A kidney metabolizable ratiometric raman spectrum detection system and a probe thereof

Abstract

The present application relates to a kind of renal metabolism ratio type raman spectrum detection system and its probe, the probe can be catalyzed by the β-glucuronidase overexpressed in tumor microenvironment, promote 1,6-elimination reaction to initiate self-destruction degradation process, cause signal response group to separate from AuNCs surface;Subsequently, AuNCs pass through glomerular basement membrane and be discharged with urine outside the body, and by means of hydrochloric acid hydroxylamine reduction effect one-step growth is high SERS active nanogold star (AuNSs), further quantitative test is carried out using raman means.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection, specifically to a ratiometric Raman spectroscopy detection system and its probe that can be metabolized by the kidney. Background Technology

[0002] Liquid biopsy, as a new technology for rapid cancer screening, uses a non-invasive method to collect samples from patients (blood, urine, saliva, and cerebrospinal fluid, etc.). By analyzing the chemical information related to the disease in the samples, it enables disease diagnosis and assessment. Compared with traditional tissue biopsies, liquid biopsy has the following advantages: non-invasiveness, high repeatability, early diagnosis, dynamic monitoring, and overcoming the influence of tumor heterogeneity. The unique enzyme spectrum is an important characteristic and intrinsic stimulating factor that distinguishes the tumor microenvironment from normal tissue. In the extracellular matrix and tumor cells of the tumor microenvironment, there are often abnormally expressed intracellular enzymes, such as matrix metalloproteinases (MMPs), esterases, β-glucuronidase (GUSB), and granzyme B. Therefore, utilizing the biocatalytic mechanisms contained in the unique enzyme spectrum database of the tumor microenvironment to achieve specific recognition and response has become a new approach to solving the challenges of early cancer screening and efficacy evaluation. Compared to enzyme-activated small molecule probes, enzyme-activated nanoprobes are more likely to achieve targeted enrichment at tumor sites. On the one hand, nanoprobes enable real-time imaging of tumors in situ through fluorescent labeling; on the other hand, exogenous biomarkers can be designed using enzyme activation strategies, achieving specific enzyme responses at tumor sites and then being excreted in the body through body fluids, feces, and other metabolites, thereby transforming in vivo tumor diagnosis into in vitro detection of exogenous biomarkers.

[0003] Unlike fluorescence microscopy, which relies on exogenous fluorescent dye molecular labeling, Raman spectroscopy depends on the inelastic light scattering of the sample itself. Raman spectroscopy can provide intrinsic vibrational fingerprint information such as sample composition and molecular symmetry, and is widely used in the identification of chemical components, structural conformation, and intermolecular interactions. The chemical bonds of various biomolecules within cells can generate characteristic Raman vibrational signals, such as those at 2850 cm⁻¹. -1 The Raman scattering at this point is attributed to the CH stretching vibration of the methylene group, and most of the methylene components in cells belong to lipids. Based on this, the 2850 cm⁻¹... -1A label-free imaging strategy for lipids was developed using band design. However, methylene groups in proteins and carbohydrate molecules within cells can generate background interference signals, affecting the accuracy of the test results. Therefore, high-resolution identification of components with similar chemical groups using Raman spectroscopy presents a significant challenge. Cellular Raman spectra show that naturally occurring molecules within cells (also known as endogenous molecules) are concentrated in the 1800-2800 cm⁻¹ band. -1 There are no Raman vibration signals within this range, and this region can be defined as the Raman silent region; while exogenous biological orthogonal reporter groups (such as alkynyl, cyano, carbon-deuterium bonds and azides) have specific Raman vibration spectra in the silent region.

[0004] However, due to the influence of variables such as laser power, substrate surface roughness, and molecular structure, the signal intensity repeatability of SERS probes is poor, making it difficult for traditional SERS probes to achieve quantitative analysis and detection of trace substances. At the in vivo level, the aggregation and uneven distribution of small amounts of SERS probes in benign tissue sites can affect the accuracy of test results. Although the washing step can maximize the contrast between tumor tissue and normal tissue, it cannot completely eliminate background signal interference caused by non-specific adsorption of probes. In addition, the stability of the laser source and the working distance between the sample and the lens or detector also affect the Raman signal intensity. Therefore, negative controls or multiple Raman reporter molecules are usually introduced to improve accuracy, but this design requires multiple nanoparticles or Raman reporter molecules, making the detection method complex and expensive.

[0005] Therefore, developing a precise and quantitative Raman spectroscopy detection method for complex systems, thereby enabling non-invasive early cancer detection and diagnosis, is of great clinical and industrial value. Summary of the Invention

[0006] This invention addresses current technological bottlenecks by providing a renally metabolizable ratiometric Raman detection system and its probe. A stimulus-responsive Raman ratiometric gold nanocluster probe (Glu-RR-AuNCs) detection system is designed. This probe is catalyzed by β-glucuronidase overexpressed in the tumor microenvironment, triggering a 1,6-elimination reaction that initiates a self-degradation process, causing the signal-responsive group 1-propynylbenzene to detach from the AuNCs surface. Subsequently, the AuNCs cross the glomerular basement membrane and are excreted in urine, where they undergo a one-step growth process using hydroxylamine hydrochloride reduction to become highly SERS-active gold nanoparticles (AuNSs), which can then be quantitatively detected using Raman spectroscopy. Differences in β-glucuronidase activity lead to varying degrees of detachment of 1-propynylbenzene from the AuNCs surface, resulting in variations in the Raman response signal intensity. Simultaneously, the internal standard reference group attached to the nanoprobe does not detach from the AuNCs surface, and the Raman intensity of the internal standard reference group remains unchanged. Quantitative detection of β-glucuronidase activity is achieved through the change in the ratio between the two signals, ultimately enabling precise early diagnosis and treatment monitoring of cancer through a novel liquid biopsy strategy. Specifically, as follows... Figure 1 As shown.

[0007] More specifically, targeting the specific hydrolytic characteristics of β-glucuronidase, this invention first explored a modular synthetic route for β-glucuronidase substrates with high selectivity that can self-degrade via enzymatic activation of the 1,6-elimination reaction. Through the derivatization and modification of multicolor Raman silencing region groups, a series of β-glucuronidase substrates were designed and synthesized. Further screening and optimization yielded the β-glucuronidase substrate Glu-RR molecule with an internal standard reference to response signal Raman peak intensity ratio of 1:1. Based on this, this invention prepared ratiometric Raman probes Glu-RR-AuNCs for the in vitro detection of β-glucuronidase activity. β-Glucuronidase specifically cleaves the Glu-RR on the surface of the Glu-RR-AuNCs probe. Hydrolysis of the glycosidic bonds in the Glu-RR molecule causes the sugar group and the 1-propynylbenzene (a signal response group) to detach from the AuNCs surface, while the cysteine ​​residue linked to the internal standard signal group remains stably anchored to the AuNCs surface via a gold-sulfur bond. This results in a ratio change in the number of Raman response groups to the internal standard reference groups on the AuNCs surface. Subsequently, under the action of gold growth medium, AuNCs can rapidly and controllably grow into AuNSs structures, and their highly active SERS effect greatly amplifies the ratiometric Raman signal. Since the β-glucuronidase activity does not affect the Raman signal intensity of the internal standard reference group, I... 2154 As β-glucuronidase activity increases, the Raman responsive group signal intensity I... 2230 Gradually weakening, leading to a decrease in the Raman ratio (I 2154 -I 2230 ) / I 2154Gradually increases. However, in the absence of β-glucuronidase, (I 2154 -I 2230 ) / I 2154 No significant changes were observed. Through a series of characterization and validation processes, a ratiometric quantitative detection of β-glucuronidase activity was achieved in vitro. Furthermore, the direct reading strategy of the Raman silencing region group avoids interference from the external environment, ensuring the accuracy and repeatability of the quantitative results.

[0008] Furthermore, this invention proposes a novel strategy to convert in vivo tumor microenvironment enzyme activity into quantitatively detectable Raman signals in urine. The Glu-RR-AuNCs probe can be specifically hydrolyzed by β-glucuronidase overexpressed in the tumor microenvironment, subsequently metabolized and excreted through renal clearance. Finally, the ratiometric Raman signal in urine is detected using the gold-SERS method to quantitatively detect β-glucuronidase activity in the tumor microenvironment. Using this method, long-term monitoring of an MDA-MB-231 tumor mouse model established the relationship between tumor volume and (I... 2154 -I 2230 ) / I 2154 The linear correlation between them was established. Ultimately, by using highly sensitive detection of ratiometric Raman signals in urine, the efficacy of the conventional chemotherapy drug doxorubicin was initially achieved.

[0009] This invention provides a renally metabolizable ratiometric Raman spectroscopy detection system, comprising:

[0010] a) A light source module that emits near-infrared light;

[0011] b) Surface-enhanced Raman scattering nanoparticles, wherein the nanoparticles are renally metabolizable ratiometric Raman scattering nanoparticles that specifically respond to β-glucuronidase, and wherein the nanoparticles are applied to mammals.

[0012] c) Urine collection and processing module, wherein the urine collection and processing module is used to collect urine after the above-mentioned nanoparticles are applied, and to further process the nanoparticles in the urine into nanostar particles;

[0013] d) Acquisition module, which acquires the surface-enhanced Raman scattering (SERS) spectra of the aforementioned nanostar particles;

[0014] e) The information processing module establishes a Raman ratio signal by comparing the Raman intensities of the signal response reporter group and the internal standard reference group, and determines the concentration level of β-glucuronidase in the tissue of this mammal.

[0015] The mammal in question is a mammal with a tumor, preferably a human.

[0016] The surface-enhanced Raman scattering nanoparticles are connected to an internal standard reference signal molecule or group, as well as a signal response reporter molecule or group in response to β-glucuronidase.

[0017] The acquisition module acquires the comparison signal reporting group I. 2230 and internal standard reference group I 2154 Raman intensity.

[0018] In the information processing module, the Raman ratio signal is (I 2154 -I 2230 ) / I 2154 ratio.

[0019] The surface-enhanced Raman scattering nanoparticles are precious metal nanoparticles.

[0020] In the urine collection and processing module, the long gold method is used to further process the nanoparticles in the urine into nanostar particles.

[0021] Before applying surface-enhanced Raman scattering nanoparticles, the Raman intensity ratio of the internal standard reference group to the signal response reporter group was 1:1.

[0022] The information processing module further includes processing the enzyme activity distribution of β-glucuronidase at the tumor site.

[0023] The detection system of this invention is used for tumor diagnosis, detection, and / or monitoring of the efficacy of chemotherapy drugs.

[0024] The tumor is one of the following: lung cancer, breast cancer, stomach cancer, colorectal cancer, thyroid cancer, liver cancer, melanoma, pancreatic cancer, bile duct cancer, bladder cancer, kidney cancer, prostate cancer, head and neck cancer, brain tumor, glioma, hematologic malignancy, or lymphoma. Preferably, it is lung cancer, breast cancer, stomach cancer, colorectal cancer, thyroid cancer, liver cancer, melanoma, pancreatic cancer, bile duct cancer, bladder cancer, kidney cancer, or prostate cancer.

[0025] The present invention also provides a surface-enhanced Raman scattering nanoparticle comprising a biocompatible metal nanocore, wherein a renal-clearable functional ligand and a β-glucuronidase substrate Raman reporter molecule or group are attached to the surface of the nanocore.

[0026] The surface-enhanced Raman scattering nanoparticles can specifically respond to the enzymatic hydrolysis of β-glucuronidase in tumor tissue.

[0027] The β-glucuronidase substrate Raman reporter molecule or group has the structure of formula I.

[0028] R1 is selected from CN,

[0029] R2 is selected from H, C1-10 alkyl, or phenyl.

[0030]

[0031] The structure of Formula I is linked to the metal nucleus surface by forming a metal-sulfur bond through cysteine.

[0032] The renally clearable functional ligand is selected from glutathione, which forms a metal-thiol bond with the surface of the metal nucleus.

[0033] The biocompatible metal is a precious metal, wherein the precious metal is gold or / or silver, preferably gold.

[0034] The nanoparticles have a particle size of 1-10 nm, preferably 2-5 nm.

[0035] The nanoparticles are nanoclusters.

[0036] The nanoparticles are also attached to a contrast agent or radioactive isotope suitable for medical imaging, wherein the contrast agent for medical imaging is selected from magnetic resonance imaging (MRI), positron emission tomography (PET), calculated single-photon emission tomography (SPECT), computed tomography (CT), ultrasound imaging (UL), optical imaging (OI), photoacoustic imaging (PI), fluorescence fluoroscopy, or fluorescence imaging.

[0037] The contrast agent is an MRI contrast agent or a radiographic contrast agent selected from paramagnetic ions of chromium (III), manganese (II), iron (III), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III).

[0038] The present invention also provides a kit comprising the above-mentioned nanoparticles or nanoparticle probes.

[0039] This invention also provides a method for preparing surface-enhanced Raman scattering nanoparticles, which involves mixing tetrachloroauric acid hydrate with deionized water, then adding a reduced glutathione solution and a compound containing the I structure to the reaction solution at room temperature, followed by heating and reacting in the dark to obtain a Glu-RR-AuNCs nanoparticle solution.

[0040] The advantages and beneficial effects of this invention are as follows:

[0041] 1) Through a modular design approach, it is easy to programmatically design specific enzyme recognition substrates starting from protease detection. By derivatizing and modifying Raman silencing groups, Glu-RR with an internal standard reference to response Raman signal intensity ratio of 1:1 was successfully synthesized and screened. More importantly, the Raman silencing groups give the probe excellent anti-interference capabilities, making it suitable for the specific detection of GUSB in complex systems.

[0042] 2) A new method for evaluating GUSB activity was established, enabling the detection of Raman ratios in artificial urine samples. This internal standard Raman probe is more suitable for qualitative and quantitative detection in complex environments.

[0043] 3) A method for urine liquid biopsy using artificial tumor markers was developed, enabling early cancer diagnosis, progression monitoring, and prognostic assessment. By utilizing renal clearance, the Raman spectroscopy results of AuNCs in urine allow for highly specific, quantitative, and qualitative assessment of GUSB enzyme activity in the tumor microenvironment. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the operation of the renal-metabolizable ratio-type Raman detection system of the present invention.

[0045] Figure 2 These are the Raman spectra of compound 6 (A) and compound 17 (B).

[0046] Figure 3 The probe characterization before and after gold growth: Glu-RR-AuNSs were prepared by gold growth of Glu-RR-AuNCs under the action of gold growth solution (NH2OH (20mM) and HAuCl4 (150μM)); (A) UV absorption spectra of Glu-RR-AuNCs (black line) and Glu-RR-AuNSs (red line). The inset is a photograph of AuNCs (light yellow) and AuNSs (blue); (B) Changes in hydrated particle size before and after gold growth.

[0047] Figure 4 The TEM-related results are as follows: (A) Electron micrograph of Glu-RR-AuNCs, scale bar 10 nm; (B) Electron micrograph of Glu-RR-AuNSs, scale bar 200 nm; (C) HAADF-STEM image of Glu-RR-AuNSs and elemental distribution map of Au (yellow) and Si (indigo blue), scale bar 50 nm.

[0048] Figure 5 These are Raman spectra of Glu-RR-AuNCs (black line) and Glu-RR-AuNSs (red line) under the same testing conditions.

[0049] Figure 6The Raman spectra of Glu-RR-AuNCs in urine were quantitatively detected using the gold-SERS method. (A) Raman spectra of Glu-RR-AuNCs after gold engraving at different concentrations; (B) Raman spectra of Glu-RR-AuNCs and their corresponding 2154 cm⁻¹. -1 Calibration curves between Raman intensities.

[0050] Figure 7 (A) Gold-RRR-AuNCs incubated with different concentrations of GUSB for 10 minutes - SERS spectra; (B) Ratio of GUSB concentration to its corresponding Raman intensity (I 2154 -I 2230 ) / I 2154 Linear correlation between them.

[0051] Figure 8 This is the kinetics of GUSB determination by long-gold SERS. (A) Raman spectrum of GUSB (200 U / mL) after incubation with Glu-RR-AuNCs over time; (B) Raman intensity ratio (I 2154 -I 2230 ) / I 2154 Changes over time.

[0052] Figure 9 (A) Raman spectra of Glu-RR-AuNCs under the action of 200 U / mL enzyme (GUSB or β-galactosidase); (B) Raman intensity ratio of Glu-RR-AuNCs (I 2154 -I 2230 ) / I 2154 .

[0053] Figure 10 The results are as follows: (A) Pharmacokinetic characterization of Glu-RR-AuNCs administered via tail vein, with the gold content of Glu-RR-AuNCs in the blood fitted using a two-compartment pharmacokinetic model; (B) Distribution of gold content in major organs of healthy nude mice 1 h or 24 h after tail vein injection of Glu-RR-AuNCs; (C) Distribution of gold content in tumors and major organs of MDA-MB-231 tumor model mice 1 h or 24 h after tail vein injection of Glu-RR-AuNCs (n=3).

[0054] Figure 11 It is (A) MDA-MB-231 tumor model mouse (1000mm) 3 (a) Raman spectra of urine at different time points after tail vein injection of Glu-RR-AuNCs probe; (b) Raman spectra of urine at different time points after tail vein injection of Glu-RR-AuNCs probe in healthy mice; (c) Raman ratio in urine of MDA-MB-231 subcutaneous xenograft model mice (red line) and healthy mice (black line) (I)2154 –I 2230 ) / I 2154 Curve showing how it changes over time.

[0055] Figure 12 (A) Calculation of renal metabolic clearance using both ICP-MS and gold-sERS methods. (B) Correlation between the gold-sERS method and ICP-MS, two methods for quantifying gold content.

[0056] Figure 13 These are silver-stained Raman mapping images of tumor sections from MDA-MB-231 tumor-bearing mice, along with H&E staining images of adjacent sections. White dashed lines mark the boundaries between the tumor (T) and normal tissue (H).

[0057] Figure 14 This is a representative silver-stained Raman mapping image of a healthy mouse kidney tissue section.

[0058] Figure 15 These are photographs of three representative groups of MDA-MB-231 tumor-bearing mice at different time points (days 3, 6, 9, 12, 15, 18, 21, 24, and 27). The inset shows a photograph of the tumor ex vivo after day 27.

[0059] Figure 16 This is a ratiometric Raman probe used to monitor the growth process of MDA-MB-231 tumors in urine. (A) Raman spectra of urine at different monitoring days; (B) Tumor volume (blue) and (I) 2154 –I 2230 ) / I 2154 Correlation between (black)

[0060] Figure 17 It is an MDA-MB-231 tumor (100, 400 and 1000 mm). 3 Immunofluorescence staining images: cell nucleus (blue), β-glucuronidase (green) stained with rabbit anti-β-glucuronidase antibody and fluorescently labeled secondary antibody.

[0061] Figure 18 MDA-MB-231 tumors of different sizes (100, 400, and 1000 mm) 3 Raman mapping image of ).

[0062] Figure 19 It is the change in body weight of MDA-MB-231 tumor model mice during long-term monitoring. Detailed Implementation

[0063] Example 1:

[0064] Synthesis and characterization of Raman reporter molecules (Glu-RR) of β-glucuronidase substrates

[0065]

[0066] Synthesis of Compound 1: Under argon protection, aluminum powder (3.41 g, 126.41 mmol) and ultra-dry tetrahydrofuran (25 mL) were added sequentially to a dry three-necked flask connected to a condenser and a constant-pressure dropping funnel. The mixture was stirred at room temperature for ten minutes, followed by the addition of a catalytic equivalent of HgCl2 and stirring for another 0.5 h. Five drops of a tetrahydrofuran / toluene mixture (10.9 mL, 126.41 mmol) in 3-bromopropyne (22 mL, 10:1 v / v) was added dropwise through the constant-pressure dropping funnel to initiate the reaction. The solution quickly turned black. After the reaction solution refluxed, the 3-bromopropyne mixture was slowly added dropwise over 1 h. The mixture was then allowed to react at room temperature for 2 h, followed by heating to 65 °C and reacting for another 4 h. After natural cooling to room temperature, an ultra-dry tetrahydrofuran (40 mL) solution containing 3.25 g (19.45 mmol) of 4-hydroxy-3-nitrobenzaldehyde was added dropwise through a constant-pressure dropping funnel at 0 °C. The reaction was carried out at 0 °C for 2 h, then transferred to room temperature and stirred for another 12 h. The reaction was monitored by TLC. After the reaction was completed, the reaction mixture was slowly added dropwise to a large amount of ice water to quench the reaction. The pH of the solution was adjusted to about 3.0 with 1N HCl. The resulting mixture was extracted three times with ethyl acetate. The organic phases were combined, evaporated to dryness, and redispersed in dichloromethane. The mixture was then extracted three times with 50 mL of 1N NaOH aqueous solution. The aqueous phases were combined, and the pH of the solution was adjusted back to about 3.0 with 1N HCl under ice bath conditions. The mixture was then extracted three times with 100 mL of dichloromethane. The combined organic phases were washed three times with saturated brine. Finally, the different degrees of product ionization at different pH values ​​were used to achieve two-phase-swapping purification. The crude product was dried over anhydrous magnesium sulfate and then purified by silica gel column chromatography (eluent ratio of petroleum ether: ethyl acetate = 5:1) to give product 1 (3.48 g, 16.8 mmol, 86%).

[0067] 1 H NMR (400MHz, CDCl3) δ = 10.57 (s, 1H), 8.16 (d, J = 2.2Hz, 1H), 7.65 (dd, J = 8.7, J = 2.2Hz, 1H), 7.17 ( d,J=8.7Hz,1H),4.89(t,J=6.2Hz,1H),2.69-2.61(m,2H),2.46(s,1H),2.11(t,J=2.6Hz,1H)ppm. 13C NMR (101MHz, CDCl3) δ = 154.3, 135.3, 135.1, 133.1, 122.2, 120.0, 79.9, 71.8, 70.8, 29.0ppm.

[0068] HRMS(ESI): m / z calcd for C 10 H 13 N₂O₄[M+NH₄] + :225.0429; found:225.0537.

[0069]

[0070] Synthesis of Compound 2: Under argon atmosphere, 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTTA, 1.38 mL, 5.07 mmol) and silver carbonate (7.39 g, 26.79 mmol) were added to anhydrous acetonitrile (10 mL) and stirred for 2 h. Then, under ice bath conditions, methyl 1-bromo-2,3,4-tri-O-acetyl-α-D-glucuronide (2.88 g, 7.24 mmol) and Compound 1 (1 g, 4.83 mmol) were added to the system. The reaction was then allowed to proceed at room temperature for 6 h, and the reaction was monitored by TLC. After the reaction was complete, the reaction was quenched with 1N HCl, filtered through diatomaceous earth, and the filtrate was extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent volume ratio of petroleum ether:ethyl acetate = 3:1) to give a pale yellow solid, Product 2 (1.53 g, 2.92 mmol, 60.56%).

[0071] 1 H NMR (400MHz, CDCl3) δ = 7.72 (d, J = 7.3Hz, 1H), 7.47 (dd, J = 5.9, 2.8Hz, 1H), 7.23 (d, J = 8.6Hz, 1H), 5.17 (dd, J = 17.7, 7 .3Hz,4H),4.76(s,1H),4.19(d,J=8.9Hz,1H),3.58(s,3H),3.26(s,1H),2.48(s,2H),1.95(s,4H),1.91(s,6H)ppm. 13 C NMR (101MHz, CDCl3) δ = 170.0, 169.6, 169.5, 166.9, 148.3, 131.6, 122.6, 119. 0,99.4,79.9,72.1,71.7,71.1,70.5,70.5,70.1,68.7,53.0,29.1,20.4ppm.

[0072] HRMS(ESI): m / z calcd for C 23 H 29 N2O 13 [M+NH4] + :541.1670; found:541.1653.

[0073]

[0074] Synthesis of Compound 3: Under argon atmosphere, Compound 2 (800 mg, 1.53 mmol) and 4-nitrophenol chloromethyl ester (616 mg, 3.06 mmol) were dissolved in ultradry dichloromethane (3.5 mL). Pyridine (300 μL, 3.74 mmol) was added dropwise under ice bath. After reacting for half an hour, the reaction was continued at room temperature for 1.5 h, and the reaction was monitored by TLC. After the reaction was completed, the reaction was quenched by adding saturated sodium bicarbonate aqueous solution (20 mL). The mixture was extracted three times with dichloromethane, washed with saturated brine (30 mL), dried over anhydrous magnesium sulfate, filtered, and evaporated to dryness. The crude product was purified by silica gel column chromatography (eluent ratio of petroleum ether: ethyl acetate = 2:1) to finally obtain a white solid product 3 (1.03 g, 1.50 mmol, 97.88%).

[0075] 1 H NMR (400MHz, CDCl3) δ = 8.28 (d, J = 9.1Hz, 2H), 7.94 (d, J = 2.2Hz, 1H), 7.64 (dd, J = 8.7, 2.3Hz, 1H), 7.42 (d, J = 8.6Hz, 1H), 7.40-7.35 (m, 2H), 5.81 (t, J =6.6Hz,1H),5.40-5.26(m,4H),4.24(d,J=8.7Hz,1H),3.74(s,3H),2.97- 2.83(m,2H),2.13(s,3H),2.10(t,J=2.6Hz,1H),2.07(d,J=4.1Hz,6H)ppm. 13 CNMR(101MHz, CDCl3)δ=171.1,170.0,169.3,169.2,166.7,155.2,151.4,149.4,145.5,141.0,133.4,13 2.3,132.2,125.3,123.6,123.5,121.7,119.7,99.4,77.3,72.5,70.9,70.1,68.6,53.0,26.2,20.5ppm.

[0076] HRMS(ESI): m / z calcd for C 30 H 32N3O 17 [M+NH4] + :706.1732; found:706.1725.

[0077]

[0078] Synthesis of Compound 4: Under argon protection, L-cysteine ​​(6 g, 24.97 mmol) was added to 1N sodium hydroxide aqueous solution (55 mL, 55 mmol). Di-tert-butyl dicarbonate (11.99 g, 54.93 mmol) was slowly added dropwise to the reaction system under ice bath conditions (2 h). The reaction was allowed to proceed overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, saturated potassium hydrogen sulfate solution was added to the reaction system to adjust the pH to 2. The mixture was extracted three times with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and the crude product obtained after rotary evaporation of the organic phase was purified by silica gel column chromatography (eluent ratio of dichloromethane:methanol = 15:1) to finally obtain a white solid product 4 (10.6 g, 24.06 mmol, 96.37%).

[0079] 1 H NMR (400MHz, MeOD) δ = 4.44-4.40 (m, 2H), 3.25 (dd, J = 13.9, 4.6Hz, 2H), 2.97 (dd, J = 13.9, 9.1Hz, 2H), 1.46 (s, 18H) ppm. 13 C NMR (101MHz, MeOD) δ = 172.8, 156.5, 79.4, 53.5, 40.5, 26.9ppm.

[0080] HRMS(ESI): m / z calcd for C 16 H 29 N₂O₈S₂[M+H] + :441.1365; found:441.1348.

[0081]

[0082] Synthesis of Compound 5: Under argon protection, Compound 4 (1 g, 2.27 mmol) and p-aminobenzonitrile (600 mg, 5.11 mmol) were dissolved in dichloromethane (10 mL) and stirred for ten minutes. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl, 900 mg, 5.79 mmol), 1-hydroxybenzotriazole (HOBt, 600 mg, 5.11 mmol), and triethylamine (600 μL, 6.81 mmol) were added sequentially to the system. The mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, 20 mL of saturated sodium bicarbonate aqueous solution was added to quench the reaction. The mixture was extracted three times with dichloromethane, washed with saturated brine, dried over anhydrous magnesium sulfate, and then evaporated to dryness. The crude product was then purified by silica gel column chromatography (eluent ratio of petroleum ether: ethyl acetate = 10:1) to give a pale yellow crystalline product 5 (1.2 g, 1.87 mmol, 82%).

[0083] 1 H NMR (400MHz, CDCl3) δ = 9.49 (s, 2H), 7.74-7.66 (m, 4H), 7.57-7.52 (m, 4H), 5.62 (d, J = 9. 7Hz, 2H), 4.89 (td, J=10.1, 4.4Hz, 2H), 3.01 (qd, J=14.7, 7.5Hz, 4H), 1.44 (s, 18H) ppm. 13 C NMR (101MHz, CDCl3) δ = 168.9, 156.2, 141.5, 133.1, 120.5, 118.6, 107.8, 80.9, 55.8, 47.2, 28.4ppm.

[0084] HRMS(ESI): m / z calcd for C 30 H 37 N6O6S2[M+H] + :641.2216; found:641.2201.

[0085]

[0086] Synthesis of Compound 6: Under argon protection, Compound 5 (3.2 g, 5 mmol) was dissolved in dichloromethane (11.5 mL), and trifluoroacetic acid (TFA, 5.27 mL, 80 mmol) was added dropwise to the reaction system under ice bath conditions. The mixture was stirred for 1 h, and the reaction was monitored by TLC during the process. After the reaction was completed, the reaction was quenched by adding saturated sodium bicarbonate aqueous solution (40 mL), extracted three times with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the crude product was purified by silica gel column chromatography (eluent ratio of dichloromethane:methanol = 360:1) to obtain a pale yellow solid product 6 (1.2 g, 3.09 mmol, 61%).

[0087] 1 H NMR (400MHz, MeOD) δ = 9.90 (s, 2H), 7.75-7.71 (m, 4H), 7.63 (dd, J = 8.9, 2.5Hz, 4H), 3.87 (dd, J =8.1,3.9Hz,2H),3.34(dd,J=13.9,3.9Hz,2H),2.98(dd,J=13.9,8.1Hz,2H),1.25(s,4H)ppm. 13 C NMR (101MHz, CDCl3) δ = 165.1, 153.5, 133.9, 121.2, 113.9, 96.1, 38.7ppm.

[0088] HRMS(ESI): m / z Calcd for C 20 H 24 N7O2S2[M+NH4] + 458.1433; found: 458.1511.

[0089]

[0090] Synthesis of Compound 7: Under argon protection, Compound 6 (750 mg, 1.7 mmol) and Compound 3 (2.58 g, 3.75 mmol) were dissolved in ultradry N,N-dimethylformamide (DMF, 16 mL). Then, 1-hydroxybenzotriazole (HOBt, 460 mg, 3.4 mmol) and pyridine (4 mL, 1.7 mmol) were added sequentially to the reaction system. The mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, 1N HCl (40 mL) was added to quench the reaction. The mixture was extracted three times with ethyl acetate, washed with saturated brine, dried over anhydrous magnesium sulfate, and the crude product was purified by silica gel column chromatography (eluent ratio of dichloromethane:methanol = 40:1) to obtain a yellow solid product 7 (2.36 g, 1.53 mmol, 90%).

[0091] 1H NMR (400MHz, CDCl3) δ = 7.82 (td, J = 6.0, 1.7Hz, 4H), 7.70-7.58 (m, 4H), 7.54-7.3 4(m,6H),5.46-5.37(m,4H),5.18(dt,J=9.4,3.8Hz,4H),4.79(t,J=6.3Hz,2H), 4.51(d,J=9.8Hz,2H),3.67(s,6H),3.28-2.92(m,4H),2.82(d,J=9.5Hz,2H),2. 58-2.54(m,4H),2.23(t,J=2.7Hz,2H),2.02(s,6H),1.98(d,J=6.6Hz,12H)ppm. 13 C NMR (101MHz, CDCl3)δ=170.0,169.2,167.8,166.7,155.7,149.1,141.5,141.0,134.3,133.4,131.6,129.9,123.1 ,119.8,108.0,99.6,72.5,70.9,70.1,68.6,56.3,53.1,36.0,31.9,29.7,29.3,27.2,22.7,20.6,20.5,14.1ppm.

[0092] HRMS(ESI): m / z calcd for C 34 H 34 N4O 15 S[M+2H 2+ ] / 2:770.1741; found:770.1667.

[0093]

[0094] Synthesis of Compound 8: Under argon protection, iodobenzene (0.98 mL, 8.71 mmol), Compound 3 (2 g, 2.9 mmol), cuprous iodide (9%, 110 mg, 580 μmol), and palladium dichloride bis(triphenylphosphine) dichloride (Pd(PPh3)2Cl2, 3%, 38.31 mg, 87.14 μmol) were added sequentially to dry toluene (16 mL). Triethylamine (400 μL, 2.9 mmol) was then slowly added dropwise to the reaction system. The mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, a saturated sodium bicarbonate aqueous solution (30 mL) was added to quench the reaction. The mixture was extracted three times with ethyl acetate, washed with saturated brine, dried over anhydrous magnesium sulfate, and the crude product was purified by silica gel column chromatography (eluent ratio of petroleum ether:ethyl acetate = 1.5:1) to obtain a pale yellow solid, product 8 (1.96 g, 2.56 mmol, 88.25%).

[0095] 1 H NMR (400MHz, CDCl3) δ = 8.22 (d, J = 8.7Hz, 2H), 8.01 (s, 1H), 7.75-7.68 (m, 1H), 7.46 (d, J = 8.7Hz, 1H), 7.36-7.26 (m, 7H), 5.94 ( t,J=7.7Hz,1H),5.40-5.32(m,4H),4.34(d,J=5.9Hz,1H),3.71(s,3H),3.12(h,J=10.8Hz,2H),2.10(s,3H),2.06(s,6H)ppm. 13 CNMR(101MHz, CDCl3)δ=170.0,169.4,169.3,166.8,155.2,151.6,145.4,133.6,132.3,132.1,131.6,128.4,125.3, 123.7,123.5,122.6,121.8,119.4,99.3,84.3,83.1,83.0,77.4,77.3,72.4,70.9,70.1,68.6,53.0,27.3,20.5ppm.

[0096] HRMS(ESI): m / z calcd for C 36 H 36 N3O 17 [M+NH4] + :782.2045; found:782.2039.

[0097]

[0098] Synthesis of Compound 9: Under argon protection, Compound 4 (2 g, 4.54 mmol) and 4-ethynylaniline (1.6 g, 13.62 mmol) were dissolved in dichloromethane (25 mL) and stirred for ten minutes. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl, 1.91 g, 9.99 mmol), 1-hydroxybenzotriazole (HOBt, 1.35 g, 9.99 mmol), and triethylamine (1.39 mL, 9.99 mmol) were added to the system sequentially. The mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, 1N HCl (40mL) was added to quench the reaction. The product was extracted three times with dichloromethane, washed three times with saturated sodium bicarbonate aqueous solution and saturated brine, dried over anhydrous magnesium sulfate, and the crude product was purified by silica gel column chromatography (eluent ratio of petroleum ether: dichloromethane = 6:1) to obtain white crystalline product 9 (2.71g, 4.24mmol, 93%).

[0099] 1 H NMR (400MHz, CDCl3) δ = 9.40 (s, 2H), 7.63-7.58 (m, 4H), 7.48-7.43 (m, 4H), 5.65 (d ,J=9.8Hz,2H),4.97(td,J=10.1,4.1Hz,2H),3.14-2.98(m,6H),1.49(s,18H)ppm. 13 CNMR (101MHz, CDCl3) δ = 168.5, 156.1, 137.9, 132.8, 120.3, 118.2, 83.3, 80.6, 77.0, 55.6, 47.4, 28.4ppm.

[0100] HRMS(ESI): m / z calcd for C 32 H 39 N4O6S2[M+H] + 639.2233; found: 639.2351.

[0101]

[0102] Synthesis of Compound 10: Under argon protection, Compound 9 (1 g, 1.57 mmol) was dissolved in dichloromethane (5 mL). Trifluoroacetic acid (TFA, 1.6 mL, 25 mmol) was added dropwise to the reaction system under ice bath conditions. The reaction system rapidly turned black. Stirring continued for 1 h, with TLC monitoring during this period. After the reaction was complete, saturated sodium bicarbonate aqueous solution (40 mL) was added to quench the reaction. The mixture was extracted three times with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and the crude product was purified by silica gel column chromatography after rotary evaporation. Compound 10 could not be obtained. LC-MS analysis of the reaction system did not reveal any ion peaks of the target product.

[0103]

[0104] Synthesis of Compound 11: Under argon protection, p-iodoaniline (5 g, 22.83 mmol), cuprous iodide (CuI, 9%, 391 mg, 2.05 mmol), tetrakis(triphenylphosphine)palladium (Pd(PPh3)4, 3%, 791.41 mg, 684 μmol), and trimethylsilylacetylene (3.55 mL, 25.11 mmol) were added sequentially to dry toluene (25 mL). Then, triethylamine (3.17 mL, 22.83 mmol) was slowly added dropwise to the reaction system. The mixture was stirred overnight at room temperature in the dark, and the reaction was monitored by TLC. After the reaction was complete, the insoluble matter was removed by filtration, and the reaction was quenched by adding saturated sodium bicarbonate aqueous solution (30 mL). The product was extracted three times with dichloromethane, washed with saturated brine, dried over anhydrous magnesium sulfate, and the crude product was purified by silica gel column chromatography (eluent ratio of petroleum ether: ethyl acetate = 6:1) to obtain a white transparent solid product 11 (3.86 g, 20.39 mmol, 89%).

[0105] 1 H NMR (400MHz, CDCl3) δ = 7.16 (d, J = 8.6Hz, 2H), 6.40 (d, J = 8.5Hz, 2H), 3.69 (s, 2H), 0.15 (s, 9H) ppm. 13 C NMR (101MHz, CDCl3) δ = 146.8, 133.1, 114.3, 111.9, 91.2, 0ppm.

[0106] HRMS(ESI): m / z calcd for C 11 H 16 NSi[M+H] + :190.1052; found:190.1046.

[0107]

[0108] Synthesis of Compound 12: Under argon protection, Compound 4 (2 g, 4.54 mmol), N-hydroxy-7-azabenzotriazole (HOAT, 1.36 g, 9.99 mmol), and 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (HATU, 3.8 g, 9.99 mmol) were added sequentially to dry N,N-dimethylformamide (DMF, 25 mL). After reacting at room temperature for 10 minutes, N,N-diisopropylethylamine (DIPEA, 1.83 mL, 9.99 mmol) was added dropwise to the reaction system under ice bath conditions, and the reaction was continued under ice bath conditions for another 10 minutes. Compound 11 (2.06 g, 10.9 mmol) was dissolved in N,N-dimethylformamide (DMF, 5 mL) and added dropwise to the reaction system under ice bath conditions. After 10 minutes, the reaction was allowed to proceed to room temperature for 8 hours, and the reaction was monitored by TLC. After the reaction was complete, 1N citric acid aqueous solution (30mL) was added to quench the reaction. The product was extracted three times with ethyl acetate, washed with saturated sodium bicarbonate aqueous solution and saturated brine respectively, dried with anhydrous magnesium sulfate, and the crude product was purified by silica gel column chromatography (eluent ratio of petroleum ether: ethyl acetate = 6:1) to obtain white solid powder product 12 (3.51g, 4.48mmol, 98%).

[0109] 1 H NMR (400MHz, CDCl3) δ = 9.20 (s, 2H), 7.37 (t, J = 8.8Hz, 4H), 7.27-7.13 (m, 4H), 5.56 (t, J = 9.8H z,2H),4.74(s,2H),2.83(q,J=11.9,9.9Hz,4H),1.29–1.15(m,18H),0.05-0.07(m,18H)ppm. 13 C NMR (101MHz, CDCl3) δ = 168.6, 156.2, 137.7, 132.6, 120.2, 119.2, 104.7, 93.9, 80.6, 55.6, 47.3, 28.4, 0ppm.

[0110] HRMS(ESI): m / z calcd for C 38 H 55 N4O6S2Si2[M+H] + :783.3102; found:783.3093.

[0111]

[0112] Synthesis of Compound 13: Under argon protection, Compound 12 (156 mg, 0.2 mmol) was dissolved in ethyl acetate (4 mL), and then 4 M hydrochloric acid gas-ethyl acetate solution (2 mL, 8 mmol) was added dropwise to the reaction system in an ice bath. The reaction was carried out in an ice bath for 1 h, during which the reaction was monitored by TLC. After the reaction was completed, recrystallization with ice-cold diethyl ether failed to yield product 13. LC-MS analysis of the reaction system did not reveal any ion peaks of the target product.

[0113]

[0114] Synthesis of Compound 14: Under argon protection, p-iodoaniline (2.5 g, 11.41 mmol), cuprous iodide (CuI, 9%, 195.65 mg, 1.03 mmol), tetrakis(triphenylphosphine)palladium (Pd(PPh3)4, 3%, 396 mg, 342 μmol), and triisopropylsilylacetylene (3.55 mL, 25.11 mmol) were added sequentially to dry toluene (25 mL). Triethylamine (1.59 mL, 11.41 mmol) was then slowly added dropwise to the reaction system. The mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, 1N citric acid aqueous solution (30mL) was added to quench the reaction. The product was extracted three times with ethyl acetate, washed with saturated brine, dried over anhydrous magnesium sulfate, and the crude product was purified by silica gel column chromatography (eluent ratio of petroleum ether: ethyl acetate = 6:1) to obtain a light yellow solid product 11 (2.08g, 10.99mmol, 96.25%).

[0115] 1 H NMR (400MHz, CDCl3) δ = 7.29 (d, J = 8.5Hz, 2H), 6.58 (d, J = 8.4Hz, 2H), 3.78 (s, 2H), 1.12 (s, 21H) ppm. 13 C NMR (101MHz, CDCl3) δ = 146.6, 133.4, 114.5, 113.1, 107.9, 87.5, 18.7, 11.4ppm.

[0116] HRMS(ESI): m / z calcd for C 17 H 28 NSi[M+H] + :274.1991; found:274.2715.

[0117]

[0118] Synthesis of Compound 15: Under argon protection, Compound 4 (1.2 g, 2.72 mmol), N-hydroxy-7-azabenzotriazole (HOAT, 816 mg, 5.99 mmol), and 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (HATU, 2.28 g, 5.99 mmol) were added sequentially to dry N,N-dimethylformamide (DMF, 20 mL). After reacting at room temperature for 10 minutes, N,N-diisopropylethylamine (DIPEA, 1.14 mL, 6.54 mmol) was added dropwise to the reaction system under ice bath conditions, and the reaction was continued under ice bath conditions for another 10 minutes. Then, a solution of Compound 14 (1.56 g, 5.72 mmol) in N,N-dimethylformamide (DMF, 3 mL) was added dropwise at 0 °C. After reacting for 10 minutes, the temperature was raised to room temperature and the reaction was continued for 8 hours. The reaction was monitored by TLC. After the reaction was complete, 1N citric acid aqueous solution (30mL) was added to quench the reaction. The product was extracted three times with ethyl acetate, washed with saturated sodium bicarbonate aqueous solution and saturated brine respectively, dried with anhydrous magnesium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent volume ratio of petroleum ether: ethyl acetate = 4:1) to obtain white solid powder product 12 (2.47g, 2.6mmol, 95.30%).

[0119] 1 H NMR (400MHz, CDCl3) δ = 9.38 (s, 2H), 7.58 (m, 4H), 7.43 (d, J = 8.7Hz, 4H), 5.62 (d, J = 9.8Hz, 2H), 4.99 (d, J = 8.3Hz, 2H), 3.10 (dd, J=14.8, 4.0Hz, 2H), 3.00 (dd, J=14.7, 11.0Hz, 2H), 1.49 (s, 18H), 1.12 (s, 42H) ppm. 13 C NMR (101MHz, CDCl3) δ = 168.6, 156.2, 137.6, 132.8, 120.4, 106.8, 90.4, 80.7, 55.7, 47.8, 28.6, 18.8, 11.5ppm.

[0120] HRMS(ESI): m / z calcd for C 50 H 79 N4O6S2Si2[M+H] + :951.4980; found:951.4964.

[0121]

[0122] Synthesis of Compound 16: Under argon protection, Compound 12 (2 g, 2.1 mmol) was dissolved in ethyl acetate (20 mL), and 4M hydrochloric acid gas-ethyl acetate solution (10.51 mL, 42.04 mmol) was added dropwise to the reaction system at 0 °C. The reaction was stirred in an ice bath for 1 h, during which the reaction was monitored by TLC. The reaction system went through a process of becoming clear and then cloudy. After the reaction was completed, ice-cold diethyl ether was added for recrystallization to give yellow crystalline product 16 (1.23 g, 1.64 mmol, 77.89%).

[0123] 1 H NMR (400MHz, CDCl3) δ = 9.66 (s, 2H), 7.55 (m, 4H), 7.44 (d, J = 8.7Hz, 4H), 3.82 (m, 2H), 3.3 4(dd,J=14.2,3.9Hz,2H),2.95(dd,J=14.7,11.0Hz,2H),1.89(s,4H),1.13(s,42H)ppm. 13 C NMR (101MHz, DMSO-D6) δ = 166.3, 146.6, 139.0, 133.3, 132.5, 121.0, 120.1, 119.8, 119.6, 118.3, 8 9.6,53.2,51.8,20.7,20.4,20.2,19.8,19.5,19.2,18.8,18.6,18.3,12.4,11.8,11.5,10.7ppm.

[0124] HRMS(ESI): m / z calcd for C 40 H 63 N4O2S2Si2[M+H] + :751.3931; found:751.3907.

[0125]

[0126] Synthesis of Compound 17: Under argon protection, Compound 16 (1 g, 1.33 mmol) and Compound 8 (2.24 g, 2.93 mmol) were dissolved in ultradry N,N-dimethylformamide (DMF, 15 mL). Then, 1-hydroxybenzotriazole (HOBt, 395.71 mg, 2.93 mmol) and pyridine (2 mL, 850 μmol) were added sequentially to the reaction system. The mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, 1N HCl (20 mL) was added to quench the reaction. The mixture was extracted three times with ethyl acetate, washed with saturated brine, dried over anhydrous magnesium sulfate, and the crude product was purified by silica gel column chromatography (eluent ratio of dichloromethane:methanol = 120:1) to obtain a white solid product 17 (2.43 g, 1.21 mmol, 91.17%).

[0127] 1 H NMR (400MHz, CDCl3) δ=9.29(s,2H),7.88(d,J=27.1Hz,2H),7.50(m,12H),7.28(m,10H),6.24(s,2H),5.86(dt,J=58.8,6.8Hz,2H),5.28(dd,J=38 .6,11.7Hz,8H),5.04-4.85(m,2H),4.23(d,J=8.1Hz,2H),3.69(d,J=15. 6Hz,6H),3.17-2.79(m,8H),2.10(s,6H),2.05(s,12H),1.13(s,42H)ppm. 13 C NMR(101MHz, CDCl3)δ=169.9,169.3,169.2,166.7,155.7,148.8,137.6, 135.0,132.8,131.5,131.4,128.3,127.2,123.1,122.5,119.6,106.5,9 9.3,90.2,84.2,83.4,72.3,72.2,70.9,70.0,68.6,56.1,52.9,52.8,31 .8,,30.8,29.6,20.4,19.0,18.9,18.7,18.6,18.6,14.1,11.9,11.2ppm.

[0128] HRMS(ESI): m / z calcd for C 100 H 120 N7O 30 S2Si2[M+NH4] + :2019.7093; found:2019.7054.

[0129]

[0130] Synthesis of Compound 18: Under argon protection, Compound 17 (500 mg, 249.71 μmol) was dissolved in methanol (10 mL) and stirred in an ice bath for ten minutes. Anhydrous lithium hydroxide (LiOH, 105.24 mg, 4.39 mmol) was weighed, dissolved in 10 mL of ultrapure water, and added dropwise to the reaction system in an ice bath. The reaction was continued in an ice bath for 0.5 h, and the reaction was monitored by TLC. During this process, the color of the reaction system changed from colorless to yellow and then back to colorless. After the reaction was completed, an appropriate amount of activated cation exchange resin (IRC-120) was added and reacted for 20 min. The final system was weakly acidic (pH ~5). After filtration and evaporation to dryness, the crude product was purified by silica gel column chromatography (eluent ratio of dichloromethane:methanol = 45:1) to obtain a white solid product 18 (140.2 mg, 81.41 μmol, 32.60%).

[0131] 1 H NMR (400MHz, DMSO-D6) δ = 10.92 (s, 2H), 8.60 (s, 2H), 7.97 (s, 2H), 7.82-7.72 (m, 4H), 7.58 (d, J = 8. 6Hz,4H),7.46(d,J=8.5Hz,4H),7.42-7.30(m,10H),6.04(d,J=3.8Hz,2H),5.93-5.82(m,2H),5.26 (d,J=3.4Hz,2H),4.95(dd,J=3.9,1.7Hz,2H),4.94-4.83(m,2H),4.13-3.46(m,6H),3.27-2.90(m, 4H),2.90-2.67(m,4H),1.82-1.73(m,2H),1.35-1.30(m,2H),1.27-1.23(m,2H),1.11(s,42H)ppm. 13 C NMR (101MHz, DMSO-D6)δ=163.2,162.2,153.2,150.0,148.3,146.1,140.5,140 .2,139.9,138.5,136.6,135.4,132.9,131.6,129.0,128.5,127.3,127.1,124 .4,124.2,123.5,123.0,122.1,119.6,118.3,114.2,114.0,113.7,110.0,106 .9,95.5,91.3,87.9,82.8,70.0,67.5,66.3,29.5,19.2,19.0,11.9,11.2ppm.

[0132] HRMS(ESI): m / z calcd for C 43 H 51 N3O 30 S1Si[M+2H 2+ ] / 2 861.2884; found:861.4740.

[0133] Example 2:

[0134] Raman characterization

[0135] During the synthesis process, Raman spectroscopy was used to test and collect the Raman spectra of different compounds under the same test conditions, with particular attention paid to the Raman peaks in the biological silencing region (Table 1). Raman testing revealed that although compound 6 also had non-overlapping internal standard reference and Raman response peaks, the intensity difference between the two peaks was too large due to the difference in the cross-sectional area of ​​the Raman reporter molecule. Figure 2 A), which is not conducive to subsequent applications. Subsequently, we used the Sonogashira coupling reaction to obtain compounds with different alkyne modifications, thus obtaining compound 17. By measuring its silent region signal, we found that the Raman peaks of the internal standard reference and the response signal of compound 17 were completely separated, and the intensity ratio was 1:1 (…). Figure 2 B), and then the acetyl and methyl ester protecting groups were removed to obtain the target product 18 (Glu-RR), which was used for subsequent research.

[0136] Table 1. Raman shifts of the synthesized compounds

[0137]

[0138] Example 3:

[0139] Preparation and characterization of ratiometric Raman probes

[0140] Tetrachloroauric acid hydrate (HAuCl4·H2O, 300 μL, 20 mM) was mixed thoroughly with 2.25 mL of deionized water. Then, reduced glutathione solution (GSH, 405 μL, 20 mM) and Glu-RR (compound 18) (45 μL, 20 mM) were added dropwise to the reaction solution at room temperature. After stirring at room temperature for ten minutes, the temperature was raised to 70 °C and the reaction was continued in the dark for 24 hours to obtain a light yellow Glu-RR-AuNCs solution. The Glu-RR-AuNCs were further purified by centrifugation (8000 rpm, 20 min) using an ultrafiltration tube (MWCO = 10 kDa, 15 mL), and washed three times with PBS (10 mM). Finally, a Glu-RR-AuNCs stock solution (20 μM) was prepared and stored at 4 °C for long-term study.

[0141] The synthesized Glu-RR and glutathione (GSH) were modified onto the surface of gold nanoclusters using a one-pot method to obtain the probe Glu-RR-AuNCs. GSH acts as both a reducing agent and a stabilizer for the gold nanoclusters. The advantage of this synthetic method is that it allows for the one-pot binding of the responsive probe Glu-RR and the functional ligand GSH onto the surface of gold nanoclusters via a simple and rapid interaction between gold and thiols. Since large-sized nanoparticles, represented by gold, possess high SERS activity, AuNCs (approximately 2 nm) were chosen as gold seed particles for subsequent growth into gold nanostars (AuNSs) for Raman signal amplification. Simultaneously, the GSH-modified gold nanoclusters exhibit excellent renal clearance properties.

[0142] The results showed that the UV-Vis absorption spectrum of Glu-RR-AuNCs had a shoulder peak at ~400 nm. Figure 3 A) No surface plasmon resonance (SPR) was observed (characteristic absorption peak of larger AuNPs, typically around 520 nm). The molecular-like absorption of Glu-RR-AuNCs can be attributed to the discrete electronic states of the ultra-small nanoparticles. AuNCs were converted into gold nanostars (AuNSs) by mixing Glu-RR-AuNCs with a gold growth solution (HAuCl4 and NH2OH·HCl). During this process, the solution color changed from light yellow to light blue, and a distinct ultraviolet absorption peak (568 nm) was observed. Dynamic light scattering (DLS) data also showed that the hydrated particle size increased from 2 nm to 65 nm. Figure 3 B). These results verify that gold nanoclusters can achieve a controllable growth process under the action of gold growth solution. At the same time, the SPR phenomenon after gold growth indicates the formation of AuNSs, which have a super strong SERS effect.

[0143] Figure 4 Electron microscopy results for A showed that the particle size of Glu-RR-AuNCs was 2 nm, while the size of the obtained Glu-RR-AuNSs after the regrowth process was approximately 60 nm. Figure 4 B). The elemental mapping results were obtained using the triisopropylsilyl group on the Glu-RRs molecule. Figure 4 C) revealed that gold and silicon elements were uniformly distributed on the surface of Glu-RR-AuNSs, verifying that Glu-RRs were retained on the surface of AuNSs during the gold growth process.

[0144] Example 4

[0145] Long gold Faraman characterization

[0146] Raman detection method for gold growth: 1 μL of freshly prepared gold growth solution (hydroxylamine hydrochloride (NH2OH·HCl, 0.4M) and tetrachloroauric acid hydrate (HAuCl4, 0.15M)) was added to a Glu-RR-AuNCs solution (25 μL). After vortexing at room temperature for three minutes, the reaction solution turned blue. 1 μL of sodium citrate aqueous solution (5%) was added as a stabilizer to improve the stability and monodispersity of the solution.

[0147] After the gold deposition process, the solution sample was encapsulated in a 0.5 mm diameter capillary tube and then subjected to Raman spectroscopy using a RenishawinVia confocal Raman microscope. The Raman spectroscopy conditions for this type of liquid sample were: a 633 nm laser as the excitation source, a laser power of 2 mW, a 50× objective lens, a 10 s integration time, and a collection depth of 2000-2500 cm⁻¹. -1 Raman spectra within the wavelength range. The standard deviation was calculated using three parallel samples, and the Raman spectrum of each sample was acquired at least three times.

[0148] By comparing the Raman spectra of the Glu-RR-AuNCs probe before and after gold growth ( Figure 5 It can be observed that, under the same concentration and testing conditions, Glu-RR-AuNSs exhibits values ​​of 2154 and 2230 cm⁻¹. -1 The characteristic peak signals of the two Raman silencing regions correspond to the two Raman silencing groups: (2-phenylacetylene)triisopropylsilane (TIPS acetylene, internal standard group) and 1-propynebenzene (signal reporter group). Meanwhile, Glu-RR-AuNCs showed no Raman silencing signal peaks, which indirectly confirms that the discrete electronic states of Glu-RR-AuNCs exhibit low SERS activity.

[0149] Example 5

[0150] Feasibility analysis of Raman urinalysis

[0151] Glu-RR-AuNCs stock solution was concentrated using ultrafiltration and redispersed in pre-collected urine from healthy mice to prepare test samples with different Glu-RR-AuNCs concentrations (0-250 pmol, 25 μL). Each Glu-RR-AuNCs sample underwent gold growth in a gold growth solution, allowing for the testing and collection of a series of Raman spectra of Glu-RR-AuNCs. The limit of detection (LOD) for Glu-RR-AuNCs in the urine samples was 3σ, where σ = S0 / S; S0 is the limit of detection (LOD) of the blank sample at 2154 cm⁻¹. -1 The standard deviation of the Raman intensity at the given location; the slope of the calibration curve is S.

[0152] To evaluate the feasibility of using a gold growth-based SERS method for the quantitative detection of Glu-RR-AuNCs, gold growth and Raman spectroscopy characterization were performed on Glu-RR-AuNCs at different concentrations in urine. Notably, the Raman spectra of different concentrations of Glu-RR-AuNCs showed significant differences at 2154 and 2230 cm⁻¹. -1 The Raman intensity remains consistent at the location ( Figure 6 A) indicates that Glu-RRs molecules and gold nanoclusters maintain good chemical stability during gold growth. (The data was obtained at 2154 cm⁻¹). -1 The intensity of the internal standard Raman peak was used to quantitatively detect Glu-RR-AuNCs in urine, such as... Figure 6 As shown in B, its linear range is 1.25-250 pmol(r) 2 =0.996), LOD was 0.53 pmol (25 μL urine, 21.2 nM AuNCs).

[0153] Example 6

[0154] Probe responsiveness to enzymes

[0155] To detect the sensitivity of the probe to β-glucuronidase, Glu-RR-AuNCs (100 pmol, 25 μL) were incubated with different concentrations of β-glucuronidase from *E. coli* (final concentration 0-200 U / mL) at 37°C for ten minutes. Then, 25 μL of ice-cold methanol was added to terminate the enzymatic hydrolysis reaction. After ultrafiltration, the samples were redispersed in 25 μL of 1×PBS, and the Raman spectra of each sample were measured using the gold induction method. The final Raman intensity ratio (IL) was used. 2154 -I 2230 ) / I 2154 Quantify the enzymatic digestion ability of GUSB on probes.

[0156] Enzyme reaction kinetics analysis

[0157] Glu-RR-AuNCs (100 pmol, 25 μL) and GUSB (final concentration 200 U / mL) were co-incubated at 37 °C. At specific time points, 25 μL of ice-cold methanol was added to terminate the GUSB enzymatic hydrolysis. The supernatant after ultrafiltration was redispersed in 25 μL of 1×PBS, and Raman spectroscopy was performed using the gold nanoparticle method to monitor the enzymatic hydrolysis of the Glu-RR-AuNCs probe (n=3) per minute.

[0158] The results showed that under the action of β-glucuronidase (GUSB), the hydrolysis of the glycosidic bonds in the Glu-RR molecule triggered a self-immolative self-destruction reaction, thereby achieving the dissociation of the signal reporter group on the surface of Glu-RR-AuNCs. Simultaneously, the internal standard group remained anchored on the AuNCs surface. By comparing the signal reporter group (I... 2230 ) and internal standard group (I 2154 The Raman intensity of ) was used to establish the Raman ratio signal (I) 2154 -I 2230 ) / I 2154 Linear relationship with GUSB concentration.

[0159] like Figure 7 As shown in Figure A, with the increase of GUSB concentration, 2230 cm⁻¹ -1 The Raman signal intensity at that location gradually weakened. Meanwhile, the internal standard at 2154 cm⁻¹... -1 The Raman intensity at that location remains relatively stable. We can utilize the Raman intensity ratio (IL) 2154 -I 2230 ) / I 2154 To achieve accurate quantitative determination of GUSB enzyme activity. For example... Figure 7 As shown in B, the Raman intensity ratio (I) 2154 -I 2230 ) / I 2154 It exhibits a good linear relationship within the enzyme activity range of 25-200 U / mL (r 2 =0.998), and LOD was 3.8 U / mL. These results demonstrate that the design of ratiometric Raman probes enables quantitative monitoring of enzyme activity during enzyme-catalyzed reactions.

[0160] Subsequently, the ratio changes in the enzyme kinetics of the Glu-RR-AuNCs probe were investigated using the gold-SERS method. Figure 8 A) Under the action of 200 U / mL GUSB, the probe reached 2230 cm⁻¹. -1 The Raman signal disappeared at 12 min, indicating that the Glu-RR molecules on the surface of Glu-RR-AuNCs were completely enzymatically hydrolyzed by GUSB. Figure 8 B further confirms the Raman intensity ratio (I) 2154 -I 2230 ) / I 2154 Suitable for enzyme kinetic analysis of Glu-RR-AuNCs.

[0161] Example 7

[0162] Specificity study of probes

[0163] Glu-RR-AuNCs were incubated with GUSB (200 U / mL) or β-galactosidase (β-Gal) in PBS buffer (10 mM, pH 7.4) at 37 °C for 12 h. Raman spectroscopy was then performed using the gold-based method. The Raman intensity ratio (Ig) was used to determine the results. 2154 -I 2230 ) / I 2154 The specific enzymatic response of Glu-RR-AuNCs was evaluated. All samples were measured at least three times under the same test parameters.

[0164] To verify the probe's ability to specifically recognize enzyme digestion, Glu-RR-AuNCs were co-incubated with 200 U / mL GUSB and β-galactosidase (β-Gal), respectively, followed by Raman spectroscopy measurements using the gold-SERS method. The results are as follows: Figure 9 As shown in A, 2154cm -1 The Raman signal intensity at the site did not change significantly, indicating that a stable gold-sulfur bond was formed between the cysteine ​​residue adjacent to the internal standard signal group and Glu-RR-AuNCs, fixing the internal standard group on the gold surface. Meanwhile, after GUSB enzymatic digestion at 2230 cm⁻¹... -1 The Raman intensity was significantly reduced at the treatment site, while no significant change in the Raman ratio signal was observed after β-Gal treatment. Figure 9 B). This result confirms that Glu-RR-AuNCs can be specifically triggered by GUSB to self-immolative groups, thereby achieving Raman intensity ratio (I). 2154 -I 2230 ) / I 2154 A significant increase.

[0165] Example 8

[0166] Pharmacokinetics and major organ distribution studies

[0167] To investigate the pharmacokinetics of Glu-RR-AuNCs in mice, Glu-RR-AuNCs solution (20 μM, 100 μL, 2 mg / kg) was first injected via the tail vein into MDA-MB-231 tumor-bearing mice (n=3). Under isoflurane anesthesia, Glu-RR-AuNCs were injected via the tail vein, and blood samples (~50 μL / mouse) were collected from the tail tip at selected time points (2 min, 5 min, 15 min, 30 min, 1 h, 3 h, 5 h, 8 h, 12 h, and 24 h). The collected blood samples were immediately mixed thoroughly with PBS buffer (2% EDTA, 100 μL) containing anticoagulant, centrifuged (4500 g, 5 min, 4 °C) to remove blood cells. After serum was treated with aqua regia, the Au content was determined by ICP-MS. By fitting the Au content data to a classic two-compartment pharmacokinetic model, the distribution half-life (t0.05) of Glu-RR-AuNCs in mouse blood can be calculated. 1 / 2α And elimination half-life (t) 1 / 2β ).

[0168] To investigate the organ distribution of the probe, Glu-RR-AuNCs solution (20 μM, 100 μL, 2 mg / kg body weight) was injected intravenously into MDA-MB-231 tumor mice or healthy mice. Mice (n=3) were sacrificed 1 h or 24 h after intravenous injection, and samples of major organs (heart, liver, spleen, lung, kidney, brain, and stomach), tissues (tumor, muscle, and fat), and blood were collected and weighed sequentially. All samples were then treated with aqua regia for 7 days, and the gold content in the samples was quantified by ICP-MS to calculate the gold content in each collected sample.

[0169] The pharmacokinetics of Glu-RR-AuNCs were investigated using the MDA-MB-231 subcutaneous tumor model. Figure 10 As shown in Figure A, the Au content in the blood decreased rapidly within the first 15 minutes, followed by a slow decline, consistent with a classic two-compartment pharmacokinetic model. The distribution half-life (t0) is also shown. 1 / 2α and elimination half-life (t) 1 / 2β The mean values ​​were 6.12±0.81 min and 5.18±0.90 h, respectively, indicating that Glu-RR-AuNCs had sufficient time to be effectively enriched at the tumor site.

[0170] Subsequently, the distribution of Glu-RR-AuNCs in major organs of healthy nude mice was examined at two time points: 1 and 24 hours after tail vein injection. The results showed that Glu-RR-AuNCs had relatively high levels in the kidney (8.96±0.14% ID / g) and blood (6.97±0.04% ID / g) 1 hour later. Figure 10 (B) After 24 hours, the gold content of Glu-RR-AuNCs in the blood significantly decreased to 1.32 ± 0.10% ID / g, while maintaining a high gold content in the kidneys (8.84 ± 0.14% ID / g), indicating that Glu-RR-AuNCs have high renal clearance efficiency in healthy mice. Furthermore, compared to traditional nanoprobes, Glu-RR-AuNCs accumulated less in the liver, suggesting that they possess the ability to evade RES clearance.

[0171] To evaluate the tumor-targeting ability and renal clearance efficiency of the same Glu-RR-AuNCs dose in MDA-MB-231 tumor model mice. Figure 10 As shown in Figure C, the tumor model mice exhibited similar organ distribution to healthy mice, while the tumor sites at 1 and 24 hours showed high gold content, at 7.28 ± 0.13% ID / g and 7.82 ± 0.21% ID / g, respectively. The GSH on the surface of the Glu-RR-AuNCs probes endowed them with high tumor targeting specificity. Therefore, Glu-RR-AuNCs, with their high tumor targeting and high renal clearance, show great promise for applications in urine detection in in vivo biomedicine.

[0172] Example 9

[0173] Feasibility of detecting cancer in urine

[0174] In vivo study of renal clearance capacity: to a tumor volume of 1000 mm 3 Female nude mice (11 weeks old, n=6) that underwent subcutaneous xenograft transplantation of MDA-MB-231 were injected intravenously with Glu-RR-AuNCs solution (20 μM, 100 μL, 2 mg / kg body weight). The mice were then placed in metabolic cages, and urine was collected at selected time points (1, 2, 3, 6, 9, 12, and 24 h). After bladder emptying, the collected urine volume was recorded. The Au content in the urine was quantified using both ICP-MS and the Changjin-SERS method, allowing for the calculation of the renal clearance rate of the Glu-RR-AuNCs probe. The accuracy of the ICP-MS and Changjin-SERS methods was assessed using the Pearson correlation coefficient (PCC).

[0175] The results are as follows Figure 11As shown in Figure A, Raman spectroscopy results at different time points indicate that the internal standard reference signal intensity (IL) in tumor model mice after tail vein injection... 2154 ) and response signal strength (I 2230 All of these gradually decreased over time, while compared to I 2154 I 2230 The attenuation was more pronounced, with no significant Raman signal intensity after 3 hours. 2154cm -1 The decrease in Raman intensity at 2230 cm⁻¹ is attributed to renal clearance, while the overexpression of GUSB enzymes in the tumor microenvironment causes Raman-responsive groups to leave the AuNCs surface, leading to the reduction in Raman intensity at 2230 cm⁻¹. -1 The Raman intensity at the location decreases until it disappears. In healthy mice, I... 2154 and I 2230 It also shows a downward trend over time. Figure 1 B).

[0176] By further plotting the Raman signal ratio (I) 2154 -I 2230 ) / I 2154 The curves showing changes over time reveal a significant difference in tumor-bearing mice in the experimental group (I...). 2154 -I 2230 ) / I 2154 The Raman ratio value increases and reaches equilibrium at 6 hours. Figure 11 C). No Raman ratio change was observed in the 1-hour urine of healthy mice, and the ratio remained relatively stable during long-term monitoring, indicating that the probe has good stability in healthy mice. This also indirectly verifies that the 1-hour ratio change in tumor model mice is caused by the overexpression of GUSB in the tumor microenvironment, demonstrating excellent tumor specificity.

[0177] The feasibility of using an internal standard reference signal to quantitatively detect the renal clearance of Glu-RR-AuNCs was then investigated. A 2154 cm² renal clearance was established. -1 The linear relationship between the Raman internal standard reference signal intensity and gold content can convert the Raman spectra at different time points into the gold content in urine, such as... Figure 12 As shown, the renal clearance rate at different time points was highly consistent with the results of ICP-MS (PCC = 0.994). Compared with ICP-MS, which requires cumbersome pretreatment processes such as nitration, the Raman detection method designed in this invention can achieve rapid determination of gold content in urine using a portable handheld Raman spectrometer, without the need for professional operation or complex sample pretreatment processes, thus significantly improving detection efficiency.

[0178] To further verify that the changes in ratiometric Raman signal originated from the enzymatic digestion of GUSB, the tumor sites at 2154 cm and 2230 cm were further analyzed.-1 The Raman characteristic peaks. Silver staining effectively enhanced the Raman signals of endogenous and exogenous molecules near AuNCs in tumor tissue (1 h pi). For example... Figure 13 The imaging results show that the tumor tissue is located at 2154 and 2230 cm. -1 Both channels exhibited significant Raman signals, which were undetectable in adjacent normal tissue. The tumor boundaries delineated by Raman mapping were highly consistent with the results of adjacent HE sections. Notably, at 2154 cm... -1 The Raman signal of the red channel is stronger than that of the 2230cm channel. -1 The green channel indicates that GUSB in the tumor can undergo in-situ enzymatic hydrolysis, leading to the removal of Raman-responsive groups from the AuNC surface, thus reflecting the enzymatic activity distribution of GUSB in the tumor site. Therefore, (I 2154 -I 2230 ) / I 2154 Ratio channels can achieve higher tumor-to-normal ratios (TNR), which are of great value for precise tumor resection.

[0179] Furthermore, by performing silver staining-Raman mapping tests on kidney tissue sections from healthy mice, we were able to find that the AuNC probe was mainly distributed in the renal tubules of the kidney tissue. Figure 14 ), 2154 and 2230cm -1 The two channels have similar Raman intensities, (I 2154 -I 2230 ) / I 2154 The absence of a significant signal in the ratio channel indicates that the Glu-RR-AuNCs probe maintains the integrity of its structure and surface chemical modifications during renal metabolic clearance in healthy mice. These results confirm that Glu-RR-AuNCs can be specifically activated by GUSB at the tumor site, subsequently metabolized and excreted via renal clearance. Raman spectroscopy analysis of urine using the gold-SERS method ultimately allows for the use of urinary AuNCs products as a quantifiable tumor marker for cancer diagnosis.

[0180] Example 10

[0181] Long-term urine monitoring in MDA-MB-231 tumor mice

[0182] To evaluate the applicability of the long-term urinary SERS method for monitoring tumor growth, female balb / c nude mice (7 weeks old, n=6) were subcutaneously inoculated with 1.5 × 10⁻⁶ mmol / L on day 0. 6MDA-MB-231 cells were administered via tail vein injection every three days with a dose of Glu-RR-AuNCs solution (20 μM, 100 μL, 2 mg / kg body weight). Urine was then collected for 1 hour using a mouse metabolic cage, and Raman spectra were collected using gold-SERS technology. The Raman intensity ratio (IL) was ultimately calculated. 2154 -I 2230 ) / I 2154 A correlation was established between the two and tumor volume.

[0183] Through monitoring mice for up to 27 days after tumor implantation, we found that as the tumors grew in mice ( Figure 15 ), 2230cm in urine -1 The Raman signal intensity at that location is significantly reduced ( Figure 16 A). For example Figure 16 As shown in B, (I) 2154 –I 2230 ) / I 2154 It showed a positive correlation with tumor volume (PCC = 0.997). On day 3 of tumor bearing in mice, the tumor diameter was ~4 mm (34.96 ± 2.49 mm). 3 When ), it is possible to detect (I) in urine. 2154 –I 2230 ) / I 2154 The change (3.68±0.12%) indicates that Raman ratio changes can be used to detect smaller tumors in the early stages of cancer. The ratio Raman signal change of Glu-RR-AuNCs is positively correlated with the degree of GUSB hydrolysis of Glu-RRs. Therefore, we infer that in the MDA-MB-231 tumor mouse model, GUSB enzyme activity is positively correlated with tumor volume; that is, the larger the tumor volume in the mouse, the higher the enzyme activity in the tumor microenvironment, and the higher the efficiency of glycosidic bond hydrolysis, which can then be detected significantly in urine. 2154 –I 2230 ) / I 2154 change.

[0184] Subsequently, immunofluorescence experiments were used to verify the above hypothesis. The immunofluorescence intensity gradually increased with tumor size, indicating that GUSB activity is indeed correlated with tumor volume. Figure 17 Similarly, such as Figure 18 The in-situ silver staining Raman mapping shown indicates that (I 2154 –I 2230 ) / I 2154 The channel signal intensity gradually increased with tumor growth, further verifying that the activity of GUSB enzyme in the tumor microenvironment is positively correlated with tumor growth.

[0185] Furthermore, during the long-term monitoring period of 27 days, there was no significant difference in the trend of body weight change between MDA-MB-231 tumor model mice and healthy mice. Figure 19 This further validates that Glu-RR-AuNCs have good biocompatibility.

[0186] The above description is merely an embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A ratiometric Raman spectroscopy detection system capable of renal metabolism, characterized in that, include: a) A light source module emitting near-infrared light; b) Surface-enhanced Raman scattering nanoparticles, wherein the nanoparticles are renally metabolizable ratiometric Raman scattering nanoparticles that specifically respond to β-glucuronidase, and the nanoparticles are applied to mammals, and the surface of the surface-enhanced Raman scattering nanoparticles is connected to an internal standard reference group and a signal response group that responds to β-glucuronidase; c) A urine collection and processing module, which is used to collect urine after the application of the above nanoparticles and further process the nanoparticles in the urine into nanostar particles; d) An acquisition module for acquiring the surface-enhanced Raman scattering spectrum of the above nanostar particles. e) The information processing module establishes a Raman ratio signal by comparing the Raman intensities of the signal response reporter group and the internal standard reference group, and determines the concentration level of β-glucuronidase in the tissue of this mammal. The nanoparticles are gold nanocluster probes Glu-RR-AuNCs, where Glu-RR is a compound of the following formula. ； Among them, β-glucuronidase can specifically cleave Glu-RR molecules, causing the signal response reporter group 1-propynylbenzene to detach from the surface of AuNCs, and the internal standard reference group (2-phenylacetylene) triisopropylsilane is stably anchored on the surface of AuNCs through gold-sulfur bonds.

2. The detection system according to claim 1, characterized in that, The mammal in question is a mammal with a tumor.

3. The detection system according to claim 2, characterized in that, The acquisition module obtains the comparison signal response report group I. 2230 and internal standard reference group I 2154 Raman intensity.

4. The detection system according to claim 3, characterized in that, In the information processing module, the Raman ratio signal is (I 2154 -I 2230 ) / I 2154 ratio.

5. The detection system according to claim 1, characterized in that, In the urine collection and processing module, the long gold method is used to further process the nanoparticles in the urine into nanostar particles.

6. The detection system according to claim 1, characterized in that, Before applying surface-enhanced Raman scattering nanoparticles, the Raman intensity ratio of the internal standard reference group to the signal response reporter group was 1:

1.

7. The detection system according to claim 1, characterized in that, The information processing module further includes processing the enzyme activity distribution of β-glucuronidase at the tumor site.

8. The detection system according to claim 1, characterized in that, This detection system is used for tumor diagnosis, detection, and / or monitoring of the efficacy of chemotherapy drugs.

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