Preparation of plasmonic-enhanced fluorescent sensor based on silver nanocubes and its application in detection of berberine hydrochloride

Abstract

The application discloses a preparation method of a plasmonic-enhanced fluorescence sensor based on silver nanocubes and application of the plasmonic-enhanced fluorescence sensor in detection of berberine hydrochloride, and the preparation method comprises the following steps: reacting isopropyl alcohol, TEOS and ammonia in a silver nanocube solution to obtain silver nanocubes coated with silica; reacting the silver nanocubes coated with silica with APTMS to obtain silver nanocubes coated with silica and modified with amino groups; reacting cadmium chloride, sodium tellurite and mercaptoacetic acid in an aqueous solution to obtain cadmium telluride quantum dots; grafting the cadmium telluride quantum dots to the surface of the silver nanocubes coated with amino-modified silica by adding EDC; and adding EDC to modify aptamers to the surface of the cadmium telluride quantum dots to obtain the plasmonic-enhanced fluorescence sensor. The plasmonic-enhanced fluorescence sensor prepared by the application has good stability, high specificity and high sensitivity, can be used for quantitative detection of berberine hydrochloride, and is more efficient, cost-saving and accurate.

Description

Technical Field

[0001] This invention belongs to the technical field of plasma-enhanced fluorescence, specifically relating to a method for preparing a plasma-enhanced fluorescence coupled aptamer sensor based on silver nanocubes and its application in the detection of berberine hydrochloride. Background Technology

[0002] Berberine hydrochloride (BH) is a bioactive alkaloid extracted from various medicinal plants such as Coptis chinensis and Berberis vulgaris. It possesses broad-spectrum properties including antibacterial, antifungal, hypotensive, antioxidant, antipyretic, antidiabetic, and neuroprotective effects, and has wide applications in clinical treatment. However, excessive use of Berberine hydrochloride may lead to adverse reactions such as drug resistance, jaundice, and gastrointestinal burden. Therefore, a rapid and effective detection method is urgently needed. Various detection methods have been proposed, including chemiluminescence, spectrophotometry, colorimetry, electrochemical methods, capillary electrophoresis, and high-performance liquid chromatography. While these methods have shown accuracy and effectiveness under specific conditions, they often require expensive equipment and complex procedures. Therefore, developing cost-effective, easy-to-operate, fast-response, and highly selective methods is urgently needed for drug quality control and clinical analysis.

[0003] Functionalizing sensors using affinity-based receptors is an effective method to eliminate false positives and false negatives, ultimately achieving precise and selective detection of targets. Aptamers are oligonucleotides or peptide molecules generated through an in vitro selection process using Systematic Evolution of Ligands (SELEX) for exponential enrichment. They can bind to specific targets with high selectivity, affinity, and specificity, sometimes even surpassing antibodies. These unique properties make them widely applicable in biosensing. Jiang et al. developed a functionalized Fe3O4 magnetic nanoparticle as a solid-phase extraction adsorbent for the selective extraction of berberine from cortexphellodendri[J]. Journal of Separation Science, 2017, 40: 2933-2940. However, the application of these existing technologies is limited to the purification of BH and has not been applied to the detection of BH. Furthermore, there are currently no reports on their application in fluorescent sensors, or on the use of PEF sensors modified with BH aptamers for the selective detection of BH. Summary of the Invention

[0004] Objective of the Invention: To address the problems existing in the prior art, this invention provides a method for preparing a plasmonic-enhanced fluorescence-coupled aptamer sensor based on silver nanocubes. The fluorescence sensor prepared by this invention enhances the luminescence intensity of cadmium telluride quantum dots through plasmonic-enhanced fluorescence technology and exhibits a concentration-dependent "on-off" characteristic for berberine hydrochloride, making it suitable as a specific berberine concentration indicator. The probe prepared by this invention effectively overcomes the shortcomings of existing berberine detection methods, such as complex operation, expensive instruments, or significant susceptibility to environmental interference.

[0005] The present invention also provides the application of the prepared plasmon-enhanced fluorescence sensor in the detection of berberine hydrochloride.

[0006] Technical Solution: To achieve the above objectives, the present invention provides a method for preparing a plasmonic-enhanced fluorescence-coupled aptamer sensor based on silver nanocubes, comprising the following steps:

[0007] (1) Prepare silver nanocube solution by adding sodium sulfide, polyvinylpyrrolidone and silver nitrate to ethylene glycol solution;

[0008] (2) Isopropanol, TEOS and ammonia were reacted in a silver nanocube solution to obtain a silica-coated silver nanocube solution.

[0009] (3) APTMS was reacted with silica-coated silver nanocubes to obtain an amino-modified silica-coated silver nanocube solution.

[0010] (4) Cadmium chloride, sodium tellurite, and mercaptoacetic acid were reacted in an aqueous solution to obtain cadmium telluride quantum dots;

[0011] (5) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to cadmium telluride quantum dots and mix well, then graft cadmium telluride quantum dots onto the surface of aminated silica-coated silver nanocubes.

[0012] (6) After adding EDC to the product of step (5) and mixing it, add the aptamer solution and react to obtain the aptamer-modified plasma-enhanced fluorescence sensor.

[0013] In step (1), ethylene glycol is added, and the mixture is heated in an oil bath to 140-160°C. After heating for 0.5-2 hours, sodium sulfide is added. After 2-20 minutes, polyvinylpyrrolidone is added. After another 2-20 minutes, silver nitrate is added to the mixture. The mixture is reacted for 2-20 minutes. After the reaction is completed, the mixture is quenched in an ice-water bath. After washing, the precipitate is dispersed in water. The volume ratio of ethylene glycol, polyvinylpyrrolidone, and silver nitrate is 5-20:2-4:1.

[0014] Preferably, in step (1), ethylene glycol is heated to 150°C and held for 1 hour, sodium sulfide ethylene glycol solution is added and held for 2 minutes, PVP is added and held for 8-9 minutes, silver nitrate is added and reacted for 10-15 minutes. The size of the silver nanocubes is determined by controlling the reaction time. The volume ratio of ethylene glycol, PVP, and silver nitrate is 10:3:1.

[0015] In step (2), isopropanol, ultrapure water and ammonia are added to the container, and the synthesized silver nanocube solution is added to the above mixture; TEOS is added and reacted for 1-4 hours; after washing, the precipitate is dispersed in water; the volume ratio of isopropanol, ultrapure water, silver nanocubes, ammonia and TEOS is 30-60:3-6:3-6:1-2:1-20.

[0016] Preferably, in step (2), after adding TEOS to the container, the reaction proceeds for 2 hours, followed by washing with ethanol, centrifugation three times, and then dissolving in water. The volume ratio of isopropanol, ultrapure water, silver nanocubes, ammonia, and TEOS is 50:5:5:1:1-20. Silicon layers of different thicknesses are synthesized by controlling the amount of TEOS added.

[0017] In step (3), APTMS is added to the silica-coated silver nanocube solution, stirred at room temperature for 0.5-4 h, then heated at 25-70°C for 0.5-4 h, and then stirred at room temperature for 0.5-8 h. After washing with water and centrifuging three times, the final volume of the mixture is adjusted to 10 mL with water to obtain amino-modified silica-coated silver nanocubes (Agcubes@SiO2-NH2). The volume ratio of the silica-coated silver nanocube solution (Agcubes@SiO2) to APTMS is 100-2000:1.

[0018] Preferably, in step (3), after adding APTMS, the mixture is stirred at room temperature for 1 hour, then heated at 65°C for 1 hour, and then stirred at room temperature for 4 hours. The volume ratio of Agcubes@SiO2 to APTMS is 2000:1.

[0019] In step (4), cadmium chloride is dissolved in water, then mercaptoacetic acid is added, the pH is adjusted to 7-12, the mixture is stirred vigorously for 1-20 minutes, an aqueous solution of Na2TeO3 is added, stirring is continued for 1-20 minutes, and the pH of the solution is maintained in the range of 10.5-11.0 during this period, then NaBH4 is added, and the obtained solution is sonicated for 1-20 minutes, and finally refluxed; the molar ratio of cadmium chloride: mercaptoacetic acid: Na2TeO3 is 1-4:2-8:1.

[0020] Preferably, in step (4), the pH of the solution is adjusted to 10.8 with NaOH, then the mixture is stirred vigorously for 5 minutes, followed by the addition of a mixed solution of Na2TeO3, and stirring is continued for 5 minutes, while maintaining the pH of the solution in the range of 10.5 to 11.0. Then NaBH4 is added, and the resulting solution is sonicated for 3 minutes. Finally, it is refluxed in an oil bath at 120°C for 12 hours. The molar ratio of cadmium chloride, mercaptoacetic acid, and Na2TeO3 is 2:4.8:1.

[0021] In step (5), EDC is added to the cadmium telluride quantum dot solution (CdTe QDs), and after placing it in an ice bath for 10-30 minutes, the amino-modified silica-coated silver nanocube solution is added to the mixture, and the mixture is reacted at room temperature for 0.5-8 hours. The volume ratio of the cadmium telluride quantum dot solution, EDC, and amino-modified silica-coated silver nanocube solution is 1-10:1:1-10.

[0022] Preferably, in step (5), EDC is added to CdTe QDs, and the mixture is placed in an ice bath. After 20 minutes, Agcubes@SiO2-NH2 is added to the mixture, and the reaction is carried out at room temperature for 4 hours. The volume ratio of CdTe QDs, EDC, and Agcubes@SiO2-NH2 is 5:1:5.

[0023] In step (6), EDC solution is added to the product of step (5) for reaction, followed by sonication, and then aptamer solution is added. The volume ratio of the product of step (5), EDC and aptamer is 5-20:1-4:1.

[0024] Preferably, the volume ratio of EDC to aptamer in step (6) is 10:2:1.

[0025] Preferably, the aptamer in step (6) is 5′-NH2-AACATAAATATTAAATTATGT-3′.

[0026] The plasmonic-enhanced fluorescence aptamer sensor based on silver nanocubes, as described in this invention, is prepared by the method for preparing such a sensor.

[0027] The present invention relates to the application of the silver nanocube-based plasmonic-enhanced fluorescence-coupled aptamer sensor in the detection of berberine hydrochloride.

[0028] Furthermore, the present invention relates to the application of the silver nanocube-based plasmonic-enhanced fluorescence-coupled aptamer sensor in the preparation of a berberine hydrochloride detection tool.

[0029] Preferably, the application of the silver nanocube-based plasmonic-enhanced fluorescence-coupled aptamer sensor in the detection of berberine hydrochloride in urine is described.

[0030] This invention prepares a plasmonic-enhanced fluorescence-coupled aptamer sensor based on silver nanocubes. Step (2) controls the silicon layer thickness, step (3) prepares for subsequent grafting, step (5) enhances fluorescence and improves sensitivity, and step (6) modifies the aptamer to improve sensor selectivity. Ultimately, the prepared fluorescence sensor exhibits good stability, high specificity, and high sensitivity, making it suitable for the quantitative detection of berberine hydrochloride with greater efficiency, cost-effectiveness, and accuracy.

[0031] This invention develops a PEF sensor based on the combination of silver nanocubes (Agcubes) and aptamers (Agcubes@SiO2-QDs-Apt) for the detection and analysis of berberine hydrochloride (BH). Specifically, silica-coated Agcubes (Agcubes@SiO2) are used as the plasmonic nanostructure and further functionalized with amino groups. Then, red-emitting CdTe quantum dots are covalently grafted onto the amino-functionalized Agcubes@SiO2 to achieve fluorescence enhancement. The berberine hydrochloride aptamer (BH-Apt) is covalently grafted onto the carboxyl groups of the CdTe quantum dots to selectively recognize BH. As a control, silver nanoparticles were prepared, and their fluorescence enhancement effects on CdTe quantum dots were compared with those of Agcubes. Three-dimensional finite-difference time-domain (3D-FDTD) simulation results show that the maximum electric field strength of Agcubes is 4.3 V / m, higher than that of Agspheres (2.9 V / m). Meanwhile, unmodified CdTe quantum dots were synthesized and their application as a control in BH analysis was explored. The fluorescence enhancement factor of Agcubes@SiO2-QDs (3.4 times) was higher than that of Agspheres@SiO2-QDs (2.1 times). In addition, BH in tablets and human urine was detected using an Agcubes-based PEF sensor.

[0032] This invention applies PEF technology to the detection of berberine hydrochloride. Multi-faceted Agcubes are used as the plasma substrate to enhance the fluorescence intensity of CdTe QDs, thereby improving the sensitivity of fluorescence detection of berberine hydrochloride. Furthermore, the sensor surface is modified with aptamers that recognize berberine hydrochloride to enhance selectivity. Silica is used as a spacer to control the distance between the plasma substrate and the fluorescent material. Silver nanocubes have more hot spots than silver nanoparticles, resulting in greater fluorescence enhancement.

[0033] This invention successfully synthesized a PEF sensor modified with Agcubes as plasmon resonance modulators for signal amplification and detection of biomolecules such as BH. Maximum fluorescence enhancement was achieved by controlling the silicon shell thickness between CdTe QDs and Agcubes. Experimental and 3D-FDTD simulation results show that Agcubes are a superior PEF material compared to silver nanospheres, as the enhanced fluorescence signal is due to the increased electric field intensity at the multiple sharp corners of the Agcubes. This interaction is spontaneous, with hydrophobic forces playing a crucial role in the quenching process. Furthermore, the PEF sensor exhibits high selectivity, sensitivity, and accuracy for BH analysis, with a detection limit as low as 87.3 nM. The recovery rate of BH in real samples using this method ranged from 98.25% to 102.05%. These findings may pave the way for the development of PEF fluorescence sensors targeting various biomolecules.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0035] 1. The plasma-enhanced fluorescence near-infrared fluorescence sensor prepared in this invention uses near-infrared fluorescent cadmium telluride quantum dots as the fluorescence source, which has the advantages of deep tissue penetration, low background fluorescence interference and low light damage.

[0036] 2. The plasmonic enhanced fluorescence near-infrared fluorescence sensor prepared in this invention uses silver nanocubes as plasmons. Compared with silver nanospheres, silver nanocubes have a multi-point structure and a greater electric field enhancement, thereby obtaining greater fluorescence enhancement.

[0037] 3. The plasma-enhanced fluorescence near-infrared fluorescence sensor prepared in this invention uses a silicon dioxide layer to control the distance between the plasma element and the fluorescent material, thereby achieving fluorescence enhancement. At the same time, the modification of the silicon dioxide layer improves the biocompatibility and stability of the near-infrared sensor.

[0038] 4. The plasma-enhanced fluorescence sensor prepared in this invention exhibits high selectivity, sensitivity, and accuracy for BH analysis, with a detection limit as low as 87.3 nM. The method achieves a recovery rate of 98.25%–102.05% for BH in real samples. These findings may pave the way for the development of PEF fluorescence sensors targeting various biomolecules.

[0039] 5. The plasma-enhanced fluorescence near-infrared fluorescence sensor of the present invention has a simple and easy preparation process, and the prepared plasma sensor has good stability. It can be used for the in vitro detection of berberine hydrochloride. At the same time, the preparation process is simple and easy to carry out and is easy to scale up. Attached Figure Description

[0040] Figure 1 The ultraviolet-visible spectrum of the Agcubes prepared in this invention;

[0041] Figure 2 The UV-Vis spectra of Agcubes@SiO2 prepared in this invention under different silica thicknesses;

[0042] Figure 3 The UV-Vis spectrum (a) of the Agcubes prepared for this invention; the excitation (b) and emission (c) spectra of the CdTe quantum dots;

[0043] Figure 4 (A) TEM image of Agcubes, (B) TEM image of Agcubes@SiO2, (C) TEM image of Agcubes@SiO2-QDs prepared for this invention;

[0044] Figure 5 TEM images of Agcubes@SiO2 shells prepared for this invention with thicknesses of 3.4 nm (A), 8.6 nm (B), 13.6 nm (C), 19.4 nm (D), 23.2 nm (E), and 31.6 nm (F);

[0045] Figure 6 Fluorescence spectra of Agcubes@SiO2-QDs prepared in this invention under different silicon shell thicknesses;

[0046] Figure 7 This is a TEM image of etched Agcubes@SiO2 prepared according to the present invention;

[0047] Figure 8 Etched Agcubes@SiO2-QDs prepared according to the present invention;

[0048] Figure 9 The fluorescence intensity decay of quantum dots (a) and Agcubes@SiO2-QDs (b) prepared for this invention;

[0049] Figure 10 3D-FDTD simulation of Agcubes (A) and silver nanospheres (Agspheres) prepared in this invention;

[0050] Figure 11 (A) The fluorescence intensity of the PEF sensor prepared for this invention changes over time. (B) The fluorescence intensity of the PEF sensor at different temperatures (15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃). (C) The fluorescence intensity of the PEF sensor changes with pH. (D) The fluorescence intensity change of the PEF sensor after incubation in 10 μM BH solution;

[0051] Figure 12(A) Fluorescence spectra of Agcubes@SiO2@QDs-Apt prepared for this invention at different concentrations of BH (0 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 150 μM). (B) Fluorescence spectra of QDs-Apt at different concentrations of BH (0 μM, 1 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 150 μM). (C) Linear fitting of FL intensity of Agcubes@SiO2@QDs-Apt with BH concentration. (D) Linear fitting of fluorescence intensity of QDs-Apt with BH concentration;

[0052] Figure 13 (A) Fluorescence response of Agcubes@SiO2-QDs-Apt prepared for this invention after co-incubation with jatorrhizine (50 μM), palmatine (50 μM), berberrubine (50 μM), and BH (50 μM). (B) Fluorescence response in the absence or presence of BH (C) BH At a concentration of 50 μM, Agcubes@SiO2-QDs-Apt coexists with other potentially coexisting substances (C coexistence Fluorescence response after incubation at 500 μM;

[0053] Figure 14 Fluorescence quenching of Agcubes@SiO2-QDs-Apt and Agcubes@SiO2-QDs prepared by this invention after adding different concentrations of BH;

[0054] Figure 15 FT-IR spectra of Agcubes (a), Agcubes@SiO2 (b), Agcubes@SiO2-NH2 (c), QDs (D), Agcubes@SiO2-QDs (e), and Agcubes@SiO2-QDs-Apt (f) prepared for this invention;

[0055] Figure 16 The Zeta potentials of Agcubes (a), Agcubes@SiO2 (b), Agcubes@SiO2-NH2 (c), QDs (d), Agcubes@SiO2-QDs (e), and Agcubes@SiO2-QDs-Apt (f) prepared for this invention;

[0056] Figure 17 DLS of Agcubes (A); Agcubes@SiO2 (B); Agcubes@SiO2-QDs (C); Agcubes@SiO2-QDs-Apt (D) prepared for this invention;

[0057] Figure 18 XPS spectra of Agcubes (a), Agcubes@SiO2 (b), Agcubes@SiO2-NH2 (c), QDs (d), Agcubes@SiO2-QDs (e), and Agcubes@SiO2-QDs-Apt (f) prepared for this invention;

[0058] Figure 19 The XPS spectrum of CdTe quantum dots prepared in this invention shows the Cd states.

[0059] Figure 20 A comparison of HPLC and PEF methods for the detection of BH;

[0060] Figure 21 The ultraviolet-visible spectrum of Agspheres;

[0061] Figure 22 The image shows a TEM image of Agspheres@SiO2 with a shell thickness of 13.3 nm.

[0062] Figure 23 The fluorescence spectrum of Agspheres@SiO2-QDs is shown, with a shell thickness of 13.3 nm. Detailed Implementation

[0063] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0064] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0065] The BH-Apt sequence is 5'-NH2-AACATAAATATTAAATTATGT-3'. It was purchased from Shanghai Sangon Biotech Co., Ltd. The following substances were also present: cadmium chloride (CdCl2), thioglycolic acid (TGA), sodium hydroxide (NaOH), sodium tellurite (Na2TeO3), sodium borohydride (NaBH4), ethylene glycol (EG) 98%, non-hydrated sodium hydrosulfide (Na2S·9H2O), poly(vinylpyrrolidone) (PVP, MW 55000), silver nitrate (AgNO3, 99.8%), ethanol (CH3CH2OH), isopropanol, ammonium hydroxide, and tetraethyl orthosilicate (TEOS, C8H). 20O4Si, 99.99%, (3-aminopropyl)trimethoxysilane (APTMS, 98%), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), triethylamine, potassium hydrogen phosphate (KH2PO4), sodium 1-heptanesulfonate (C7H 15 O3SNa), acetonitrile (C2H3N), and HPLC (≥99.9%) were all purchased from Shanghai McLean Co., Ltd., China.

[0066] Example 1

[0067] 1. Preparation of silver nanocubes

[0068] 30 mL of ethylene glycol was added to a 250 mL three-necked round-bottom flask and magnetically stirred at 250 rpm. The mixture was heated to 150 °C in an oil bath and maintained at this temperature until the reaction was complete. Nitrogen gas was introduced to remove water from the ethylene glycol. After heating for 1 h, 360 μL of freshly prepared sodium sulfide ethylene glycol solution (Na₂S·9H₂O, 3 mM) was added. After reacting for 2 min, 9 mL of polyvinylpyrrolidone (PVP) ethylene glycol solution (20 mg / μL) was added. After reacting for 8-9 min, 3 mL of silver nitrate ethylene glycol solution (282 mM) was added to the mixture, and the reaction was allowed to proceed for 15 min. At the end of the reaction, the mixture was quenched in an ice-water bath, washed three times by centrifugation with ethanol, and then the entire precipitate was dispersed in 24 mL of ultrapure water to obtain a silver nanocube (Agcubes) solution.

[0069] 2. Synthesis of silica-coated silver nanocubes (Agcubes@SiO2)

[0070] In a 25 mL flask, 10 mL of isopropanol, 1 mL of ultrapure water, and 200 μL of ammonia (25%) were added. Then, 1 mL of the synthesized silver nanocubes solution was added to the mixture. Different volumes (0.1 mL, 0.5 mL, 1 mL, 2 mL, 3 mL, and 4 mL) of ethanol solution of tetraethyl orthosilicate (TEOS) (1.71 mM) were added, and the reaction was carried out at room temperature for 2 h. After washing with ethanol and centrifuging three times, the precipitate was diluted with ultrapure water to a final volume of 10 mL to obtain the Agcubes@SiO2 solution.

[0071] 3. Preparation of amino-functionalized silicon dioxide-coated silver nanocubes (Agcubes@SiO2-NH2)

[0072] Add 5 μL of (3-aminopropyl)trimethoxysilane (APTMS) to 10 mL of Agcubes@SiO2 solution, stir at room temperature for 1 h, then heat and stir at 65 °C for 1 h. Then, stir the mixture at room temperature for 4 h. Wash with water, centrifuge three times, and dilute the precipitate to a final volume of 10 mL with ultrapure water to obtain the Agcubes@SiO2-NH2 solution.

[0073] 4. Synthesis of CdTe quantum dots

[0074] 91.34 mg of cadmium chloride was dissolved in 100 mL of water, then 36 μL of mercaptoacetic acid was added, and the pH of the solution was adjusted to 10.8 with 0.1 M NaOH. The mixture was then stirred vigorously for 5 minutes. Next, 100 mL of an aqueous solution containing 19.72 mg of Na₂TeO₃ was added to the above mixture, and the resulting solution was stirred for another 5 minutes, maintaining the pH between 10.5 and 11.0 during this time. Then, 160 mg of NaBH₄ was added, and the resulting solution was sonicated for 3 minutes. Finally, the solution was refluxed in an oil bath at 120 °C for 12 h to obtain the CdTe QDs solution.

[0075] 5. In a 25 mL round flask, add 5 mL of CdTe QDs and 1 mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (0.1 μM, ultrapure water). After incubating on ice for 20 min, add 5 mL of Agcubes@SiO2-NH2 to the mixture and react at room temperature for 4 h to obtain Agcubes@SiO2-QDs solution.

[0076] 6. Add 5 mL of the above-obtained Agcubes@SiO2-QDs and 1 mL of EDC solution (0.1 μM, ultrapure water) to a 25 mL round-bottom flask, sonicate for 20 min, then add 0.5 mL of BH-Apt (0.1 mol / L) and stir at room temperature for 2 h to obtain Agcubes@SiO2-QDs-Apt, which is the fluorescence sensor (PEF) of this invention.

[0077] The preparation method of QDs-Apt is the same as that of Agcubes@SiO2-QDs-Apt, except that in step (6), Agcubes@SiO2-QDs is directly replaced with an equal amount of CdTe QDs.

[0078] Example 2

[0079] UV-Vis spectra of Agcubes solution and Agcubes@SiO2 solution prepared in Example 1

[0080] Take 2 mL of the Agcubes solution and Agcubes@SiO2 solution prepared in Example 1 respectively for ultraviolet detection.

[0081] UV-Vis Spectroscopy: The UV-Vis spectrum of the above solution was measured. The UV-Vis spectrum was obtained using pure water as a reference solution, scanning the wavelength range of 300 nm–750 nm. The resulting Agcubes UV-Vis spectrum (see...) Figure 1 The extinction spectrum of Agcubes exhibits a strong and narrow plasmon resonance peak at 432 nm, indicating a small size distribution of the nanoparticles. Furthermore, two additional surface plasmon resonance modes are present at 352 nm and 380 nm. The UV-Vis spectrum of the Agcubes@SiO2 solution was obtained by scanning the wavelength range of 300 nm–900 nm (see...). Figure 2 After modification with silica, the surrounding medium of Agcubes changed from water (refractive index 1.33) to silica (refractive index 1.46). This caused a red shift in the absorption peak of Agcubes. Figure 2 This indicates that redshift can be used as an indirect indicator of shell thickness.

[0082] Example 3

[0083] Example 1: Overlay diagram of the UV absorption peak of Agcubes and the fluorescence emission peak of CdTe QDs.

[0084] The UV-Vis spectrum was scanned using pure water as a reference solution, covering the wavelength range of 300 nm to 750 nm. Fluorescence spectroscopy was performed on the Agcubes and CdTe QDs solutions. Fluorescence emission was measured using 400 nm excitation, with a slit width of 5 nm / 5 nm between excitation and emission, and a voltage of 700 V. The UV-Vis spectrum of the Agcubes and CdTe QDs solutions was then analyzed. The fluorescence excitation peak of the CdTe QDs was... Figure 3 Curve b) Fluorescence emission peak ( Figure 3 Curve c) and the UV absorption peak of Agcubes ( Figure 3 By combining curve a) and comparing them, a spectral overlap diagram is obtained (see...). Figure 3 ), Figure 3 The results show that the UV absorption peak and the fluorescence emission peak partially overlap, indicating that, theoretically, Agcubes can enhance the fluorescence intensity of CdTe QDs.

[0085] Example 4

[0086] Transmission electron microscopy (TEM) images of Agcubes, Agcubes@SiO2, and Agcubes@SiO2-QDs prepared in Example 1.

[0087] Two copper meshes were picked up with tweezers and immersed in Agcubes, Agcubes@SiO2, and Agcubes@SiO2-QDs solutions, respectively. They were then removed and placed in disposable petri dishes lined with filter paper. After the copper meshes dried, they were placed into the sample injector of a transmission electron microscope (TEM). The TEM images were obtained (see...). Figure 4 ). Figure 4 The image shows that Agcubes has a cubic structure with a side length of approximately 60 nm. Figure 4 Figure b shows the presence of silicon dioxide on the Agcubes surface, with a thickness of approximately 13.6 nm. This indicates the successful synthesis of Agcubes@SiO2. Figure 4 The image shows that CdTe quantum dots are uniformly distributed on the surface of Agcubes@SiO2, indicating the successful synthesis of Agcubes@SiO2-QDs.

[0088] Example 5

[0089] Transmission electron microscopy (TEM) images of Agcubes@SiO2 with different silicon layer thicknesses obtained in Example 1

[0090] The distance between CdTe quantum dots and Agcubes was controlled by the thickness of the silica spacer layer. Agcubes@SiO2-QDs were prepared by adding different amounts of TEOS (0.1 mL, 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL) to the reaction, corresponding to a silicon shell thickness of 3.4 nm. Figure 5 A), 8.6nm ( Figure 5 B), 13.6nm ( Figure 5 C), 19.4nm Figure 5 D), 23.2nm ( Figure 5 E), 31.6nm ( Figure 5 F).

[0091] Example 6

[0092] Fluorescence emission spectrum of Agcubes@SiO2-QDs solution prepared in Example 1.

[0093] Agcubes@SiO2-QDs were synthesized according to step (5) of Example 1, and fluorescence emission spectra of Agcubes@SiO2-QDs with different silicon layer thicknesses were prepared.

[0094] Fluorescence spectroscopy testing: Fluorescence emission spectra of the Agcubes@SiO2-QDs solution were measured. The fluorescence emission spectra were measured with 400 nm excitation, an excitation / emission slit width of 5 nm / 5 nm, and a voltage of 700 V. The obtained fluorescence emission spectra (see...) Figure 6The experimental results showed that the maximum fluorescence enhancement (3.4 times) was obtained when the silicon shell thickness was 13.6 nm. Subsequently, the sensor was prepared using 1 mL of TEOS added in Example 1. The QDs-Apt and Agspheres@SiO2-QDs were also prepared using the above-mentioned optimal concentration.

[0095] Example 7

[0096] In Example 1, the enhancement effect was achieved by using Agcubes@SiO2 with NaCl-etched silver cores as a blank control.

[0097] The fluorescence enhancement factor (EF) plays a crucial role in assessing the PEF phenomenon. Its calculation formula is EF = I... cpl / I ref , where I cpl I represents the fluorescence intensity of the fluorophore coupled to the nanoparticles. ref Ideally, the reference fluorescence intensity should reflect the fluorescence intensity of the fluorophore coupled to the nanoparticle under the same additional conditions, to provide a corresponding reference intensity.

[82] However, due to the lack of a unified standard, fluorescence enhancement factors often exhibit significant variations within the same system. In practical procedures, measurements are frequently performed from independent fluorophore solutions. However, this approach can introduce errors due to the challenge of perfectly replicating all relevant variables in another sample. To address this issue, one approach involves functionalizing silica spheres of similar size to the plasma nanoparticles with the same concentration of fluorophores as a reference intensity. However, differences between the nanostructures on anisotropic nanostructures and the silica shell coating still exist. A more convenient approach involves using strong etchants (such as cyanide ions or nitric acid) to dissolve the metal core, producing coreless nanoshells or “etched” nanoparticles. Another approach utilizes cyanide ions to dissolve the metal nanoparticles; while this method provides a more representative control sample (with the same nanoparticle and fluorophore concentrations, and the same fluorophore surface coverage), these strong etchants may release fluorescent material from the nanoparticle surface by cleaving dye-nanoparticle bonds. For example, in the case of silver-silicon core-shell nanoparticles, researchers have demonstrated the use of “mild” etchants, such as chloride ions. Therefore, to verify the fluorescence enhancement effect of Agcubes@SiO2 on quantum dots, the influence of the silicon layer on the fluorescence of QDs was first eliminated. In this embodiment, saturated sodium chloride was used to dissolve the silver core of Agcubes@SiO2. Specifically, the Agcubes@SiO2 solution prepared in Example 1 was soaked in saturated sodium chloride for 24 hours, washed, and centrifuged three times. The SiO2 formed after etching Agcubes@SiO2 was used as a control sample. TEM images of the etched nanoparticles strongly confirmed the success of the etching process. Figure 7Following steps 3-5 of Example 1, SiO2 formed by etching Agcubes@SiO2 was synthesized into SiO2-QDs. The FL intensity of the synthesized SiO2-QDs was consistent with that of CdTe quantum dots. Figure 8 These results indicate that silica has no effect on the fluorescence intensity of CdTe quantum dots. In this example, QDs with the same concentration as SiO2-QDs were also prepared (step 4 of Example 1), and their fluorescence spectra were measured (according to Example 6). Furthermore, the above EF was calculated based on the fluorescence intensity of SiO2-QDs. The silica shell proposed by this method does not affect the fluorescence intensity of the QDs themselves, which also proves that the enhancement of fluorescence intensity of Agcubes@SiO2-QDs with a silicon layer thickness of 13.6 nm prepared in step 5 of Example is caused by Agcubes. Figure 6 This result demonstrates that the silica shell can be used as a fluorescence blank control to calculate the fluorescence enhancement factor after the introduction of silver nanocubes.

[0098] Example 8

[0099] The CdTe QDs solution and Agcubes@SiO2-QDs solution prepared in Example 1 were subjected to quantum yield calculation and fluorescence lifetime testing.

[0100] Rhodamine 6G (with quantum yield, Rhodamine 6G was used as a standard substance for measuring quantum yield. CdTe quantum dots and Agcubes@SiO2-QDs were dispersed in an aqueous solution after being dissolved in ethanol. The solutions were then diluted to different concentrations (5 μM, 4 μM, 3 μM, 2 μM, and 1 μM), calculated based on the molar amounts of Cd and Te added during the synthesis of the CdTe quantum dots. The maximum absorbance of each solution was measured, and the fluorescence intensity at the maximum emission wavelength of 626 nm was recorded using a full-spectrum scan. Finally, the fluorescence quantum yield could be calculated using the following formula:

[0101]

[0102] in, For standard matter quantum yield, To test the quantum yield of matter, A S and A X The maximum absorbance of the standard substance and the test substance are respectively, F S and F X The fluorescence intensities of the standard substance and the test substance at specific wavelengths are represented by n. X and n X These are the refractive indices of the solvents for the standard substance and the test substance, respectively.

[0103] The fluorescence lifetimes of CdTe QDs solutions and Agcubes@SiO2-QDs solutions were measured using a transient / steady-state fluorescence spectrometer, FluoroMax-4 (HORIBA, Japan). The bi-exponential model used for the fluorescence lifetime measurement is described by the following formula:

[0104]

[0105] Where τ1 and τ2 represent time constants, and α1 and α2 represent the amplitudes of the fast and slow components, respectively. The average lifetime of τ is calculated as follows:

[0106]

[0107] Table 1 and Figure 9 The results showed that the fluorescence lifetime of Agcubes@SiO2-QDs decreased from 41.31 ns to 32.69 ns compared to the fluorescence intensity of CdTe QDs. Simultaneously, with Rhodamine 6G (Φ = 0.95) as the standard fluorophore, the fluorescence quantum yield of Agcubes@SiO2-QDs increased from 26.93% for CdTe QDs to 86.34%. These results indicate that the coupling of plasma Agcubes with quantum dots affects the decay path of quantum dots and enhances both radiative and non-radiative decay rates. The increased quantum yield further demonstrates the plasma-enhanced fluorescence effect.

[0108] Table 1. Fluorescence characteristics of the PEF sensor

[0109]

[0110] Example 9

[0111] The electric field distribution of silver nanocubes was simulated using the finite-difference time-domain (FDTD) simulation software.

[0112] In 3ds Max, silver nanocubes and silver nanospheres with a side length of 60 nm were modeled. In the finite-difference time-domain simulation, Ag(Silver)-Johnson and Christy were selected as the materials for the Ag structure. The simulation time was set to 1000 seconds, the simulation temperature to 300 K, and a perfectly matched layer (PML) boundary condition was used. A covering mesh was introduced on the silver nanostructures, with a maximum mesh step size of 1 nm in the X, Y, and Z directions. To establish circularly polarized light, two total field scattering (TFSF) sources perpendicular to the polarization direction were added to the simulation. The incident light field intensity was set to 1 V / m, and the incident light wavelength was 400 nm. A frequency domain field profile monitor was added to visualize the electromagnetic field information. Agcubes@SiO2-QDs (4.6 V / m, Figure 10The electric field strength of A) is significantly higher than that of non-cubic Agspheres@SiO2-QDs (2.9 V / m). Figure 10 B). The above results show that the stronger the LSPR, the stronger the fluorescence enhancement. These results also confirm that Agcubes have a square scattering cross section, making them excellent candidates for PEF sensors.

[0113] Agspheres@SiO2-QDs were prepared using the method described in Example 1, except that the synthesized silver nanocubes solution was replaced with a silver nanospheres solution (the absorbance of the added Agspheres was controlled to be consistent with that of the Agcubes). Specifically, 35 mg of silver nitrate was dissolved in 100 mL of deionized water and heated to boiling. Then, 4 mL (1%) of sodium citrate was added to the mixture, and the mixture was boiled for 1 h to obtain the silver nanospheres solution. The UV absorption peak of the synthesized Agspheres was located at 430 nm. Figure 21 The synthesized Agspheres@SiO2 TEM image shows a shell thickness of 13.3 nm. Figure 22 The fluorescence spectrum of Agspheres@SiO2-QDs Figure 23 This indicates that Agspheres, acting as plasmon polarons, enhanced the fluorescence of QDs by 2.1 times, and Agcubes@SiO2-QDs by 3.4 times. Figure 6 The fluorescence enhancement factor of Agspheres@SiO2-QDs was 2.1 times higher than that of non-cubic Agspheres@SiO2-QDs. Figure 23 )

[0114] Example 10

[0115] Stability study of the fluorescence sensor (PEF) prepared in Example 1

[0116] Before applying the PEF sensor, the fluorescence stability of the PEF sensor was investigated. Figure 11 A indicates that the PEF sensor remains stable after being stored at room temperature for 120 hours. Furthermore, the effect of temperatures ranging from 15°C to 50°C on the PEF sensor was investigated. Figure 11 B). The sensor exhibited excellent stability at different temperatures after incubation for 10 minutes. The pH stability of the PEF sensor was tested in the range of 3–11. The results showed that the FL intensity of the PEF sensor decreased under strong acid and strong alkaline conditions. Figure 11C). The PEF sensor exhibited the highest fluorescence intensity at pH 7, therefore pH 7 was selected as the optimal detection pH. The effect of incubation time between the PEF sensor and BH was also investigated. 1 mL of 50 μM BH was incubated with 1 mL of the PEF sensor prepared in Example 1 at room temperature for 20 min. Fluorescence spectroscopy was performed as in Example 6, and the sensor's fluorescence tended to stabilize. Figure 11 D indicates that all quenchers have occupied the binding sites, and the reaction is complete. Therefore, 20 minutes was chosen as the incubation time.

[0117] Example 11

[0118] The fluorescence emission patterns and line graphs of the fluorescence sensor prepared in Example 1 after incubation with different concentrations of berberine hydrochloride

[0119] An Agcubes-free control sensor (QDs-Apt) was prepared as a comparative reference according to the method of Example 1. 1 mL of different concentrations of BH was incubated with either the 1 mL LPEF sensor or the QDs-Apt prepared in Example 1 at room temperature for 20 min, and fluorescence spectroscopy was measured as in Example 6. It is noteworthy that in BH solutions of the same concentration, Agcubes@SiO2-QDs-Apt… Figure 12 A) compared to QDs-Apt( Figure 12 B) exhibited more pronounced fluorescence quenching. Furthermore, a linear relationship between FL intensity and BH concentration was established. The linear relationship between the sensor's FL intensity and BH concentration was fitted as y = 0.0975x + 0.0289(Agcubes@SiO2-QDs-Apt, Figure 12 C), y=0.0277x+0.0234(QDs-Apt, Figure 12 D), where y represents F0 / F-1, x represents the BH concentration in μM, F0 represents the original FL intensity of the sensor, and F represents the FL of the sensor during incubation with BH. The linear detection ranges of Agcubes@SiO2@QDs-Apt and QDs-Apt are 0.1–100 μM and 1–100 μM, respectively. Compared with Agcubes@SiO2-QDs-Apt (87.3 nM), the detection limit of QDs-Apt (898.9 nM) (calculated according to 3σ / k, where σ is the relative standard deviation of the blank sample (12 times), and k is the slope of the experimentally determined linear equation) is improved by 10 times, demonstrating that the PEF sensor prepared in this invention has higher sensitivity.

[0120] Example 12

[0121] Study on the selectivity and interference of the fluorescence sensor prepared in Example 1

[0122] The selectivity of the PEF sensor was evaluated by its response to similar compounds such as jatrorrhizine hydrochloride, palmatine chloride, and berberrubine. One mL of the PEF sensor prepared in Example 1 was incubated with 1 mL of similar compounds at 50 μM or higher for 20 minutes, and the fluorescence intensity of the solution was measured according to the method in Example 6. The results clearly showed that the PEF sensor exhibited higher fluorescence quenching to BH than these similar compounds. Figure 13 A). Furthermore, the anti-interference capability of the PEF sensor was also studied. Figure 13 B). The levels of relevant amino acids (proline (Pro), threonine (Thr), glutamic acid (Glu), leucine (Leu), and phenylalanine (Phe)) and metal ions (Ca) in BH tablets were examined at concentrations 10 times higher than the BH concentration. 2+ Mg 2+ Na + K + ) and common excipients (starch, sucrose, glucose, magnesium stearate, sodium carboxymethyl starch (CMS), dextrin, and corn starch). The results showed that these substances did not exhibit significant interference in the BH assay. Figure 13 (B) This means that potential interferences may exist in biological samples but will not affect the sensitivity and selectivity of the PEF sensor. Therefore, the developed sensor shows good application prospects in detecting BH in raw materials and drug formulations.

[0123] Example 13

[0124] Verification diagram of the selectivity effect of the fluorescent sensor aptamer prepared in Example 1

[0125] To further verify that the high selectivity of the PEF sensor is due to the aptamer on the PEF sensor, a PEF sensor without BH aptamer modification (Agcubes@SiO2-QDs) was prepared as a control sensor. 1 mL of 50 μM BH was incubated for 20 minutes with both the 1 mL PEF sensor prepared in Example 1 and the PEF sensor without aptamer modification (using the method of Example 1, without adding the aptamer in step (6)). The fluorescence intensity of the solutions was then measured. The results showed that fluorescence quenching was more pronounced in the PEF sensor containing the BH aptamer. Figure 14 This demonstrates the high affinity and specificity of the BH aptamer for BH.

[0126] Example 14

[0127] Infrared spectra of the freeze-dried powders of the Agcubes solution, Agcubes@SiO2 solution, Agcubes@SiO2-NH2 solution, CdTeQDs solution, Agcubes@SiO2-QDs solution, and Agcubes@SiO2-QDs-Apt solution prepared in Example 1 were analyzed.

[0128] To further characterize the composition of the PEF sensor, Fourier transform infrared spectroscopy (FT-IR) was employed. Figure 15 E). With Agcubes ( Figure 15 a) Compared to 1089cm -1 The new peak at that location is attributed to the symmetrical bending vibration of Si-O-Si on Agcubes@SiO2. Figure 15 b). After amino modification on Agcubes@SiO2-NH2 ( Figure 15 c), at 3449cm -1 The peak value at 1600 cm⁻¹ corresponds to the stretching vibration of the NH bond. -1 The peak at 1660 cm⁻¹ corresponds to the bending vibration of the NH bond. The characteristic carboxyl peaks of CdTe quantum dots are located at 1660 cm⁻¹. -1 1574cm -1 1473cm -1 and 1426cm -1 ( Figure 15 d) This confirmed the successful fabrication of CdTe quantum dots. When quantum dots were modified on Agcubes@SiO2, the quantum dots at 1649 cm⁻¹... -1 and 1569cm -1 A peak appeared at ( ) Figure 15 e) indicates the presence of amide bonds, demonstrating successful modification of CdTe quantum dots onto the Agcubes@SiO2-QDs surface. Following the reaction with Agcubes@SiO2-QDs, the reaction at 1736 cm⁻¹... -1 (C=O) and 1255cm -1 A characteristic peak appears at (O=P-OH). Figure 15 The results confirmed that the BH aptamer was successfully modified on Agcubes@SiO2-QDs-Apt.

[0129] Example 15

[0130] The zeta potentials of the Agcubes solution, Agcubes@SiO2 solution, Agcubes@SiO2-NH2 solution, CdTeQDs solution, Agcubes@SiO2-QDs solution and Agcubes@SiO2-QDs-Apt prepared in Example 1 were detected.

[0131] The zeta potential of the PEF sensor was studied to determine its composition. Figure 16 The Zeta potential of Agcubes is -(34.17±0.96) mV. Figure 16 a). After coating the Agcubes surface with silicon dioxide, the zeta potential of Agcubes@SiO2 is -(30.02±1.95) mV. Figure 16 b). Subsequently, after amination treatment of Agcubes@SiO2 with APTMS, the Zeta potential of Agcubes@SiO2-NH2 increased to -(6.13±0.38) mV. Figure 16 c). The increase in the Zeta potential of Agcubes@SiO2-NH2 is due to the protonation of amino groups on the Agcube@SiO2-NH2 surface. The Zeta potential of CdTe quantum dots is -(37.66±0.93) mV. Figure 16 d). Therefore, the Zeta potential of Agcubes@SiO2-QDs drops to -(30.18±1.50)mV. Figure 16 e), indicating that CdTe quantum dots were successfully grafted onto Agcubes@SiO2-QDs. Furthermore, the Zeta potential of Agcubes@SiO2-QDs-Apt decreased to -(32.84±1.36) mV. Figure 16 f) confirmed that negatively charged nucleic acids were attached to the surface of Agcubes@SiO2-QDs-Apt.

[0132] Example 16

[0133] The Agcubes, Agcubes@SiO2-NH2, Agcubes@SiO2-QDs, and Agcube@SiO2-QDs-Apt prepared in Example 1 were subjected to dynamic light scattering (DLS).

[0134] Further exploration using dynamic light scattering (DLS) revealed that Agcubes, Agcubes@SiO2-NH2, Agcubes@SiO2-QDs, and Agcube@SiO2-QDs-Apt were well dispersed in aqueous solution, with DLS diameters of 73.26 nm, 99.66 nm, 157.2 nm, and 158.9 nm, respectively. Figure 17 The diameter of the PEF sensor also increased during the synthesis process, indicating that the PEF sensor was successfully prepared.

[0135] Example 17

[0136] The fluorescence sensor prepared in Example 1 was subjected to X-ray photoelectron spectroscopy analysis.

[0137] The chemical compositions of the freeze-dried Agcubes, Agcubes@SiO2, Agcubes@SiO2-NH2, CdTe QDs, Agcubes@SiO2-QDs, and Agcubes@SiO2-QDs-Apt were characterized by XPS analysis. Figure 18 As shown, the characteristic peaks of Ag 3d, Si 2p, N 1s, Cd 3d, and P 2p are 369 eV, 103.6 eV, 399.15 eV, 404.35 eV, and 134.55 eV, respectively. The appearance of these characteristic peaks indicates the elemental composition of Agcubes@SiO2-QDs-Apt.

[0138] Example 18

[0139] X-ray photoelectron spectroscopy analysis was performed on the CdTe quantum dots prepared in Example 1.

[0140] To determine the elemental composition and orbital states present in CdTe QDs, XPS spectra of lyophilized CdTe QDs were tested. Figure 19 The peaks of Cd at 404.9 eV and 411.6 eV are respectively 3d. 5 / 2 and 3D 3 / 2 Orbital state. The peak separation at 6.7 eV corresponds to 3d. 5 / 2 and 3D 3 / 2 Spin-orbit splitting occurs between orbital states. These values ​​are close to the previously reported binding energy of Cd. These peaks in Cd confirm the bonding between Cd and Te, thus forming CdTe quantum dots.

[0141] Example 21

[0142] The fluorescence sensor prepared in Example 1 was used for a spike recovery experiment.

[0143] The BH in berberine hydrochloride tablets, compound berberine tablets, and urine was analyzed using a PEF sensor (Table 2). n tablets of berberine hydrochloride and compound berberine tablets were taken, sugar coating removed, and ground into powder. The powder was dissolved in V volumes of water, where M is the relative molecular mass of berberine hydrochloride. The fluorescence method developed in this experiment was applied to the above-prepared solution for detection (according to the method in Example 11). The measured fluorescence intensity was substituted into the standard curve (F / F0 = 0.0296 + 0.0962C) to calculate the detection concentration C. The average drug content (mg / tablet) was calculated using the following formula:

[0144]

[0145] The extract was diluted n times, and 0.5 mL of the extract and 0.5 mL of the LPEF sensor were mixed and reacted for 20 min. The change in fluorescence intensity was then detected. BH was recovered by spiking with BH standard samples and detected in urine. The results showed that the BH content in berberine hydrochloride tablets and compound berberine tablets was 98.85 mg / tablet and 52.33 mg / tablet, respectively, consistent with the labeled amount. The recovery rate was 98.25%–102.05%, with high precision (relative standard deviation (RSD) = 1.99%–4.81%).

[0146] Table 2. BH content in actual samples determined using a PEF sensor.

[0147]

[0148] Example 20

[0149] The berberine hydrochloride solution was detected by high performance liquid chromatography.

[0150] To further verify the accuracy of the PEF sensor, high-performance liquid chromatography (HPLC) was used to detect BH. The HPLC conditions were as follows: octadecylsilane-bonded silica gel was used as the stationary phase; 0.01 mol / L ammonium dihydrogen phosphate solution (adjusted to pH 2.8 with phosphoric acid)-acetonitrile (75:25) was used as the mobile phase; the detection wavelength was 345 nm; and the injection volume was 10 μL. The chromatographic residence time was 8.1 min. The results from the PEF sensor were consistent with those from HPLC. Figure 20 These results collectively demonstrate the applicability of PEF-based methods for the sensitive and accurate detection of BH in real-world samples.

[0151] Example 21

[0152] The sensor of this invention is compared with other detection methods.

[0153] Table 3 Comparison of various BH detection methods

[0154]

[0155] [1]Nie LR,Song H,Yohannes A,Liang SW,Yao S.Extraction in cholinium-based magnetic ionic liquid aqueous two-phase system for the determination ofberberine hydrochloride in Rhizoma coptidis[J].RSC Advances,2018,8(44):25201-9.

[0156] [2]Gao W H,Lin S Y,Jia L,Guo X K,Chen X G,Hu Z D.Analysis ofprotoberberine alkaloids in several herbal drugs and related medicinalpreparations by non-aqueous capillary electrophoresis[J].Journal ofSeparation Science,2005,28(1):92-7.

[0157] [3]Hu Z,Xie M,Yang D,Chen D,Jian J,Li H,et al.A simple,fast,andsensitive colorimetric assay for visual detection of berberine in humanplasma by NaHSO4-optimized gold nanoparticles[J].RSC Advances,2017,7(55):34746-54.

[0158] [4]He X,Li X,Feng S,Li X,Nong C.Fabrication ofphotoelectrochemicalsensor based on Fe-Doped MoSe2 for the sensitive detection ofberberinehydrochloride[J].Journal ofThe Electrochemical Society,2021,168(5):056523.

[0159] Comparing PEF sensors with existing detection methods: Compared to HPLC, PEF sensors have a detection range (0-119 μM) and detection limit (68.45 nM) that are close to those of HPLC, but are more convenient in terms of operation and time, requiring no expensive equipment or specialized technicians. Compared to colorimetric methods based on gold nanoparticles (Au NPs), although colorimetric methods are more convenient to observe with the naked eye, they are far inferior to PEF sensors in terms of detection limit (710 nM) and detection range (0-4.5 μM). Compared to photoelectrochemical sensors based on the photosensitive material Fe-doped MoSe2 (FeMoSe2) (0.025–15.0 μM), they have a wider detection range. However, the lifespan of photoelectrochemical sensors may be affected by wear and corrosion of the photoelectrode material, requiring periodic replacement or maintenance. Furthermore, none of the above sensors modified the specific recognition element. The PEF sensor in this invention is based on aptamer modification. The binding between the aptamer and the target molecule is highly specific; therefore, the aptamer sensor has high selectivity and can accurately detect the target molecule without being affected by other interfering substances. In summary, compared to (Table 3), the proposed detection method has a significant advantage in LOD. This method is simple, low-cost, and has great potential for practical application.

Claims

1. A method for preparing a plasmonic-enhanced fluorescence-coupled aptamer sensor based on silver nanocubes for the detection of berberine hydrochloride, characterized in that, Includes the following steps: (1) Prepare silver nanocube solution by adding sodium sulfide, polyvinylpyrrolidone solution and silver nitrate solution to ethylene glycol solution; (2) Isopropanol, TEOS and ammonia were reacted in a silver nanocube solution to obtain a silica-coated silver nanocube solution; (3) APTMS was reacted with silica-coated silver nanocubes to obtain an amino-modified silica-coated silver nanocube solution; (4) Cadmium chloride, sodium tellurite, and mercaptoacetic acid are reacted in an aqueous solution to obtain cadmium telluride quantum dots; (5) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) solution to the cadmium telluride quantum dot solution and mix well, then graft the cadmium telluride quantum dots onto the surface of aminated silica-coated silver nanocubes. (6) After adding EDC solution to the product of step (5) and mixing well, add aptamer solution and react to obtain aptamer-modified plasma-enhanced fluorescence coupled aptamer sensor; In step (1), the ethylene glycol solution is heated in an oil bath to 140-160°C for 0.5-2 hours. Sodium sulfide is then added, and after 2-20 minutes, polyvinylpyrrolidone solution is added. After another 2-20 minutes, silver nitrate solution is added to the mixture, and the reaction is allowed to proceed for 2-20 minutes. After the reaction is complete, the mixture is quenched in an ice-water bath, washed, and the precipitate is dispersed in water. The volume ratio of ethylene glycol, polyvinylpyrrolidone solution, and silver nitrate solution is 5-20:2-4:

1. In step (3), APTMS is added to the silica-coated silver nanocube solution, stirred at room temperature for 0.5-4 h, then heated at 25-70°C for 0.5-4 h, and then stirred at room temperature for 0.5-8 h; the volume ratio of the silica-coated silver nanocube solution to APTMS is 100-2000:

1. In step (6), the aptamer is 5′-NH2-AACATAAATATTAAATTATGT-3′.

2. The preparation method according to claim 1, characterized in that, In step (2), isopropanol, ultrapure water and ammonia are added to the container, and the synthesized silver nanocube solution is added to the above mixture; TEOS solution is added, and the reaction is carried out for 1-4 h; after washing, the precipitate is dispersed in water; the volume ratio of isopropanol, ultrapure water, silver nanocube solution, ammonia and TEOS solution is 30-60:3-6:3-6:1-2:1-20.

3. The preparation method according to claim 1, characterized in that, In step (4), cadmium chloride is dissolved in water, then mercaptoacetic acid is added, the pH is adjusted to 7-12, the mixture is stirred vigorously for 1-20 minutes, an aqueous solution of Na2TeO3 is added, stirring is continued for 1-20 minutes, and the pH of the solution is maintained in the range of 10.5-11.0 during this period, then NaBH4 is added, and the obtained solution is sonicated for 1-20 minutes, and finally refluxed. The molar ratio of cadmium chloride, mercaptoacetic acid and Na2TeO3 is 1-4:2-8:

1.

4. The preparation method according to claim 1, characterized in that, In step (5), EDC solution is added to the cadmium telluride quantum dot solution, and after placing it in an ice bath for 10-30 min, the amino-modified silica-coated silver nanocube solution is added to the mixture, and the reaction is carried out at room temperature for 0.5-8 hours. The volume ratio of the cadmium telluride quantum dot solution, EDC solution, and amino-modified silica-coated silver nanocube solution is 1-10:1:1-10.

5. The preparation method according to claim 1, characterized in that, In step (6), EDC solution is added to the product of step (5), followed by sonication, and then aptamer solution is added for reaction. The volume ratio of the product of step (5), EDC solution and aptamer solution is 5-20:1-4:

1.

6. A plasmonic-enhanced fluorescence aptamer sensor based on silver nanocubes prepared by the method described in claim 1.

7. The application of the silver nanocube-based plasmonic fluorescence coupled aptamer sensor as described in claim 6 in the detection of berberine hydrochloride.

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