 12%, the color of the solution begins to turn yellow. This may be because when the concentration of H2O2 is too high, excessive OH is generated, which not only destroys the catalytic sites of AuNFs, but also causes TMB to be over-oxidized to form yellow diimine, resulting in a yellow solution. Therefore, 8% H2O2 is finally selected for subsequent experiments, but the concentration of H2O2 can achieve the response technical effect when it is 6%-12%.

[0124] 4. Optimization of different concentrations of TMB: TMB, as a substrate for catalytic oxidation, can directly participate in the reaction to generate blue oxidation products (oxTMB), and its concentration determines the strength of the sensor signal. Therefore, optimizing the concentration of TMB is an important step to increase the performance of the sensor.

[0125] Experimental method: as shown in Example 2-2, the difference is that the concentration gradient of TMB is set to 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 12 mM, and the experimental results are shown in Figure 12

[0126] Experimental results and analysis: as shown in Figure 12 With the increase of TMB concentration in the range of 1 mM-8 mM, more and more TMB substrates are oxidized to oxTMB. Therefore, the absorbance signals and electrochemical signals of the blank group and the signal group increase. When the concentration of TMB increases to 8 mM, the signal difference between the blank group and the signal group reaches the maximum (red dotted line box area), indicating that the performance of the sensor reaches the best at this concentration. When the concentration of TMB exceeds 8 mM, although the signals of the blank group and the signal group are slightly increasing, the difference tends to be stable. And the color of the colorimetric solution has little difference when observed by naked eye. Therefore, the concentration of TMB is selected as 8 mM in the subsequent experiments. But the concentration of TMB solution ≥6 mM can achieve the response technical effect.

[0127] 5. Optimization of secondary incubation temperature: Temperature is one of the important factors affecting the performance of the sensor. Temperature can indirectly affect the performance of the sensor by affecting the catalytic activity of AuNFs, the rate of TMB oxidation reaction, and the recognition ability of SPA to the target. Therefore, it is extremely important to optimize the reaction temperature of the sensor.

[0128] Experimental method: as shown in Example 2-2, the difference is that the secondary incubation temperature is set to 20℃, 30℃, 40℃, 50℃, 60℃, and the experimental results are shown in Figure 13

[0129] Experimental results and analysis: as shown in Figure 13 ​​As shown, the absorbance signal of the blank group gradually increased in the temperature range of 10-40℃, while the signal group gradually decreased. This is because as the temperature rises, the catalytic activity of AuNFs and the oxidation rate of TMB gradually increase, causing the solution color and absorbance signal of the blank group to increase continuously. When Kana is added (signal group), as the temperature rises, the binding efficiency between SPA and the target increases, resulting in an increase in the number of SPA1-Kana-SPA2 sandwich structures, which inhibits the catalytic performance of AuNFs. When the temperature increases to 40℃, the difference in solution color and absorbance signal between the blank group and the signal group is the largest (at the red dotted line area), indicating that the sensitivity of the sensor reaches the optimum at this time. When the temperature exceeds 40℃, the difference between the blank group and the signal group decreases, which may be because the temperature is too high at this time, affecting the stability of the aptamer structure and the catalytic activity of AuNFs (Bai et al., 2022). Similarly, the electrochemical signal value continues to rise in the temperature range of 10-40℃, reaching a maximum at 40℃. Therefore, 40℃ is chosen as the reaction temperature for subsequent experiments, but good technical effects can be achieved within the range of 20-50℃.

[0130] 6. Secondary incubation time optimization: The aggregation of AuNFs relies on the ternary sandwich structure formed by SPA and Kana, and the incubation time of Kana will directly affect the number of ternary sandwich structures. Therefore, it is necessary to optimize the incubation time of Kana.

[0131] Experimental method: As shown in Example 2-2, except that the secondary incubation time was set to 0 min, 20 min, 60 min, 80 min, 100 min, 120 min, 150 min, and 180 min, and the experimental results are shown in Figure 14 .

[0132] Experimental results and analysis: As shown in Figure 14 , since the blank group does not add Kana, its signal value remains stable, and as the Kana incubation time increases from 10 min to 120 min, the signal group's absorption peak signal gradually decreases, i.e., the difference between the absorbance signal and the electrochemical signal of the blank group and the signal group gradually increases. When the incubation time reaches 120 min, the difference reaches the maximum, indicating that the performance of the sensor reaches the best at this time. When it exceeds 120 min, the signal begins to gradually decrease, which may be because at this time, the target and SPA incubate for too long, causing the target to dissociate or the conformation of SPA to change. Therefore, 120 min is finally chosen as the incubation time of Kana for subsequent experiments. However, incubation times within the range of 100-150 min can achieve corresponding technical effects.

[0133] Test Example 5 Sensor performance evaluation

[0134] 1. Linear range

[0135] Experimental method: The aptamer sensor based on flower-shaped gold nanoelectrode constructed in Example 2-2 was photographed by camera to visualize the photo, and the absorbance of the solution was measured by UV-Vis spectrophotometer (the maximum absorption wavelength of oxTMB is at 652 nm). The linear relationship between the absorbance of the solution at 652 nm and the concentration of Kana was established. The prepared AuNFs electrode was used to measure the electrochemical signal of oxTMB by chronoamperometry in the electrochemical workstation, and the peak shape change was observed. After background correction and blank subtraction, the electrochemical signal value was recorded, and the linear relationship between the electrochemical signal value and the concentration of Kana was established. The voltage was set to 100 mV, and the amperometric response time was 60 s. The experimental results are shown in Figure 15 .

[0136] Experimental results and analysis: Under the optimal experimental conditions, 10 different concentrations of Kana were measured, and the linear range was evaluated. As shown in Figure 15 (A), as the concentration of Kana gradually increased from 50 nm to 3000 nm, the color of the solution changed from deep blue to light blue. The measurement of oxTMB was performed using a UV-Vis spectrophotometer, and the results are shown in Figure 15 (B). It can be seen that the absorbance at 652 nm decreases with the increase of Kana concentration, and the change of Kana concentration in the range of 100 nM-3000 nM shows a good linear relationship with the absorbance value. As shown in Figure 15 (C), the linear equation is y = -0.14525x + 0.53776 (R 2 = 0.99827), where x represents the logarithm of the concentration of Kana, and y represents the absorbance signal measured at 652 nm by UV-Vis spectrophotometer. According to the formula 3σ / k, the detection limit (LOD) of this method is calculated to be 0.51 nM. At the same time, the current signal of oxTMB was detected by chronoamperometry, and the results are shown in Figure 15 (D), which is consistent with the results of absorbance determination. The electrochemical signal decreases with the increase of Kana concentration, and the change of Kana concentration in the range of 50 nM-2000 nM shows a good linear relationship with the electrochemical signal. As shown in Figure 15 (E), the linear equation is y = -3.0653x + 1.06809 (R 2 = 0.99768), where x represents the concentration of Kana, and y represents the current intensity. According to the formula 3σ / k, the LOD is calculated to be 0.074 nM.

[0137] The dual-mode sensor constructed in this study was compared with other dual-mode sensors for detecting Kana, and the results are shown in Table 2. The colorimetric / electrochemical dual-mode sensor constructed in this experiment has simple operation steps, low detection limit and wide linear range, and can meet the detection requirements of Kana in water samples.

[0138] Table 2 Comparison of performance of Kana detection methods in this study and other methods

[0139]

[0140] Among them, reference 5 is Liu S, Wang Y, Xiang F, et al. 2024. Fluorescent and polarity-switchable photoelectrochemical dual-mode homogeneous sensing platform for ultrasensitive kanamycin detection based on EXO III-driven signal amplification [J]. Microchimica Acta, 191:602.

[0141] Reference 6 is Lee H B, Son S E, Ha C H, et al. 2024. Dual-mode colorimetric and photothermal aptasensor for detection of kanamycin using flocculent platinum nanoparticles [J]. Biosensors and Bioelectronics, 249:116007.

[0142] Reference 7 is Gao X, Sun Z, Wang X, et al. 2022. Construction of a dual-model aptasensor based on G-quadruplexes generated via rolling circle amplification for visual / sensitive detection of kanamycin [J]. Science of The Total Environment, 839:156276.

[0143] Reference 8 is 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.

[0144] 2. Selectivity: Because the composition of the solution to be tested is often complex during the actual sample detection process, in order to verify whether the sensor can play a good role in identifying Kana in actual application, tobramycin (Tobramycin), gentamicin (Gentamicin), streptomycin (Streptomycin), amikacin (Amikacin), netilmicin (Netilmicin) and other five kinds of aminoglycoside antibiotics similar in nature to Kana were evaluated. Among them, the concentration of the target substance Kana was 1 μM, and the concentration of other interfering ions was 10 μM.

[0145] Experimental method: as shown in Example 2-2, the difference is that a certain concentration of tobramycin (Tobramycin), gentamicin (Gentamicin), streptomycin (Streptomycin), amikacin (Amikacin), netilmicin (Netilmicin), Kana and their mixed solution were added to the centrifuge tube containing 100 μL SPA1-AuNFs probe and 100 μL SPA2-AuNFs probe, respectively. The experimental method is as shown in Figure 16 .

[0146] Experimental results and analysis: the detection results are shown in Figure 16 . Only when Kana exists in the water sample to be tested, the absorbance of ultraviolet-visible spectrometry at 652 nm will decrease sharply. This is because SPA1 and SPA2 can only specifically recognize Kana when it exists, and form a SPA1-Kana-SPA2 ternary sandwich structure with Kana, so as to inhibit the catalytic activity of AuNFs. It is proved that the aptamer sensor constructed by the method has good selectivity to Kana, and can be applied in the detection of actual water.

[0147] 3. Reproducibility: AuNFs, as an important part of the sensor, the reproducibility of its synthesis directly determines whether the performance of the sensor is stable.

[0148] Experimental method: six groups of AuNFs with multiple branches were synthesized respectively, and the sensors were constructed according to the construction method of Example 2-2, and the experimental results are shown in Figure 17 .

[0149] Experimental results and analysis: As shown in Figure 17 (A), the catalytic performance of six groups of SPA-AuNFs synthesized under the same experimental conditions was evaluated. It was found that there was little difference in the color of the six groups of solutions, and the absorbance of the solutions at 652 nm and the chronoamperometry of the solutions were measured using a UV-visible spectrophotometer, and it was found that the detection results showed the same results. Among them, the relative standard deviation (RSD) of the two methods was 1.3% and 2.2%, respectively, indicating that the AuNFs synthesized in this experiment had high reproducibility.

[0150] Subsequently, the reproducibility of the sensor for detecting Kana was evaluated, as shown in Figure 17 (B). Using the six groups of AuNFs synthesized under optimal conditions to detect 1 μM Kana, it was found that there was little difference in the color of the six groups of solutions. The absorbance of the solutions at 652 nm and the chronoamperometry of the solutions were measured using a UV-visible spectrophotometer, and it was found that the detection results showed the same results. Among them, the relative standard deviation (RSD) of the two methods was 2.8% and 2.5%, respectively, indicating that the sensor had good reproducibility for detecting Kana in this experiment.

[0151] 4. Stability: In order to verify the stability of the sensor, AuNFs stored in the refrigerator for different times were used to detect 1 μM Kana under optimal conditions using a UV-visible spectrophotometer and chronoamperometry, respectively.

[0152] Experimental method: As shown in Example 2-2, except that the synthesized AuNFs were placed in a refrigerator at 4°C for different days (1 day, 3 days, 5 days, 10 days, 15 days, 20 days, 25 days, 30 days) to construct the sensor, and the experimental results are shown in Figure 18 .

[0153] Experimental results and analysis: As shown in Figure 18 , when observed by colorimetric visual observation, the color of the solutions stored for 1-20 days was basically the same, and then the color-developed solution was measured, and the detection results were basically consistent. When stored for 30 days, the performance of the sensor could still maintain more than 85% through detection, indicating that the dual-mode sensor had high stability.

[0154] Test Example 6 Actual sample detection

[0155] In order to prove the feasibility and practicability of the application of the constructed electrochemical aptamer sensor in actual water bodies, the sensor was used to detect water samples from three different regions after spiking (spiking concentrations were 10 nM, 100 nM and 500 nM).

[0156] Experimental method: The samples for actual water detection were collected from Minjiang River (taken from Dujiangyan), mineral water (a certain brand) and Ink Lake (taken from Chengdu University of Technology), respectively. Firstly, the water samples were spiked with the concentration of 10 nM, 100 nM and 500 nM, respectively. After spiking, the water samples were centrifuged at 8000 rpm using a high-speed centrifuge, and the centrifugation time was set to 10 min, in order to remove larger impurities. Then, the actual water samples were filtered using a 0.22 μm needle filter to remove smaller impurities. Finally, the Kana in the spiked samples was detected according to the detection steps of the constructed dual-mode sensor (i.e. Kana in Example 2-2 was replaced by the sample to be detected), and the experimental results are shown in Table 3.

[0157] Experimental results and analysis: The results are shown in Table 3. The recovery rate of the spiked sample was in the range of 88.3% to 103.4%, and the relative standard deviation (RSD) of three parallel experiments was less than 5%, which proved that the accuracy and precision of the analysis method were high, and it had high reliability, and could be used for the determination of Kana in actual water samples.

[0158] Table 3: Spiked detection results of actual water samples

[0159]

[0160]

[0161] Finally, it should be noted that the above examples are only preferred embodiments of the present application, and the protection scope of the present application is not limited to the above examples. Any technical solutions falling within the scope of the present application are within the protection scope of the present application. It should be pointed out that, for ordinary skilled persons in the art, improvements and refinements without departing from the principles of the present application are also considered to be within the protection scope of the present application.

Claims

1. Use of flower-like gold nanoparticles in the electrochemical-colorimetric dual-mode sandwich-type aptasensor for the detection of Kana, characterized in that, Comprising the following steps: S1. Respectively mix the thiolated SPA1 solution and SPA2 solution with the flower-shaped gold nano solution to obtain SPA1-AuNFs probe solution and SPA2-AuNFs probe solution linked by Au-S bond; S2. Add the sample to be tested to the mixed solution of SPA1-AuNFs probe solution and SPA2-AuNFs probe solution for incubation, after the incubation, add H2O2 solution and TMB solution, mix and incubate again, then respectively measure the absorbance of the solution by ultraviolet-visible spectrophotometer and determine the electrochemical signal value by chronoamperometry, to obtain the concentration of Kana in the sample to be tested.

2. Use according to claim 1, characterized in that, The flower-shaped gold nano solution is prepared by seed growth method.

3. Use according to claim 1, characterized in that, The seed growth method is specifically: (1) Mix HAuCl4 solution with ultrapure water, stir and heat to vigorous boiling, quickly add sodium citrate solution, mix and stir until the solution color changes from blue to purple and finally gradually changes to red, then continue to heat to obtain colloidal gold seed solution; (2) Drop AuNPs solution into ultrapure water, then drop colloidal gold seed solution and sodium citrate solution into the mixture, mix and stir to heat to 50℃, then add hydroquinone solution while cooling, stir until the color changes from black gray to blue, continue to stir and cool, centrifuge to obtain flower-shaped gold nano.

4. Use according to claim 3, characterized in that, The concentrations of HAuCl4 solution, sodium citrate solution and AuNPs solution are all 1%, w / v.

5. The use according to claim 1, characterized in that, The concentrations of SPA1 solution and SPA2 solution in S1 are both greater than or equal to 1 μM.

6. The use according to claim 1, characterized in that, The concentration of H2O2 solution in S2 is 6%-12%.

7. Use according to claim 1, characterized in that, The concentration of TMA solution in S2 is ≥6 mM.

8. The use according to claim 1, characterized in that, The secondary incubation temperature in S2 is 20-50℃.

9. The use according to claim 1, characterized in that, The secondary incubation time in S2 is 100-150 min.

10. The use according to claim 1, characterized in that, The concentration of SPA1 solution in S1 is 1.2 μM, the concentration of SPA2 solution is 1 μM, the concentration of H2O2 solution in S2 is 8%, the concentration of TMB solution is 8 mM, the secondary incubation temperature is 40℃, and the secondary incubation time is 120 min.

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