Application of gold-silver core-shell nanorod in SERS (Surface Enhanced Raman Scattering) detection of active oxygen

Abstract

The invention discloses an application of a gold-silver core-shell nanorod in SERS (Surface Enhanced Raman Scattering) detection on active oxygen, the shell layer material of the gold-silver core-shell nanorod is silver, the central core material is a gold nanorod, and the middle layer material between the shell layer and the central core is a Raman signal molecule. The gold-silver core-shell nanorod serves as an SERS nanoprobe, the dual signal enhancement effect of gold-silver alloy on Raman signal molecules is combined, SERS signals are reduced through gradual etching of a silver layer in the cell apoptosis process by means of the strong oxidizing property of ROS, the change of the Raman signals generated after the ROS etches a silver shell is monitored, and the SERS detection accuracy is improved. A new thought for detecting intracellular and extracellular active oxygen is introduced, and the process of increasing the active oxygen content is intuitively and simply reflected, so that the change of the intracellular and extracellular active oxygen in the cell apoptosis process is monitored in real time, and the function of an active oxygen signal in a cell signal channel is known.

Description

Technical Field

[0001] The invention relates to the technical field of Raman spectroscopy detection and analysis, and in particular to application of a gold-silver core-shell nanorod in SERS detection of active oxygen. Background Art

[0002] Patulin (PAT), also known as patulin, is a toxic metabolite mainly produced by fungi such as Aspergillus and Penicillium. The toxin is widely distributed in nature, especially common in spoiled and moldy vegetables, fruits, nuts, grains and their products, especially in rotten apples and their processed products such as apple juice and cider. In view of the fact that apples are a common fruit in our daily diet, the potential harm of PAT is particularly significant. PAT mainly enters the human body through the digestive tract, and is distributed to various tissues and organs throughout the body through the blood circulation, causing adverse effects on the kidneys, liver, intestines and immune system, and can cause ulcers, shortness of breath, pulmonary congestion and other serious discomfort symptoms in a short period of time.

[0003] Apoptosis is an important mechanism of cell self-destruction, involving mitochondria-mediated apoptosis signal transduction, death receptor signaling pathway, and endoplasmic reticulum-mediated apoptosis signal transduction. Studies have shown that fungal toxins to which PAT belongs exert their toxic effects by inducing cellular oxidative damage, among which reactive oxygen species (ROS)-mediated oxidative stress plays an important role in this process.

[0004] Traditional methods for detecting ROS include colorimetry, mass spectrometry, and electrochemical sensors. Colorimetry is based on the color change produced by the reaction of ROS with specific reagents. Although it is accurate and reliable, it has limited sensitivity, and there is a risk of error in the detection of low-concentration ROS. It is also easily affected by environmental interference and its accuracy is affected. Mass spectrometry ionizes ROS in the sample and accelerates it into the mass spectrometer, and determines its type and content based on the mass-to-charge ratio. It is suitable for the study of the synthesis and metabolism of ROS in biological systems. However, it has high requirements for samples and complex pretreatment, which increases the complexity and time consumption of the experiment. Electrochemical sensors react with ROS by modifying electrochemically active substances, causing voltage or current changes to quantify ROS concentrations. They are widely used in biological sample detection, but their performance is greatly affected by environmental factors and experimental conditions need to be strictly controlled.

[0005] In summary, given the widespread existence and serious harm of PAT, as well as the limitations of existing ROS detection technology, it is urgent to develop an efficient, sensitive and easy-to-operate ROS detection method. Summary of the invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide an application of gold-silver core-shell nanorods in SERS detection of active oxygen.

[0007] The above object of the present invention is achieved through the following technical solutions:

[0008] The present invention provides an application of a gold-silver core-shell nanorod in SERS detection of active oxygen. The shell material of the gold-silver core-shell nanorod is silver, the central core material is a gold nanorod, and the intermediate layer material between the shell layer and the central core is a Raman signal molecule. In the process of SERS detection of active oxygen by the gold-silver core-shell nanorod, the active oxygen etches the silver in the gold-silver core-shell nanorod, and the SERS signal of the gold-silver core-shell nanorod changes.

[0009] The present invention prepares high-performance nanoprobes (i.e., gold-silver core-shell nanorods), designs nanorods with a sandwich structure, wherein the core is a gold nanorod, the shell is silver, and the sandwich is a Raman signal molecule. By utilizing the strong oxidizing effect of active oxygen on the etching of metals, a highly sensitive SERS nanoprobe is designed.

[0010] Furthermore, the Raman signal molecule is selected from one or more of 4-mercaptobenzonitrile (4-Mercaptobenzonitrile, MBN), 4-mercaptobenzoic acid (4-MBA), 2-nitrobenzoic acid (2-NBA) and 4-mercaptobenzaldehyde.

[0011] The Raman signal molecules can be linked to the inner gold through Au-S covalent bonds.

[0012] Furthermore, the outer side of the shell layer of the gold-silver core-shell nanorods is modified with a polyethylene glycol (PEG) derivative containing a thiol group.

[0013] In a specific embodiment, the gold-silver core-shell nanorods are composed of an innermost gold core, a Raman signal molecule sandwiched in the middle, an outer silver shell, and an outermost PEG derivative containing a thiol group. The PEG derivative containing a thiol group can improve the stability of the nanoprobe, while reducing the toxicity of the metal in the nanoprobe to cells, improving its biocompatibility, and making it easier to enter cells.

[0014] Furthermore, the thiol-containing polyethylene glycol derivative is selected from one or more of methoxy polyethylene glycol thiol (SH-mPEG), bisthiol polyethylene glycol (SH-PEG-SH), phospholipid-polyethylene glycol-thiol (DSPE-PEG-SH) and thiol-polyethylene glycol-octadecyl (C18-PEG-SH).

[0015] Furthermore, the particle size of the gold-silver core-shell nanorods is 35-45 nm.

[0016] Furthermore, the preparation method of the gold-silver core-shell nanorods comprises the following steps:

[0017] (1) adding chloroauric acid (HAuCl4) solution and sodium borohydride (NaBH4) solution to a hexadecyltrimethylammonium halide solution in sequence to react and obtain a gold seed (Au NPs) solution;

[0018] (2) mixing a silver nitrate (AgNO3) solution, a chloroauric acid solution and a hexadecyltrimethylammonium halide solution, then adding a sodium hydroxide (NaOH) solution and a hydrogen peroxide (H2O2) aqueous solution and stirring to mix evenly, and then adding the gold seed solution obtained in step (1) to obtain a gold nanorod (Au NRs) solution;

[0019] (3) adding the Raman signal molecule solution to the gold nanorod solution obtained in step (2), and mixing them evenly to obtain a gold nanorod (Au NRs) solution containing Raman signal molecules;

[0020] (4) Adding silver nitrate (AgNO3) solution, ascorbic acid (AA) solution and ammonia water (NH3·H2O) to the gold nanorod solution containing Raman signal molecules obtained in step (3) to react and obtain the gold-silver core-shell nanorods.

[0021] The invention adopts a sodium borohydride reduction method to synthesize gold seeds, and then uses hydrogen peroxide as a reducing agent to synthesize gold nanorods using a seed growth method.

[0022] Furthermore, in step (1), the concentration of the hexadecyltrimethylammonium halide solution is 0.05-0.5M, the concentration of the chloroauric acid solution is 10-100mM, the concentration of the sodium borohydride solution is 0.1-1M, and the volume ratio of the hexadecyltrimethylammonium halide solution, the chloroauric acid solution and the sodium borohydride solution is (300-500):(4-6):(4-6).

[0023] Furthermore, in steps (1) and (2), the hexadecyltrimethylammonium halide is hexadecyltrimethylammonium bromide (CTAB) and / or hexadecyltrimethylammonium chloride (CTAC).

[0024] Furthermore, in step (2), the concentration of the silver nitrate solution is 0.1-0.5M, the concentration of the chloroauric acid solution is 10-100mM, the concentration of the hexadecyltrimethylammonium halide solution is 0.05-0.5M, and the volume ratio of the silver nitrate solution, the chloroauric acid solution and the hexadecyltrimethylammonium halide solution is (1-2):(20-30):(500-1500).

[0025] Furthermore, in step (2), the concentration of the sodium hydroxide solution is 1-2M, the concentration of the hydrogen peroxide aqueous solution is 20-40%, and the volume ratio of the chloroauric acid solution, the sodium hydroxide solution and the hydrogen peroxide aqueous solution is (200-300):(50-150):(5-10).

[0026] Furthermore, in step (2), the volume ratio of the chloroauric acid solution to the gold seed solution is (20-30):(2-5).

[0027] Furthermore, in step (3), the concentration of the Raman signal molecule solution is 500-1500 μM, and the volume ratio of the gold nanorod solution to the Raman signal molecule solution is (500-1500):(5-10).

[0028] Furthermore, in step (4), the concentration of the silver nitrate solution is 0.5-3 mM, the concentration of the ascorbic acid solution is 5-25 mM, and the volume ratio of the gold nanorod solution containing Raman signal molecules, the silver nitrate solution, the ascorbic acid solution and the ammonia solution is (30-50):(30-50):(1-3):(5-10).

[0029] In a specific embodiment, the method for preparing the gold-silver core-shell nanorods specifically comprises the following steps:

[0030] (1) Adding chloroauric acid (HAuCl4) solution to hexadecyltrimethylammonium halide solution, and then rapidly injecting sodium borohydride solution under stirring, stirring for 2-5 minutes, exhausting the generated hydrogen, and placing in a water bath at 25-30°C for 2-6 hours to obtain a gold seed (Au NPs) solution;

[0031] (2) mixing a silver nitrate (AgNO3) solution, a chloroauric acid solution and a hexadecyltrimethylammonium halide solution, then adding a sodium hydroxide (NaOH) solution and a hydrogen peroxide (H2O2) aqueous solution and stirring for 2-5 minutes to mix evenly, then adding a gold seed solution, stirring and reacting for 30-50 seconds, placing the obtained mixed solution in a 25-30°C water bath for 3-6 hours, then centrifuging and washing for multiple times and then redissolving the precipitate in an equal volume of ultrapure water to obtain a gold nanorod (Au NRs) solution;

[0032] (3) Adding the Raman signal molecule solution to the gold nanorod solution, oscillating and mixing, incubating overnight, and centrifuging and washing to remove unbound Raman signal molecules to obtain a gold nanorod solution containing Raman signal molecules.

[0033] (4) adding silver nitrate solution, ascorbic acid solution and ammonia water to the gold nanorod solution containing Raman signal molecules, vortexing and mixing them evenly to stabilize the reaction, thereby obtaining the gold-silver core-shell nanorods.

[0034] Furthermore, the method for modifying the outer side of the shell layer of the gold-silver core-shell nanorods with a polyethylene glycol derivative containing a thiol group comprises the following steps: adding a solution of a polyethylene glycol derivative containing a thiol group to a solution of the gold-silver core-shell nanorods to obtain a gold-silver core-shell nanorod whose outer side of the shell layer is modified with a polyethylene glycol derivative containing a thiol group.

[0035] The polyethylene glycol derivatives containing thiol groups can improve the stability and biocompatibility of gold-silver core-shell nanorods. The prepared gold-silver core-shell nanorods with the outer shell modified with the polyethylene glycol derivatives containing thiol groups can be used as SERS nanoprobes for SERS detection.

[0036] Furthermore, the gold-silver core-shell nanorod solution is prepared by centrifugally resuspending the gold-silver core-shell nanorods in an equal volume of water.

[0037] Furthermore, the concentration of the thiol-containing polyethylene glycol derivative solution is 10-1000 μM, and the volume ratio of the gold-silver core-shell nanorod solution to the thiol-containing polyethylene glycol derivative solution is (500-1000):(0.5-2).

[0038] Furthermore, the thiol-containing polyethylene glycol derivative is selected from one or more of methoxy polyethylene glycol thiol (SH-mPEG), bisthiol polyethylene glycol (SH-PEG-SH), phospholipid-polyethylene glycol-thiol (DSPE-PEG-SH) and thiol-polyethylene glycol-octadecyl (C18-PEG-SH).

[0039] Furthermore, the method for performing SERS detection on active oxygen using the gold-silver core-shell nanorods comprises the following steps:

[0040] The gold and silver core-shell nanorods are diluted in a basic culture medium, incubated with cells to be tested, the basic culture medium is discarded and washed, a toxin solution dissolved in the basic culture medium is added for co-culture, the basic culture medium dissolved with toxins is discarded and washed, the cells are stained and fixed, and SERS spectral detection and SERS imaging are performed to obtain the detection results of intracellular reactive oxygen species;

[0041] Alternatively, the gold-silver core-shell nanorods are mixed with a hydrogen peroxide solution for reaction, and after centrifugation, SERS spectrum detection and SERS imaging are performed to obtain the detection result of in vitro active oxygen.

[0042] Furthermore, the gold-silver core-shell nanorods are diluted in a basic culture medium. When the gold-silver core-shell nanorods are mixed with the basic culture medium, the concentration of the Au element in the obtained culture medium is 20-100 μM, preferably 60-70 μM. This concentration range can maintain higher cell activity and achieve better detection effects.

[0043] Furthermore, the basic culture medium is preferably DMEM basic culture medium.

[0044] Furthermore, the toxins in the toxin solution include patulin, T-2 toxin, deoxynivalenol (DON), aflatoxin B1 (AFB1), and HT-2 toxin.

[0045] When cells begin to undergo apoptosis under toxin induction, the reactive oxygen species in the cytoplasm increases compared to normal conditions, thereby monitoring the reactive oxygen species during intracellular apoptosis.

[0046] Furthermore, the concentration of the toxin solution preferably corresponds to the IC 50 This concentration range can ensure that the toxin has a certain toxicity to the cells being tested and maintain a certain cell viability.

[0047] Furthermore, the concentration of the toxin solution is 20-100 μM, preferably 60-70 μM.

[0048] The detection method of the present invention is based on surface enhanced Raman scattering (SERS), and the SERS nanoprobe (i.e., gold-silver core-shell nanorod) is easily absorbed by cells so as to realize the dynamic detection of reactive oxygen in living cells. The SERS nanoprobe presents a sandwich structure, consisting of an innermost gold core, a Raman signal molecule sandwiched in the middle, and an outer silver shell. In order to improve its biocompatibility, a polyethylene glycol derivative containing a thiol group is also modified on the outermost side. Compared with gold nanorods, gold-silver core-shell nanorods not only increase the localized surface plasmon resonance (LSPR) effect of silver metal, but the coupling of gold and silver further enhances the Raman signal.

[0049] In the presence of a large amount of active oxygen, the outer silver shell is gradually etched, causing the silver shell to become thinner, the SERS signal to weaken, and the coupling between the gold core and the silver shell to weaken, further reducing the Raman signal intensity of the Raman signal molecule.

[0050] Toxins induce cell apoptosis and produce a large amount of reactive oxygen species. Based on this principle, the detection and imaging of intracellular reactive oxygen species can be achieved.

[0051] Beneficial effects of the present invention:

[0052] 1. The present invention encapsulates a layer of silver on the outside of the gold nanorods and places the Raman signal molecules in the gap between the gold core and the silver shell. The prepared gold-silver core-shell nanorods can superimpose the SERS enhancement effects of gold and silver and the coupling enhancement effect between the two, which can greatly improve the intensity of the SERS signal.

[0053] 2. The SERS nanoprobe of the present invention combines the dual signal enhancement effect of gold-silver alloy on Raman signal molecules, and the gradual etching of the silver layer during cell apoptosis causes the SERS signal to decrease, which is different from the detection mechanism using chemical reactions of ROS. The present invention uses the strong oxidizing property of ROS to monitor the changes in the Raman signal generated after etching the silver shell, introduces a new idea for detecting intracellular and extracellular reactive oxygen species, intuitively and simply reflects and monitors the process of increasing reactive oxygen content, thereby realizing real-time monitoring of changes in intracellular and extracellular reactive oxygen species during cell apoptosis, which is conducive to understanding the role of reactive oxygen signals in cell signaling pathways.

[0054] 3. The SERS nanoprobe designed in the present invention is easy to prepare and has high stability. Its detection limit covers the common concentration range of reactive oxygen species in cells, and exhibits a linear range (100nM to 1000nM) and high sensitivity (LOD is 43.78nM) for detecting reactive oxygen species. It can be used efficiently and conveniently for imaging and detecting intracellular ROS.

[0055] 4. The SERS nanoprobe of the present invention has the characteristics of low toxicity and no damage to cells, is easy to load into cells, can detect the content of reactive oxygen species inside and outside cells respectively, and can intuitively reflect the changes of reactive oxygen species and intracellular substances in combination with cell imaging technology. Its synthesis and detection costs are relatively low, and it has practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The UV-visible absorption spectrum and TEM image of Au NRs in Example 1; wherein a is the UV-visible absorption spectrum and b is the TEM image.

[0057] Figure 2 is the Raman spectrum of Au-MBN NRs in Examples 1-6; wherein a is the Raman spectrum of Au-MBN NRs, and b is 1065 cm -1 Raman intensity data at .

[0058] Figure 3 The Raman spectra of Au / MBN / Ag NRs in Example 1 and Examples 7-11 are shown in Figure 1. Wherein, a is the Raman spectra of Au / MBN / Ag NRs, and b is 1065 cm -1 Raman intensity data at .

[0059] Figure 4These are the UV spectra and Raman spectra of Au NRs, Au-MBN NRs, Au / MBN / Ag NRs and Au / MBN / Ag NRs@PEG in Example 1, and the TEM image of the SERS nanoprobe prepared in Example 1; wherein a is the UV spectrum, b is the Raman spectrum, and c is the TEM image of the SERS nanoprobe prepared in Example 1.

[0060] Figure 5 Graph showing the stability test results of the SERS nanoprobe prepared in Example 1; wherein, a is a UV-visible spectrum graph for testing salt stability, b is a UV-visible spectrum graph for testing pH stability, c is a Raman spectrum graph for testing the stability of the SERS nanoprobe in buffer, and d is a Raman spectrum graph for testing the stability of the SERS nanoprobe in culture medium.

[0061] Figure 6 The results of the SERS nanoprobe prepared in Example 1 for in vitro reactive oxygen species detection are shown in Figure 1; a is a UV spectrum, b is a Raman spectrum, and c is a 1065 cm -1 a is the Raman intensity data diagram at , and d is the TEM image of the SERS nanoprobe after in vitro etching.

[0062] Figure 7 This is a graph showing the detection results of the cytotoxicity of the SERS nanoprobe prepared in Example 1.

[0063] Figure 8 Graph showing the detection results of the uptake of the SERS nanoprobes prepared in Example 1 by HepG2 cells; wherein, a is a standard curve graph of Au concentration and CPS, and b is a graph showing the change in the number of SERS nanoprobes entering the cells measured by ICP-MS when the SERS nanoprobes were co-incubated with cells for different time periods (0-10h).

[0064] Fig. 9 This is the SERS detection imaging image of the SERS nanoprobe prepared in Example 1 in living cells.

[0065] Fig.10 The SERS nanoprobe prepared in Example 1 is used for the quantitative detection of reactive oxygen species in living cells; a is the Raman spectra of the SERS nanoprobe in cells after adding patulin and incubating for different time periods, and b is the Raman spectra at 1065 cm -1 Raman intensity data at . DETAILED DESCRIPTION

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0067] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0068] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0069] Example 1

[0070] A method for preparing gold / silver core-shell nanorods Au / MBN / Ag NRs@PEG (SERS nanoprobe) comprises the following steps:

[0071] (1) Au NPs were synthesized by NaBH4 reduction method: 50 μL HAuCl4 solution (20 mM) was added to 4 mL CTAB solution (0.1 M), and then 48 μL freshly prepared NaBH4 solution (0.1 M) was quickly injected under stirring. The mixture was stirred for 2 min, the generated hydrogen was discharged, and the mixture was placed in a 28 °C water bath for 2 h to obtain Au NPs solution.

[0072] (2) Au NRs were synthesized by seed growth method using H2O2 as a reducing agent: 10 μL AgNO3 solution (0.1M) and 250 μL HAuCl4 solution (20 mM) were added to 10 mL CTAB solution (0.1M), followed by 100 μL NaOH solution (1M) and 5 μL H2O2 aqueous solution (30%, w / w) and stirred for 2 min to mix evenly, and the solution changed from yellow to colorless. Then 30 μL Au NPs solution prepared by S1 was quickly added and stirred for 30 s. The obtained mixed solution was placed in a 28°C water bath for 2 h, and then the precipitate was redissolved in an equal volume of ultrapure water after centrifugation and washing several times to obtain Au NRs solution.

[0073] (3) Add 8 μL of MBN solution (1 mM) to 1 mL of Au NRs solution, shake and mix, incubate overnight, and centrifuge to remove unbound MBN to obtain an Au-MBN NRs solution.

[0074] (4) Add 400 μL of AgNO 3 solution (1 mM), 20 μL of AA solution (10 mM), and 80 μL of NH 3 · H 2 O to 400 μL of Au-MBN NRs solution, vortex to mix evenly, incubate for 15 min to stabilize the reaction, and resuspend in an equal volume of ultrapure water by centrifugation to obtain Au / MBN / Ag NRs solution.

[0075] (5) Add 1 μL of 100 μmol / L mPEG-SH solution to 1 mL of Au / MBN / Ag NRs solution, incubate overnight and then centrifuge to obtain the SERS nanoprobe (Au / MBN / Ag NRs@PEG); resuspend the SERS nanoprobe in an equal volume of ultrapure water to obtain a SERS nanoprobe solution.

[0076] The UV-visible absorption spectrum of Au NRs in Example 1 was tested using a UV absorption spectrometer. Figure 1 As shown in (a), Au NRs produced two absorption peaks, including a longitudinal absorption peak at 730 nm and a transverse absorption peak at 530 nm.

[0077] The Au NRs in Example 1 were characterized by transmission electron microscopy (TEM). Figure 1 As shown in (b), AuNRs have a well-defined rod-like morphology, demonstrating the successful synthesis of the rod-like structure.

[0078] Example 2

[0079] A method for preparing a SERS nanoprobe is basically the same as that of Example 1, except that in step (3), 2 μL of MBN solution (1 mM) is added to 1 mL of Au NRs solution, the mixture is shaken and mixed, incubated overnight, and centrifuged to remove unbound MBN to obtain an Au-MBN NRs solution.

[0080] Example 3

[0081] A method for preparing a SERS nanoprobe is basically the same as that of Example 1, except that in step (3), 4 μL of MBN solution (1 mM) is added to 1 mL of Au NRs solution, the mixture is shaken and mixed, incubated overnight, and centrifuged to remove unbound MBN to obtain an Au-MBN NRs solution.

[0082] Example 4

[0083] A method for preparing a SERS nanoprobe is basically the same as that of Example 1, except that in step (3), 6 μL of MBN solution (1 mM) is added to 1 mL of Au NRs solution, the mixture is shaken and mixed, incubated overnight, and centrifuged to remove unbound MBN to obtain an Au-MBN NRs solution.

[0084] Example 5

[0085] A method for preparing a SERS nanoprobe is basically the same as that of Example 1, except that: in step (3), 10 μL of MBN solution (1 mM) is added to 1 mL of Au NRs solution, the mixture is shaken and mixed, incubated overnight, and centrifuged to remove unbound MBN to obtain an Au-MBN NRs solution.

[0086] Example 6

[0087] A method for preparing a SERS nanoprobe is basically the same as that of Example 1, except that in step (3), 12 μL of MBN solution (1 mM) is added to 1 mL of Au NRs solution, the mixture is shaken and mixed, incubated overnight, and centrifuged to remove unbound MBN to obtain an Au-MBN NRs solution.

[0088] The Raman spectra of the Au-MBN NRs in test examples 1-6 are as follows: Figure 2 As shown in the figure, with the increase of the amount of MBN solution added, the Raman signal gradually increased. At 8 μL, the enhancement effect of Au NRs on the MBN Raman signal reached saturation and then decreased slightly. Therefore, the optimal amount of MBN solution added was 8 μL.

[0089] Example 7

[0090] A method for preparing a SERS nanoprobe is basically the same as that in Example 1, except that: in step (4), 100 μL of AgNO3 solution (1 mM), 20 μL of AA solution (10 mM) and 80 μL of NH3·H2O are added to 400 μL of Au-MBN NRs solution, vortexed to mix evenly, incubated for 15 min to stabilize the reaction, and resuspended in an equal volume of ultrapure water by centrifugation to obtain an Au / MBN / Ag NRs solution.

[0091] Example 8

[0092] A method for preparing a SERS nanoprobe is basically the same as that in Example 1, except that: in step (4), 200 μL of AgNO3 solution (1 mM), 20 μL of AA solution (10 mM) and 80 μL of NH3·H2O are added to 400 μL of Au-MBN NRs solution, vortexed to mix evenly, incubated for 15 min to stabilize the reaction, and resuspended in an equal volume of ultrapure water by centrifugation to obtain an Au / MBN / Ag NRs solution.

[0093] Example 9

[0094] A method for preparing a SERS nanoprobe is basically the same as that in Example 1, except that: in step (4), 300 μL of AgNO3 solution (1 mM), 20 μL of AA solution (10 mM) and 80 μL of NH3·H2O are added to 400 μL of Au-MBN NRs solution, vortexed to mix evenly, incubated for 15 min to stabilize the reaction, and resuspended in an equal volume of ultrapure water by centrifugation to obtain an Au / MBN / Ag NRs solution.

[0095] Example 10

[0096] A method for preparing a SERS nanoprobe is basically the same as that in Example 1, except that: in step (4), 500 μL of AgNO3 solution (1 mM), 20 μL of AA solution (10 mM) and 80 μL of NH3·H2O are added to 400 μL of Au-MBN NRs solution, vortexed to mix evenly, incubated for 15 min to stabilize the reaction, and resuspended in an equal volume of ultrapure water by centrifugation to obtain an Au / MBN / Ag NRs solution.

[0097] Embodiment 11

[0098] A method for preparing a SERS nanoprobe is basically the same as that in Example 1, except that: in step (4), 600 μL of AgNO3 solution (1 mM), 20 μL of AA solution (10 mM) and 80 μL of NH3·H2O are added to 400 μL of Au-MBN NRs solution, vortexed to mix evenly, incubated for 15 min to stabilize the reaction, and resuspended in an equal volume of ultrapure water by centrifugation to obtain an Au / MBN / Ag NRs solution.

[0099] The Raman spectra of Au / MBN / Ag NRs in Example 1 and Examples 7-11 are shown in FIG. Figure 3 As shown, with the increase in the amount of AgNO3 solution added, the Raman signal intensity gradually increases and eventually tends to be stable, the enhancement effect of silver reaches a saturation state, and a stably enhanced Raman signal is obtained. Therefore, the addition amount of AgNO3 solution is preferably 400 μL.

[0100] Test Example 1

[0101] The UV spectra and Raman spectra of Au NRs, Au-MBN NRs, Au / MBN / Ag NRs and Au / MBN / Ag NRs@PEG in Example 1 were tested. The test results are as follows: Figure 4 As shown, from Figure 4 It can be seen in Figure a that after being coated with a silver shell, the longitudinal plasma absorption peak of gold blue-shifts from around 730nm to around 520nm, and the characteristic absorption peak of silver appears around 400nm. Figure 4As can be seen in figure b, the intensity comparison of the MBN Raman signal before and after enhancement by gold and gold-silver alloy is that AuNRs themselves do not have a Raman signal, but after connecting to MBN, a Raman signal of a certain intensity is obtained. After depositing the silver layer, the Raman signal of MBN is greatly enhanced, and a nanoprobe with a high-intensity SERS signal is obtained.

[0102] The SERS nanoprobe prepared in Example 1 was characterized by transmission electron microscopy (TEM). Figure 4 As shown in middle c, it can be clearly seen that the gold core is covered with a layer of silver shell, which has the morphological characteristics of the gold and silver shell structure, confirming that a SERS nanoprobe with a gold core and silver shell structure was obtained.

[0103] Test Example 2

[0104] The SERS nanoprobe prepared in Example 1 was subjected to a stability test.

[0105] (1) Characterization of the Salt Stability of SERS Nanoprobes by UV-Vis Spectroscopy

[0106] The SERS nanoprobe solution was centrifuged and resuspended in solutions containing different NaCl concentrations (100 mM, 200 mM, 300 mM, 400 mM, and 500 mM), vortexed and mixed, and allowed to stand for 6 h before the UV-visible spectrum of the SERS nanoprobe was detected by UV-visible absorption spectrometer. The test results are shown in Figure 2. Figure 5 As shown in (a), the prepared SERS nanoprobe still has good optical stability under high salt concentration and is suitable for the intracellular environment.

[0107] (2) Characterization of pH stability of SERS nanoprobes by UV-visible spectroscopy

[0108] The SERS nanoprobe solution was centrifuged and resuspended in aqueous solutions with different pH values ​​(pH = 5, 6, 7, 8, 9), vortexed and mixed, and allowed to stand for 6 hours before the UV-visible spectrum of the SERS nanoprobe was detected by UV-visible absorption spectrometer. The results are as follows: Figure 5 As shown in middle b, the prepared SERS nanoprobe can maintain good stability within the pH range of common physiological environments and can be used for subsequent intracellular experiments.

[0109] (3) Raman detection to evaluate the stability of SERS nanoprobes in buffer and culture medium

[0110] The two groups of SERS nanoprobe solutions were centrifuged and resuspended in PBS buffer solution (0.01 M, pH = 7.4) and DMEM basic culture medium for incubation for 0 h, 6 h, 12 h, 24 h and 36 h, respectively, and the SERS signals of MBN were detected. The detection results are shown in Figure 5 Medium c and Figure 5As shown in Figure d, it can be seen that in the environment of PBS buffer and DEME culture medium, the Raman signal of the prepared SERS nanoprobe is not greatly affected when incubated for different time periods, and the buffer and culture medium will not interfere with subsequent signal detection.

[0111] Test Example 3

[0112] The SERS nanoprobe prepared in Example 1 was used for in vitro reactive oxygen species detection, and H2O2 aqueous solutions of different concentrations were used to verify the feasibility of the SERS nanoprobe for reactive oxygen species sensing.

[0113] The detection method is as follows: add H2O2 aqueous solution of different concentrations to the Au / MBN / Ag NRs@PEG nanoprobe solution and mix thoroughly, so that the concentration of H2O2 in the mixed solution is 0, 10, 100, 200, 400, 600, 800, 1000, 1500 nM, wait for 6 hours for the reaction to be complete to obtain the test solution. Measure the ultraviolet-visible absorption spectrum (UV-vis) with a wavelength range of 450-1000 cm -1 The liquid to be tested was dropped onto a glass slide wrapped with tin foil and dried before measuring SERS. The excitation wavelength was 785 nm and the scanning range was 400-2500 cm -1 .

[0114] Test results such as Figure 6 As shown, from Figure 6 From the UV spectrum of a, we can see that with the increase of H2O2 concentration, the characteristic peak of silver at 400nm blue-shifts, and the absorption peak intensity also decreases. At the same time, the characteristic absorption peak of AuNRs gradually recovers above 700nm, indicating that in the process of increasing H2O2 concentration, the active oxygen continuously etches the silver shell on the surface, causing its UV peak to blue-shift and intensity to decrease, and finally etching back to AuNRs. The SERS nanoprobe after in vitro etching (H2O2 concentration of 1000nM) is characterized, as shown in Figure 2. Figure 6 As shown in Figure d, after the outer silver shell is etched away, its appearance is consistent with that of the gold nanorods before being wrapped with the silver shell. Figure 6 As can be seen in b and c, the Raman signal intensity of the SERS probe decreases with the increase of H2O2 concentration. On the one hand, the thinning of the silver layer causes the Raman signal intensity to decrease. On the other hand, the coupling between silver and gold is also weakened, further reducing the SERS intensity of the Raman signal molecule. -1 The SERS changes at the position have a good linear relationship, and the regression equation is Y = 20554.77-13.91X (R 2=0.9950), and LOD is 43.78 nM. Compared with previous reports, the SERS nanoprobe prepared in the present invention has higher sensitivity.

[0115] Test Example 4

[0116] The CCK-F kit was used to detect the cytotoxicity of the SERS nanoprobe prepared in Example 1, and because the concentration of the SERS nanoprobe in the SERS nanoprobe solution was difficult to measure, the Au concentration was used instead of the concentration of the SERS nanoprobe.

[0117] The detection method is: 1×10 4 After incubating HepG2 cells overnight and allowing them to adhere to the wall, SERS nanoprobe solutions with different Au concentrations (20μM, 40μM, 60μM, 80μM, 100μM) were added and cultured for 6, 12, and 24 hours. 100μL CCK-F reagent solution was added and placed in a cell culture incubator to react in the dark for 30 minutes, and then the fluorescence intensity was measured immediately, with an excitation wavelength of 494nm and an emission wavelength of 517nm. When evaluating cell viability, cells without drug administration were calculated as a blank control group.

[0118] Test results such as Figure 7 As shown, it is explained that even if high concentration of nanoprobes are co-incubated with cells for 24 hours, the survival rate of HepG2 cells can still be maintained above 90%, proving that the SERS nanoprobe designed in the present invention has low toxicity, good biocompatibility, and is suitable for the cell environment.

[0119] Test Example 5

[0120] The Au / MBN / AgNRs@PEG (SERS nanoprobe) solutions with different Au concentrations were digested with aqua regia overnight, diluted through the membrane, and the response value (CPS) was measured using an inductively coupled plasma mass spectrometer (ICP-MS). A standard curve was drawn based on the Au concentration and the response value (CPS), as shown in Figure 8 As shown in a.

[0121] ICP-MS was used to evaluate the uptake of the SERS nanoprobes prepared in Example 1 by HepG2 cells. The test method was as follows: HepG2 cells were cultured in a 6-well plate for 12 hours until they adhered to the wall, and a SERS nanoprobe solution with an Au concentration of 60 μM was added. After incubation for different periods of time, the culture medium was discarded, and the HepG2 cells were washed and digested to make them fall off the bottom of the dish, and then collected and counted by centrifugation. Aqua regia was added to digest the cells overnight, and the final sample was obtained after dilution through the membrane and analyzed using ICP-MS, thereby calculating the number of SERS nanoprobes that entered the cells. The test results are shown in Figure 2. Figure 8As shown in middle b, as the co-incubation time of SERS nanoprobes and cells increases, the amount of SERS nanoprobes taken into cells gradually increases and reaches saturation around 8-10h. 4 The Au content of each cell is about 152nM.

[0122] Test Example 6

[0123] The SERS nanoprobe prepared in Example 1 was used for the quantitative detection of reactive oxygen species in living cells. HepG2 cells (3×10 4 After overnight incubation in a laser confocal microplate until adhered, the SERS nanoprobes diluted in the basic medium were added and co-cultured with the adhered HepG2 cells for 8 hours. The basic medium was then discarded, and 60 μM patulin was added, and the cells were co-incubated for 0, 1, 2, 3, 4, and 5 hours, respectively, and finally fixed with 4% paraformaldehyde.

[0124] The SERS spectrum and SERS image of a single cell were obtained using a micro-Raman imaging spectrometer. The excitation wavelength was set to 785 nm, the power was 5 mW, the spot diameter was 1 μm, and the exposure time was 1 s. The results are shown in Figure 2. Fig. 9 As shown, Bright is the cell image under bright field, SERS is the corresponding Raman imaging image, and Overlay is the superposition processing of the first two images. Fig. 9 It was shown that as the toxin incubation time increased, the degree of cell apoptosis deepened and the intensity of intracellular SERS signal decreased.

[0125] The excitation wavelength was set to 785 nm, the power was 5 mW, the spot diameter was 1 μm, and the exposure time was 1 s. The Raman spectra at different points were collected and the average value was taken as the SERS spectrum result. The results are shown in the figure. Fig.10 shown. Fig.10 The collected Raman spectra also show that reactive oxygen species etched the silver shell of the nanoprobe. As the reactive oxygen content increased, the detected SERS signal gradually weakened.

[0126] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art should understand that other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. An application of gold and silver core-shell nanorods in SERS detection of active oxygen, characterized in that: The shell material of the gold-silver core-shell nanorods is silver, the central core material is gold nanorods, and the intermediate layer material between the shell layer and the central core is a Raman signal molecule; in the process of SERS detection of active oxygen by the gold-silver core-shell nanorods, the active oxygen etches the silver in the gold-silver core-shell nanorods, and the SERS signal of the gold-silver core-shell nanorods changes.

2. The use according to claim 1, characterized in that: The Raman signal molecule is selected from one or more of 4-mercaptobenzonitrile, 4-mercaptobenzoic acid, 2-nitrobenzoic acid and 4-mercaptobenzaldehyde.

3. The use according to claim 1, characterized in that: The outer side of the shell layer of the gold-silver core-shell nanorod is modified with a polyethylene glycol derivative containing a thiol group.

4. The use according to claim 3, characterized in that: The polyethylene glycol derivative containing a thiol group is selected from one or more of methoxy polyethylene glycol thiol, dithiol polyethylene glycol, phospholipid-polyethylene glycol-thiol and thiol-polyethylene glycol-octadecyl.

5. The use according to claim 1 or 2, characterized in that: The preparation method of the gold-silver core-shell nanorods comprises the following steps: (1) adding a chloroauric acid solution and a sodium borohydride solution to a hexadecyltrimethylammonium halide solution in sequence to react to obtain a gold seed solution; (2) mixing the silver nitrate solution, the chloroauric acid solution and the hexadecyltrimethylammonium halide solution, then adding the sodium hydroxide solution and the hydrogen peroxide solution and stirring to mix evenly, and then adding the gold seed solution obtained in step (1) to obtain a gold nanorod solution; (3) adding the Raman signal molecule solution to the gold nanorod solution obtained in step (2), and mixing them evenly to obtain a gold nanorod solution containing Raman signal molecules; (4) adding silver nitrate solution, ascorbic acid solution and ammonia water to the gold nanorod solution containing Raman signal molecules obtained in step (3) to react and obtain the gold-silver core-shell nanorods.

6. The use according to claim 5, characterized in that: In step (3), the concentration of the Raman signal molecule solution is 500-1500 μM, and the volume ratio of the gold nanorod solution to the Raman signal molecule solution is (500-1500):(5-10).

7. The use according to claim 5, characterized in that: In step (4), the concentration of the silver nitrate solution is 0.5-3 mM, the concentration of the ascorbic acid solution is 5-25 mM, and the volume ratio of the gold nanorod solution containing Raman signal molecules, the silver nitrate solution, the ascorbic acid solution and the ammonia solution is (30-50):(30-50):(1-3):(5-10).

8. The use according to claim 5, characterized in that: A solution of a polyethylene glycol derivative containing a thiol group is added to the solution of the gold-silver core-shell nanorods to obtain gold-silver core-shell nanorods with the outer shell layer modified with the polyethylene glycol derivative containing a thiol group.

9. The use according to claim 1, characterized in that: The method for performing SERS detection on active oxygen by gold-silver core-shell nanorods comprises the following steps: The gold and silver core-shell nanorods are diluted in a basic culture medium, incubated with cells to be tested, the basic culture medium is discarded and washed, a toxin solution dissolved in the basic culture medium is added for co-culture, the basic culture medium dissolved with toxins is discarded and washed, the cells are stained and fixed, and SERS spectral detection and SERS imaging are performed to obtain the detection results of intracellular reactive oxygen species; Alternatively, the gold-silver core-shell nanorods are mixed with a hydrogen peroxide solution for reaction, and after centrifugation, SERS spectrum detection and SERS imaging are performed to obtain the detection result of in vitro active oxygen.

10. The use according to claim 9, characterized in that: The gold-silver core-shell nanorods are diluted in a basic culture medium, and the concentration of Au element in the obtained culture medium is 20-100 μM.

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