A bimodal sensor and methods of making and using the same

Abstract

The application discloses a bimodal sensor and a preparation method and application thereof, and the bimodal sensor comprises Fe3O4@TCPP which is surface-modified with mercury ion aptamer; the Fe3O4@TCPP comprises Fe3O4 and tetra (4-carboxyphenyl) porphyrin which is wrapped on the surface of the Fe3O4. The nano probe has low cost, good anti-interference and selectivity, can realize specific identification of mercury ions in food, and then realizes output of fluorescence / colorimetric spectral bimodal detection results, achieves the purpose of self-correction detection, and improves the selectivity, accuracy and efficiency of detection.

Description

Technical Field

[0001] This invention relates to the field of food analysis technology, and in particular to a dual-modal sensor, its preparation method, and its application. Background Technology

[0002] Mercury is one of the most toxic heavy metal ions. Mercury ions (Hg) 2+ Mercury can accumulate in vital organs through the food chain, potentially leading to chronic diseases such as brain damage and kidney failure. Due to its high water solubility, the most common stable form of mercury is the solvated divalent mercury ion (Hg). 2+ Various technologies have been developed for the detection of mercury in food, such as atomic absorption spectrometry, electrochemiluminescence, and inductively coupled plasma mass spectrometry.

[0003] Hg 2+ It can interact with two thymine (T) residues in DNA to form a stable T-Hg. 2+ -T structure. Due to its highly selective binding, DNA structure-based sensors can selectively detect Hg. 2+ This approach offers advantages such as low cost and convenience, leading to the development of various sensors based on this strategy. However, DNA-based single-modal sensors exhibit relatively low sensitivity compared to other methods.

[0004] In recent years, magnetic nanomaterials have developed rapidly and have been widely used in fields such as biology and medicine. Among them, magnetic Fe3O4 nanoparticles possess excellent superparamagnetism and can be used for the separation and enrichment of target substances. Surface modification based on traditional nanomaterials can efficiently catalyze the oxidation of chromogenic substrates, producing chromogenic products even with trace amounts of the target substance. Compared with traditional colorimetric methods, the sensing performance of mercury has been significantly improved due to the excellent catalytic ability and signal amplification characteristics of nanozymes. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a dual-modal sensor, its fabrication method, and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a nanoprobe comprising Fe3O4@TCPP with a surface modified with a mercury ion aptamer; wherein the Fe3O4@TCPP comprises Fe3O4 and tetrakis(4-carboxyphenyl)porphyrin coated on the surface of the Fe3O4.

[0007] In this invention, the nanoprobe utilizes the strong magnetism of Fe3O4 to enhance the catalytic activity of the nanozyme, which is a mimicry of enzymes. In some embodiments, the mass ratio of Fe3O4 to tetrakis(4-carboxyphenyl)porphyrin is (2-7):(1-5), such as (3-6):(1.5-4).

[0008] In some embodiments, the mass molar ratio of Fe3O4@TCPP to the mercury ion aptamer is (0.1-1.0) mg:(10-200) µmol.

[0009] In some embodiments, the nucleotide sequence of the mercury ion aptamer includes any of the following: The nucleotide sequence is 5'-TTT TTT TTT TTT TTT TTT TT-3' (shown in SEQ ID NO.1), with an amino group modified at its 5' end; The nucleotide sequence is 5'-TTTTTTTTTTGGGGGGGAAAAAAAA-3' (shown in SEQ ID NO.2), with an amino group modified at its 5' end; The nucleotide sequence is 5'-TTCTGTGGGTCTGTTCGGG-3' (shown in SEQ ID NO.3), with an amino group modified at its 5' end.

[0010] In some embodiments, the average particle size of the nanoprobe is 100-500 nm, such as 150-450 nm, 200-400 nm, or 250-350 nm.

[0011] A second aspect of the present invention provides a method for preparing the nanoprobe, comprising the following steps: S1: Fe3O4@TCPP was prepared by reacting Fe3O4, a surfactant, and tetrakis(4-carboxyphenyl)porphyrin. S2: The nanoprobe was prepared by coupling Fe3O4@TCPP with a mercury ion aptamer.

[0012] In some embodiments, the mass ratio of Fe3O4 to surfactant is (2-7):(1-4), such as (3-6):(1.5-3).

[0013] In some embodiments, the surfactant includes at least one selected from polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), hexadecyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium chloride (CTAC), sodium dodecylbenzenesulfonate (SDBS), polydiallyl dimethylammonium chloride (PDDA), and polysorbate. In this invention, the surfactant promotes the encapsulation of Fe3O4 particles by TCPP.

[0014] In some embodiments, in S1, the reaction temperature is 80-120°C; the reaction time is 1-3 hours.

[0015] In some embodiments, in S1, the reaction solvent of the reaction includes at least one of N,N-dimethylformamide, anhydrous ethanol, and water (such as deionized water); such as a mixed solution of N,N-dimethylformamide and anhydrous ethanol; such as a mixed solution of N,N-dimethylformamide and anhydrous ethanol in a volume ratio of 1:(1-5) or (2-4):1.

[0016] In some embodiments, S1: Fe3O4, a surfactant, and tetrakis(4-carboxyphenyl)porphyrin are reacted, and the reaction product is washed to obtain Fe3O4@TCPP; in this invention, washing the reaction product can remove the surfactant.

[0017] In some embodiments, S1: Fe3O4, a surfactant, and tetrakis(4-carboxyphenyl)porphyrin are dissolved in a reaction solvent, ultrasonicated, and then reacted. The reaction product is washed and dried to obtain Fe3O4@TCPP. The ultrasonic power is 60-100W, and the ultrasonic time is 20-40min.

[0018] In some embodiments, the washing is performed using at least one of anhydrous ethanol and water (such as deionized water).

[0019] In some embodiments, the drying temperature is 50-150°C, such as 70-110°C; and the drying time is 1-5 hours, such as 1.5-3 hours.

[0020] In some embodiments, in step S2, Fe3O4@TCPP is activated with an activator and then coupled with a mercury ion aptamer to obtain the nanoprobe after sealing. The activator includes a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and an N-hydroxysuccinimide solution (EDC / NHS). The volume ratio of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution to the N-hydroxysuccinimide solution (EDC / NHS) is (0.5~3):1, such as (0.8~2):1.

[0021] In some embodiments, in S2, Fe3O4@TCPP is dissolved in a buffer solution, sonicated, and then activated.

[0022] In some embodiments, the mass-to-volume ratio of Fe3O4@TCPP to the buffer solution is (2-10) mg / (2-10) mL, such as (3-8) mg / (3-8) mL.

[0023] In some embodiments, the reaction time of the coupling reaction in S2 is 8-24 hours, such as 10-20 hours.

[0024] In some embodiments, in S2, the concentration of the aptamer is 0.1~2.0 mmol / L, such as 0.2~1.0 mmol / L.

[0025] In some embodiments, the blocking includes using a bovine serum albumin solution; the mass concentration of the bovine serum albumin solution is 0.5% to 2.0%, such as 1.0% to 2.0%; and the blocking time is 30-90 min, such as 40-80 min.

[0026] A third aspect of the present invention provides a dual-modal sensor comprising the aforementioned nanoprobe.

[0027] In some embodiments, the dual-modal sensor further includes a solvent, such as water or a buffer solution (e.g., Tris-HCl solution).

[0028] In some embodiments, the concentration of the nanoprobe in the dual-modal sensor is 10~2000 μmol / L.

[0029] A fourth aspect of the present invention provides a kit for detecting mercury ions, comprising the nanoprobe or the dual-modal sensor and a colorimetric reagent.

[0030] In some embodiments, the colorimetric reagent comprises hydrogen peroxide and a colorimetric substrate; the colorimetric substrate comprises at least one of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, o-phenylenediamine (OPD), 3,3'-diaminobenzidine, 4-chloro-1-naphthol, and 3-amino-9-ethylcarbazole.

[0031] In some embodiments, the concentration of the chromogenic substrate is 3.0-8.0 mmol / L, such as 4.0-7.0 mmol / L; and the amount of the chromogenic substrate used in the kit is 100-400 μL.

[0032] In some embodiments, the concentration of hydrogen peroxide is 1.0-50.0 mmol / L, such as 10-50.0 mmol / L; and the amount of hydrogen peroxide used in the kit is 100-300 μL.

[0033] In some embodiments, the nanoprobe is prepared into a solution using a buffer solution with a concentration of 1-4 mg / mL; the amount of nanoprobe solution used in the kit is 50-300 μL.

[0034] A fifth aspect of the present invention provides an application of the aforementioned nanoprobe, the aforementioned dual-modal sensor, or the aforementioned reagent kit for detecting mercury ions in mercury ion detection.

[0035] A sixth aspect of the present invention provides a method for detecting mercury ions, comprising the following steps: Mercury ions can be detected using the aforementioned nanoprobe, the aforementioned dual-modal sensor, or the aforementioned mercury ion detection kit.

[0036] In some embodiments, the method for detecting mercury ions includes the following steps: taking a sample solution to be tested, a chromogenic substrate, hydrogen peroxide and a nanoprobe, mixing them, and then detecting the absorbance and / or fluorescence intensity of the solution.

[0037] In the nanoprobe of this invention, Fe3O4 provides both an iron source and magnetic separation functionality. 3+ When (provided by Fe3O4) coordinates to the central cavity of TCPP, a metalloporphyrin (Fe-TCPP) is formed, endowing the entire nanomaterial with peroxidase mimicry (nanozyme) activity. Through TCPP coating and coordination with iron, a high-performance Fe-TCPP nanozyme catalytic center is formed, catalyzing the reaction of chromogenic substrates with hydrogen peroxide to produce substances with dual-modal signals (colorimetric and fluorescent). An amino-containing mercury ion aptamer is covalently immobilized on the nanomaterial surface as a specific Hg recognition agent. 2+ Molecular switch. Hg 2+ Binding to the aptamer triggers a conformational change, thereby modulating the catalytic activity of the nanozyme. The entire system can achieve specific recognition of mercury ions in food, thus realizing dual-modal detection results output of fluorescence / colorimetric spectroscopy, achieving the purpose of self-calibrating detection, and improving the selectivity, accuracy and efficiency of detection.

[0038] In some embodiments, the sample to be tested includes at least one of mercury-containing wastewater, soil, and food.

[0039] In some embodiments, the concentration of mercury ions in the sample to be tested is 0.01 μg / L-10 g / L, such as 0.01 μg / L-1 g / L, 0.01 μg / L-100 mg / L, 0.01 μg / L-100 μg / L, etc.

[0040] In some embodiments, the absorbance of the detection solution includes the absorbance of the detection solution at 400-450 nm, such as 417 nm.

[0041] In some embodiments, the fluorescence intensity of the detection solution includes the fluorescence intensity of the detection solution at 550-600 nm, such as 569 nm.

[0042] The beneficial effects of this invention are: The nanoprobe of this invention has the advantages of low cost, high sensitivity, and good anti-interference and selectivity.

[0043] The nanoprobes of this invention are easy to prepare.

[0044] The nanoprobe of this invention can specifically identify mercury ions in food, thereby achieving dual-modal detection results output of fluorescence / colorimetric spectroscopy, achieving the purpose of self-calibrating detection, and improving the selectivity, accuracy and efficiency of detection. Attached Figure Description

[0045] Figure 1 The images show scanning electron microscope (SEM) images of pure Fe3O4 (a), pure TCPP-Fe (b), and Fe3O4@TCPP (Fe) (c) and transmission electron microscope (TEM) images of Fe3O4@TCPP (Fe) synthesized in Example 1 of this invention.

[0046] Figure 2 This is the fluorescence emission spectrum response diagram of the Fe3O4@TCPP (Fe) @Apt probe for quantitative analysis of mercury ions in Example 2 of the present invention.

[0047] Figure 3 This is the working curve of the Fe3O4@TCPP (Fe)@Apt probe for quantitative detection of mercury ions by fluorescence emission spectroscopy in Example 2 of the present invention.

[0048] Figure 4 This is the UV-Vis absorption spectrum response of the Fe3O4@TCPP (Fe) @Apt probe for quantitative analysis of mercury ions in Example 2 of the present invention.

[0049] Figure 5 This is the working curve of the Fe3O4@TCPP (Fe)@Apt probe for quantitative detection of mercury ions by ultraviolet-visible absorption spectroscopy in Example 2 of the present invention.

[0050] Figure 6 This is a bar chart showing the selective fluorescence emission spectrum response of the Fe3O4@TCPP (Fe)@Apt probe to mercury ions in Example 3 of the present invention.

[0051] Figure 7 This is a bar chart showing the fluorescence emission spectrum response of the Fe3O4@TCPP (Fe)@Apt probe to mercury ions in Example 3 of the present invention.

[0052] Figure 8 This is a bar chart showing the selective UV-Vis spectrophotometric response of the Fe3O4@TCPP (Fe) @Apt probe to mercury ions in Example 3 of this invention.

[0053] Figure 9This is a bar chart showing the UV-Vis spectrophotometric response of the Fe3O4@TCPP (Fe)@Apt probe to mercury ions in Example 3 of the present invention. Detailed Implementation

[0054] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0055] In the following examples, the nucleotide sequence of the mercury ion aptamer is shown in SEQ ID NO.1.

[0056] Example 1 This embodiment prepares a probe, and the specific process is as follows: S1: Preparation of Fe3O4@TCPP (Fe) material: 50 mg of Fe3O4, 20 mg of polyvinylpyrrolidone, and 30 mg of tetrakis(4-carboxyphenyl)porphyrin were added to the inner liner of a polytetrafluoroethylene reactor and dissolved in 27 mL of N,N-dimethylformamide and 9 mL of anhydrous ethanol solution. The mixture was sonicated for 30 min. The mixture was then heated at 100 °C for 2 h in a high-temperature oven. After the reaction was completed, the mixture was cooled to room temperature and removed from the oven. It was then washed with anhydrous ethanol and dried in a vacuum drying oven at 60 °C for 12 h. S2: Fe3O4@TCPP (Fe)@Apt probe was prepared by modifying the material with an aptamer (Apt): 5 mg of Fe3O4@TCPP material was added to 5 mL of Tris-HCl solution and sonicated for 30 min. Then, 0.5 mL of a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide solution (EDC / NHS) was added and incubated for 60 min. Next, 0.1 mL of a 0.5 μmol / L aptamer solution was added and incubated for 12 h. Finally, 1 mL of 1.5% ( w / v The probe was blocked with bovine serum albumin solution for 60 min, centrifuged to remove the supernatant, and then reconstituted with 5 mL of Tris-HCl to obtain the final Fe3O4@TCPP (Fe) @Apt probe.

[0057] Figure 1 Scanning electron microscope (SEM) images of pure Fe3O4 (a), pure TCPP-Fe (b), and Fe3O4@TCPP(Fe) (c) and transmission electron microscope (TEM) image of Fe3O4@TCPP(Fe) (d) are shown.

[0058] Depend on Figure 1 As can be seen from the scanning electron microscope (SEM), Fe3O4 particles exhibit a cubic / polyhedral morphology with uniform size. This is a typical characteristic of pure Fe3O4 nanoparticles. SEM of TCPP-Fe reveals large, amorphous aggregates with a rough surface and no obvious crystal boundaries. SEM of Fe3O4@TCPP (Fe) shows that, compared to pure Fe3O4, the particle morphology changes from "regular cubic" to irregular aggregates, but still retains a certain "granularity" (distinguishing it from the large, amorphous particles of pure TCPP-Fe); simultaneously, the particle size is slightly larger and the surface is rougher. TEM of Fe3O4@TCPP (Fe) reveals areas of light and dark contrast (bright areas correspond to the Fe3O4 core, and dark areas correspond to the TCPP (Fe) shell). This further confirms the successful synthesis of the probe precursor Fe3O4@TCPP (Fe).

[0059] Example 2 This embodiment uses the probe from Example 1 to establish a mercury ion standard curve. The specific process is as follows: Establish a fluorescence standard curve: Mix 50 μmol / L Fe3O4@TCPP (Fe)@Apt probe solution with standard mercury ion solutions at concentrations of 0.02 μg / L, 0.1 μg / L, 0.3 μg / L, 0.5 μg / L, 1.0 μg / L, 1.5 μg / L, and 2.0 μg / L at a ratio of 2:1. v / v After mixing and incubating for 30 minutes, 500 μL of Tris-HCl solution at pH 7.4, 200 μL of 5.0 mmol / L OPD, and 100 μL of 30.0 mmol / L H2O2 were added, and the mixture was reacted for 20 minutes. The supernatant was magnetically separated and added to a cuvette. The intensity of the characteristic absorption peak of the fluorescence emission spectrum of the probe relative to the concentration of the standard mercury ion solution was obtained, and a fluorescence standard curve was established. The intensity of the characteristic absorption peak at 569.0 nm of the fluorescence emission spectrum of the Fe3O4@TCPP (Fe) @Apt probe relative to the concentration of the standard mercury ion solution was obtained, and a fluorescence standard curve was established.

[0060] The fluorescence standard detection curve and quantitative detection working curve of Fe3O4@TCPP (Fe) @Apt probe with mercury ion concentration are shown below. Figure 2 , Figure 3 As shown, the Fe3O4@TCPP (Fe)@Apt probe exhibits a good linear relationship with mercury ion concentration in the range of 0.02 to 2.0 μg / L, and the correlation coefficient of the working curve is [data missing]. R 2 The value is 0.9989, and the linear equation is y. F0-F =720.31x + 265.37, detection limit is 0.01 μg / L.

[0061] Establish a colorimetric standard curve: Mix 50 μmol / L Fe3O4@TCPP (Fe)@Apt probe solution and standard mercury ion solutions with concentrations of 0.05 μg / L, 0.5 μg / L, 0.7 μg / L, 1.0 μg / L, 2.0 μg / L, 3.0 μg / L, and 5.0 μg / L at a ratio of 2:1 (…). v / v After mixing and incubating for 30 minutes, 500 μL of Tris-HCl solution at pH 7.4, 200 μL of 5.0 mmol / L OPD, and 100 μL of 30.0 mmol / L H2O2 were added, and the mixture was reacted for 20 minutes. The supernatant was then magnetically separated and added to a cuvette. The intensity of the characteristic absorption peak in the UV-Vis spectrum of the relationship between the probe concentration and the standard mercury ion solution concentration was obtained, and a colorimetric standard curve was established. The intensity of the characteristic absorption peak at 417.0 nm in the UV-Vis spectrum of the relationship between the Fe3O4@TCPP (Fe) @Apt probe concentration and the standard mercury ion solution concentration was obtained, and a colorimetric standard curve was established.

[0062] The colorimetric standard detection curve and quantitative detection working curve of Fe3O4@TCPP (Fe) @Apt probe with mercury ion concentration are shown below. Figure 4 , Figure 5 As shown, the Fe3O4@TCPP (Fe)@Apt probe exhibits a good linear relationship with mercury ion concentration in the range of 0.05 to 5.0 μg / L, and the correlation coefficient of the working curve is [data missing]. R 2 The value is 0.9986, and the linear equation is y. A0-A =0.071x + 0.096, the detection limit is 0.02 μg / L.

[0063] Example 3 This embodiment uses the probe from Example 1 to detect interference in mercury ion detection. The specific process is as follows: Configure different metals (K) at a concentration of 100.0 μg / L. + Mg 2+ Na + Ca 2+ Ba 2+ Cu 2+ Co 2+ Ni 2+ Al 3+ Cr 3+Ten metal ion solutions and a 1.0 μg / L mercury ion solution were used. A 50 μmol / L Fe3O4@TCPP (Fe)@Apt probe solution prepared in Example 1 was mixed with 100.0 μg / L solutions of different metals at a ratio of 2:1 (v / v) and incubated for 30 minutes. Then, 500 μL of Tris-HCl solution (pH 7.4), 200 μL of 5.0 mmol / L OPD, and 100 μL of 30.0 mmol / L H2O2 were added, and the reaction proceeded for 20 minutes. The Fe3O4@TCPP (Fe)@Apt probe and K were measured. + Mg 2+ Na + Ca 2+ Ba 2+ Cu 2 + Co 2+ Ni 2+ Al 3+ Cr 3+ Fluorescence emission spectrum and UV-Vis absorption spectrum of metal ion solutions. For example... Figure 6 and Figure 8 The selective fluorescence emission spectrum and UV-Vis absorption spectrum response histogram of the Fe3O4@TCPP (Fe)@Apt probe are shown.

[0064] Prepare a mixed solution of different metals at a concentration of 100.0 μg / L and mercury ions at a concentration of 1.0 μg / L (K). + +Hg 2+ Mg 2+ +Hg 2+ Na + +Hg 2+ Ca 2+ +Hg 2+ Ba 2+ +Hg 2+ Cu 2+ +Hg 2+ Co 2+ +Hg 2+ Ni 2+ +Hg 2+ Al 3+ +Hg 2+ Cr 3+ +Hg 2+Ten mixed metal ions were used. A 50 μmol / L Fe3O4@TCPP (Fe)@Apt probe solution prepared in Example 1 was mixed with 100.0 μg / L solutions of different metals at a ratio of 2:1 (v / v) and incubated for 30 minutes. Then, 500 μL of Tris-HCl solution at pH 7.4, 200 μL of 5.0 mmol / L OPD, and 100 μL of 30.0 mmol / L H2O2 were added, and the reaction proceeded for 20 minutes. The Fe3O4@TCPP (Fe)@Apt probe and K were then measured. + +Hg 2+ Mg 2+ +Hg 2+ Na + +Hg 2+ Ca 2+ +Hg 2+ Ba 2+ +Hg 2+ Cu 2+ +Hg 2+ Co 2+ +Hg 2+ Ni 2+ +Hg 2+ Al 3+ +Hg 2+ Cr 3+ +Hg 2+ Fluorescence emission and UV-Vis absorption spectra of mixed metal ion solutions. For example... Figure 7 and Figure 9 The figure shows the anti-interference fluorescence emission spectrum and UV-Vis absorption spectrum response histogram of the Fe3O4@TCPP (Fe)@Apt probe. The graph uses a different histogram than Hg. 2+ Metal ions are used as representatives.

[0065] The relationship between the fluorescence emission spectra and UV-Vis absorption spectra of the Fe3O4@TCPP (Fe) @Apt probe and mercury ions, and the tested metal ions, is shown in the figure. Figures 6 to 9 .

[0066] Figure 6 , Figure 7 This is a bar chart showing the fluorescence emission spectrum response of the interfering substances and mercury ions. Figure 8 , Figure 9This is a UV-Vis spectrophotometric response histogram of the interfering substances and mercury ions. The tests show that the fluorescence emission and UV-Vis absorption spectra of the Fe3O4@TCPP(Fe)@Apt probe in solutions of 10 metal ions are very weak, while the fluorescence emission and UV-Vis absorption spectra of mercury ions are significantly enhanced. Moreover, the presence of metal ions does not interfere with the recognition of mercury ions. Therefore, in the presence of the tested metal ions, Fe3O4@TCPP(Fe)@Apt exhibits good selectivity and anti-interference properties for the recognition of mercury ions.

[0067] Example 4 This embodiment uses the probe from Example 1 to detect food. The specific process is as follows: Actual samples of milk, cheese, dairy beverages, rice, wheat flour, and peanuts were spiked. The spiked samples were prepared by adding 0.1 μg / L, 0.5 μg / L, and 1.0 μg / L mercury ion standard solutions to the sample. The steps for determining the concentration of mercury ions in the sample are as follows: Before spiking, the milk, cheese, and dairy beverage samples were pretreated. The pretreatment process was as follows: To remove easily adsorbed proteins from the milk, cheese, and dairy beverage, 2.5 mL of each of the purchased skim milk, yogurt, and dairy beverage were spiked with 1.0 mL of trichloroacetic acid (30%). v / v The samples were treated by vortexing for 5 min to remove proteins. Then, they were centrifuged at 10000 rpm for 15 min, and the supernatant was collected and filtered through a 0.22 μm nylon membrane. For rice, wheat flour, and peanuts, a microwave digestion method was used for sample pretreatment to reduce matrix interference. 1.0–4.0 g of each sample was weighed and placed in an Erlenmeyer flask of the digestion apparatus. Several glass beads were added, along with 45.0 mL of nitric acid and 10.0 mL of sulfuric acid. The Erlenmeyer flask was rotated to prevent localized carbonization. After attaching the condenser, the mixture was heated at a low temperature until foaming began, at which point heating was stopped. After foaming stopped, the mixture was refluxed for 2 hours. If the solution turns brown during heating, add 5.0 mL of nitric acid and continue reflux for 2 hours until the sample is completely dissolved and turns pale yellow or colorless. After cooling, carefully add 20.0 mL of water from the top of the condenser and continue heating and refluxing for 10 minutes. After cooling, rinse the condenser with an appropriate amount of water and add the rinsing solution to the digestion solution. Filter the digestion solution through glass wool into a 100 mL volumetric flask. Wash the conical flask and filter with a small amount of water and add the rinsing solution to the volumetric flask. Add water to the mark and mix well for later use.

[0068] The samples were then spiked, and fluorescence emission spectroscopy and UV-Vis absorption spectroscopy were used for detection. Three consecutive tests were performed, and the average value and relative standard deviation of the fluorescence characteristic peak at 569 nm were calculated for the three data points. These values ​​were then substituted into the mercury ion concentration fluorescence emission spectroscopy standard curve to obtain the mercury ion concentration in the spiked samples. The calculated sample spike recovery rates are shown in Table 1, with relative standard deviations ranging from 0.4% to 2.3%. Similarly, the average value and relative standard deviation of the UV-Vis absorption peak at 417 nm were calculated for the three data points, and these values ​​were substituted into the mercury ion concentration UV-Vis spectrophotometric standard curve to obtain the mercury ion concentration in the spiked samples. The calculated sample spike recovery rates are shown in Table 1, with relative standard deviations ranging from 0.1% to 0.7%. It can be seen that the detection results from fluorescence emission spectroscopy and UV-Vis absorption spectroscopy can achieve self-calibration, improving the accuracy of the detection.

[0069] Table 1. Dual-modal detection of mercury ions in food using ultraviolet and fluorescence methods

[0070] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A nanoprobe, characterized in that: The Fe3O4@TCPP is surface-modified with a mercury ion aptamer; the Fe3O4@TCPP comprises Fe3O4 and tetrakis(4-carboxyphenyl)porphyrin coated on the surface of the Fe3O4; The nucleotide sequence of the mercury ion aptamer is shown in SEQ ID NO.1, with an amino group modified at its 5' end.

2. The nanoprobe according to claim 1, characterized in that: The mass ratio of Fe3O4 to tetrakis(4-carboxyphenyl)porphyrin is (2-7):(1-5).

3. The nanoprobe according to claim 1, characterized in that: The molar ratio of Fe3O4@TCPP to the mercury ion aptamer is (0.1-1.0) mg:(10-200) µmol.

4. A method for preparing the nanoprobe according to any one of claims 1-3, characterized in that: Includes the following steps: S1: Fe3O4@TCPP was prepared by reacting Fe3O4, a surfactant, and tetrakis(4-carboxyphenyl)porphyrin. S2: The nanoprobe was prepared by coupling Fe3O4@TCPP with a mercury ion aptamer.

5. A dual-modal sensor, characterized in that: Including the nanoprobe as described in any one of claims 1-3.

6. A kit for detecting mercury ions, characterized in that: It includes the nanoprobe according to any one of claims 1-3 or the dual-modal sensor according to claim 5, and a colorimetric reagent.

7. The application of the nanoprobe according to any one of claims 1-3, the dual-modal sensor according to claim 5, or the kit for detecting mercury ions according to claim 6 in the detection of mercury ions.

8. A method for detecting mercury ions, characterized in that: Includes the following steps: Mercury ions can be detected using the nanoprobe described in any one of claims 1-3, the dual-modal sensor described in claim 5, or the reagent kit for detecting mercury ions described in claim 6.

9. The method for detecting mercury ions according to claim 8, characterized in that: The method for detecting mercury ions includes the following steps: taking the sample solution to be tested, a chromogenic substrate, hydrogen peroxide, and a nanoprobe, mixing them, and then detecting the absorbance and / or fluorescence intensity of the solution.

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