Preparation method and application of capsule-shaped internal standard gap Raman probe

Abstract

The invention relates to a preparation method and application of a capsule-shaped internal standard gap Raman probe. The preparation method comprises the following steps: firstly, preparing a gold nanorod by using a seed mediated growth method in which 5-bromosalicylic acid participates, and then preparing a capsule-shaped gold-core silver-shell nanorod embedded with a Raman signal molecule 4-NBT; finally, preparing the capsule-shaped internal standard gap core-shell nanoprobe by utilizing an electrochemical replacement method, wherein the Raman intensity of the capsule-shaped gold-core silver-shell nanoprobe at 1331 cm <-1 > is obviously improved; by introducing a capsule-shaped gap structure, a controllable nano gap (1-15nm) is formed between a core-shell outer layer and a capsule shell, so that a local electromagnetic field is doubly superposed on a core-shell interface and a gap region, and a synergistic enhancement effect is generated; by adopting an internal standard method, relatively high stability and accuracy can be kept in a long-time measurement or continuous monitoring process, and the method has the advantages of high environmental interference resistance and good wide-spectrum detection compatibility; the method is applied to the technical fields of in-vitro diagnosis, biomedical Raman imaging and biological anti-counterfeiting.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical sensing and imaging, and relates to a preparation method and application of a capsule-shaped internal standard gap Raman probe. Background Art

[0002] Surface-enhanced Raman scattering (SERS) is a powerful vibrational spectroscopy technique widely used for ultrasensitive and nondestructive molecular detection. Raman detection is closely related to Raman reporters, compounds used to label and identify specific molecules in Raman detection. These molecules possess unique scattering peaks that can be clearly distinguished in Raman spectra, enabling the detection and identification of target molecules. The selection and placement of Raman reporters are crucial in constructing SERS nanoprobes or tags. However, existing studies have mostly used Raman reporter molecules modified on the exterior of nanoparticles [Bi, CX; Yin, XL; Zhao, HY. Synthesis of Au@Ag core-shell nanocubes with finely tuned shell thicknesses for surface-enhanced Raman spectroscopic detection. Rsc Advances 2024, 14, 20145-20151]. This can lead to issues with Raman signal stability and intensity. One approach to addressing these issues is to modify Raman reporters within nanoparticles or materials. Directly modifying Raman reporter molecules inside nanoparticles may affect their interaction with lasers, resulting in a weakening of the Raman signal. Nanoscale gaps can significantly enhance Raman signals. When laser light is irradiated on nanoparticles with a gap structure, the electromagnetic field in the gap will be greatly enhanced, which is of great significance for improving the sensitivity and accuracy of detection. Currently, the gap Raman probes reported in the literature include petal-shaped gap-enhanced Raman probes [Zhang, YQ; Gu, YQ; He, J.; Thackray, BD; Ye, J. Ultrabright gap-enhanced Raman tags for high-speed bioimaging. Nature Communications 2019, 10]. The main feature is that the probe core is a petal-shaped gold nanoparticle, the outer layer is wrapped with a gold shell, and a subnanometer (1-2nm) gap is formed between the core and the shell, in which Raman signal molecules (such as 4-NBT) are embedded. This structure generates a "hot spot" through the electromagnetic field coupling effect, significantly enhancing the Raman signal, with an enhancement factor of up to 5×10 9The disadvantage is that the reaction process requires multiple centrifugation purifications and the reaction needs to be subjected to intense ultrasonic treatment. There are also egg yolk-shell nanorods [Zhu, J.; Zhang, S.; Weng, GJ; Li, JJ; Zhao, JW The morphology regulation and plasmonic spectral properties of Au@AuAg yolk-shell nanorods with controlled interior gap. Spectrochimica Acta Part a-Molecular and Biomolecular Spectroscopy 2020, 236], which have a three-layer structure of an inner gold nanorod core, an intermediate gap, and an outer AuAg alloy shell. The internal gap and shell thickness can be adjusted by parameters. However, the shell shape changes from a regular capsule to a polygon as the initial morphology of the Ag shell changes, and the morphological uniformity of the nanoparticles is poor. Peanut-shaped interstitial nanoparticles [Kuthala, N.; Shanmugam, M.; Kong, XY; Chiang, C.S.; Hwang, K.C.S.] are designed for plasmonic field-field / field-lattice coupling-enhanced NIR-II photodynamic therapy using core-gap-shell gold nanopeanuts. Nanoscale Horizons 2022, 7] and consist of a gold nanorod (AuNR) core, a gap, and an AuAg alloy shell. The gap can be filled with a high-dielectric-constant NaCl aqueous solution. By adjusting the dielectric constant of the interstitial medium, a significant red shift of the plasmon absorption band is achieved, with the extinction coefficient at 1064 nm increased by 12.6 times, enhancing near-infrared light absorption. However, synthesizing core-gap-shell nanostructures with uniform gap distance and shape is difficult, and irregular gaps may affect plasmonic coupling efficiency. No gap-shaped internal standard Raman probes have been reported that maintain a capsule shape. Therefore, developing a method for preparing capsular internal standard gap Raman probes is of great importance. Summary of the Invention

[0003] In order to overcome the defects of the above-mentioned prior art, the purpose of the present invention is to provide a method for preparing a capsule-shaped internal standard gap Raman probe and its application, firstly, using the seed-mediated growth method with the participation of pentabromosalicylic acid to prepare gold nanorods, then preparing capsule-shaped gold core-silver shell nanorods embedded with the Raman signal molecule 4-NBT, and finally preparing capsule-shaped internal standard gap core-shell nanoprobe by electrochemical replacement method; the capsule-shaped gold core-silver shell nanoprobe is prepared at 1331 cm -1 The Raman intensity increased significantly.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] A capsule-shaped internal standard gap Raman probe comprises the following raw materials in a proportion: 4-13 mL of a 0.08-0.2 mM cetyltrimethylammonium bromide (CTAB) solution; 200-500 mL of a 0.12-0.55 M cetyltrimethylammonium bromide (CTAB) solution; 100-200 mL of a 0.06-0.1 mM cetyltrimethylammonium bromide (CTAB) solution; 5.05-10.2 mL of a 23-28 mM HAuCl4 solution; 50-200 μL of a 0.8-1.2 mM HAuCl4 solution; and 600-100 mL of a 8-12 mM NaHB4 solution. 00μL; 5-bromosalicylic acid (analytical grade >98.00%) 0.5-0.8g; 2-15mL of 2-6mM AgNO3 solution; 75-400μL of 0.008-0.02M AgNO3 solution; 1.5-3mL of 0.04-0.1M ascorbic acid solution; 100-500μL of 0.08-0.2M ascorbic acid solution; 50-200μL of 0.08-0.12M ascorbic acid solution; 160-320μL of 0.02-20mM signal molecule 4-NBT solution; 150-700μL of 0.08-0.2M NaOH solution.

[0006] A method for preparing a capsule-shaped internal standard gap Raman probe comprises the following steps:

[0007] Step 1, prepare a seed solution; take 2-8 mL of a 0.08-0.2 mM hexadecyltrimethylammonium bromide (CTAB) solution and mix it with 50-200 μL of a 23-28 mM HAuCl4 solution; then add 600-1000 μL of an 8-12 mM NaHB4 solution, stir evenly at room temperature until the solution color changes to brown, to obtain a seed solution, and keep it warm at 30-50 ° C for later use;

[0008] Step 2, prepare gold nanorod solution; take 200-500mL of 0.12-0.55M hexadecyltrimethylammonium bromide (CTAB) solution, add 0.5-0.8g of 5-bromosalicylic acid, dissolve by ultrasonication at 50-100kHZ for 5-10min, and keep warm in a water bath at 30-50℃; then add 2-6mM After 2-15mL of AgNO3 solution is shaken and mixed, it is allowed to stand for 10-20min, and then 5-10mL of HAuCl4 solution with a concentration of 23-28mM and 145-150mL of ultrapure water are added thereto, and stirred evenly at room temperature; then 1.5-3mL of ascorbic acid solution with a concentration of 0.04-0.1M is added, and after the solution becomes colorless, 300-600μL of the seed solution obtained in step 1 is added, the bottle mouth is sealed, and the solution is kept at 30-50℃ and allowed to stand for 5-12h to obtain a gold nanorod solution; 100-300mL of the gold nanorod solution is taken and centrifuged at 8000-10000rpm / min at room temperature for 10-15min; after removing the supernatant, 100-200mL of hexadecyltrimethylammonium bromide (CTAB) solution with a concentration of 0.06-0.1mM is added, and the longitudinal absorption peak intensity of the gold nanorod solution is adjusted to 1.0;

[0009] Step 3, prepare capsule-shaped internal standard Raman signal molecule 4-NBT gold core silver shell nanorods; take 10-20mL of gold nanorod solution with a longitudinal absorption peak intensity of 1.0, add 160-320μL of signal molecule 4-NBT solution with a concentration of 0.02-20mM, and stir at room temperature 10000-12000rpm / min for 10-20min to connect the signal molecule 4-NBT to the surface of the gold nanorods; then, add 75-400μL of AgNO3 solution with a concentration of 0.008-0.02M, 100-500μL of ascorbic acid solution with a concentration of 0.08-0.2M, and 100-500μL of NaOH solution with a concentration of 0.08-0.2M in sequence; after mixing evenly, let it stand in a water bath at 60-75℃ for 5-60min to grow a silver layer to obtain capsule-shaped internal standard Raman signal molecule 4-NBT gold core silver shell nanorods;

[0010] Step 4. Prepare capsule-shaped internal standard gap Au@AuAg nanoprobe: take 2-5mL of 0.08-0.2M hexadecyltrimethylammonium bromide (CTAB) solution in a test tube, add 50-200μL of 0.8-1.2mM HAuCl4 solution, 50-200μL of 0.08-0.12M ascorbic acid solution and 50-200μL of 0.08-0.2M NaOH solution in sequence, and finally add 2-5mL of the capsule-shaped internal standard Raman signal molecule 4-NBT gold core silver shell nanorod solution obtained in step 3. After mixing evenly, keep warm in a water bath at 30-50℃ and let stand for 8-12h to obtain capsule-shaped internal standard gap Au@Au Ag nanoparticles.

[0011] The length of the gold nanorods in the gold nanorod solution obtained in step 2 is 30-100 nm.

[0012] In step 3, 4-NBT molecules are chemically adsorbed onto gold nanorods through Au-S bonds, and then a silver layer is grown to form a capsule-shaped gold core-silver shell structure, achieving capsule-shaped shaping.

[0013] The thickness of the silver layer of the gold core silver shell nanorods of the internal standard Raman signal molecule 4-NBT obtained in step 3 is 1-30 nm.

[0014] In step 4, the NaOH solution is an alkaline regulating agent for silver layer replacement etching, and the NaOH solution can be replaced by potassium hydroxide solution or ammonia water.

[0015] The gap size of the capsule-shaped internal standard gap Au@Au Ag nanoparticles prepared in step 4 is 1-15 nm.

[0016] A capsule-shaped internal standard gap Raman probe is prepared from raw materials with the component ratio or the preparation method of the capsule-shaped internal standard gap Raman probe.

[0017] A capsule-shaped internal standard gap Raman probe is used in the fields of in vitro diagnosis, biomedical Raman imaging, and biological anti-counterfeiting technology.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. Nanostructure Innovation: Capsule-like Gap Design Achieves Synergistic Signal Enhancement

[0020] 1. Core-shell-gap dual coupling effect

[0021] The electromagnetic field enhancement of traditional core-shell nanoparticles (such as Au@Ag) mainly depends on the core-shell interface. However, the present invention introduces a capsule-like gap structure to form a controllable nanogap (1-15nm) between the core-shell outer layer and the capsule shell, so that the local electromagnetic field is doubly superimposed at the core-shell interface and the gap region, producing a synergistic enhancement effect.

[0022] 2. Internal standard molecules have advantages in anti-interference of Raman signals

[0023] Reduce external interference and improve measurement stability. Because the internal standard method measures specific peaks within the sample, these peaks are highly stable and less susceptible to changes in external conditions. This allows the internal standard method to maintain high stability and accuracy during long-term measurements or continuous monitoring.

[0024] 2. Preparation process optimization: green controllability and batch stability

[0025] 1. Improvement of seed solution synthesis process

[0026] In step 1, the 5-bromosalicylic acid reduction method replaces the traditional silver nitrate-assisted growth method. Through the synergistic effect of bromide ions (Br-) and 5-bromosalicylic acid, the aspect ratio of the gold nanorods can be precisely controlled. Compared to existing technologies, the present invention improves product uniformity and avoids structural distortion during the subsequent coating process.

[0027] 2. Improved controllability of core-shell coating

[0028] In step 2, AgNO3 and NaOH are added stepwise, and the reaction temperature (65°C) and stirring rate are controlled to achieve uniform growth of the silver shell thickness. The traditional co-precipitation method is prone to cracking of the silver shell due to uneven ion diffusion. However, the present invention uses the gradient reduction effect of ascorbic acid (ascorbic acid) to cause the orderly deposition of silver atoms along the axis of the gold nanorods, reducing the roughness of the core-shell interface.

[0029] 3. Breakthrough in capsule packaging technology

[0030] Step 3 employs a low-temperature dynamic self-assembly method (30°C water bath), using CTAB micelle templates to guide the epitaxial growth of gold atoms within the core-shell structure, forming a closed capsule structure. Compared to traditional high-temperature reduction methods, this method avoids thermal decomposition of the internal standard molecule and provides controllable capsule shell porosity, effectively protecting the internal standard while allowing the free diffusion of small molecule targets.

[0031] 3. Performance advantages: improved sensitivity, stability and applicability

[0032] 1. Strong ability to resist environmental interference

[0033] The physical encapsulation of the core-shell structure by the capsule shell prevents the erosion of the core by external oxidants, biological macromolecules, etc.

[0034] 2. Has wide spectrum detection compatibility

[0035] The adjustability of the capsule gap (controlled by changing the HAuCl4 feed amount) makes it adaptable to target molecules of different sizes (such as small drug molecules, proteins, and nucleic acids).

[0036] 4. Application scenarios have been expanded

[0037] This probe can be effectively used in:

[0038] 1. In vivo real-time monitoring: The bioinertness of the capsule structure supports in situ detection of the tumor microenvironment;

[0039] 2. Multi-component simultaneous analysis: By designing different internal standard molecules, multiple markers can be detected together;

[0040] 3. Portable rapid testing equipment: High stability supports long-term storage and use in the field or POCT scenarios.

[0041] In summary, the capsule-shaped internal standard gap Raman probe and its preparation method provided by the present invention significantly improve the performance of surface-enhanced Raman scattering (SERS) probes through structural design and process innovation; they break through the bottlenecks of existing technologies in sensitivity, anti-interference and preparation efficiency, and provide innovative tools for precision medical diagnosis and trace analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 TEM image of gold nanorods.

[0043] Figure 2 TEM image of gold-silver core-shell nanorods with internal standard signal molecules.

[0044] Figure 3 Capsule-shaped internal standard interstitial Au@AuAg nanoparticles.

[0045] Figure 4 These are the absorption spectra of three types of nanoparticles: gold nanorods, gold-silver core-shell nanorods with internal standard signal molecules, and capsule-shaped interstitial gold-silver core-shell nanoparticles with internal standard molecules.

[0046] Figure 5 Raman spectrum of capsule-shaped internal standard interstitial Au@AuAg nanoparticles.

[0047] Figure 6 Absorption spectra (a) and corresponding (bf) TEM images of gold core-silver shell nanorods synthesized with internal standard 4-NBT in different volumes of AgNO3 solution and their corresponding (gk) silver shell thickness distribution.

[0048] Figure 7 Absorption spectra (a) and corresponding (bf) TEM images of Au@AuAg capsule-shaped internal gap nanoparticles prepared with different Ag shell thicknesses and their corresponding (gk) internal gap thickness distributions. DETAILED DESCRIPTION

[0049] The technical solution of the present invention will be described in detail below through specific embodiments:

[0050] This invention employs a "shaping followed by displacement etching" approach to prepare encapsulated internal standard gap Raman probes. First, gold nanorods are prepared using a seed-mediated growth method involving pentabromosalicylic acid. Next, encapsulated gold core-silver shell nanorods embedded with the Raman signal molecule 4-NBT are prepared. Finally, an electrochemical displacement method is used to prepare encapsulated internal standard gap core-shell nanoprobes, which are then characterized by Raman spectroscopy. The specific steps are as follows:

[0051] Example 1

[0052] A method for preparing a capsule-shaped internal standard gap Raman probe specifically comprises the following steps:

[0053] 1) Prepare a seed solution; mix 2 mL of a 0.08 mM cetyltrimethylammonium bromide (CTAB) solution with 50 μL of a 23 mM HAuCl4 solution; then add 600 μL of an 8 mM NaHB4 solution and stir evenly at room temperature until the solution changes color to brown to obtain the seed solution. Keep the mixture at 30°C until ready for use;

[0054] 2) Preparation of gold nanorod solution; 100 mL of 0.12 M hexadecyltrimethylammonium bromide (CTAB) solution was added, 0.5 g of 5-bromosalicylic acid was added, ultrasonically dissolved at 50 kHz for 5 min, and kept warm in a water bath at 30°C; 2 mL of 2 mM AgNO3 solution was then added and shaken to mix, then allowed to stand for 10 min, 5 mL of 23 mM HAuCl4 solution and 145 mL of ultrapure water were added thereto, and stirred evenly at room temperature; 1.5 mL of 0.04 M ascorbic acid solution was then added, and after the solution became colorless, 300 μL of the seed solution obtained in step 1 was added, the bottle was sealed, and allowed to stand at 30°C for 5 h; a gold nanorod solution ( Figure 1 TEM image of gold nanorods); 100 mL of the gold nanorod solution was centrifuged at 8000 rpm / min for 10 min at room temperature; after removing the supernatant, 100 mL of a 0.06 mM hexadecyltrimethylammonium bromide (CTAB) solution was added to adjust the longitudinal absorption peak intensity of the gold nanorod solution to 1.0;

[0055] 3) Preparation of capsule-shaped gold core silver shell nanorods containing the internal standard Raman signal molecule 4-NBT; 10 mL of a gold nanorod solution having a longitudinal absorption peak intensity of 1.0 was added thereto 160 μL of a signal molecule 4-NBT solution having a concentration of 0.02 mM, and stirred at room temperature at 10,000 rpm / min for 10 min to connect the signal molecule 4-NBT to the surface of the gold nanorods; then, 75 μL of a 0.008 M AgNO3 solution, 100 μL of a 0.08 M ascorbic acid solution, and 100 μL of a 0.08 M NaOH solution were added in sequence; after uniform mixing, the mixture was placed in a 60°C water bath and allowed to stand for 5 min to grow a silver layer, thereby obtaining capsule-shaped gold core silver shell nanorods containing the internal standard Raman signal molecule 4-NBT; Figure 2 TEM image of gold and silver core-shell nanorods containing internal standard signal molecules;

[0056] 4) Preparation of capsule-shaped internal standard interstitial Au@Au Ag nanoprobes: Take 2-5 mL of 0.08 M hexadecyltrimethylammonium bromide (CTAB) solution in a test tube, add 50 μL of 0.8 mM HAuCl4 solution, 50 μL of 0.08 M ascorbic acid solution and 50 μL of 0.08 M NaOH solution in sequence, and finally add 2 mL of the capsule-shaped internal standard Raman signal molecule 4-NBT gold core silver shell nanorod solution obtained in step 3. After mixing evenly, keep warm in a 30°C water bath and let stand for 8 hours to obtain capsule-shaped internal standard interstitial Au@AuAg nanoparticles. Figure 3 It is a capsule-shaped internal standard interstitial gold and silver core-shell nanoparticle; Figure 4 Absorption spectra of three nanoparticles: gold nanorods, gold-silver core-shell nanorods with internal standard signal molecules, and capsule-shaped internal standard interstitial gold-silver core-shell nanoparticles; Figure 5 Raman spectrum of capsule-shaped internal standard interstitial Au@AuAg nanoparticles.

[0057] Example 2

[0058] A method for preparing a capsule-shaped internal standard gap Raman probe specifically comprises the following steps:

[0059] 1) Prepare a seed solution; mix 3 mL of a 0.1 mM cetyltrimethylammonium bromide (CTAB) solution with 100 μL of a 24 mM HAuCl4 solution; then add 650 μL of a 9 mM NaHB4 solution and stir evenly at room temperature until the solution changes color to brown to obtain the seed solution. Keep the mixture at 35°C until ready for use.

[0060] 2) Preparation of gold nanorod solution: 200 mL of 0.2 M hexadecyltrimethylammonium bromide (CTAB) solution was added to 0.55 g of 5-bromosalicylic acid, and ultrasonically dissolved at 60 kHz for 6 min, and then kept warm in a 35°C water bath; then 5 mL of 3 mM AgNO3 solution was added and shaken to mix, and then allowed to stand for 12 min, and then 6 mL of 24 mM HAuCl4 solution and 146 mL of ultrapure water were added thereto, and stirred at room temperature; then the concentration of Prepare a 0.05M ascorbic acid solution (1.7 mL). After the solution becomes colorless, add 350 μL of the seed solution obtained in step 1, seal the bottle, and incubate at 35°C for 6 h to obtain a gold nanorod solution. Take 150 mL of the gold nanorod solution and centrifuge at 8500 rpm / min for 11 min at room temperature. After removing the supernatant, add 120 mL of a 0.07 mM hexadecyltrimethylammonium bromide (CTAB) solution and adjust the longitudinal absorption peak intensity of the gold nanorod solution to 1.0.

[0061] 3) Preparation of capsule-shaped gold core silver shell nanorods containing the internal standard Raman signal molecule 4-NBT; 15 mL of a gold nanorod solution having a longitudinal absorption peak intensity of 1.0 was added thereto 180 μL of a 0.1 mM signal molecule 4-NBT solution, and the mixture was stirred at room temperature at 11,000 rpm / min for 12 min to attach the signal molecule 4-NBT to the surface of the gold nanorods; then, 100 μL of a 0.01 M AgNO3 solution, 200 μL of a 0.1 M ascorbic acid solution, and 200 μL of a 0.1 M NaOH solution were added in sequence; after uniform mixing, the mixture was placed in a 65°C water bath and allowed to stand for 10 min to grow a silver layer, thereby obtaining capsule-shaped gold core silver shell nanorods containing the internal standard Raman signal molecule 4-NBT;

[0062] 4) Preparation of capsule-shaped internal standard interstitial Au@AuAg nanoprobes: Take 2-5 mL of 0.1 M hexadecyltrimethylammonium bromide (CTAB) solution in a test tube, add 80 μL of 0.9 mM HAuCl4 solution, 80 μL of 0.09 M ascorbic acid solution and 80 μL of 0.09 M NaOH solution in sequence, and finally add 2.5 mL of the capsule-shaped internal standard Raman signal molecule 4-NBT gold core silver shell nanorod solution obtained in step 3. After mixing evenly, keep warm in a 35°C water bath and let stand for 9 hours to obtain capsule-shaped internal standard interstitial Au@Au Ag nanoparticles.

[0063] Example 3

[0064] A method for preparing a capsule-shaped internal standard gap Raman probe specifically comprises the following steps:

[0065] 1) Prepare a seed solution; take 5 mL of a 0.12 mM cetyltrimethylammonium bromide (CTAB) solution and mix it with 150 μL of a 25 mM HAuCl4 solution; then add 750 μL of a 10 mM NaHB4 solution and stir evenly at room temperature until the solution turns brown to obtain the seed solution. Keep the mixture at 40°C until ready for use;

[0066] 2) Preparation of gold nanorod solution: 300 mL of 0.3 M hexadecyltrimethylammonium bromide (CTAB) solution was added to 0.7 g of 5-bromosalicylic acid, and ultrasonically dissolved at 70 kHz for 7 min, and then kept warm in a 40°C water bath; then 8 mL of 4 mM AgNO3 solution was added and shaken to mix, and then allowed to stand for 17 min, and then 7 mL of 25 mM HAuCl4 solution and 147 mL of ultrapure water were added thereto, and stirred at room temperature; then the concentration of Prepare a 0.06M ascorbic acid solution (2 mL). After the solution becomes colorless, add 400 μL of the seed solution obtained in step 1, seal the bottle, and incubate at 40°C for 7 h to obtain a gold nanorod solution. Take 175 mL of the gold nanorod solution and centrifuge at 9000 rpm / min for 12 min at room temperature. After removing the supernatant, add 170 mL of a 0.08 mM hexadecyltrimethylammonium bromide (CTAB) solution and adjust the longitudinal absorption peak intensity of the gold nanorod solution to 1.0.

[0067] 3) Preparation of capsule-shaped gold core silver shell nanorods containing the internal standard Raman signal molecule 4-NBT; 17 mL of a gold nanorod solution having a longitudinal absorption peak intensity of 1.0 was added to 200 μL of a 1 mM signal molecule 4-NBT solution, and the mixture was stirred at room temperature at 11500 rpm / min for 17 min to attach the signal molecule 4-NBT to the surface of the gold nanorods; then, 150 μL of a 0.012 M AgNO3 solution, 300 μL of a 0.12 M ascorbic acid solution, and 300 μL of a 0.12 M NaOH solution were added in sequence; after mixing evenly, the mixture was placed in a 67°C water bath and allowed to stand for 15 min to grow a silver layer, thereby obtaining capsule-shaped gold core silver shell nanorods containing the internal standard Raman signal molecule 4-NBT;

[0068] 4) Preparation of capsule-shaped internal standard interstitial Au@AuAg nanoprobes: Take 3.5 mL of 0.12 M hexadecyltrimethylammonium bromide (CTAB) solution in a test tube, add 135 μL of 1.0 mM HAuCl4 solution, 150 μL of 0.1 M ascorbic acid solution and 135 μL of 0.1 M NaOH solution in sequence, and finally add 3.5 mL of the capsule-shaped internal standard Raman signal molecule 4-NBT gold core silver shell nanorod solution obtained in step 3. After mixing evenly, keep warm in a 40°C water bath and let stand for 10 hours to obtain capsule-shaped internal standard interstitial Au@AuAg nanoparticles.

[0069] Example 4

[0070] A method for preparing a capsule-shaped internal standard gap Raman probe specifically comprises the following steps:

[0071] 1) Prepare a seed solution; mix 7.5 mL of a 0.16 mM cetyltrimethylammonium bromide (CTAB) solution with 180 μL of a 27 mM HAuCl4 solution; then add 900 μL of an 11 mM NaHB4 solution and stir evenly at room temperature until the solution changes color to brown to obtain the seed solution. Keep the mixture at 45°C until ready for use;

[0072] 2) Preparation of gold nanorod solution: 400 mL of 0.4 M hexadecyltrimethylammonium bromide (CTAB) solution was added, 0.75 g of 5-bromosalicylic acid was added, ultrasonically dissolved at 80 kHz for 9 min, and kept warm in a 45°C water bath; then 13 mL of 5 mM AgNO3 solution was added and shaken to mix, and the mixture was allowed to stand for 18 min. Then, 9 mL of 27 mM HAuCl4 solution and 148 mL of ultrapure water were added, and the mixture was stirred at room temperature; then, 1 mL of 5 mM HAuCl4 solution was added and the mixture was stirred at room temperature. Prepare a 0.08M ascorbic acid solution (2.8 mL). After the solution becomes colorless, add 550 μL of the seed solution obtained in step 1, seal the bottle, and incubate at 45°C for 10 h to obtain a gold nanorod solution. Take 275 mL of the gold nanorod solution and centrifuge at 9800 rpm / min for 14 min at room temperature. After removing the supernatant, add 180 mL of a 0.08 mM hexadecyltrimethylammonium bromide (CTAB) solution and adjust the longitudinal absorption peak intensity of the gold nanorod solution to 1.0.

[0073] 3) Preparation of capsule-shaped gold core silver shell nanorods containing the internal standard Raman signal molecule 4-NBT; 18 mL of a gold nanorod solution having a longitudinal absorption peak intensity of 1.0 was added to 300 μL of a 10 mM signal molecule 4-NBT solution, and the mixture was stirred at room temperature at 11500 rpm / min for 18 min to attach the signal molecule 4-NBT to the surface of the gold nanorods; then, 350 μL of a 0.018 M AgNO3 solution, 450 μL of a 0.18 M ascorbic acid solution, and 450 μL of a 0.18 M NaOH solution were added in sequence; after uniform mixing, the mixture was placed in a 72°C water bath and allowed to stand for 50 min to grow a silver layer, thereby obtaining capsule-shaped gold core silver shell nanorods containing the internal standard Raman signal molecule 4-NBT;

[0074] 4) Preparation of capsule-shaped internal standard interstitial Au@AuAg nanoprobes: Take 2.5 mL of 0.18 M hexadecyltrimethylammonium bromide (CTAB) solution in a test tube, add 150 μL of 1.1 mM HAuCl4 solution, 180 μL of 0.11 M ascorbic acid solution and 200 μL of 0.11 M NaOH solution in sequence, and finally add 4 mL of the capsule-shaped internal standard Raman signal molecule 4-NBT gold core silver shell nanorod solution obtained in step 3. After mixing evenly, keep warm in a 45°C water bath and let stand for 11 hours to obtain capsule-shaped internal standard interstitial Au@AuAg nanoparticles.

[0075] Example 5

[0076] A method for preparing a capsule-shaped internal standard gap Raman probe specifically comprises the following steps:

[0077] 1) Prepare a seed solution; mix 8 mL of a 0.2 mM cetyltrimethylammonium bromide (CTAB) solution with 200 μL of a 28 mM HAuCl4 solution; then add 1000 μL of a 12 mM NaHB4 solution and stir evenly at room temperature until the solution changes color to brown to obtain the seed solution. Keep the mixture at 50°C until ready for use;

[0078] 2) Preparation of gold nanorod solution: 500 mL of 0.55 M hexadecyltrimethylammonium bromide (CTAB) solution was added, 0.8 g of 5-bromosalicylic acid was added, and ultrasonic dissolution was performed at 100 kHz for 10 min, and the mixture was kept warm in a 50°C water bath; 15 mL of 6 mM AgNO3 solution was then added and shaken to mix, and the mixture was allowed to stand for 20 min. 10 mL of 28 mM HAuCl4 solution and 150 mL of ultrapure water were then added, and the mixture was stirred at room temperature; the mixture was then added Add 3 mL of 0.1 M ascorbic acid solution, and after the solution becomes colorless, add 600 μL of the seed solution obtained in step 1, seal the bottle, and incubate at 50°C for 12 hours to obtain a gold nanorod solution; take 300 mL of the gold nanorod solution, centrifuge at 10,000 rpm / min at room temperature for 15 minutes; after removing the supernatant, add 200 mL of 0.1 mM hexadecyltrimethylammonium bromide (CTAB) solution, and adjust the longitudinal absorption peak intensity of the gold nanorod solution to 1.0;

[0079] 3) Preparation of capsule-shaped gold core silver shell nanorods containing the internal standard Raman signal molecule 4-NBT; 20 mL of a gold nanorod solution having a longitudinal absorption peak intensity of 1.0 was added to 320 μL of a 20 mM signal molecule 4-NBT solution, and the mixture was stirred at room temperature for 20 min at 12,000 rpm / min to attach the signal molecule 4-NBT to the surface of the gold nanorods; then, 400 μL of a 0.02 M AgNO3 solution, 500 μL of a 0.2 M ascorbic acid solution, and 500 μL of a 0.2 M NaOH solution were added in sequence; after uniform mixing, the mixture was placed in a 75°C water bath and allowed to stand for 60 min to grow a silver layer, thereby obtaining capsule-shaped gold core silver shell nanorods containing the internal standard Raman signal molecule 4-NBT;

[0080] 4) Preparation of capsule-shaped internal standard interstitial Au@AuAg nanoprobes: Take 5 mL of 0.2 M hexadecyltrimethylammonium bromide (CTAB) solution in a test tube, add 180 μL of 1.2 mM HAuCl4 solution, 200 μL of 0.12 M ascorbic acid solution and 200 μL of 0.12 M NaOH solution in sequence, and finally add 5 mL of the capsule-shaped internal standard Raman signal molecule 4-NBT gold core silver shell nanorod solution obtained in step 3. After mixing evenly, keep warm in a 50°C water bath and let stand for 12 hours to obtain capsule-shaped internal standard interstitial Au@AuAg nanoparticles.

[0081] Experimental verification:

[0082] During the synthesis of gold core silver shell nanorods in step 3, the amounts of other reagents were kept constant, and gold core silver shell nanorods with different silver shell thicknesses were prepared by adjusting the volume of AgNO3 solution (75 μL, 125 μL, 175 μL, 225 μL, 275 μL). Figure 6 As shown in (a), when the volume of the AgNO3 solution initially increases, the longitudinal LSPR absorption peak of the Au-Ag interface in the long wavelength region shows a blue shift and its intensity increases, and a new absorption peak is observed at 345nm. As the volume of the AgNO3 solution further increases, the intensity of the new absorption peak continues to increase and red shifts. Figure 6 The transmission electron microscopy (TEM) characterization results of (bf) show that the prepared gold core silver shell nanorods exhibit good monodispersity and size uniformity, and present a clearly identifiable capsule-shaped core-shell structure. In addition, by counting the silver shell thickness of the gold core silver shell nanoparticles in the TEM images, the silver shell thickness distribution diagram under different AgNO3 solution volumes was drawn ( Figure 6Quantitative data analysis showed that when the volumes of the AgNO3 solutions were 75 μL, 125 μL, 175 μL, 225 μL, and 275 μL, respectively, the average silver shell thicknesses of the prepared gold-core-silver-shell nanorods were 3.4 nm, 5.4 nm, 6.4 nm, 8.9 nm, and 9.3 nm, respectively.

[0083] Next, the regulation mechanism of the silver shell thickness on the morphology of Au@AuAg capsule-shaped internal standard gap nanoparticles was investigated. In the synthesis process of Au@AuAg capsule-shaped internal standard gap nanoparticles in step 4, the volume of HAuCl4 solution was kept constant at 200μL, and a series of gold core silver shell nanorods were prepared with 75μL, 125μL, 175μL, 225μL and 275μL of AgNO3 solution respectively, and used as sacrificial templates to construct Au@AuAg capsule-shaped internal standard gap nanoparticles. Figure 7 As can be seen from the absorption spectrum of (a), as the thickness of the silver shell increases, the longitudinal localized surface plasmon resonance (LSPR) absorption peak of the Au@AuAg capsule-shaped internal standard interstitial nanoparticles not only shows a blue shift, but also its intensity gradually increases. At the same time, the distance between the transverse and longitudinal LSPR absorption peaks gradually narrows. Transmission electron microscopy characterization results ( Figure 7 (bf)) clearly shows that the internal gap size of Au@AuAg capsule-shaped internal standard gap nanoparticles gradually increases as the silver shell thickness increases. Figure 7 Statistical analysis of the internal gap thickness distribution data in (gk) revealed that the average internal gap thicknesses of the prepared Au@AuAg capsular internal standard gap nanoparticles were 1.8nm, 3.0nm, 3.7nm, 5.2nm, and 7.2nm, respectively, when the silver shell thicknesses were 3.4nm, 5.4nm, 6.4nm, 8.9nm, and 9.3nm. Notably, the AuAg alloy shell maintained a stable capsular geometry throughout the entire control process.

[0084] The present invention discloses a method for preparing and applying a capsule-shaped internal standard gap Raman probe. First, gold nanorods are prepared by a seed-mediated growth method involving pentabromosalicylic acid; then, capsule-shaped gold core-silver shell nanorods embedded with the Raman signal molecule 4-NBT are prepared; finally, an electrochemical replacement method is used to prepare a capsule-shaped internal standard gap core-shell nanoprobe. The capsule gold core-silver shell nanoprobe is prepared at 1331 cm -1 The Raman intensity increased significantly.

[0085] The above content is only for explaining the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A capsule-shaped internal standard gap Raman probe, characterized in that: The invention comprises raw materials with the following component proportions: 4-13 mL of a 0.08-0.2 mM hexadecyltrimethylammonium bromide (CTAB) solution; 200-500 mL of a 0.12-0.55 M hexadecyltrimethylammonium bromide (CTAB) solution; 100-200 mL of a 0.06-0.1 mM hexadecyltrimethylammonium bromide (CTAB) solution; 5.05-10.2 mL of a 23-28 mM HAuCl4 solution; 50-200 μL of a 0.8-1.2 mM HAuCl4 solution; 600-1000 μL of a 8-12 mM NaHB4 solution; and 5- Bromosalicylic acid (analytical grade >98.00%) 0.5-0.8 g; 2-15 mL of 2-6 mM AgNO3 solution; 75-400 μL of 0.008-0.02 M AgNO3 solution; 1.5-3 mL of 0.04-0.1 M ascorbic acid solution; 100-500 μL of 0.08-0.2 M ascorbic acid solution; 50-200 μL of 0.08-0.12 M ascorbic acid solution; 160-320 μL of 0.02-20 mM signaling molecule 4-NBT solution; 150-700 μL of 0.08-0.2 M NaOH solution.

2. A method for preparing a capsule-shaped internal standard gap Raman probe, characterized in that: The following steps are involved: Step 1, prepare a seed solution; take 2-8 mL of a 0.08-0.2 mM hexadecyltrimethylammonium bromide (CTAB) solution and mix it with 50-200 μL of a 23-28 mM HAuCl4 solution; then add 600-1000 μL of an 8-12 mM NaHB4 solution, stir evenly at room temperature until the solution color changes to brown, to obtain a seed solution, and keep it warm at 30-50 ° C for later use; Step 2, prepare gold nanorod solution; take 200-500mL of 0.12-0.55M hexadecyltrimethylammonium bromide (CTAB) solution, add 0.5-0.8g of 5-bromosalicylic acid, dissolve by ultrasonication at 50-100kHZ for 5-10min, and keep warm in a water bath at 30-50℃; then add 2-6mM After 2-15mL of AgNO3 solution is shaken and mixed, it is allowed to stand for 10-20min, and then 5-10mL of HAuCl4 solution with a concentration of 23-28mM and 145-150mL of ultrapure water are added thereto, and stirred evenly at room temperature; then 1.5-3mL of ascorbic acid solution with a concentration of 0.04-0.1M is added, and after the solution becomes colorless, 300-600μL of the seed solution obtained in step 1 is added, the bottle mouth is sealed, and the solution is kept at 30-50℃ and allowed to stand for 5-12h to obtain a gold nanorod solution; 100-300mL of the gold nanorod solution is taken and centrifuged at 8000-10000rpm / min at room temperature for 10-15min; after removing the supernatant, 100-200mL of hexadecyltrimethylammonium bromide (CTAB) solution with a concentration of 0.06-0.1mM is added, and the longitudinal absorption peak intensity of the gold nanorod solution is adjusted to 1.0; Step 3, prepare capsule-shaped internal standard Raman signal molecule 4-NBT gold core silver shell nanorods; take 10-20mL of gold nanorod solution with a longitudinal absorption peak intensity of 1.0, add 160-320μL of signal molecule 4-NBT solution with a concentration of 0.02-20mM, and stir at room temperature 10000-12000rpm / min for 10-20min to connect the signal molecule 4-NBT to the surface of the gold nanorods; then, add 75-400μL of AgNO3 solution with a concentration of 0.008-0.02M, 100-500μL of ascorbic acid solution with a concentration of 0.08-0.2M, and 100-500μL of NaOH solution with a concentration of 0.08-0.2M in sequence; after mixing evenly, let it stand in a water bath at 60-75℃ for 5-60min to grow a silver layer to obtain capsule-shaped internal standard Raman signal molecule 4-NBT gold core silver shell nanorods; Step 4. Prepare capsule-shaped internal standard gap Au@AuAg nanoprobe: take 2-5mL of 0.08-0.2M hexadecyltrimethylammonium bromide (CTAB) solution in a test tube, add 50-200μL of 0.8-1.2mM HAuCl4 solution, 50-200μL of 0.08-0.12M ascorbic acid solution and 50-200μL of 0.08-0.2M NaOH solution in sequence, and finally add 2-5mL of the capsule-shaped internal standard Raman signal molecule 4-NBT gold core silver shell nanorod solution obtained in step 3. After mixing evenly, keep warm in a water bath at 30-50℃ and let stand for 8-12h to obtain capsule-shaped internal standard gap Au@AuAg nanoparticles.

3. The method for preparing a capsule-shaped internal standard gap Raman probe according to claim 2, characterized in that: The length of the gold nanorods in the gold nanorod solution obtained in step 2 is 30-100 nm.

4. The method for preparing a capsule-shaped internal standard gap Raman probe according to claim 2, characterized in that: In step 3, 4-NBT molecules are chemically adsorbed onto gold nanorods through Au-S bonds, and then a silver layer is grown to form a capsule-shaped gold core-silver shell structure, achieving capsule-shaped shaping.

5. The method for preparing a capsule-shaped internal standard gap Raman probe according to claim 2, characterized in that: The thickness of the silver layer of the gold core silver shell nanorods of the internal standard Raman signal molecule 4-NBT obtained in step 3 is 1-30 nm.

6. The method for preparing a capsule-shaped internal standard gap Raman probe according to claim 1, characterized in that: In step 4, the NaOH solution is an alkaline regulating agent for silver layer replacement etching, and the NaOH solution can be replaced by potassium hydroxide solution or ammonia water.

7. The method for preparing a capsule-shaped internal standard gap Raman probe according to claim 1, characterized in that: The gap size of the capsule-shaped internal standard gap Au@Au Ag nanoparticles prepared in step 4 is 1-15 nm.

8. A capsule-shaped internal standard gap Raman probe, characterized in that: The invention is prepared by the raw material of claim 1 or the preparation method of the capsule-shaped internal standard gap Raman probe according to any one of claims 2 to 7.

9. The capsule-shaped internal standard gap Raman probe according to claim 8 is used in the fields of in vitro diagnosis, biomedical Raman imaging, and biological anti-counterfeiting technology.

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