Preparation of plasma enhanced fluorescence sensor based on gold nanocage and application of plasma enhanced fluorescence sensor in noradrenaline detection

Abstract

The invention discloses preparation of a plasma enhanced fluorescence sensor based on a gold nanocage and application of the plasma enhanced fluorescence sensor in noradrenaline detection. The preparation method comprises the following steps: preparing a silver nanocube solution; reacting the silver nanocubes and chloroauric acid in a polyvinylpyrrolidone aqueous solution to obtain a gold nanocage solution; adding polyethylene glycol into the gold nanocage solution for reaction to obtain a polyethylene glycol modified gold nanocage solution; mixing a chloroauric acid aqueous solution and a bovine serum albumin aqueous solution, and adding an alkali reagent for reaction to obtain a gold nano-cluster solution; and adding the gold nanocluster, EDC and NHS into a polyethylene glycol modified gold nanocage solution, and reacting to obtain the plasma enhanced fluorescence sensor. The prepared fluorescence sensor for plasma enhanced fluorescence is good in stability, strong in specificity and high in sensitivity, can be used for detection of noradrenaline actual samples and living cells, and is more efficient, cost-saving and more accurate.

Description

Technical Field

[0001] The invention belongs to the technical field of plasma enhanced fluorescence, and in particular relates to the preparation of a plasma enhanced fluorescence sensor based on gold nanocages and the application of the sensor in norepinephrine detection. Background Art

[0002] Norepinephrine (NE), as a core catecholamine neurotransmitter, plays a pivotal role in regulating various physiological and pathophysiological processes such as neural conduction, metabolic balance and organ function. Impairment of NE transmission not only leads to impaired cognition and arousal, but is also closely related to a series of neurodegenerative diseases and mental disorders, such as depression, attention deficit hyperactivity disorder (ADHD), Alzheimer's disease (AD) and Parkinson's disease (PD). Although NE is crucial in neuronal signal transduction and physiological function, there are still many challenges in achieving sensitive and convenient detection of NE in living cells or drug analysis. Traditional detection methods, such as high performance liquid chromatography (HPLC) and capillary electrophoresis, not only require complex sample pretreatment and professional operation skills, but also rely on sophisticated and expensive instruments and equipment. These limitations seriously restrict their accuracy and convenience in NE detection in drugs, and also fail to achieve dynamic monitoring of NE under physiological conditions.

[0003] In contrast, real-time fluorescence imaging technology provides a new, efficient and non-invasive method for tracking NE in living cells. In the prior art, a water-soluble sulfonate red anthocyanin fluorophore based on the "protection-deprotection" mechanism has been developed for fluorescence imaging of NE. It has attracted much attention due to its low background fluorescence interference, strong deep tissue penetration, and low photodamage to biological media. However, there are also some natural defects, such as low quantum yield (usually less than 10%) and poor photostability, which limit their further imaging or sensing applications in biological media.

[0004] Therefore, it is particularly important to design a plasmonic fluorescence-enhanced sensor for the detection of norepinephrine to achieve more sensitive and rapid detection. Such a sensor is expected to overcome the limitations of existing fluorescent dyes and provide a new solution for the detection of NE in living cells or drug analysis. Summary of the invention

[0005] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a method for preparing a plasma-enhanced fluorescence sensor based on gold nanocages. The fluorescence sensor prepared by the present invention improves the luminescence intensity of gold nanoclusters through plasma-enhanced fluorescence technology, has a norepinephrine concentration-dependent "on-off" characteristic, and can be used as a specific norepinephrine concentration indicator. The probe prepared by the present invention can effectively solve the shortcomings of the existing norepinephrine detection, such as complex operation, expensive instruments, or greater environmental interference.

[0006] The invention also provides application of the prepared plasma enhanced fluorescence sensor in norepinephrine detection.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the preparation method of a plasmon-enhanced fluorescence sensor based on gold nanocages (Aucages) described in the present invention comprises the following steps:

[0008] (1) Adding sodium sulfide, polyvinyl pyrrolidone, and silver nitrate to an ethylene glycol solution to prepare a silver nanocube (Aucages) solution;

[0009] (2) reacting a silver nanocube solution and a chloroauric acid solution in a polyvinyl pyrrolidone solution to obtain a gold nanocage (Aucages) solution;

[0010] (3) adding polyethylene glycol to the gold nanocage solution to obtain a polyethylene glycol-modified gold nanocage solution (Aucages@PEG);

[0011] (4) mixing an aqueous solution of chloroauric acid and an aqueous solution of bovine serum albumin, and adding an alkali reagent to react to obtain a gold nanocluster solution (AuNCs);

[0012] (5) Adding gold nanocluster solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide to the polyethylene glycol-modified gold nanocage solution, the reaction yields a gold nanocage-based plasmon enhanced fluorescence sensor (PEF sensor).

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

[0014] Preferably, in step (1), ethylene glycol is heated to 150°C for 1 hour, sodium sulfide ethylene glycol solution is added for 2 minutes, PVP is added for 8-9 minutes, silver nitrate is added and reacted for 10-15 minutes, and 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), the polyvinyl pyrrolidone aqueous solution is heated to boiling, and then the silver nanocube solution is added. After the reaction for 10-20 minutes, the chloroauric acid aqueous solution is added dropwise thereto to react. After impurities are removed, the precipitate is washed and dispersed after washing. The volume ratio of the polyvinyl pyrrolidone, the silver nanocube and the chloroauric acid is 80-150:5-20:1.

[0016] Preferably, in step (2), a polyvinyl pyrrolidone aqueous solution is added to the container, and then heated to 100°C in a water bath environment and kept boiling for 5-30 minutes. Thereafter, the synthesized silver nanocubes are added for 5-15 minutes. Then, a chloroauric acid aqueous solution is added, during which the reaction time and the amount of chloroauric acid aqueous solution added are precisely controlled to achieve effective regulation of the size of the gold nanocage. At the same time, the localized surface plasmon resonance (LSPR) peak of the gold nanocage is measured to further monitor and determine the relevant properties of the gold nanocage; after washing, the precipitate is dispersed in water; the volume ratio of the polyvinyl pyrrolidone solution to the silver nanocube is 80-150:1, and the amount of chloroauric acid added is such that the plasma resonance (LSPR) peak reaches 654nm to stop the reaction.

[0017] Furthermore, in step (2), a polyvinyl pyrrolidone aqueous solution is added to the container, and then heated to 100°C in a water bath environment and kept boiling for 10 minutes. Thereafter, the synthesized silver nanocubes are added for 10 minutes. Then, a chloroauric acid aqueous solution is added, during which the reaction time and the amount of chloroauric acid aqueous solution added are precisely controlled to achieve effective regulation of the size of the gold nanocage. After centrifugal washing, the precipitate is dispersed in water; the volume ratio of the polyvinyl pyrrolidone aqueous solution, the chloroauric acid aqueous solution, and the silver nanocubes is 100:15:1.

[0018] Wherein, in step (3), the gold nanocage solution is stirred in an ice bath in the dark, polyethylene glycol is added and the reaction is continued in an ice bath in the dark for 10-15 hours; the volume ratio of the gold nanocage solution to ethylene glycol is 25-75:1.

[0019] Preferably, in step (3), 10 mL of Auages ​​is added to the flask, stirred at 250 rpm in an ice bath in the dark, and 200 μL of PEG aqueous solution is added to react for 12 h; the volume ratio of the gold nanocage solution (Aucages) to PEG is 50:1.

[0020] Wherein, in step (4), a chloroauric acid solution and a bovine serum albumin solution are mixed, and an alkali reagent is added to react to obtain a gold nanocluster solution; the volume ratio of the chloroauric acid, bovine serum albumin and the alkali reagent is 5-15:5-15:1.

[0021] Preferably, in step (4), 10 mM chloroauric acid solution and 50 mg / mL BSA solution are mixed, and 1 mol / L NaOH solution is added to react to obtain a gold nanocluster (Au NCs) solution; the volume ratio of chloroauric acid: BSA: NaOH is 5-15:5-15:1.

[0022] Furthermore, in step (4), 10 mM chloroauric acid solution and 50 mg / mL BSA solution are mixed, and 1 mol / L NaOH solution is added to react to obtain a gold nanocluster (Au NCs) solution; the volume ratio of chloroauric acid: BSA: NaOH is 10:10:1.

[0023] In step (5), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC solution is added to the gold nanocluster solution, stirred in an ice bath for 10-20 minutes, then N-hydroxysuccinimide NHS solution is added and stirred at room temperature for 10-20 minutes, and then the polyethylene glycol-modified gold nanocage solution is added and reacted at room temperature for 3-4 hours, wherein the volume ratio of the gold nanocluster solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and polyethylene glycol-modified gold nanocage solution is 3-6:1:1:3-6.

[0024] Preferably, in step (5), the Au NCs solution is added to a round-bottom flask under an ice bath, followed by the addition of an EDC solution (5-10 mM), stirred for 10 min, and then the addition of an NHS solution (5-10 mM) and stirred at room temperature for 20 min, and then the previously prepared Auages@PEG is added. The AuNCs solution, EDC, NHS, Auages@PEG 5K The volume ratio of the solution is 3-6:1:1:3-6.

[0025] Preferably, the Au NCs solution in step (5) is added to a round-bottom flask under an ice bath, followed by the addition of EDC solution (5-10 mM), stirring for 10 min, and then adding NHS solution (5-10 mM) and stirring at room temperature for 20 min, and then adding the previously prepared AuCages@PEG 5K The AuNCs solution, EDC, NHS, Auages@PEG 5K The volume ratio of the solution is 5:1:1:5.

[0026] The plasmon-enhanced fluorescence sensor based on gold nanocages is prepared by the method for preparing the plasmon-enhanced fluorescence sensor based on gold nanocages of the present invention.

[0027] The gold nanocage-based plasma enhanced fluorescence sensor has a hollow gold nanocage with unique surface plasma properties. When the fluorescent substance maintains an appropriate distance from the gold nanocage, the gold nanocage can enhance the fluorescence of the fluorescent dye.

[0028] Application of the gold nanocage-based plasmon-enhanced fluorescence sensor of the present invention in the preparation of reagents or tools for norepinephrine detection.

[0029] Furthermore, the gold nanocage-based plasmon-enhanced fluorescence sensor of the present invention is used in the preparation of reagents or tools for detecting norepinephrine bitartrate.

[0030] The invention provides an application of the gold nanocage-based plasmon-enhanced fluorescence sensor in the preparation of reagents or tools for realizing fluorescence imaging of norepinephrine in living cells.

[0031] Furthermore, the gold nanocage-based plasmon-enhanced fluorescence sensor can be used as a tool for detecting norepinephrine in living PC-12 cells.

[0032] The present invention uses silver nanocubes (Agcubes) as templates and prepares gold nanocages (Aucages) with hollow structures through electric dipole displacement reaction. The system of the present invention proposes the preparation of gold nanocage solutions for the first time. Gold nanocages have unique surface plasma characteristics. When the fluorescent substance maintains an appropriate distance from the gold nanocage, the gold nanocage can enhance the fluorescence of the fluorescent dye. Using polyethylene glycol to control the distance between the gold nanocage and the fluorescent substance can improve its stability and enhance its biocompatibility.

[0033] The gold nanoclusters (Au NCs) selected in the present invention have deep tissue penetration characteristics, low background fluorescence interference and minimal light damage, and are expected to achieve in vivo imaging of norepinephrine. The present invention is the first to develop a plasmon-enhanced fluorescence sensor based on gold nanocages and use it for accurate detection of norepinephrine bitartrate and fluorescence imaging of living cells. It not only has deep tissue penetration characteristics, low background fluorescence interference and minimal light damage, but also solves the technical problems of low photoluminescence intensity and poor biocompatibility of existing near-infrared fluorescent dyes.

[0034] A plasma-enhanced fluorescence sensor based on gold nanocages (Aucages) prepared by the present invention can be used for accurate detection of norepinephrine bitartrate and fluorescence imaging of living cells. The plasma-enhanced fluorescence sensor uses the plasma enhancement effect of Auages ​​to effectively enhance the fluorescence intensity of Au NCs and amplify the fluorescence signal, thereby effectively improving the sensitivity of detection. Secondly, the use of Au NCs can avoid the self-interference of the organism, thereby improving the accuracy of recognition. In addition, the plasma-enhanced fluorescence sensor has high selectivity, sensitivity and accuracy for NE analysis, and the detection limit is as low as 0.47μM. The method has a detection recovery rate of NE in actual samples of 98.25% to 102.05%, and can also detect norepinephrine in living cells PC-12.

[0035] The preparation process of the fluorescent sensor of the present invention is simple and easy to carry out, and is easy to mass produce. The preparation method of the plasma enhanced fluorescent sensor of the present invention is: using silver nanocubes and HAuCl 4 Through the galvanic replacement reaction between the two groups, a gold nanocage with a porous wall and a hollow nanostructure was prepared. The thiol- and amino-functionalized polyethylene glycol was connected to the gold nanocage and Au NCs through gold-sulfur bonds and amide bonds, respectively, to control the distance between the gold nanocage and Au NCs to achieve fluorescence enhancement. Due to the electron transfer effect, Au NCs specifically recognize norepinephrine. As the concentration of norepinephrine increases, the fluorescence of the probe will gradually increase, forming an "on-off" response with a norepinephrine concentration-dependent fluorescence signal. Further, the monitoring of norepinephrine levels in living PC-12 cells is achieved.

[0036] The present invention achieves significant enhancement of the fluorescence signal through the synergistic effect of the localized surface plasmon resonance (LSPR) effect of gold nanocages (Au NCs) and the fluorescence characteristics of gold nanoclusters (Au NCs), and triggers the "on-off" response of the fluorescence signal through the electron transfer effect, thereby achieving specific recognition of norepinephrine. Specifically: 1. LSPR-coupled fluorescence enhancement mechanism: The porous hollow structure of the gold nanocage can produce a strong electromagnetic field enhancement effect. When Au NCs maintain an appropriate distance from its surface, its excited state energy is transferred to the plasma mode of Auages ​​through non-radiative dipole-dipole interaction, thereby significantly improving the fluorescence quantum yield (from 18.26% to 35.64%). 2. PEG-mediated distance regulation: The distance between the gold nanocage and AuNCs is controlled by the molecular chain length of thiol-amino bifunctionalized polyethylene glycol (PEG), which not only avoids fluorescence quenching caused by too close distance (such as direct contact between unmodified AuCages and AuNCs), but also ensures the effective enhancement of fluorescence by the plasma field. 3. Specific recognition mechanism: Through the electron transfer effect, the "on-off" response of the fluorescence signal is triggered to specifically detect norepinephrine.

[0037] The LSPR effect of Auages ​​in the sensor prepared by the present invention is combined with the fluorescence characteristics of AuNCs to have a significant synergistic effect, achieving higher sensitivity than AuNC alone (detection limit 0.47μM). The molecular weight of PEG (such as 5k Da) optimizes the spacing, avoids energy transfer quenching, and improves biocompatibility (cell survival rate>95%), achieving precise regulation. At the same time, the selectivity of PEF sensors for NE is significantly better than that of other catecholamines (such as adrenaline and dopamine), and the specificity is improved. Compared with other nanostructures used for plasmon applications, such as gold nanoparticles, silver nanoparticles and silver nanocubes, the structure prepared by the present invention shows unique advantages by precisely controlling the molar ratio of reactants, so that its different localized surface plasmon resonance (LSPR) peaks can be more conveniently located in the near-infrared (NIR) spectral region. On the other hand, compared with the silica shell prepared by the traditional sol-gel method, the strategy of using polyethylene glycol (PEG) as a spacer is not only easy to control, but also can accurately obtain the required spatial length by flexibly adjusting the molecular weight of PEG. In addition, the shell material prepared by this method showed excellent biocompatibility, and the cell viability was maintained above 90%, further highlighting its potential in the field of biological applications.

[0038] The porous hollow structure of the gold nanocage of the present invention can produce a strong electromagnetic field enhancement effect. By precisely controlling the molar ratio of the reactants, it is easier to make different localized surface plasmon resonance (LSPR) peaks of the gold nanocages (Aucages) fall into the near infrared (NIR) range.

[0039] The present invention controls the distance between the LSPR peak and the plasma substrate and the fluorophore by controlling the variable method. Using Auages ​​with different LSPR wavelengths (536nm / 654nm / 754nm), the fluorescence is enhanced by 3.5 times only at 654nm, which has the greatest fluorescence enhancement, proving the criticality of matching the LSPR with the AuNCs emission peak. At the same time, PEG molecular weight screening is performed, and the fluorescence intensity of 1000 / 5000 / 10000Da is compared. The enhancement effect is optimal at 5000Da, verifying the necessity of spacing regulation.

[0040] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0041] 1. The plasma enhanced fluorescence sensor prepared by the present invention uses near-infrared fluorescent gold nanoclusters as the fluorescence source, which has the advantages of deep tissue penetration, low background fluorescence interference and low light damage.

[0042] 2. The plasma enhanced fluorescence sensor prepared by the present invention uses gold nanocages as plasmons. Gold nanocages are a new and promising type of hollow nanostructures with porous walls. They have multi-pointed structures and hollow structures, which give them strong electric field enhancement characteristics and more significant fluorescence enhancement effects. At the same time, the preparation of gold nanocages shows unique advantages by precisely controlling the molar ratio of reactants, so that its different localized surface plasmon resonance (LSPR) peaks can be more conveniently located in the near-infrared (NIR) spectral region.

[0043] 3. The plasma enhanced fluorescence sensor prepared by the present invention uses polyethylene glycol (PEG) to control the distance between the plasma unit and the fluorescent substance. It is not only easy to control, but also the distance can be accurately controlled by flexibly adjusting the PEG molecular weight to achieve optimal fluorescence enhancement. At the same time, the modification of polyethylene glycol improves the biocompatibility and stability of the sensor.

[0044] 4. The plasma enhanced fluorescence sensor prepared by the present invention has high selectivity, sensitivity and accuracy for norepinephrine (NE) analysis, with a detection limit as low as 0.47 μM. The detection recovery rate of NE in actual samples by this method is 98.06% to 105.34%. At the same time, these findings pave the way for the development of PEF fluorescence sensors for various biomolecules.

[0045] 5. The plasma enhanced fluorescence sensor prepared by the present invention has high cell activity between 0-10 μM, indicating good biocompatibility and can realize fluorescence imaging of norepinephrine in living cells, which is of great significance for the diagnosis of neurodegenerative diseases.

[0046] 6. The preparation process of the plasma enhanced fluorescence sensor of the present invention is simple and easy, and the prepared sensor has good stability. At the same time, the preparation process is simple and easy, and it is easy to mass produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Auages ​​and Auages@PEG prepared by the present invention 5K Transmission electron microscopy image.

[0048] Figure 2 Overlapping graph of fluorescence emission peaks of AuCages and Au NCs with different plasma resonance wavelengths prepared by the present invention

[0049] Figure 3 The fluorescence emission spectra of the plasmon-enhanced fluorescence sensor prepared in the present invention using Auages ​​with different plasmon resonance wavelengths as the plasmon substrate (A) and modified with polyethylene glycol of different molecular weights (B).

[0050] Figure 4 Auages ​​(a) and Auages@PEG prepared in the present invention 5K (b), AuNCs(c), AuCages@PEG 5K -DLS of AuNCs (d);

[0051] Figure 5 Au NCs solution (I) and ion-enhanced fluorescence sensor Auages@PEG prepared by the present invention 5K -Fluorescence lifetime diagram of AuNCs(Ⅱ);

[0052] Figure 6 3D-FDTD simulation of the gold nanocages (Aucages) prepared by the present invention;

[0053] Figure 7 (A) Changes in fluorescence intensity of the plasma enhanced fluorescence sensor prepared by the present invention over time. (B) FL intensity of the plasma enhanced fluorescence sensor at different temperatures (15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C). (C) Changes in fluorescence intensity of the plasma enhanced fluorescence sensor with pH. (D) Changes in fluorescence intensity of the plasma enhanced fluorescence sensor after incubation in NE solution;

[0054] Figure 8(A) Fluorescence spectra of the plasmon-enhanced fluorescence sensor prepared in the present invention at different concentrations of NE (0μM, 1μM, 10μM, 20μM, 40μM, 60μM, 80μM, 100μM). (B) Fluorescence spectra of AuNCs at different concentrations of NE (0μM, 1μM, 10μM, 20μM, 40μM, 60μM, 80μM, 100μM). (C) AuCages@PEG 5K -Linear fitting of the FL intensity of AuNCs and NE concentration. (D) Linear fitting of the fluorescence intensity of AuNCs and NE concentration;

[0055] Fig. 9 (A) Fluorescence response of the plasmon-enhanced fluorescence sensor prepared in the present invention after incubation with NE (40 μM), epinephrine (40 μM), dopamine (40 μM) and catechol (40 μM). (B) Fluorescence response of the plasmon-enhanced fluorescence sensor prepared in the present invention after incubation with NE (40 μM), epinephrine (40 μM), dopamine (40 μM) and catechol (40 μM). NE =50 μM), the plasmon enhanced fluorescence sensor and other substances that may coexist (C coexistence =500 μM) after incubation;

[0056] Fig.10 Auages ​​(a) and Auages@PEG prepared in the present invention 5K (b), Au NCs(c), AuCages@PEG 5K -FT-IR spectrum of Au NCs (d);

[0057] Fig.11 Auages ​​(a) and Auages@PEG prepared in the present invention 5K (b), Au NCs(c), AuCages@PEG 5K -Zeta potential of Au NCs (d);

[0058] Fig.12 The ion-enhanced fluorescence sensor Auages@PEG prepared by the present invention 5K -Comparison of AuNCs and HPLC for the detection of norepinephrine bitartrate injection

[0059] Fig.13 The ion-enhanced fluorescence sensor Auages@PEG of different concentrations prepared by the present invention 5K - Cytotoxicity of Au NCs in PC-12 cells;

[0060] Fig.14 The ion-enhanced fluorescence sensor Auages@PEG prepared by the present invention 5K-Laser confocal fluorescence imaging of Au NCs in PC-12 cells.

[0061] Fig.15 The ion-enhanced fluorescence sensor Auages@PEG prepared by the present invention 5K -Comparison of fluorescence intensity of Au NCs and AuNCs in PC-12 cells. DETAILED DESCRIPTION

[0062] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0063] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0064] L-Norepinephrine (NE) and rhodamine 6G (R6G) were purchased from Shanghai Aladdin Reagent Co., Ltd., China. Epinephrine, dopamine, uric acid, citric acid, glycine, L-cysteine, vitamin C, vitamin B6, ethylene glycol, sodium sulfide (Na 2 S·9H 2 O), silver nitrate (AgNO 3 ), 1-ethyl-3-[3-dimethylaminopropylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), ferrous chloride (FeCl 2 ), zinc chloride (ZnCl 2 ), magnesium chloride (MgCl 2 ) and copper chloride (CuCl 2 ) were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. Sodium chloride (NaCl) and potassium chloride (KCl) were purchased from China National Pharmaceutical Chemical Reagent Co., Ltd. Anhydrous calcium chloride (CaCl 2 ) was purchased from Shanghai Hushi Co., Ltd., China. Polyethylene glycol (NH 2 -PEG-SH, M W 1000, 5000, 10000Da) were purchased from Guangzhou Tianke Co., Ltd., China. Polyvinylpyrrolidone (PVP, M W 40000) was purchased from Shanghai Aladdin Reagent Co., Ltd., China. Phosphate buffered saline (PBS, pH 7.4), DMEM medium, fetal bovine serum (FBS) and Cell Counting Kit-8 (CCK-8) were purchased from Jiangsu KeyGen Biotech Co., Ltd.

[0065] Example 1

[0066] 1. Preparation of Silver Nanocubes (Agcubes)

[0067] 30 mL of ethylene glycol was added to a 250 mL three-necked round-bottom flask, and the mixture was stirred at 250 rpm under magnetic stirring. The mixture was heated to 150 °C in an oil bath and the temperature was maintained until the reaction was completed. Nitrogen was introduced to remove the water in the ethylene glycol. After heating for 1 h, 360 μL of freshly prepared sodium sulfide solution (Na 2 S·9H 2 O, 3mM,), after reacting for 2 minutes, 9mL of polyvinyl pyrrolidone (PVP) solution (20mg / μL) dissolved in ethylene glycol was added, and after reacting for 8-9 minutes, 3mL of silver nitrate (AgNO 3 ) solution (282 mM) was added and reacted 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 all the precipitates were dispersed in 24 mL of ultrapure water to obtain a silver nanocube (Agcubes) solution.

[0068] 2. Synthesis of Auages

[0069] Add 100 mL of polyvinyl pyrrolidone (PVP) (1.5 mg dissolved in 1 mL of water) aqueous solution to the flask, heat to 100 ° C until light boiling for 10 min, add 1 mL of silver nanocubes (Agcubes) prepared in step (1) and heat for 10 min, then slowly add chloroauric acid aqueous solution (0.2 mM) with a syringe and observe the color change, determine its peak position by LSPR spectrum, stop heating and terminate the reaction when the plasma resonance peak is 654 nm, and add about 17 mL of chloroauric acid aqueous solution. Then use saturated sodium chloride solution to dissolve and remove the AgCl impurities generated in the reaction, wash and centrifuge twice, and adjust the volume to 50 mL to obtain a gold nanocage solution (Aucages654).

[0070] If you want to synthesize a gold nanocage solution (Aucages 536) with a plasma resonance wavelength of 536 nm, just replace the amount of chloroauric acid added in step (2) with 13 mL.

[0071] If you want to synthesize a gold nanocage solution (Aucages 754) with a plasma resonance wavelength of 754 nm, just replace the amount of chloroauric acid added in step (2) with 21 mL.

[0072] 3. Synthesis of Polyethylene Glycol-Modified Gold Nanocages (Aucages@PEG)

[0073] Add 10 mL of Aucalis prepared in step 2 to the flask, stir at 250 rpm in an ice bath away from light, and add 200 μL (4 μM) PEG 5k The reaction was continued overnight.

[0074] If you want to synthesize polyethylene glycol with a molecular weight of 1K to modify gold nanocages PEG 1k (Aucages@PEG 1k ) Simply replace the PEG in step (3) 5k Replacement with PEG 1k , repeat steps (3)(4)(5). (Aucages@PEG 1k )

[0075] If you want to synthesize polyethylene glycol with a molecular weight of 10K to modify gold nanocages PEG 1k (Aucages@PEG 1k ) Simply replace the PEG in step (3) 5k Replacement with PEG 10k , repeat steps (3)(4)(5). (Aucages@PEG 10k )

[0076] 4. Synthesis of gold clusters (Au NCs)

[0077] Add 5 mL of 10 mM chloroauric acid aqueous solution to 5 mL of 50 mg mL -1 After stirring the BSA aqueous solution for 2 min, 0.5 mL of 1 mol L -1 The mixture was stirred at 37 °C for 12 h.

[0078] 5. Synthesis of Plasmon Enhanced Fluorescence Sensor (Aucages@PEG-AuNCs)

[0079] 5 mL of Au NCs was added to a round-bottom flask under an ice bath, followed by 0.5 mL (10 mM) of EDC solution, stirred for 10 min, and then 0.5 mL (10 mM) of NHS solution was added and stirred at room temperature for 20 min. Then 5 mL of Aucage@PEG prepared in step (3) was added and reacted at room temperature for 4 h to obtain a plasmon enhanced fluorescence sensor (PEF sensor, Auages@PEG-AuNCs).

[0080] Meanwhile, in step (5), a gold nanocage solution with a plasmon resonance wavelength of 536 nm, a gold nanocage solution with a plasmon resonance wavelength of 654 nm, or a gold nanocage solution with a plasmon resonance wavelength of 754 nm is used to obtain different plasmon-enhanced near-infrared fluorescence sensors (Aucages536@PEG 5k-Au NCs), (Aucages654@PEG 5k -Au NCs) and (Aucages754@PEG 5k -Au NCs). Meanwhile, in step (5), a gold nanocage solution with a plasma resonance wavelength of 654 nm and a polyethylene glycol with a molecular weight of 1K are used to modify the gold nanocage PEG 1k , polyethylene glycol molecular weight 5K modified gold nanocage PEG 5k Or polyethylene glycol molecular weight 10K modified gold nanocage PEG 10k The Au nanocage solution was used to obtain different plasmon-enhanced near-infrared fluorescence sensors (Aucages654@PEG 1k -Au NCs), (Aucages654@PEG 5k -AuNCs) and (Aucages654@PEG 10k -Au NCs). The following examples used this condition to prepare the ion-enhanced fluorescence sensor Auages654@PEG 5K -AuNCs were tested in subsequent examples.

[0081] Example 2

[0082] Example 1: Transmission electron microscopy images of Aucalis and Agcubes@PEG

[0083] Use tweezers to pick up two copper meshes and place them on Auages654 and Auages654@PEG respectively. 5K After soaking in the solution, take it out and put it into a disposable culture dish lined with filter paper. After the copper mesh is dried, put it into the sample injector of the transmission electron microscope. The transmission electron microscope image (see Figure 1 ). Figure 1 The middle a shows that Aucalis 654 has a hollow cubic structure with a side length of about 64.28 nm. Figure 1 Figure b shows that there is PEG on the surface of Auages654, and the thickness is about 5.3nm. This indicates that Auages654@PEG 5K Successfully synthesized.

[0084] Example 3

[0085] Example 1 Overlapping graph of the ultraviolet absorption peak of Auages ​​and the fluorescence emission peak of AuNCs

[0086] Take 2 mL of Auages ​​solution for UV detection, and take 2 mL of Au NCs solution for fluorescence detection.

[0087] UV-Visible spectrum test: Use pure water as the reference solution and scan the spectrum within the wavelength range of 300nm-700nm.

[0088] Fluorescence spectrum test: Take the above solutions and measure the fluorescence emission spectrum. The fluorescence emission spectrum is determined with 430nm excitation, 5nm / 5nm slit width for excitation and emission, and 700V voltage.

[0089] Figure 2 The UV absorption peaks of Auages ​​with different LSPR peaks (536nm, 654nm, and 754nm) are shown. Figure 2 Curves Ⅰ, Ⅱ, Ⅲ) and the fluorescence excitation and emission peaks of Au NCs ( Figure 2 Curves IV and V) are combined and compared to obtain the spectrum overlap diagram (see Figure 2 ), Figure 2 The results show that the emission spectra of Auages ​​with LSPR peak at 654 nm overlap the most with those of Au NCs, indicating that in theory, Auages ​​at 654 nm can achieve the maximum enhancement of the fluorescence intensity of AuNCs.

[0090] Example 3

[0091] Fluorescence emission spectrum of Auages@PEG-Au NCs solution prepared in Example 1.

[0092] Auages@PEG-Au NCs were synthesized according to the method of step (5) of Example 1, and plasmon-enhanced fluorescence sensors were prepared using Auages ​​with different LSPR peaks (536 nm, 654 nm, and 754 nm) as plasmons. At the same time, plasmon-enhanced fluorescence sensors modified with gold nanocages of polyethylene glycol with different molecular weights were prepared, and the distance between the gold nanocages and Au NCs was controlled by three different molecular weights of PEG.

[0093] Fluorescence emission spectra of Aucages@PEG-AuNCs.

[0094] Fluorescence spectrum test: Take the above Auages@PEG-AuNCs solution and measure the fluorescence emission spectrum. The fluorescence emission spectrum was excited at 430nm, the slit width of excitation and emission was 5nm / 5nm, and the voltage was 700V. The obtained fluorescence emission spectrum (see Figure 3 ), the experimental results show that the selection of Aucages 654 ( Figure 3 A curve III) as the plasma substrate to obtain the maximum fluorescence enhancement (3.5 times), while Aucalis524 ( Figure 3 A curve II) and Aucalis 754 ( Figure 3Curve A IV) as a plasma substrate shows a fluorescence quenching effect because the electric field enhanced fluorescence effect generated by Auages ​​with non-overlapping spectra cannot effectively resist the close-range resonance energy transfer between AuNCs and Auages, so it shows fluorescence quenching; at the same time, based on Auages ​​654, the plasma enhanced fluorescence sensors modified with polyethylene glycol of different molecular weights have different fluorescence enhancement effects, among which the plasma enhanced fluorescence sensor with a polyethylene glycol molecular weight of 5000 has the best fluorescence enhancement effect ( Figure 3 B curve III).

[0095] In summary, when the plasmon resonance wavelength of the gold nanocage is 654 nm and the molecular weight of polyethylene glycol is 5000, the prepared plasmon-enhanced fluorescence sensor has the best fluorescence enhancement effect. Therefore, the following examples are all prepared under this condition. Plasmon-enhanced fluorescence sensor Auages654@PEG 5K -AuNCs were tested in subsequent examples.

[0096] Example 4

[0097] The Auages654 prepared in Example 1 and Auages654@PEG with different molecular weights, such as 1000, 5000, and 10000, were added to 1K ,Aucages654@PEG 5K ,Aucages654@PEG 10K Perform dynamic light scattering (DLS) testing.

[0098] Further exploration of the findings by DLS (see Figure 4 ), Auages654 solution Auages654@PEG 1K ,Aucages654@PEG 5K ,Aucages654@PEG 10K It dispersed well in aqueous solution, and the DLS diameters were 67.25nm, 69.12nm, 75.23nm, and 90.31nm, respectively. The DLS diameter increased with the increase of molecular weight. The DLS results showed that PEG with different molecular weights was successfully grafted onto the surface of Auages654.

[0099] Example 5

[0100] The AuNCs solution prepared in Example 1 and Auages654@PEG 5K -AuNC solution for quantum yield calculation and fluorescence lifetime test

[0101] Rhodamine 6G (with quantum yield, ) is used as a standard substance for quantum yield measurement. Rhodamine 6G was dissolved in ethanol solution, and Au NCs solution and Auages654@PEG 5K -AuNC was dispersed in aqueous solution and then diluted to different concentrations (5μM, 4μM, 3μM, 2μM and 1μM). The maximum absorbance of each solution was measured, and the corresponding fluorescence intensity at the maximum emission wavelength was recorded by full spectrum scanning. Finally, the fluorescence quantum yield can be calculated using the following formula:

[0102]

[0103] in, is the quantum yield of the standard substance, To test the quantum yield of a substance, A S and A X are the maximum absorbance of the standard substance and the test substance, respectively, S and F X are the fluorescence intensities of the standard substance and the test substance at specific wavelengths, n X and n X are the refractive indices of the solvents for the standard substance and the test substance, respectively.

[0104] Au NCs solution and Auages654@PEG were tested using transient / steady-state fluorescence spectroscopy 5K -The fluorescence lifetime of AuNCs solution. The description formula of the double exponential model used in the fluorescence lifetime test is as follows:

[0105]

[0106] Among them, τ 1 and τ 2 Represents the time constant. α 1 and α 2 Represents the amplitude of the fast and slow components. The average lifespan of τ is calculated as follows:

[0107]

[0108] Figure 5 The results showed that compared with the fluorescence intensity of Au NCs, AuCages654@PEG 5K -Au NCs fluorescence lifetime was shortened from 1.19μs to 1.10μs. At the same time, when rhodamine 6G (Φ = 0.95) was used as the standard fluorophore, Auages654@PEG 5K-The fluorescence quantum yield of AuNCs increased from 18.26% of Au NCs to 35.64%. These results indicate that the coupling of plasmon AuCages with Au NCs affects the decay path of Au NCs and enhances the radiative and non-radiative decay rates of quantum dots, while the increase in quantum yield demonstrates the plasmon-enhanced fluorescence effect.

[0109] Example 6 Simulating the electric field distribution of gold nanocages using finite difference time domain (FDTD) simulation software

[0110] A gold nanocage model with a side length of 60 nm was established in 3D Max; the resulting electric field simulation diagram is shown in Figure 6 Auages ​​have a large light absorption area and better optical properties than ordinary gold nanoparticles. FDTD simulation results show that strong electromagnetic field enhancement of localized surface plasmon effect occurs inside and outside Auages, and coupling occurs between the internal and external electromagnetic fields, which may generate stronger electric fields to cause enhancement of fluorescence emission signals, confirming that Auages ​​are an outstanding candidate material for plasmon-enhanced fluorescence sensors.

[0111] Example 7

[0112] The plasmon-enhanced fluorescence sensor (Aucages654@PEG 5K -Au NCs) stability study diagram

[0113] Take the plasma fluorescence enhanced fluorescence sensor Auages654@PEG prepared in Example 1 5K -Au NCs, placed at room temperature for a period of time (0-120 h), and fluorescence detection was performed. Figure 7 A shows that the plasmon-enhanced fluorescence sensor remains stable within 120 h when stored at room temperature. 5K -Au NCs were incubated at different temperatures for 10 min (T = 15°C-50°C) and fluorescence detection was performed. Figure 7 B shows that the sensor exhibits excellent stability at different temperatures. Take the plasma fluorescence enhanced fluorescence sensor Auages654@PEG prepared in Example 1 5K -Au NCs, incubated at different pH for 10 min (pH = 3-11), and fluorescence detection was performed. The results showed that under strong acid and strong base conditions, the fluorescence intensity of the plasmon enhanced fluorescence sensor decreased ( Figure 7C). The fluorescence intensity of the plasma enhanced fluorescence sensor is the highest at pH 7, so the pH 7 buffer solution is selected as the optimal detection pH. 5K -Au NCs were mixed with norepinephrine in a volume ratio of 1:1 and incubated at room temperature for 0-120 min for fluorescence detection ( Figure 7 D), the results show that after incubation at room temperature for 20 min, the fluorescence of the sensor tends to be stable, indicating that norepinephrine occupies all the binding sites of Au NCs at this time, so 20 min was selected as the incubation time.

[0114] Example 8

[0115] Fluorescence emission graph and line graph of the plasmon enhanced fluorescence sensor (PEF) prepared in Example 1 after incubation with different concentrations of norepinephrine

[0116] Au NCs were prepared according to the method of Example 1 as a comparative reference. To further verify the fluorescence enhancement effect of the plasmon-enhanced fluorescence sensor, the blank control AuNCs were incubated with different concentrations of NE (0 μM, 1 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM) at a volume ratio of 1:1 at room temperature for 15 min, and the above solutions were taken to measure the fluorescence emission spectra ( Figure 8 A). The fluorescence emission spectrum was determined with an excitation wavelength of 430 nm, a slit width of 5 nm / 5 nm for excitation and emission, and a voltage of 700 V. As the concentration of NE increased, the fluorescence of AuNCs increased, and a relationship between FL intensity and concentration was established. The fluorescence intensity of AuNCs had a good linear relationship with the concentration of NE (R 2 =0.9983) in the concentration range of 0-60 μM, the corresponding linear equation is y=0.00615x+0.0270 (y represents F 0 / F-1; x represents the concentration of NE (μM), F 0 represents the fluorescence intensity of Au NCs in the absence of NE, and F represents the fluorescence intensity of Au NCs when the sensor is incubated with NE). The detection limit was calculated to be 1.28 μM by dividing the 3-fold standard deviation (12 times) of the blank detection by the slope of the linear curve ( Figure 8 B) Plasmon-enhanced fluorescence sensor Auages654@PEG 5K -Au NCs and different concentrations of NE (0μM, 1μM, 10μM, 20μM, 40μM, 60μM, 80μM, 100μM) were incubated at room temperature for 15 min at a volume ratio of 1:1. The above solutions were taken to measure the fluorescence emission spectra ( Figure 8C). Fluorescence emission spectrum was measured with excitation at 430 nm, slit widths of excitation and emission of 5 nm / 5 nm, and voltage of 700 V. The linear equation of fluorescence emission spectrum fitting between fluorescence intensity of plasma enhanced fluorescence sensor and NE concentration was y=0.0135x+0.0109, R 2 =0.9956, the linear range is 1-60μM, and the detection limit is 0.47μM. Compared with Au NCs, the plasmon-enhanced fluorescence sensor has a lower detection limit ( Figure 8 D) (calculated according to 3σ / k, σ is the relative standard deviation of the blank sample (12 times, k is the slope of the linear equation determined experimentally) increased by 2.74 times, proving that the plasma enhanced fluorescence sensor prepared by the present invention has higher sensitivity.

[0117] Example 9

[0118] The plasma enhanced fluorescence sensor (PEF) prepared in Example 1 and the interference study were configured with 50 μM norepinephrine, 50 μM concentration of norepinephrine structural analogs such as epinephrine, dopamine, and catecholamines. In addition, norepinephrine and other interfering substances K, Na + Mg 2+ , Fe 2+ 、Zn 2+ , Cu 2+ , Ca 2+ , uric acid, vitamin B6, citric acid, glycine, L-cysteine, vitamin C, and tartaric acid were mixed in pairs, and the concentration of norepinephrine in the mixed solution was 50 μM, and the interfering substances were all 500 μM. The gold nanocage with a plasma resonance wavelength of 654 nm and the polyethylene glycol-modified plasma enhanced fluorescence sensor with a molecular weight of 5000 prepared in Example 1 were incubated with norepinephrine, structurally similar substances, and the mixed solutions of the two in pairs at a volume ratio of 1:1 at room temperature for 15 minutes to test the fluorescence emission spectrum. The obtained fluorescence response is shown in Fig. 9 The results showed that the plasmon-enhanced fluorescence sensor selectively exhibited fluorescence enhancement after incubation with norepinephrine, and the structural analogs of norepinephrine did not significantly affect the plasmon-enhanced fluorescence sensor ( Fig. 9 A), in addition, in the presence of 10 times the interference, the fluorescence sensor can still achieve a good fluorescence response without being interfered by the interference ( Fig. 9 B). It shows that the plasma enhanced fluorescence sensor prepared by the present invention has good specificity. Therefore, the developed sensor has good application prospects in detecting NE in raw materials and drug formulations.

[0119] Example 10

[0120] The Auages654 solution and Auages654@PEG prepared in Example 1 were 5K Solution, Au NC solution, Auages654@PEG 5K -Infrared spectrum of the powder after freeze-drying of Au NCs solution.

[0121] Fourier transform infrared spectroscopy (FT-IR) was also used to demonstrate the successful preparation of the probe ( Fig.10 ). Compared with Auages654 (curve a), Auages654@PEG 5K (Curve b) peak at 2933cm -1 (CH asymmetric stretching vibration), 2873 cm -1 (CH asymmetric stretching vibration) and 1542cm -1 (CH bending vibration) increased significantly, indicating that PEG 5K Successfully coated on the surface of AuCages654. The peak of Au NCs (curve c) is at 1660 cm -1 , 1542cm -1 , 1402cm -1 The characteristic peak of amide bond indicates the successful synthesis of surface gold clusters. 5K -Au NCs (curve d) peak at 1660 cm -1 (Amide I band), 1542 cm -1 (C=O deformation vibration) 1402cm -1 The increase in (NH stretching vibration) indicates that the carboxyl groups on the surface of the gold clusters were successfully grafted onto the PEG through amide bonds. 5K on the amino group.

[0122] Embodiment 11

[0123] The Auages654 solution, Auages654@PEG solution, AuNC solution, and Auages654@PEG solution prepared in Example 1 were 5K -Au NCs solution was used to detect the zeta potential.

[0124] The zeta potential of the plasmon-enhanced fluorescence sensor was studied to determine its composition ( Fig.11 ). The zeta potential of AuCages654(a) is -(8.35±0.91)mV. PEG 5K After modification to the Auages ​​surface, Auages654@PEG 5K(b) The charge is (20.18±0.49) mV, indicating that HS-PEG carries a positive charge 5K -NH 2 The Au NCs (c) itself has a charge of -(28.15±0.47) mV and is grafted to Auages654@PEG via an amide bond. 5K After that, Aucalis654@PEG 5K The charge of -Au NCs (d) was -(25.08±0.54) mV, indicating the successful grafting of AuNCs.

[0125] Example 12

[0126] The plasma enhanced fluorescence sensor prepared in Example 1 was spiked and recovered.

[0127] Plasmon-enhanced fluorescence sensor was used to detect the drug norepinephrine bitartrate injection, and a spike recovery experiment was performed. The fluorescence method developed in this experiment was applied to norepinephrine bitartrate injection (according to the method of Example 8), and the measured fluorescence intensity was brought into the standard curve (F / F 0 =1.0109+0.0135C) to calculate the detection concentration C. The detection concentration of this method is 2.06 mg / mL, which is consistent with the labeled amount of the drug 2 mg / mL. At the same time, the recovery rate of this method is 98.06-105.34%, and the RSD is less than 5%, indicating that the current PEF sensor can effectively test actual samples and produce satisfactory results.

[0128] Table 1 Determination of NE in actual samples by plasmon enhanced fluorescence sensor

[0129]

[0130] Example 13

[0131] Detection of norepinephrine bitartrate injection by high performance liquid chromatography

[0132] In order to further verify the accuracy of the plasma enhanced fluorescence sensor, NE was detected by high performance liquid chromatography. The liquid phase conditions were as follows: octadecylsilane bonded silica gel was used as the filler; 0.14% sodium heptane sulfonate solution-methanol (65:35) (pH adjusted to 3.0±0.1 with phosphoric acid) was used as the mobile phase; the detection wavelength was 280nm; and the injection volume was 20μL. The plasma enhanced fluorescence sensor of Example 12 was consistent with the HPLC detection results ( Fig.12 ). These results together demonstrate the applicability of the PEF-based method for sensitive and accurate detection of NE in real samples, and, compared with HPLC, it does not require expensive equipment and professional technicians, and has the convenience of operation and real-time detection.

[0133] Embodiment 14

[0134] The Au NCs solution prepared in Example 1 and the plasma fluorescence enhanced fluorescence sensor Auages654@PEG 5K - Cytotoxicity diagram of AuNCs, where the plasmon resonance wavelength of gold nanocages is 654 nm.

[0135] Take the Au NCs solution prepared in Example 1 and the plasma fluorescence enhanced fluorescence sensor Auages654@PEG 5K -Au NCs were diluted to different concentrations (0-10 μM) and added to PC-12 cells (8000 cells / well) pre-cultured for 24 h and incubated for 24 h. After washing twice with sterile PBS buffer, 10 μL CCK-8 reagent was added and incubated for 2 h. The absorbance at 450 nm was detected using a microplate reader.

[0136] The obtained cytotoxicity graph is shown in Fig.13 , Fig.13 The probe was shown to be almost nontoxic to PC-12 cells in the range of 0-10 μM, indicating its good biocompatibility.

[0137] Embodiment 15

[0138] The Au NCs solution prepared in Example 1 and the plasma fluorescence enhanced fluorescence sensor Auages654@PEG 5K -Intracellular laser confocal fluorescence imaging of AuNCs, where the plasmon resonance wavelength of the gold nanocage is 654nm.

[0139] Take the Au NCs solution prepared in Example 1 and the plasma fluorescence enhanced fluorescence sensor Auages654@PEG 5K -Au NCs were added to PC-12 cells (3000 cells / well) pre-cultured for 24 hours and incubated for 2 hours. After washing twice with sterile PBS buffer, they were detected using a Nikon AX laser confocal fluorescence microscope.

[0140] The laser confocal fluorescence imaging is shown in Fig.14 , Fig.14 The results showed that red fluorescence appeared on the surface of PC-12 cells treated with AuNCs solution and plasmon fluorescence enhanced fluorescence sensor, both of which can label NE in living cells.

[0141] Image J was used to perform fluorescence data statistics on the obtained confocal fluorescence images. Fig.15 , Fig.15The results showed that the fluorescence of the plasmon-enhanced fluorescence sensor on the cell surface was significantly enhanced by 3.12 times compared with that of PC-12 treated with Au NCs, which also indicated that AuCages had a plasmon fluorescence enhancement effect on AuNCs, making the detection sensitivity of NE in living cells higher. Therefore, the probe has a good fluorescence enhancement effect and can be successfully applied to cell imaging to improve detection efficiency.

[0142] Example 16

[0143] Plasma fluorescence enhanced fluorescence sensor Auages654@PEG of the present invention 5K -Au NCs (the plasmon resonance wavelength of gold nanocages is 654 nm) were compared with other existing detection methods for NE detection, and the structure is shown in Table 3.

[0144] Table 3 Comparison of various NE detection methods

[0145]

[0146] [1]Hollow dummy template imprinted boronate-modified polymers forextraction of norepinephrine,epinephrine and dopamine prior to quantitation by HPLC[J].Microchim.Acta 186(2019)686,https: / / doi.org / 10.1007 / s00604-019-3801-2.

[0147] [2]Bimetallic MOFs-based electrodes for the simultaneous electrochemical detection of epinephrine and norepinephrine[J].J.Electrochem.Soc.171(2024)097501–097501.https: / / doi.org / 10.1149 / 1945-7111 / ad6c80.

[0148] [3]A highly sensitive electrochemical sensor for simultaneousvoltammetric determination of noradrenaline,acetaminophen,xanthine andcaffeine based on aflavonoid nanostructured modified glassy carbon electrode[J].Sens.Actuators,B.192(2014)634–641.https: / / doi.org / 10.1016 / j.snb.2013.11.006.

[0149] [4]A colorimetric probe for the selective detection of norepinephrinebased on adouble molecular recognition with functionalized gold nanoparticles[J].ACS Appl.Nano Mater.2(2019)1367–1373.https: / / doi.org / 10.1021 / acsanm.8b02254.

[0150] [5]A wide-color-varying ratiometric nanoprobe for detection ofnorepinephrine in urine samples[J].Anal.Chim.Acta.1039(2018)124–131.https: / / doi.org / 10.1016 / j.aca.2018.07.043.

[0151] [6]Molecularly imprinted polymers on dual-color quantum dots forsimultaneous detection ofnorepinephrine and epinephrine[J].Sens.Actuators,B.229(2016)38–46.https: / / doi.org / 10.1016 / j.snb.2016.01.113.

[0152] [7]A lighting up NIR fluorescent sensing assay for norepinephrine and its application in the imaging of depressed mice brain[J].Sens.Actuators,B.417(2024)136182.https: / / doi.org / 10.1016 / j.snb.2024.136182.

[0153] The PEF sensor prepared by the present invention is compared with the reported detection method. Compared with the HPLC method, the PEF sensor is easy to operate, can achieve real-time detection, and does not require expensive equipment and professional technicians. Compared with the electrochemical method, the detection range of the PEF sensor is similar, but the service life of the electrochemical sensor may be affected by the wear of the sensing material and requires regular replacement and maintenance. Compared with the fluorescence analysis method using PNE as a probe (its maximum emission wavelength (E M ) is 527nm), the PEF sensor has a longer E M The maximum emission wavelength of QDs@MIPs (E M ) is the same as the PEF sensor; however, its stability is easily affected by the environment. The maximum emission wavelength (E M ) is in the near-infrared (NIR) range and has been applied to mouse brain research, but its limit of detection (LOD) is higher than that of the PEF probe. Therefore, the prepared PEF sensor has low cost and high sensitivity, and has great potential for clinical application.

Claims

1. A method for preparing a plasmon-enhanced fluorescence sensor based on gold nanocages, characterized in that: The steps include: (1) adding sodium sulfide, polyvinyl pyrrolidone, and silver nitrate into an ethylene glycol solution to prepare a silver nanocube solution; (2) reacting the silver nanocube solution and the chloroauric acid solution in a polyvinyl pyrrolidone solution to obtain a gold nanocage solution; (3) adding polyethylene glycol to the gold nanocage solution to react and obtain a polyethylene glycol-modified gold nanocage solution; (4) mixing the chloroauric acid solution and the bovine serum albumin solution, and adding an alkaline reagent to react to obtain a gold nanocluster solution; (5) Adding a gold nanocluster solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide to a polyethylene glycol-modified gold nanocage solution, the reaction yields a gold nanocage-based plasmon-enhanced fluorescence sensor.

2. The preparation method according to claim 1, characterized in that: 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 h, sodium sulfide is added, and after waiting for 2-20 min, polyvinyl pyrrolidone is added. After another 2-20 min, silver nitrate is added to the mixture, and the mixture is reacted for 2-20 min. After the reaction is completed, the mixture is immersed in an ice water bath to quench, and the precipitate is dispersed in water after washing; the volume ratio of the ethylene glycol, polyvinyl pyrrolidone and silver nitrate is 5-20:2-4:

1.

3. The preparation method according to claim 1, characterized in that: In step (2), the polyvinyl pyrrolidone solution is heated to boiling, and then the silver nanocube solution is added. After reacting for 10-20 minutes, a chloroauric acid solution is added dropwise thereto to react. After removing impurities, the solution is washed, and after washing, the precipitate is dispersed. The volume ratio of the polyvinyl pyrrolidone to the silver nanocube is 80-150:

1.

4. The preparation method according to claim 1, characterized in that: In step (3), the gold nanocage solution is stirred in an ice bath away from light, polyethylene glycol is added and the reaction is continued for 10-15 hours; the volume ratio of the gold nanocage solution to ethylene glycol is 25-75:

1.

5. The preparation method according to claim 1, characterized in that: In step (4), the chloroauric acid solution and the bovine serum albumin solution are mixed, and an alkali reagent is added to react to obtain a gold nanocluster solution; the volume ratio of the chloroauric acid, the bovine serum albumin and the alkali reagent is 5-15:5-15:

1.

6. The preparation method according to claim 1, characterized in that: In step (5), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added to the gold nanocluster solution, stirred in an ice bath for 10-20 minutes, then N-hydroxysuccinimide is added and stirred at room temperature for 10-20 minutes, and then the polyethylene glycol-modified gold nanocage solution is added and reacted at room temperature for 3-4 hours. The volume ratio of the gold nanocluster solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and polyethylene glycol-modified gold nanocage solution is 3-6:1:1:3-6.

7. A plasmon-enhanced fluorescence sensor based on gold nanocages prepared by a method for preparing a plasmon-enhanced fluorescence sensor based on gold nanocages.

8. The plasma enhanced fluorescence sensor according to claim 7, characterized in that: The gold nanocage-based plasma enhanced fluorescence sensor has a hollow gold nanocage with unique surface plasma characteristics. When the fluorescent substance maintains an appropriate distance from the gold nanocage, the gold nanocage can enhance the fluorescence of the fluorescent dye.

9. Use of the gold nanocage-based plasmon-enhanced fluorescence sensor according to claim 7 in the preparation of a norepinephrine detection reagent or tool.

10. A use of the gold nanocage-based plasmon-enhanced fluorescence sensor according to claim 7 in the preparation of reagents or tools for realizing fluorescence imaging of norepinephrine in living cells.

Images