Composite nano material as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of in-situ detection and preparation of composite nanomaterials, and particularly relates to a composite nanomaterial as well as a preparation method and application thereof. The nano material is a functional nano composite material which is loaded with gold nanoparticles and probe molecules prepared through organic synthesis and is coated with a hyaluronic acid film. The nano-material carrier is prepared by taking potassium permanganate and potassium oxalate as raw materials; the Raman active molecular probe is modified by the gold nanoparticles through a gold-sulfur bond forming principle. The preparation process is simple, the cost is low, the operation is easy, the prepared nano composite material has high sensitivity, high selectivity and excellent biocompatibility, simultaneous detection of SERS channels of two biomarkers, namely GSH and APN, can be realized, and the application prospect is wide. The method can be used for accurately, efficiently and quickly distinguishing normal cells from tumor cells clinically by detecting biomarkers with different contents in the normal cells and the tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of in-situ detection and composite nanomaterial preparation technology, specifically relating to a composite nanomaterial, its preparation method and application. Background Technology

[0002] Due to their uncontrolled proliferation, invasion, and metastasis, tumor cells have become a major global health threat. The harmful mechanisms of tumors include: excessive growth of the primary tumor leading to organ failure (such as liver failure caused by liver cancer), invasion of nerves or bones causing severe pain, and distant metastasis (the spread of cancer cells through the blood / lymphatic system to form new lesions, such as brain metastasis and bone metastasis) being the root cause of approximately 90% of cancer-related deaths.

[0003] Currently, the core methods for clinical tumor detection include medical imaging (such as CT, MRI, and PET-CT) and histopathological biopsy. Imaging techniques can non-invasively locate tumor lesions and assess metastasis, but their resolution is limited, making it difficult to detect tiny early tumors or trace metastatic cells (“minimal residual disease”), and sometimes it is difficult to distinguish between benign and malignant tumors. Histopathology is the “gold standard” for diagnosis, but it is an invasive procedure with the risk of sampling bias, and it can only provide a static “snapshot” of the tumor at a certain point in time, unable to reflect the tumor’s gene mutations, molecular subtypes, and evolution during treatment in real time and dynamically. These limitations mean that many tumors are discovered at an intermediate or advanced stage, missing the optimal treatment window. Therefore, accurate and real-time monitoring of the presence of early tumor cells provides an important opportunity for timely intervention and treatment.

[0004] Therefore, in-depth research and development of new detection mechanisms at the molecular level has become crucial. Cancer is essentially a genetic disease, caused by a combination of molecular events such as driver gene mutations, abnormal signaling pathways, and metabolic reprogramming. Molecular detection technologies (such as next-generation sequencing (NGS), liquid biopsy, and mass spectrometry imaging) aim to directly detect these fundamental changes. Currently, researchers are conducting extensive research on detection targets in tumor cells. Among these, glutathione (GSH) and aminopeptidase (APN) have been shown to be potential biomarkers for detecting the presence of tumor cells at the molecular level.

[0005] However, most current methods for measuring GSH and APN rely on single signal identification and focus on a single biomarker, failing to effectively reveal the overall picture. Furthermore, due to the complexity of the structure and composition of biomarkers in tumor cells, systems controlled by a single target may lack specificity, resulting in off-target effects and false positive signals. Therefore, there is an urgent need to develop a novel detection strategy that can simultaneously perform multi-signal identification and multi-component analysis.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a composite nanomaterial, its preparation method and application; the composite nanomaterial has high sensitivity, high selectivity and excellent biocompatibility, and can realize the simultaneous detection of two substances, GSH and APN, biomarkers in cells via SERS channels.

[0008] To overcome the shortcomings of the prior art, the present invention provides the following technical solution: A method for preparing composite nanomaterials includes the following steps: S1. Preparation of gold nanoparticles: Using tetrachloroauric acid as the gold source and ultrapure water as the dispersion medium, sodium citrate was added at 120~130℃ to carry out a reduction reaction; after the reaction was completed, gold nanoparticles were obtained by centrifugation. S2, Preparation of probe molecule C: ① Using 3-aminothiophenol as the raw material and DMSO as the reaction solvent, the reaction was carried out in an oil bath at 75~85℃. The product was separated and purified by column chromatography to obtain intermediate A. ② Using intermediate A and BOC-L-alanine as reaction substrates, HATU as condensation catalyst, and TEA and DMF as mixed solvents, the reaction was carried out in an oil bath at 45~55℃. After extraction and purification, intermediate B was obtained. ③ Under acidic conditions, intermediate B undergoes a deBOC reaction to obtain the target probe molecule C; S3. Preparation of Au-C complex: The probe molecule C and gold nanoparticles were separately prepared into aqueous solutions, then thoroughly mixed at a volume ratio, and a coupling reaction was carried out at room temperature. After the reaction was completed, the Au-C complex was obtained by centrifugation and washing with water. S4. Preparation of core-shell structured Au-C@MnO2 nanoparticles: Au-C complex was dispersed in ultrapure water to form a dispersion, which was placed in an ice-water bath and the pH was adjusted to 9.0-10.0 with an alkaline solution. Then, potassium permanganate solution and potassium oxalate solution were added sequentially. After standing in the ice-water bath, the mixture was transferred to a water bath at 55-65℃ for heating to obtain core-shell structured Au-C@MnO2 nanoparticles. S5, Encapsulated composite nanomaterials: Au-C@MnO2 nanoparticles were prepared into an aqueous solution, mixed with an aqueous solution of hyaluronic acid in a volume ratio, and reacted by stirring at room temperature. After centrifugation and washing with water, the composite nanomaterial was obtained.

[0009] Further, in step S1, the specific amount of raw materials used to prepare gold nanoparticles is as follows: 1 mL of 1% tetrachloroauric acid solution is mixed evenly with 100 mL of ultrapure water; sodium citrate is added in the form of a 1% aqueous solution, and the amount of sodium citrate aqueous solution added is 10 mL.

[0010] Further, in step S2, the concentration of 3-aminothiophenol in DMSO is 0.3~0.5 mol / L; And / or, the reaction time in step ① is 3-5 h, and the column chromatography eluent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 5:1; And / or, in step ②, the molar equivalent ratio of BOC-L-alanine, intermediate A, HATU, and TEA is 2.2:1:4:8, and the reaction time is 20~28h; And / or, the acidic conditions for step ③ are: TFA as solvent, dichloromethane as diluent, BOC removal reaction temperature of 45~55℃, and reaction time of 2~4h.

[0011] Furthermore, in step S3, the concentration of the aqueous solution of probe molecule C is 10. -3 The concentration of the gold nanoparticle aqueous solution was 1% (mol / L), and the volume ratio of the two was 1:1. The coupling reaction time was 25-35 min, the centrifugation speed was 10000 rpm, the centrifugation time was 6-10 min, and the number of water washes was 3.

[0012] Further, in step S4, the volume of the Au-C complex dispersion is 10 mL; the alkaline solution is a 0.08~0.12 mol / L potassium hydroxide solution; the concentration of the potassium permanganate solution is 0.008~0.012 mol / L and the volume is 70~90 μL; the concentration of the potassium oxalate solution is 0.008~0.012 mol / L and the volume is 350~450 μL; the ice-water bath standing time is 8~12 min; and the water bath heating time at 55~65℃ is 1.5~2.5 h.

[0013] Further, in step S5, the concentration of the hyaluronic acid aqueous solution is 4~6 mg / mL; the concentration of the Au-C@MnO2 nanoparticle aqueous solution is 0.8~1.2 mg / mL; the volume ratio of the two is 1:1; the reaction time is 2.5~3.5 h with stirring at room temperature; the centrifugation speed is 10000 rpm; the centrifugation time is 6~10 min; and the number of water washings is 3.

[0014] In addition, the present invention also provides a composite nanomaterial prepared by the preparation method described above; the composite nanomaterial has Au-C as the core, MnO2 as the shell, and is coated with a hyaluronic acid film on the surface, with a particle size of 45~55nm.

[0015] In addition, the present invention also provides the application of the composite nanomaterials described above in detecting biomarkers in cells and distinguishing normal cells from tumor cells, wherein the biomarkers are glutathione and aminopeptidase N.

[0016] Furthermore, the cells included normal hepatocytes (THLE) and hepatocellular carcinoma cells (HepG-2); during GSH detection, the 768 cm⁻¹ region of the SERS spectrum was monitored. -1 1349cm -1 1606cm -1 Changes in signal intensity at a certain point indicate a change in GSH content; a decrease in signal intensity indicates a change in GSH content. When detecting APN, monitoring the 941 cm⁻¹ region in the SERS spectrum... -1 The change in the intensity of the characteristic peak indicates a change in the APN content.

[0017] Furthermore, the detection limit of the composite nanomaterial for APN is 1 ng / mL, and within the range of 0~1 μg / mL, the APN concentration is related to 941 cm⁻¹. -1 The logarithm of the SERS signal strength shows a linear relationship. And / or, the reaction time of the composite nanomaterial with GSH is 8~12 min, and the subsequent reaction time with APN is 3~5 h; after the reaction, 2 μL of the reaction system is aspirated and placed on a silicon wafer for SERS spectroscopy detection.

[0018] Compared with the prior art, the technical solution of the present invention has at least the following technical effects: (1) This invention utilizes the advantages of MnO2, such as high porosity, good adsorption and good stability, to modify TMB molecules and load gold nanoparticles through stepwise modification and coat them with hyaluronic acid film to construct functionalized composite nanomaterials. The preparation process is simple, low cost, easy to operate and easy to prepare in large quantities.

[0019] (2) The composite nanomaterials prepared in this invention modify the hyaluronic acid film, which further improves the stability of the probe and avoids interference from the complex intracellular environment and false positive signals.

[0020] In summary, the preparation process of this invention is simple, low-cost, and easy to operate. The resulting composite nanomaterials exhibit high sensitivity and excellent biocompatibility, enabling SERS dual-material sensing of two biomarkers: glutathione and aminopeptidase N. The development and application of this material are expected to provide a more convenient and efficient monitoring method for distinguishing between normal cells and tumor cells, and offer new insights for the detection of biomarkers in other diseases. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the preparation process of the composite nanomaterial of the present invention; Figure 2 This is a schematic diagram illustrating the detection principle of the composite nanomaterials of the present invention; Figure 3 The image shows the scanning electron microscope (SEM) characterization of the composite nanomaterials prepared in Example 1. Figure 4 This is a diagram demonstrating the feasibility of using the composite nanomaterials prepared in Example 1 for the detection of GSH and APN. Figure 5 The image shows the SERS curve of GSH detected by the composite nanomaterials prepared in Example 1. Figure 6 The SERS spectrum curve (A) and linear correlation diagram (B) of the composite nanomaterials prepared in Example 1 for detecting APN are shown. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Those skilled in the art should understand that the embodiments described are merely illustrative of the invention and should not be considered as specific limitations thereof. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. Process parameters not specifically specified in the following embodiments are generally performed under conventional conditions.

[0023] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0024] According to a first aspect of the present invention, a method for preparing composite nanomaterials is provided, such as... Figure 1 As shown, the workflow demonstrates the complete steps from the synthesis of gold nanoparticles and probe molecules to the Au-C complex, Au-C@MnO2 core-shell particles, and finally hyaluronic acid coating, specifically including the following steps: S1. Preparation of gold nanoparticles: Using tetrachloroauric acid as the gold source and ultrapure water as the dispersion medium, sodium citrate was added at 120~130℃ to carry out a reduction reaction; after the reaction was completed, gold nanoparticles were obtained by centrifugation. The specific process is as follows: 1 mL of 1% tetrachloroauric acid solution is added to 100 mL of ultrapure water and mixed evenly; the mixture is heated to boiling at 120~130℃ (preferably 125℃) and 350 rpm, and then 10 mL of 1% sodium citrate aqueous solution (prepared by dissolving 0.1 g of sodium citrate in 10 mL of ultrapure water) is added, and the mixture is heated and stirred for 3~5 min; after the reaction is completed, the mixture is centrifuged at 10000 rpm, and the precipitate is collected to obtain gold nanoparticles (Au NPs).

[0025] S2, Preparation of probe molecule C: ① Using 3-aminothiophenol as the raw material and DMSO (dimethyl sulfoxide) as the reaction solvent, the reaction was carried out in an oil bath at 75~85℃. The product was separated and purified by column chromatography to obtain intermediate A. The specific process is as follows: 3-Aminobenzylthiophenol (concentration 0.3~0.5mol / L, preferably 0.4mol / L) is dissolved in DMSO (dimethyl sulfoxide), and the concentration of 3-aminobenzylthiophenol in DMSO is 0.3~0.5mol / L (preferably 0.4mol / L). The mixture is stirred in an oil bath at 75~85℃ (preferably 80℃) for 4 hours, and the reaction progress is monitored by TLC. After the reaction solution is cooled to room temperature, it is diluted with ultrapure water. The organic phase is extracted with ethyl acetate and dried with anhydrous sodium sulfate. The crude product is purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1, volume ratio) to obtain intermediate A (3,3-p-aminobenzylthiophenol).

[0026] ② Using intermediate A and BOC-L-alanine as reaction substrates, HATU as condensation catalyst, and TEA and DMF as mixed solvents, the reaction was carried out in an oil bath at 45~55℃. After extraction and purification, intermediate B was obtained. The specific process is as follows: 1 equivalent of intermediate A (3,3-p-aminothiophenol), 2.2~2.5 equivalents (preferably 2.2 equivalents) of BOC-L-alanine, 4~4.5 equivalents (preferably 4 equivalents) of HATU (2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate) catalyst, and 8~8.5 equivalents (preferably 8 equivalents) of TEA (triethylamine) are sequentially added to DMF (N,N-dimethylformamide). The mixture is first stirred at 0°C, then heated to 45~55°C (preferably 50°C) and reacted in an oil bath for 20~28h (preferably 24h). After quenching the reaction solution with ultrapure water, it is extracted with ethyl acetate and dried with anhydrous sodium sulfate to obtain intermediate B. ③ Under acidic conditions, intermediate B undergoes a deBOC reaction to obtain the target probe molecule C; The specific process is as follows: Using TFA (trifluoroacetic acid) as a solvent, intermediate B is placed in a dichloromethane system and stirred at 45~55℃ (preferably 50℃) for 2.5~3.5h (preferably 3h) to remove the BOC protecting group (tert-butyloxycarbonyl); after cooling the reaction solution, it is diluted with ultrapure water, and the organic phase is extracted with ethyl acetate and dried with anhydrous sodium sulfate to obtain a yellow oily probe molecule C(2R,2'R)-N,N'-(disulfonylbis(3,1-benzene))bis(2-aminopropionamide), with the following specific chemical structure: .

[0027] S3. Preparation of Au-C complex: The probe molecule C and gold nanoparticles were separately prepared into aqueous solutions, then thoroughly mixed at a volume ratio, and a coupling reaction was carried out at room temperature. After the reaction was completed, the Au-C complex was obtained by centrifugation and washing with water. The specific process is as follows: Probe molecule C is prepared into 10 using ultrapure water. -3 ~10 -2 mol / L (preferably 10) -3 A solution of gold nanoparticles (mol / L) was mixed with 200 μL of concentrated aqueous solution of gold nanoparticles (concentration of 1%) at a volume ratio of 1:1 and reacted at room temperature (25~30℃) for 25~35 min (preferably 30 min); then centrifuged at 10000 rpm for 7~9 min (preferably 8 min), washed 2~3 times (preferably 3 times) with ultrapure water, and the precipitate was collected to obtain the Au-C complex.

[0028] S4. Preparation of core-shell structured Au-C@MnO2 nanoparticles: Au-C complex was dispersed in ultrapure water to form a dispersion, which was placed in an ice-water bath and the pH was adjusted to 9.0-10.0 with an alkaline solution. Then, potassium permanganate solution and potassium oxalate solution were added sequentially. After standing in the ice-water bath, the mixture was transferred to a water bath at 55-65℃ for heating to obtain core-shell structured Au-C@MnO2 nanoparticles. The specific process is as follows: A 10 mL solution of the Au-C complex was prepared using ultrapure water and placed in an ice-water bath. 0.08–0.12 mol / L (preferably 0.1 mol / L) potassium hydroxide solution was added to adjust the pH of the system to 9.0–10.0 (preferably 9.5). Subsequently, 80 μL of 0.008–0.012 mol / L (preferably 0.01 mol / L) potassium permanganate solution and 400 μL of 0.008–0.012 mol / L (preferably 0.01 mol / L) potassium oxalate solution were added sequentially. After the mixture was allowed to stand in the ice-water bath for 8–12 min (preferably 10 min), it was transferred to a water bath at 55–65 °C (preferably 60 °C) and heated for 1.5–2.5 h (preferably 2 h) to obtain Au-C@MnO2 nanoparticles (with an Au-C complex core and a MnO2 shell). S5, Encapsulated composite nanomaterials: Au-C@MnO2 nanoparticles were prepared into an aqueous solution, mixed with an aqueous solution of hyaluronic acid in a volume ratio, and reacted by stirring at room temperature. After centrifugation and washing with water, the composite nanomaterial was obtained.

[0029] The specific process is as follows: Hyaluronic acid is dissolved in ultrapure water to prepare a hyaluronic acid solution with a concentration of 4~6 mg / mL (preferably 5 mg / mL); Au-C@MnO2 nanoparticles are dissolved in ultrapure water and sonicated to prepare a nanoparticle solution with a concentration of 0.8~1.2 mg / mL (preferably 1 mg / mL); the nanoparticle solution is added dropwise to the hyaluronic acid solution at a volume ratio of 1:1, and the mixture is stirred at room temperature for 2.5~3.5 h (preferably 3 h); then the mixture is centrifuged at 10000 rpm for 8~12 min (preferably 10 min), washed 2~3 times (preferably 3 times) with ultrapure water, and the precipitate is collected to obtain the composite nanomaterial (Au-C@MnO2 nanoparticles coated with a hyaluronic acid film).

[0030] According to a second aspect of the present invention, a composite nanomaterial is provided, which is prepared by the preparation method described above; the composite nanomaterial has Au-C as the core, MnO2 as the shell, and a hyaluronic acid film coated on the surface, with a particle size of 45~55nm.

[0031] The composite nanomaterial provided by this invention has a structure of "Au-C core—MnO2 shell—hyaluronic acid coating layer"; wherein, the Au-C core is formed by gold nanoparticles and probe molecules C connected by gold-sulfur bonds; the MnO2 shell is generated by the reaction of Au-C complex with potassium permanganate and potassium oxalate; the hyaluronic acid coating layer is formed by the reaction of Au-C@MnO2 nanoparticles with hyaluronic acid at room temperature. The functionalized composite nanomaterial provided by this invention, upon stimulation by glutathione (GSH), exhibits core-shell structure disintegration, surface TMB molecules are detached, and Raman scattering (SERS) fluorescence signal is enhanced. Simultaneously, the released gold nanoparticles, upon stimulation by aminopeptidase N (APN), will specifically generate SERS signals. By monitoring changes in the dual SERS signals, SERS dual-substance sensing of two biomarkers, glutathione and aminopeptidase N, can be achieved. The detection principle is as follows: Figure 2 As shown, after the cell internalizes the nanomaterial, the hyaluronic acid on the surface is dissolved by hyaluronidase in the cell, exposing the MnO2 shell. The MnO2 shell reacts with GSH and then disintegrates, causing the shedding of TMB molecules on the surface, which reduces the TMB signal and causes a change in the SERS spectrum. After the MnO2 shell is completely dissolved, the Au-C core inside is exposed. The amide bond of C can be recognized and cleaved by the APN in the cell, thus producing a new characteristic peak in the SERS spectrum and a corresponding signal change with the change of APN concentration.

[0032] According to a third aspect of the present invention, the application of the composite nanomaterial described above in detecting biomarkers in cells and distinguishing normal cells from tumor cells is provided, wherein the biomarkers are glutathione and aminopeptidase N.

[0033] In the above application, as a preferred embodiment, the cells include normal hepatocytes (THLE) and hepatocellular carcinoma cells (HepG-2); when detecting GSH, the 768 cm⁻¹ region of the SERS spectrum is monitored. -1 1349cm -1 1606cm -1 Changes in signal intensity at a certain point indicate a change in GSH content; a decrease in signal intensity indicates a change in GSH content. When detecting APN, monitoring the 941 cm⁻¹ region in the SERS spectrum... -1 The change in the intensity of the characteristic peak indicates a change in the APN content.

[0034] In the above applications, as a preferred embodiment, the detection limit of the composite nanomaterial for APN is 1 ng / mL, and the APN concentration is related to 941 cm⁻¹ within the range of 0~1 μg / mL. -1 The logarithm of the SERS signal strength shows a linear relationship. Optionally, the reaction time between the composite nanomaterial and GSH is 8–12 min, and the subsequent reaction time with APN is 3–5 h; after the reaction, 2 μL of the reaction system is placed on a silicon wafer for SERS spectroscopy detection. The feasibility verification of the composite nanomaterial for GSH and APN detection is as follows: Figure 4 As shown.

[0035] The present invention will now be described in detail with reference to embodiments thereof. These examples are provided by way of explanation and not by way of limitation. In fact, those skilled in the art will recognize that modifications and variations can be made to the present invention without departing from its scope or spirit. For example, a feature shown or described as part of one embodiment may be used in another embodiment to produce yet another embodiment. Therefore, it is desirable that the present invention encompass such modifications and variations that fall within the scope of the appended claims and their equivalents.

[0036] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are conventional methods, and the materials used are commercially available unless otherwise specified.

[0037] Example 1 This embodiment provides a method for preparing composite nanomaterials, including the following steps: S1. Preparation of gold nanoparticles: Using tetrachloroauric acid as raw material and ultrapure water as solvent, gold nanoparticles Au NPs were obtained by reaction at 125℃ and reduction with sodium citrate. The specific steps are as follows: mix tetrachloroauric acid with ultrapure water, heat to boiling at 125 ℃ and 350 rpm, add sodium citrate solution, continue heating and stirring for 3~5 min, and centrifuge at 10000 rpm to obtain gold nanoparticles.

[0038] S2. Preparation of probe molecule C(2R,2'R)-N,N'-(disulfonylbis(3,1-benzene))bis(2-aminopropionamide): Using 3-aminothiophenol as a raw material and DMSO as a solvent, the reaction was carried out in an oil bath at 80°C. The intermediate 3,3-p-aminothiophenol A was obtained by column chromatography. Then, using BOC-L-alanine and 3,3-p-aminothiophenol as raw materials, HATU as a catalyst, and TEA and DMF as solvents, the reaction was carried out in an oil bath at 50°C. The intermediate B was obtained by extraction. Finally, BOC was removed under acidic conditions to obtain probe molecule C(2R,2'R)-N,N'-(disulfonylbis(3,1-benzene))bis(2-aminopropionamide). The specific steps were as follows: 10 g of 3-aminothiophenol (400 mmol) was dissolved in 200 mL of DMSO and reacted in an oil bath at 80°C for 4 hours with stirring. The solution was then cooled to room temperature and diluted with ultrapure water. The organic layer was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The crude product was further purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1 v / v) to obtain compound A. One equivalent of compound A, 2.2 equivalents of BOC-L-alanine, 4 equivalents of HATU, and 8 equivalents of TEA were added sequentially to DMF and reacted at 0°C with stirring at 50°C for 24 hours. The reaction mixture was quenched with ultrapure water, extracted with ethyl acetate, and dried over anhydrous sodium sulfate to obtain compound B. Finally, the Boc protecting group of compound B was removed in dichloromethane using TFA as a solvent and stirred at 50°C for 3 hours. The solution was then cooled to room temperature and diluted with ultrapure water. After extraction with ethyl acetate, the organic layer was dried with anhydrous sodium sulfate to obtain a yellow oily product, which yielded the probe molecule (2R,2'R)-N,N'-(disulfonylbis(3,1-benzene))bis(2-aminopropionamide).

[0039] S3. Connecting probe C and gold nanoparticles: The nanomaterials obtained in step S2 and the gold nanoparticles obtained in step S1 are prepared into solutions and then thoroughly mixed. The reaction is carried out at room temperature, and Au-C is obtained by centrifugation and washing.

[0040] The specific steps are as follows: The probe molecules obtained in step S2 are prepared into 10 solutions using ultrapure water as a solvent. -3 The solution was mixed thoroughly with 200 μL of concentrated gold nanoparticles at a volume ratio of 1:1, and the reaction was carried out at room temperature for 30 min. The mixture was then centrifuged at 10,000 rpm for 8 min and washed three times with water to obtain Au-C.

[0041] S4. Preparation of core-shell materials: Au-C was placed in an ice-water bath, and the pH was adjusted to 9.5 with potassium hydroxide solution. Then, potassium permanganate solution and potassium oxalate solution were added. 80 μL of potassium permanganate and 400 μL of potassium oxalate were added. After the mixture was allowed to stand in the ice-water bath for 10 minutes, it was transferred to a 60°C water bath and heated for 2 hours to finally obtain Au-C@MnO2 nanoparticles.

[0042] The specific steps are as follows: Au-C was prepared into a 10 mL solution using ultrapure water and placed in an ice-water bath. First, 0.1 mol / L potassium hydroxide solution was added to adjust the pH to 9.5. Then, 80 μL of 0.01 mol / L potassium permanganate solution and 400 μL of 0.01 mol / L potassium oxalate solution were added sequentially. The mixture was immersed in the ice-water bath for 10 minutes, then transferred to a 60°C water bath and heated for 2 hours to obtain Au-c@MnO2 nanoparticles.

[0043] S5. Encapsulating nanomaterials: The nanoparticles obtained in step S4 are prepared into solutions with hyaluronic acid and then thoroughly mixed. The mixture is then reacted at room temperature, and the composite nanomaterials are obtained by centrifugation and washing.

[0044] The specific steps are as follows: Hyaluronic acid is dissolved in ultrapure water to obtain a hyaluronic acid solution with a concentration of 5 mg / mL; the nanomaterials prepared in step S4 are dissolved in ultrapure water and sonicated (20 kHz, 5 min) to obtain a nanomaterial solution with a concentration of 1 mg / mL; the nanomaterial solution is added dropwise to the hyaluronic acid solution at a volume ratio of 1:1, and the mixture is stirred at room temperature for 3 h. It is then centrifuged at 10000 rpm for 8 min, washed three times with water to obtain the composite nanomaterials. The composite nanomaterials are characterized by scanning electron microscopy, and the results are as follows: Figure 3 As shown, the particle size of the composite nanomaterial is 50 nm.

[0045] Application examples The composite nanomaterials and glutathione (GSH) were mixed uniformly at a 1:1 volume ratio and allowed to stand at room temperature for 10 minutes. Before Raman spectroscopy, 10 μL of 0.5 mmol / L TMB solution (prepared using 0.2 mmol / L sodium citrate buffer, pH 4.0, prepared as follows: ① Solution A: 1.05 g citric acid + 50 mL water, dissolved; ② Solution B: 1.47 g sodium citrate + 50 mL water, dissolved; ③ Mixture: 32.75 mL of Solution A + 17.25 mL of Solution B, mixed well) was added, and the reaction continued for 4 h. The feasibility of detecting GSH and APN in the composite nanomaterials prepared in Example 1 was investigated. The composite nanomaterials were placed in different GSH solutions for reaction. After 10 min, 2 μL of the reaction system was pipetted onto a silicon wafer and placed on the sample stage of a Raman spectrometer. The excitation wavelength was set to 785 nm, the laser intensity to 25%, and the integration time to 15 s. The SERS spectra were recorded, and the results are shown in the attached image. Figure 5 .Depend on Figure 5 It can be seen that after the nanomaterials react with different concentrations of GSH, the values ​​of 768, 1349, and 1606 cm⁻¹ are observed. -1 The signal strength at the location decreased significantly, indicating that the material can achieve SERS sensing of GSH.

[0046] The feasibility of detecting APN ability of the composite nanomaterials prepared in Example 1 was investigated by placing the composite nanomaterials in a 5x10⁻¹⁰ spherical environment. -3The reaction was carried out in a mol / L GSH solution. After 10 min of reaction, APN was added and the reaction continued for 4 h. After the reaction was completed, 2 μL of the reaction system was placed on a silicon wafer and placed on the sample stage of a Raman spectrometer. The excitation wavelength was set to 785 nm, the laser intensity to 25%, and the integration time to 15 s. The SERS spectrum was recorded. Figure 6 As shown. By Figure 6 It can be seen that the detection limit of the composite nanomaterial for APN is 1 ng / mL, and within the range of 0-1 μg / mL, the APN concentration is related to 941 cm⁻¹. -1 The material exhibits a good linear relationship between the logarithms of the SERS signal intensity, proving that it can achieve highly sensitive sensing of APN.

[0047] The foregoing has described and evaluated some embodiments of the present invention. It should be understood that the present invention is not limited to the specific embodiments described above. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the scope of the present invention. This does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the present invention, still fall within the protection scope of the present invention.

Claims

1. A method for preparing composite nanomaterials, characterized in that, Includes the following steps: S1. Preparation of gold nanoparticles: Using tetrachloroauric acid as the gold source and ultrapure water as the dispersion medium, sodium citrate was added at 120~130℃ to carry out a reduction reaction; after the reaction was completed, gold nanoparticles were obtained by centrifugation. S2, Preparation of probe molecule C: ① Using 3-aminothiophenol as the raw material and DMSO as the reaction solvent, the reaction was carried out in an oil bath at 75~85℃. The product was separated and purified by column chromatography to obtain intermediate A. ② Using intermediate A and BOC-L-alanine as reaction substrates, HATU as condensation catalyst, and TEA and DMF as mixed solvents, the reaction was carried out in an oil bath at 45~55℃. After extraction and purification, intermediate B was obtained. ③ Under acidic conditions, intermediate B undergoes a deBOC reaction to obtain the target probe molecule C; S3. Preparation of Au-C complex: The probe molecule C and gold nanoparticles were separately prepared into aqueous solutions, then thoroughly mixed at a volume ratio, and a coupling reaction was carried out at room temperature. After the reaction was completed, the Au-C complex was obtained by centrifugation and washing with water. S4. Preparation of core-shell structured Au-C@MnO2 nanoparticles: Au-C complex was dispersed in ultrapure water to form a dispersion, which was placed in an ice-water bath and the pH was adjusted to 9.0-10.0 with an alkaline solution. Then, potassium permanganate solution and potassium oxalate solution were added sequentially. After standing in the ice-water bath, the mixture was transferred to a water bath at 55-65℃ for heating to obtain core-shell structured Au-C@MnO2 nanoparticles. S5, Encapsulated composite nanomaterials: Au-C@MnO2 nanoparticles were prepared into an aqueous solution, mixed with an aqueous solution of hyaluronic acid in a volume ratio, and reacted by stirring at room temperature. After centrifugation and washing with water, the composite nanomaterial was obtained.

2. The preparation method according to claim 1, characterized in that, In step S1, the specific amounts of raw materials used to prepare gold nanoparticles are as follows: 1 mL of 1% tetrachloroauric acid solution is mixed evenly with 100 mL of ultrapure water; sodium citrate is added in the form of a 1% aqueous solution, and the amount of sodium citrate aqueous solution added is 10 mL.

3. The preparation method according to claim 1, characterized in that, In step S2, the concentration of 3-aminothiophenol in DMSO is 0.3~0.5 mol / L; And / or, the reaction time in step ① is 3-5 h, and the column chromatography eluent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 5:1; And / or, in step ②, the molar equivalent ratio of BOC-L-alanine, intermediate A, HATU, and TEA is 2.2:1:4:8, and the reaction time is 20~28h; And / or, the acidic conditions for step ③ are: TFA as solvent, dichloromethane as diluent, BOC removal reaction temperature of 45~55℃, and reaction time of 2~4h.

4. The preparation method according to claim 1, characterized in that, In step S3, the concentration of the aqueous solution of probe molecule C is 10. -3 The concentration of the gold nanoparticle aqueous solution was 1% (mol / L), and the volume ratio of the two was 1:

1. The coupling reaction time was 25-35 min, the centrifugation speed was 10000 rpm, the centrifugation time was 6-10 min, and the number of water washes was 3.

5. The preparation method according to claim 1, characterized in that, In step S4, the volume of the Au-C complex dispersion is 10 mL; the alkaline solution is a 0.08~0.12 mol / L potassium hydroxide solution; the concentration of the potassium permanganate solution is 0.008~0.012 mol / L and the volume is 70~90 μL; the concentration of the potassium oxalate solution is 0.008~0.012 mol / L and the volume is 350~450 μL; the standing time in the ice-water bath is 8~12 min, and the heating time in the water bath at 55~65℃ is 1.5~2.5 h.

6. The preparation method according to claim 1, characterized in that, In step S5, the concentration of the hyaluronic acid aqueous solution is 4~6 mg / mL; the concentration of the Au-C@MnO2 nanoparticle aqueous solution is 0.8~1.2 mg / mL; the volume ratio of the two is 1:1; the reaction time is 2.5~3.5 h with stirring at room temperature; the centrifugation speed is 10000 rpm; the centrifugation time is 6~10 min; and the number of water washings is 3.

7. A composite nanomaterial, characterized in that, The composite nanomaterial is prepared by the preparation method according to any one of claims 1-6; the composite nanomaterial has Au-C as the core, MnO2 as the shell, and is coated with a hyaluronic acid film on the surface, with a particle size of 45~55nm.

8. The application of the composite nanomaterial as described in claim 7 in detecting biomarkers in cells and distinguishing between normal cells and tumor cells, wherein the biomarkers are glutathione and aminopeptidase N.

9. The application according to claim 8, characterized in that, The cells included normal hepatocytes (THLE) and hepatocellular carcinoma cells (HepG-2); during GSH detection, the 768 cm⁻¹ region of the SERS spectrum was monitored. -1 1349cm -1 1606cm -1 Changes in signal intensity at a certain point indicate a change in GSH content; a decrease in signal intensity indicates a change in GSH content. When detecting APN, monitoring the 941 cm⁻¹ region in the SERS spectrum... -1 The change in the intensity of the characteristic peak indicates a change in the APN content.

10. The application according to claim 9, characterized in that, The detection limit of the composite nanomaterial for APN is 1 ng / mL. Within the range of 0–1 μg / mL, the APN concentration is related to 941 cm⁻¹. -1 The logarithm of the SERS signal strength shows a linear relationship. And / or, the reaction time of the composite nanomaterial with GSH is 8~12 min, and the subsequent reaction time with APN is 3~5 h; after the reaction, 2 μL of the reaction system is aspirated and placed on a silicon wafer for SERS spectroscopy detection.

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