A tandem ratiometric raman probe and its preparation method and application

Abstract

The application belongs to the technical field of molecular imaging, and particularly relates to a tandem ratio response Raman probe and a preparation method and application thereof. The Raman probe is a substance composed of a gold nano substrate, a dye molecule and a polymer. The gold nano substrate has good chemical stability and rich surface plasmon, and can enhance the Raman signal. The surface of the substance is connected with a Raman reporter molecule, so that the tandem response of ROS and pH can be realized, and a basic Raman peak is used as an internal reference. The response peaks of the two markers and the internal reference Raman spectrum peak are not overlapped, so that the accurate quantification of ROS and pH can be realized. The reporter molecule has pH stability when ROS does not respond, so that the pH fluctuation monitoring after the opening of ROS can be realized. The application has high substance ratio signal, good stability and high sensitivity, can image the nerve inflammation site in the brain microenvironment, and then specifically image the pH fluctuation in the brain disease environment, is beneficial to the pH monitoring of the lesion site, and has great significance for in-depth understanding of the brain function change and disease progression mechanism.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular imaging, and particularly relates to a Raman probe and a preparation method and application thereof. BACKGROUND

[0002] The pH of the brain microenvironment is critically important in regulating cell signaling, cell death, memory formation, and maintaining the functional homeostasis of neuronal electrical activity and neural circuits. Generally, the extracellular pH of the brain is maintained in the range of 7.2 to 7.4 by the action of transporters (e.g., Na + / HCO3 - co-transporters and Cl - / HCO3 - exchangers) and pH-sensitive membrane proteins expressed by neurons and glial cells (e.g., acid-sensing ion channels ASICs). Due to the sensitivity of membrane proteins such as ion channels, transporters, and ATPase pumps to pH, even a small fluctuation of 0.1 to 0.2 pH units can have a significant impact on neural state, glial cell function, and neurotransmitter regulation. Generally speaking, an increase in the microenvironment pH enhances neuronal excitability, while acidification attenuates excitability. Notably, the relationship between pH and neuronal excitability is not simple. Specifically, some neurons (e.g., chemosensitive neurons) exhibit higher excitability under acidic conditions, while other neurons (e.g., hippocampal neurons) can exhibit the opposite reaction. Moreover, both acute and chronic perturbations of the extracellular acid-base balance of the brain can significantly promote the initiation and progression of neurological diseases (e.g., schizophrenia, brain tumors, epilepsy, and aging). Given the correlation between brain pH and neural function and brain diseases, dynamic measurement of in vivo brain pH fluctuations is crucial for a deep understanding of the mechanisms of brain function changes and disease progression.

[0003] Currently, a variety of brain pH detection techniques have been developed, including electrochemical measurement, magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), and fluorescence imaging. Among them, electrochemical detection using microelectrodes is the most direct method for detecting brain pH, with good sensitivity and procedural simplicity. However, the direct interaction of rigid electrode interfaces with brain tissue can affect neuronal or glial cell activity, thereby affecting the accuracy of pH measurement and posing significant invasive risks. As a non-invasive alternative method, various magnetic resonance techniques, including 31 PMRS, 1 HMRS, and 1H MRI can provide quantitative information for pH determination. However, these techniques face challenges including slow acquisition speed, need for specialized hardware and coil equipment, and complex operating procedures. Fluorescence imaging has shown significant advantages in pH imaging, including high sensitivity, simplicity, and high spatiotemporal resolution. However, the selective physiological barrier of the central nervous system, the blood-brain barrier (BBB), hinders the effective entry of many exogenous substances, including drugs and probes, into the brain parenchyma. Therefore, although many fluorescence probes for pH detection have been developed, the challenge of crossing the BBB and achieving pH-specific optical imaging at the lesion site still exists.

[0004] Raman spectroscopy is derived from the inelastic scattering of incident light when it interacts with target molecules, which directly or indirectly provides specific spatial resolution and chemical insight into molecular structure and conformation. As an ideal alternative to molecular imaging or sensing, Raman spectroscopy overcomes the limitations of fluorescence, such as susceptibility to photobleaching and autofluorescence. In addition, the low sensitivity inherent in Raman scattering can be enhanced by surface-enhanced Raman scattering (SERS), even reaching single-molecule detection levels after adsorption to a corrugated metal surface. Importantly, SERS probes can be precisely fabricated through surface modification to improve biocompatibility, cross physiological barriers, selectively target specific cells, and even organelles. However, SERS probes often encounter problems of non-specific aggregation, uneven distribution, and susceptibility to working conditions when used in vivo, resulting in ambiguous and even inaccurate detection signals. The ratio method strategy provides a promising way to improve detection accuracy by using a signal self-calibration method.

[0005] Although the pathological mechanisms behind neurological diseases are diverse, including protein aggregation, systemic infection, aging, and genetic mutation, they share the common feature of chronic neuroinflammation. Autoimmune neurological diseases, such as multiple sclerosis, are characterized by the infiltration of peripheral inflammatory cells, while primary neurodegenerative diseases, such as Alzheimer's disease and epilepsy, are typically manifested by significant microglial and astrocyte activation. In the context of neuroinflammation, with the increase in enzyme activity, especially the activity of NADPH oxidase, mitochondrial dysfunction is exacerbated and the production of reactive oxygen species (ROS) is increased. This leads to oxidative damage and thus amplifies the inflammatory response. At the same time, neuroinflammation stimulates the synthesis and release of pro-inflammatory mediators, including cytokines, chemokines, complement components, and free radicals, ultimately leading to impaired brain function and structural changes. Studies have shown that hydrogen peroxide (H2O2) is upregulated in the context of neuroinflammation.

[0006] Accordingly, constructing a ratiometric SERS probe that can cross the blood-brain barrier and be activated by H2O2 for specific imaging of pH fluctuations in the environment of brain diseases is an important technology for in-depth understanding of the mechanisms of brain function changes and disease progression. SUMMARY

[0007] The present application aims to provide a self-ratio SERS probe with series double ratio response, high ratio signal, good stability, high brain entry efficiency, high sensitivity and good biological safety, and a preparation method and application thereof, and solve the technical problems of strong invasiveness, slow operation speed, complex equipment program and blood-brain barrier penetration barrier in the existing imaging brain pH, especially specific imaging of pH fluctuation in brain disease environment, so as to facilitate accurate monitoring of pH fluctuation in brain disease environment.

[0008] The series ratio response Raman probe provided by the present application is a substance composed of a gold nano substrate, dye molecules and polymers, and its structure is shown in the following formula V:

[0009]

[0010] Among them, A is a gold nano substrate;

[0011] X 1 The structure is shown in formula I a :

[0012]

[0013] Among them, S is connected to the gold on the surface of A through a gold-sulfur bond, R 1 and R 2 are independently hydrogen, halogen, C1-C4 alkyl, sulfonic acid group or nitro group; R 1 and R 2 are the same or different.

[0014] X 2 is L 1 -L 2 , L 1 is a group connected to the gold on the surface of A through a gold-sulfur bond, L 2 is a hydrophilic polymer group, and middle- indicates the connection between the two, and the same below;

[0015] X 3 is L 1 -L 3 -L 4 , L 1 is a group connected to the gold on the surface of A through a gold-sulfur bond, L 3 is a hydrophilic polymer group, and L 4 is a targeting group.

[0016] A is a Raman signal enhanced substrate, which is used to provide electromagnetic field hot spots to enhance the Raman signal of Raman reporter X 1 , and improve the sensitivity of detection; X 1X is a reaction molecule having H2O2 and pH dual-ratio responsiveness for providing a quantitative basis of the target marker; X 2 X is used for increasing the biocompatibility of the probe; X 3 X is used for imparting the probe with the performance of efficiently crossing the blood-brain barrier.

[0017] The A is a gold nano substrate as per the conventional art, and the shape of the A is preferably a multi-branch star shape, a polygon shape, a rod shape or a spherical shape, further preferably a multi-branch star shape or a polygon shape, and more preferably a multi-branch star shape.

[0018] The particle size of the A is 5 nm to 200 nm, preferably 10 nm to 100 nm, and more preferably 50 ± 20 nm.

[0019] The A is prepared by reacting chloroauric acid, gold seeds, silver nitrate and ascorbic acid in a solvent to form a gold-sulfur bond.

[0020] In the substance as shown in Formula V:

[0021] X 1 The molar ratio of X to A is 1 x 10 4 : 1 to 5 x 10 4 : 1, preferably 1 x 10 4 : 1 to 3 x 10 4 : 1, and more preferably 2 x 10 4 : 1.

[0022] X 2 The molar ratio of X to A is 5 x 10 4 : 1 to 50 x 10 4 : 1, preferably 10 x 10 4 : 1 to 40 x 10 4 : 1, and more preferably 15 x 10 4 : 1.

[0023] X 3 The molar ratio of X to A is 1 x 10 6 : 1 to 3 x 10 6 : 1, preferably 1.5 x 10 6 : 1 to 3.5 x 10 6 : 1, and more preferably 2 x 10 6 : 1.

[0024] Further:

[0025] The halogen is Cl, Br or I;

[0026] The C1-C4 alkyl group is a methyl group, an ethyl group, an n-propyl group, an iso-propyl group, an n-butyl group, an iso-butyl group, a sec-butyl group or a tert-butyl group, and is preferably a methyl group.

[0027] said X 2 in the structure of:

[0028] said L 1 in the structure of:

[0029]

[0030] said L 2 is a polyethylene glycol group, a polyethylene glycol derivative group, a polylysine group or a polyethylenimine group; preferably a polyethylene glycol derivative group.

[0031] said L 2 has a molecular weight of 1000-10000, preferably 4000-6000, more preferably 5000.

[0032] said L 2 is a polyethylene glycol group, in the structure of:

[0033]

[0034] said X 2 is L 1 -L 2 , when L 2 is a polyethylene glycol group, X2 is in the structure of:

[0035]

[0036] said L 3 is a maleimide group modified polyethylene glycol group, a maleimide group modified polyethylene glycol derivative group, a maleimide group modified polylysine group or a maleimide group modified polyethylenimine group, preferably a maleimide group modified polyethylene glycol derivative group.

[0037] said L 3 has a molecular weight of 1000-10000, preferably 4000-6000, more preferably 5000.

[0038] said L 3 is a maleimide group modified polyethylene glycol group, in the structure of:

[0039]

[0040] said L 4 is a blood-brain barrier crossing targeting group, preferably a targeting neuroinflammatory related receptor group, more preferably a receptor for advanced glycation end products (RAGE) targeting peptide, an anti-VCAM-1 antibody group or an anti-IL-1β antibody group, particularly preferably a RAGE targeting peptide.

[0041] said L 4 is a RAGE targeting peptide, and has an amino acid sequence of CELKVLMEKEL.

[0042] X 3 is L 1 -L 3 -L 4 , when L 3 is a polyethylene glycol group, L 4 RAGE targeting peptide, X 3 has a structure of:

[0043]

[0044] Further:

[0045] The hydrated particle size of the substance shown in formula V can be 110±30nm.

[0046] The surface charge of the substance shown in formula V can be 9±5mV.

[0047] The substance shown in formula V can have Raman effect.

[0048] The Raman spectrum formed by the substance shown in formula V has one or more characteristic peaks at 303cm -1 , 482cm -1 , 520cm -1 , 552cm -1 , 590cm -1 , 928cm -1 , 790cm -1 , 1169cm -1 and 1430cm -1 .

[0049] The Raman spectrum formed by the substance shown in formula V has characteristic peaks at 303cm -1 , 482cm -1 , 520cm -1 , 552cm -1 , 590cm -1 , 928cm -1 , 790cm -1 , 1169cm -1 and 1430cm -1 .

[0050] The Raman spectrum formed by the substance shown in formula V has characteristic peaks at 482cm -1 , 590cm -1 and 928cm -1 .

[0051] The substance shown in formula V can be used as a probe.

[0052] The substance shown in formula V can be used for detecting active oxygen, which can be superoxide anion, hydrogen peroxide, singlet oxygen, hydroxyl radical, lipid peroxidation radical, ozone, hypochlorite or hypobromite, in particular hydrogen peroxide.

[0053] The concentration of the active oxygen can be 5 μmol / L to 100 μmol / L.

[0054] The substance shown in formula V can be used for detecting the pH in the active oxygen environment. The pH range can be 2.0 to 10.0.

[0055] The substance shown in formula V can be used for detecting organs, tissues or cells, such as brain tissues or neuron cells.

[0056] The substance shown in formula V can be used for detecting organs, tissues or cells containing active oxygen, and the pH thereof, which can be neuron cells or brain tissues (such as epileptogenic foci).

[0057] The present application also provides a preparation method of the substance shown in formula V, comprising the following steps: reacting the substance X shown in formula a 1 , polymer X 2 and polymer X 3 in a solvent to obtain the substance V.

[0058] The solvent is an alcohol solvent and / or an aqueous solvent, preferably an alcohol solvent and water.

[0059] The alcohol solvent is preferably methanol and / or ethanol, more preferably methanol.

[0060] The aqueous solvent is deionized water, distilled water or pure water, more preferably deionized water.

[0061] When the solvent is a combination of an alcohol solvent and an aqueous solvent, the volume ratio of the alcohol solvent to the aqueous solvent is 1:10 to 1:500, preferably 1:50 to 1:150, more preferably 1:80 to 1:100.

[0062] The molar ratio of the substance I a to A is 1×10 4 :1 to 5×10 4 :1, preferably 1×10 4 :1 to 3×10 4 :1, more preferably 2×10 4 :1.

[0063] The polymer X 2 ​The molar ratio of the substance I to A is 1*10 3 :1~1*10 5 :1, preferably 1*10 4 :1~3*10 4 :1, more preferably 2*10 4 :1.

[0064] The molar ratio of the polymer X 3 to A is 1*10 6 :1~5*10 6 :1, preferably 1*10 6 :1~3*10 6 :1, more preferably 2*10 6 :1.

[0065] The molar ratio of the polymer X 2 to the polymer X 3 is 1:50~1:1000, preferably 1:50~1:200, more preferably 1:100.

[0066] The molar volume ratio of the A to the solvent is 1:50~1:500, preferably 1:100~1:300, more preferably 1:202.17.

[0067] The molar volume ratio of the substance I a to the solvent is (50~150):1, preferably 90:1~120:1, more preferably 98.93:1.

[0068] The molar volume ratio of the polymer X 3 to the solvent is 5000:1~15000:1, preferably 8000:1~10000:1, more preferably 9892.66:1.

[0069] The A participates in the reaction in the form of a solution of A, and the concentration of A is 1~10 nmol / L, preferably 1 nmol / L.

[0070] The substance provided by the application has good chemical stability, rich surface plasmons, and can greatly enhance the Raman signal, by taking gold nanometer as an enhancing substrate. 1ROS and pH can be realized in series response, and the Raman peak is basically unchanged as a reference. ROS response peak, pH response peak and internal reference Raman peak are not overlapped, which can realize accurate quantification of ROS and pH. Moreover, the reporter molecule has pH stability when ROS does not respond, so it can realize the monitoring of pH fluctuation after ROS is turned on. When the substance interacts with ROS, the structure of the ROS response group of the reporter molecule changes, causing changes in the corresponding Raman spectrum, and the ratio of the Raman peak of the non-response group to the characteristic peak formed by it increases (I non-response / I response ROS). On this basis, the substance turns on the pH responsiveness, and the structure of the pH response group of the reporter molecule changes at different pH, causing changes in the corresponding Raman spectrum, and the ratio of the Raman peak of the non-response group to the characteristic peak formed by it increases (I non-response / I response pH).

[0071] The substance provided by the application has tandem double responsiveness and self-ratio calibration, and has the advantages of high ratio signal, good stability, high sensitivity, etc. It can image the nerve inflammation site in the brain microenvironment, and then specifically image the pH fluctuation in the brain disease environment, which is beneficial to the pH monitoring of the lesion site, and has great significance for in-depth understanding of the mechanism of brain function change and disease progression. In addition, the targeting group connected on the substance can specifically recognize the receptor for advanced glycation end products (RAGE) highly expressed on the brain vascular endothelial cells in the nerve inflammation site, guide the image probe to cross the blood-brain barrier through transcytosis, and realize targeted enrichment and signal activation in the nerve inflammation area. In the inflammation area, the probe responds to ROS, and the Raman signal changes in a ratio type, while the ability of pH monitoring is turned on, realizing specific monitoring of pH fluctuation in the lesion site to study the correlation between brain function and brain disease.

[0072] Compared with the developed various brain pH detection technologies, the substance has the characteristics of "efficient brain entry across blood-brain barrier", "low influence on non-lesion area", "specific response in lesion area", "ROS and pH tandem double responsiveness", "self-ratio calibration", etc., which can effectively improve the signal-to-noise ratio of the lesion site, and can depict the pH fluctuation in the environment of various brain diseases to in-depth study its mechanism and develop new treatment strategies. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 The ratio type SERS probe is used for imaging H2O2 and pH fluctuation in neuron cells. Among them, A is a schematic diagram of separation, culture and Raman imaging of mouse primary neurons, B is a bright field image of patch clamp recording of neuron electrophysiology, C is a representative electrophysiology result of neurons under different stimulations, and D is the endogenous H2O2 and pH level confocal Raman imaging of mouse primary neurons under different stimulations.

[0074] Figure 2In vivo brain SERS imaging of epilepsy mouse model using HCB probe. A, Schematic diagram of in vivo brain SERS imaging of epilepsy mouse model using HCB probe. B, Simultaneous recording of neural electrical activity and SERS ratio monitoring of pH fluctuation in mouse brain in different grades of seizure. Top, Representative EEG results. Middle, Representative EEG power spectrum. Bottom, pH in mouse brain recorded by HCB probe over time.

[0075] Figure 3 SERS ratio images of mouse brain before, during and after different drug treatments for epilepsy mouse injected with SERS probe V. Left, Representative EEG results. Middle, Pseudo-color I 928 / I 482 ratio images depicting H2O2concentration. Right, Pseudo-color I 928 / I 590 ratio images showing pH value. DETAILED DESCRIPTION

[0076] The present application is further illustrated by the following examples without thereby limiting the present application to the examples described. The experimental methods in the following examples, if not otherwise specified, are selected according to the conventional methods and conditions, or according to the commercial instruction.

[0077] Example 1: Synthesis of substance I-a

[0078]

[0079] N,N-dimethylformamide (DMF, 0.5 mol) and 40 mL dichloromethane were uniformly mixed in an ice-salt bath for 30 min. 37 mL phosphorus oxychloride (0.4 mol) and 10 g cyclohexanone (I-1, 0.1 mol) were uniformly mixed and then added dropwise into the above mixture. The reaction was refluxed for 3 h, and then the reaction liquid was poured into 200 g crushed ice and cooled overnight. The yellow solid was collected by filtration to obtain the target product chloral (I-2).

[0080] 320 mg indole substance (I-3, 2 mmol) and 950 μL 2-iodoethanol (12.2 mmol) were dissolved in 15 mL o-dichlorobenzene. The mixture was refluxed at 100°C for 48 h, and the crude product was precipitated. The crude product was washed with 500 mL acetone to obtain indole ethanol (I-4).

[0081] Further, 203 mg chloral substance (I-2, 1.18 mmol) and 730 mg indole ethanol substance (I-4, 2.26 mmol) were dissolved in 50 mL n-butanol-toluene mixed solvent (C4H 10O:C7H8 = 7:3, V:V), 120°C refluxing with stirring overnight. After the system was dried, the crude product was dissolved in 5 mL of methanol, and the methanol solution was added dropwise to 150 mL of vigorously stirred ice-ether, and then filtered and dried to obtain the crude product. The crude product was dissolved in 2 mL of a 2:1 mixture of dichloromethane and methanol to obtain a solution to be purified. The solution to be purified was then separated and purified using silica gel column chromatography to obtain dark green material Cy7-Cl (I-5).

[0082] The specific operation conditions for silica gel column chromatography separation and purification are as follows:

[0083] Silica gel column: inner diameter 40 mm, length 300 mm, packed with 200-300 mesh silica gel;

[0084] Elution conditions: after the solution to be purified was loaded, 100 mL of dichloromethane was used first, followed by 500 mL of a dichloromethane-methanol mixture (CH2Cl2:CH4O = 15:1, V:V), and finally 300 mL of a dichloromethane-methanol mixture (CH2Cl2:CH4O = 5:1, V:V), with the elution rate controlled at 30 mL / min-80 mL / min.

[0085] Then triethylamine (7.34 mmol, 20 eq) and resorcinol (3.67 mmol, 10 eq) were dissolved in N,N-dimethylformamide (DMF). After stirring at room temperature for 30 min, substance I-5 (0.37 mmol, 1.0 eq) was added, and the reaction was stirred at 88°C for 6 h. After the system was dried, the crude product was dissolved in 2 mL of a 2:1 mixture of dichloromethane and methanol to obtain a solution to be purified. The solution to be purified was then separated and purified using silica gel column chromatography to obtain blue material Hem-H (I-6).

[0086] The specific operation conditions for silica gel column chromatography separation and purification are as follows:

[0087] Silica gel column: inner diameter 40 mm, length 300 mm, packed with 200-300 mesh silica gel;

[0088] Elution conditions: after the solution to be purified was loaded, 200 mL of dichloromethane was used first, followed by 1500 mL of a dichloromethane-methanol mixture (CH2Cl2:CH4O = 12:1, V:V), and finally 500 mL of a dichloromethane-methanol mixture (CH2Cl2:CH4O = 4:1, V:V), with the elution rate controlled at 30 mL / min-80 mL / min.

[0089] Substance I-6 (207 mg, 0.5 mmol), 4-bromomethylphenylboronic acid pinacol ester (193 mg, 0.65 mmol), K2CO3(83 mg, 0.6 mmol) were dissolved in anhydrous dimethylformamide (DMF, 30 mL) and reacted at 50°C for 6 h under nitrogen protection. After the system was dried, it was dissolved in 2 mL of a 2:1 mixture of dichloromethane and methanol to obtain a solution to be purified. The solution to be purified was then separated and purified using silica gel column chromatography to obtain dark blue substance Hem-HB (I-7).

[0090] The specific operation conditions for silica gel column chromatography separation and purification are as follows:

[0091] Silica gel column: inner diameter 40 mm, length 300 mm, packed with 200-300 mesh silica gel;

[0092] Elution conditions: after the solution to be purified was loaded, 100 mL of dichloromethane was used first, then 500 mL of a dichloromethane-methanol mixed solvent (CH2Cl2:CH4O = 15:1, V:V) was used, and finally 300 mL of a dichloromethane-methanol mixed solvent (CH2Cl2:CH4O = 5:1, V:V) was used, with the elution speed controlled at 30 mL / min-80 mL / min.

[0093] Finally, substance I-7 (0.18 mmol, 1.0 eq) was mixed with dimethylaminopyridine (DMAP, 0.55 mmol, 3.0 eq), lipoic acid (0.18 mmol, 1.0 eq), and dicyclohexyl carbodiimide (DCC, 0.55 mmol, 3.0 eq) in 5.0 mL of dichloromethane. The reaction was stirred at room temperature for 18 h in the dark. After the system was dried, it was dissolved in 2 mL of a 2:1 mixture of dichloromethane and methanol to obtain a solution to be purified. The solution to be purified was then separated and purified using silica gel column chromatography to obtain dark blue substance Hem-HBS (I-a).

[0094] The specific operation conditions for silica gel column chromatography separation and purification are as follows:

[0095] Silica gel column: inner diameter 40 mm, length 300 mm, packed with 200-300 mesh silica gel;

[0096] Elution conditions: after the solution to be purified was loaded, 100 mL of dichloromethane was used first, then 900 mL of a dichloromethane-methanol mixed solvent (CH2Cl2:CH4O = 20:1, V:V) was used, and finally 500 mL of a dichloromethane-methanol mixed solvent (CH2Cl2:CH4O = 5:1, V:V) was used, with the elution speed controlled at 30 mL / min-80 mL / min.

[0097] 1H NMR (400 MHz, CDC13) δ: 8.63 (d, 1H, J = 14.78 Hz), 7.87 (d, 2H, J = 8.00 Hz), 7.49-7.33 (m, 8H), 7.20 (s, 1H), 6.95 (dd, 1H, J = 8.62, 2.38 Hz), 6.87 (d, 1H, J = 2.35 Hz), 6.78 (d, 1H, J = 14.80 Hz), 5.25 (s, 2H), 5.03 (t, 2H, J = 5.10 Hz), 4.72 (t, 2H, J = 5.23 Hz), 3.46-3.41 (m, 1H), 3.13-3.08 (m, 2H), 2.89 (t, 2H, J = 6.20 Hz), 2.74 (t, 2H, J = 6.27 Hz), 2.42-2.36 (m, 1H), 2.09-2.06 (m, 2H), 1.97-1.91 (m, 2H), 1.77 (s, 6H), 1.58 (s, 6H), 1.34 (s, 12H).

[0098] ESI-MS calc. for C 48 H 57 BNO6S2[M+H] + : 818.3715, found 818.3721.

[0099] Example 2: Synthesis of Polymer III-a

[0100]

[0101] Into 5 mL of anhydrous DMF, 5 g of amino-polyethylene glycol 5000-maleimide (1.0 mmol), 309 g of thioctic acid (1.5 mmol), 570 mg of HATU (1.5 mmol), and 258 mg of N,N-diisopropylethylamine (DIPEA, 2 mmol) were added, stirred at room temperature for 24 h, then purified by dialysis using a dialysis bag with a molecular weight cut-off of 5000, using 5 L of deionized water, changing 3 times; each dialysis for 5 h, after dialysis, frozen in a refrigerator at ~80°C for 24 h, then freeze-dried in a freeze dryer to obtain polymer Lip-PEG-MAL (III-1).

[0102] Take 52 mg Lip-PEG-MAL (10 umol), dissolved in 5 mL PBS (pH = 6.8) acetonitrile mixed solution (PBS / acetonitrile = 8:2, V / V), then add 13.34 mg Cys-Angiopep2 (10 umol), stir at room temperature for 24 h, then use a dialysis bag with a molecular weight cutoff of 5000 to purify with 5 L of deionized water, change 3 times, each time dialysis for 5 h, after dialysis, freeze in the refrigerator at -80℃ for 24 h, then freeze-dried in a freeze-drier to obtain the polymer Lip-PEG-RAGE (III-a).

[0103] Example 3: Synthesis of probe HCB

[0104] Prepare a methanol solution of substance I-a with a concentration of 1 mol / L as a solvent for use.

[0105] Prepare a methanol solution of polymer II-a with a concentration of 1 mol / L as a solvent for use.

[0106] Prepare a methanol solution of polymer III-a with a concentration of 1 mol / L as a solvent for use.

[0107] Prepare 100 mL of a chloroauric acid solution with a concentration of 1 mM using deionized water, slowly add 15 mL of a 1% citric acid deionized water solution to the chloroauric acid solution, and boil for 15 min to obtain a gold seed solution with a particle size of 5-20 nm and a concentration of 0.01 nmol / L.

[0108] Prepare 200 mL of a chloroauric acid solution with a concentration of 0.3 mM using deionized water, adjust the pH of the chloroauric acid solution to acidic (pH = 2) with pH = 1 hydrochloric acid, and add 4 mL of the above gold seed solution under stirring at 650 rpm / r. Further, increase the stirring speed of the solution to 1500 rpm / r, and add 2 mL of a 3.0 mmol / L silver nitrate deionized water solution and 1.2 mL of a 100 mmol / L ascorbic acid deionized water solution, and stir for 20 s to obtain a gold nanostar solution with a particle size of 50±20 nm.

[0109] Prepare the above prepared gold nanostar solution into a gold nanostar solution with a concentration of 5.0 nmol / L using deionized water for use.

[0110] Take 200 mL of the above prepared gold nanostar solution with a concentration of 5.0 nmol / L, first add 20 μl of the methanol solution of substance I-a and react at room temperature for 5 min, then add 2 mL of the methanol solution of polymer II-a and react at room temperature for 5 min, and finally add 150 μl of the methanol solution of polymer III-a and react at room temperature for 5 h to obtain the final probe HCB solution.

[0111] The structure of probe HCB is as follows:

[0112]

[0113] wherein,

[0114] The structure of I-a' is:

[0115]

[0116] The structure of II-a' is:

[0117]

[0118] The structure of III-a' is:

[0119]

[0120] Characterization of probe HCB:

[0121] Morphology of probe: The morphology of probe HCB was completed by transmission electron microscopy experiment. 10 μL of the prepared probe deionized water solution was added dropwise on a copper mesh, dried with an infrared lamp, and observed under a transmission electron microscope. The probe HCB was in the form of a uniform nanostar with more branches, and the particle size was 50±20 nm.

[0122] Particle size of probe: The particle size of the probe was measured on a Malvern particle size analyzer by dynamic light scattering method. The prepared probe was deionized and diluted to 0.1 nmol / L. 1 mL of the sample was placed in a Malvern sample cell for testing. The hydration particle size of the probe was about 110±30 nm.

[0123] Raman spectrum of probe: We used a handheld Raman spectrometer to determine the Raman spectrum of HCB. 0.5 mL of the probe solution with a concentration of 1 nmol / L was irradiated with an external laser. The Raman spectrum after excitation by 785 nm excitation light was collected, and the collection time was 0.5 s. The characteristic peaks of the probe HCB are shown in Table 1 below.

[0124] Table 1 Characteristic peaks of probe HCB (Raman spectrum intensity unit counts).

[0125]

[0126] Example 4: Test of response performance of probe HCB to H2O2

[0127] The Raman spectra of the probe HCB in response to H2O2 were determined using a handheld Raman spectrometer. 1 mL of 1 nmol / L HCB was dissolved in 10 μL of H2O2 at concentrations of 0, 10 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 70 μmol / L, 80 μmol / L, 90 μmol / L, and 100 μmol / L. After the probe reacted with H2O2 for 1 min, the Raman spectra of the probe HCB excited by 785 nm excitation light were acquired. The characteristic peaks and their assignments are shown in Table 2 below.

[0128] Table 2: Raman spectral assignment data of probe HCB in response to H2O2 (Raman spectral intensity unit: counts).

[0129]

[0130] The intensity of the Raman spectrum characteristic peak of probe HCB was further investigated as a function of H2O2 concentration, and the results are shown in Table 3.

[0131] Table 3: Characteristic peak intensity data of probe HCB at different H2O2 concentrations (Raman spectral intensity units: counts).

[0132]

[0133] As shown in Table 3, the Raman peak attributed to phenylboronic acid (peak at 482 cm⁻¹) -1 The peak intensity of the cyclohexene cycloalkyl C-C-C peak (at 930 cm⁻¹) decreased significantly after the action of H₂O₂, while the Raman peak attributable to the symmetrical stretching of the cyclohexene cycloalkyl C-C-C was significantly reduced. -1 After H2O2 treatment, the peak intensity of the characteristic double peak (I) remained relatively stable overall. With the H2O2 concentration remaining constant, when the H2O2 concentration increased from 0 to 100 μmol / L, the intensity ratio of the characteristic double peaks (I) increased. Peak2 / I Peak1 The Raman signal intensity ratio increased from 3.99 to 93.28, an improvement of more than 20 times. (The last sentence appears to be incomplete and possibly refers to a different topic.) Peak2 / I Peak1 Plotting the signal intensity ratio against the H2O2 concentration value, within a certain range of H2O2 concentration, I Peak2 / I Peak1 The value is positively correlated with the H2O2 concentration.

[0134] Example 5: pH response performance test of probe HCB after ROS response

[0135] First, the Raman spectra of the probe HCB in response to pH were measured using a handheld Raman spectrometer. 1 mL of 1 nmol / L HCB was added to citric acid-sodium bicarbonate buffer solutions at different pH values, and Raman spectra of the probe HCB after excitation with 785 nm excitation light were collected. The characteristic peaks and their assignments in the spectra are shown in Table 4 below.

[0136] Table 4: Raman spectral assignment data of probe HCB in response to pH (Raman spectral intensity unit: counts).

[0137]

[0138] The intensity of the Raman spectral characteristic peak of the probe HCB was further investigated as a function of pH concentration, and the results are shown in Table 5.

[0139] Table 5: Characteristic peak intensity data of probe HCB at different pH values ​​(Raman spectral intensity units: counts).

[0140]

[0141] As shown in Table 5, the Raman peak attributed to phenylboronic acid (peak at 482 cm⁻¹) -1 Raman peak of benzene ring C-C bending vibration (peak at 590 cm⁻¹) -1 The stretched Raman peak of cyclohexene cycloalkyl C-C-C (peak at 930 cm⁻¹) and cyclohexene cycloalkyl C-C-C -1 The peak intensity remained relatively stable at different pH levels. When there was no H2O2, and the HCB concentration remained constant, the characteristic bimodal intensity ratio (I0.0) increased from pH 2.0 to 10.0. Peak3 / I Peak1 and I Peak3 / I Peak2 The Raman signal intensity ratio remains essentially unchanged. (The last part, "I," appears to be a typo and can be omitted.) Peak3 / I Peak1 and I Peak3 / I Peak2 Plotting the signal intensity ratio against pH value, within a certain pH range, I Peak3 / I Peak1 and I Peak3 / I Peak2 The value is not affected by pH.

[0142] Secondly, the Raman spectra of the probe HCB in response to ROS and pH were measured using a handheld Raman spectrometer. 1 mL of 1 nmol / L HCB was added to 100 μmol / L H2O2 and reacted for 1 min. Then, the mixture was added to citric acid-sodium bicarbonate buffer solutions at different pH values. Raman spectra of the probe HCB after excitation with 785 nm excitation light were collected. The characteristic peaks and their assignments are shown in Table 6 below.

[0143] Table 6: Raman spectral assignment data of probe HCB in response to pH (Raman spectral intensity unit: counts).

[0144]

[0145] The changes in the intensity of the Raman spectrum characteristic peaks of probe HCB after being treated with H2O2 with pH concentration were further investigated, and the results are shown in Table 7.

[0146] Table 7: Characteristic peak intensity data of probe HCB at different pH values ​​under the action of H2O2 (Raman spectral intensity unit: counts).

[0147]

[0148] As shown in Table 7, the Raman peak at 590 cm⁻¹ attributed to the benzene ring C-C bending vibration is... -1 The peak at 930 cm⁻¹ significantly increased with increasing pH, while the extended Raman peak attributable to cyclohexene cycloalkyl C-C-C was 930 cm⁻¹. -1 The peak intensity remained relatively stable at different pH levels. After H2O2 treatment, with the probe HCB concentration remaining constant, the characteristic bimodal intensity ratio (I0.0) increased from pH 2.0 to 10.0. Peak2 / I Peak1 The Raman signal intensity ratio decreased from 13.19 to 0.87, an improvement of more than 15 times. (The last sentence appears to be incomplete and possibly refers to a different topic.) Peak2 / I Peak1 Plotting the signal intensity ratio against pH value, within a certain pH range, I Peak2 / I Peak1 The value is positively correlated with pH.

[0149] Example 6: HCB probe imaging of neurons in different states

[0150] 24 hours prior to incubation, 14 mm diameter glass discs were placed in culture plates using sterile forceps, and 0.05% poly-L-lysine was added for coating. After coating at room temperature for 2 hours, the discs were aspirated, and the plates were rinsed three times with phosphate buffered saline (PBS) and air-dried. Brain tissue was extracted from C57BL / 6J neonatal rats within 48 hours of birth and placed in culture dishes containing pre-chilled PBS. The meninges and blood vessels were removed, and bilateral hippocampal tissue was isolated. The hippocampal tissue was transferred to papain solution (2 mg / mL, diluted with high-glucose DMEM) using sterile forceps, and an appropriate amount of DNase I was added to prevent DNA release and protein entanglement after digestion from affecting further digestion. The plates were placed in a 37°C incubator, and the plates were gently shaken every 5 minutes. After 20 minutes, digestion was terminated with all-purpose medium containing 10% FBS. The cells were gently pipetted and the cell suspension was collected through a 70 μm cell sieve. The plates were centrifuged at 1000 rpm for 5 minutes, and the supernatant was carefully aspirated, taking care not to disperse or discard the cells at the bottom. The cells were then gently and slowly pipetted to resuspend them in 10% FBS complete medium. After counting, the cells were appropriately diluted to 4 x 10⁻⁶ cells / mL. 4 / cm 2 Cells were seeded at a density of [insert density here] on poly-L-lysine-coated slides. The slides were incubated at 37°C with 5% CO2. After 4 hours, neuronal adhesion was observed, and 10% NB27 medium was added to the culture plate to maintain the balance. The next day, half the medium was replaced with NB medium. Thereafter, cell growth was observed every three days, and the medium was replaced halfway each time.

[0151] After the CTZ powder had returned to room temperature, 0.39 mg was quickly weighed and dissolved in 100 μL of DMSO to prepare a 10 mM stock solution. Using a serial dilution method, NB27 medium was added dropwise with shaking to ensure complete dissolution, resulting in a 100 μM working solution. This solution was filtered through a 0.45 μm needle filter before use. 40 μL of the original medium in the well plate was replaced with the CTZ working solution, and the plates were mixed thoroughly using the cross-hatching method to achieve a final CTZ concentration of 5 μM. Primary hippocampal neurons were cultured at 37°C in a 5% CO2 atmosphere for 48 hours. Spontaneous neuronal firing was recorded using patch-clamp, confirming the successful induction of epileptiform discharges by chronic CTZ treatment. Figure 1 As shown.

[0152] A cell culture chamber, matched to the stage of an inverted microscope, was connected to an artificial cerebrospinal fluid perfusion system. A mechanical peristaltic pump was used to fill the chamber with fluid, ensuring that the inflow and outflow rates of the perfusion system were equal. Cell slides were removed from the cell culture incubator and placed in the chamber. Under the inverted microscope, the field of view was adjusted, and pyramidal neurons with full morphology, good refractive index, and intact axons were selected for recording. Sutter blank electrodes (outer diameter OD: 1.5 mm, inner diameter ID: 0.86 mm) were used for recording, fabricated using a P-2000 glass electrode drawing instrument. After filling the electrode with fluid, air bubbles were removed. The resistance of the glass recording electrode was 3-5 MΩ. Before immersing the electrode in the liquid, apply appropriate positive pressure using a 1mL syringe to prevent electrode tip blockage. Adjust the microscope focus and bring the electrode close to the target cell. When the electrode is gently pressed against the cell, a slight indentation will be observed on the surface, and the resistance will increase (approximately 0.2-0.3 MΩ). Slowly apply negative pressure to form a high-resistance peak junction (>1 GΩ) between the electrode and the cell, rupturing the cell membrane and establishing a whole-cell electrophysiological recording state. Figure 1 As shown in the figure. Crosstalk resistance (Rs) was monitored during recording; cells with Rs greater than 60 MΩ were excluded from analysis. Cell membrane potentials were clamped to -70 mV in current-clamp mode, and stimuli of 0 and 100 pA were applied. The number of action potentials (APs) induced under different current injections was used as an indicator of neuronal excitability. Experimental data were collected using an Axon Digidata 1550 data acquisition system and a MultiClamp 700B amplifier at a sampling frequency of 10 kHz. The acquired data were analyzed using pCLAMP 10.2 software. The number of neuronal action potentials under different stimuli is shown in Table 8.

[0153] As shown in Table 8, the neurons in the DMSO control group had the lowest excitability. After CTZ stimulation, the action potential of neurons increased approximately fourfold at 100 pA. The action potential of CTZ-stimulated neurons decreased by 28.0% after TPM treatment. However, the combined treatment of TPM and NAC significantly reduced neuronal excitability, decreasing the action potential by 60.2%, approaching that of the control group. Figure 1 As shown.

[0154] 100 nmol / L HCB probe solution was added to neurons after different stimuli, and the cells were incubated at 37℃ and 5% CO2 for 2–8 h. The culture medium was discarded, and the cells were washed three times with 0.01 M PBS. Then, 1 mL of HEPES buffer was added, and SERS mapping was performed under a 63× water microscope using a confocal Raman microscope. Grating: 300 g / mm, spectral range: 0–3600 cm⁻¹ -1 Excitation light: 785nm, laser power: 5mW, step size: 0.4μm.

[0155] Table 8: Neuronal firing activity, characteristic peak signals, and intensity ratios of the two characteristic peaks in different experimental groups (Raman spectral intensity units: counts).

[0156]

[0157] Table 8 shows the characteristic peak data of the acquired Raman images. It can be seen that the four groups of cells treated with different methods showed peak values ​​at 928 cm⁻¹. -1 The intensity was almost similar at 482 cm. When neurons were stimulated with CTZ, the intensity was similar at 482 cm. -1 and 590cm -1 The intensity of the Raman peaks at all locations decreased, I 928 / I 482 and I 928 / I 590 The ratio map showed enhanced signals, indicating that our probe HBF can identify the neuroinflammation level and pH fluctuations in epileptiform neurons. In comparison, TPM treatment of epileptiform neurons... 928 / I 590 The ratio map signal was significantly lower than that of the CTZ group, recovering towards the blank control group. Additionally, after 24 hours of combined TPM and NAC treatment for epileptiform neurons, I... 928 / I 482 and I 928 / I 590 The ratio map signal was significantly lower than that of the CTZ group, but not significantly different from that of the blank control group, indicating that the probe we prepared has high response sensitivity to changes in H2O2 and pH.

[0158] Example 7: Application of probe HCB in real-time ratio SERS imaging for non-invasive recording of intracranial pH fluctuations in epileptic mice.

[0159] Establishment of a mouse model of in situ hippocampal epilepsy. Male C57 mice (4-6 weeks old) weighing 20-25 grams were anesthetized with a 5% isoflurane / oxygen mixture and fixed on a stereotaxic apparatus. Anesthesia was maintained by continuous inhalation of 1-2% isoflurane during the procedure. Kalinic acid (KA, 0.4 μg, 0.5 μL) was injected into the hippocampus of the mice over 5 minutes. The injection site parameters were: 2 mm posterior to the anterior fontanelle, 2 mm to the right of the midline, and 1.8 mm deep in the frontal lobe. After injection, the gas anesthesia was removed, and the seizure severity was observed using the following criteria: Grade 1, facial twitching and pronounced chewing; Grade 2, chewing, head nodding, and wet dog-like tremors; Grade 3, unilateral forelimb clonus; Grade 4, bilateral forelimb clonus; Grade 5, tonicity, loss of balance, and falls. Mice were classified into seizure grades 1-5 based on observation and included in subsequent experiments. Based on the above procedure, we replaced kainic acid with an equal volume of physiological saline and injected it into the hippocampus of mice, which became the physiological saline control mice.

[0160] HCB probe (120 nmol / kg) was injected via the tail vein. After 4 hours, mice were anesthetized with 2% isoflurane and fixed on the operating table. The area was disinfected and draped. The scalp was incised, muscles were separated, and a bone window (approximately 0.5 × 1 cm) was created using an electric drill to expose the surface of the cerebral cortex while preserving the dura mater. We used a handheld Raman spectrometer (Ocean Optics QE65 Pro, excitation wavelength 785 nm, time 500 ms) for imaging. The handheld Raman spectrometer was connected to a computer, and the Raman spectra were displayed in real time using Oceanview software. First, we located the epileptic focus using H2O2 imaging and found that the SERS intensity in the ipsilateral cortex was higher than that of the I... 928 / I 482 Localized increase. Conversely, I in the normal brain... 928 / I 482 The ratio remained relatively constant, and the surface HCB probe could specifically locate the epileptic focus. Subsequently, intraperitoneal injection of KA induced seizures of varying degrees, and synchronized neuronal activity led to a significant increase in spike discharges of local field potentials. We observed using HCB probe imaging that the pH value within the epileptic focus decreased during the tonic phase and then gradually recovered after the seizure ceased. Furthermore, we observed that the acidity within the epileptic focus gradually increased as the seizure intensity increased from grade 2 to grade 5. According to the calibration curve, the pH value during mouse seizures could reach 6.6 (…). Figure 2 ).

[0161] Example 8: Application of HCB probe in monitoring drug efficacy during different treatment processes in mice

[0162] Mice with successful modeling of spontaneous epileptic seizures were randomly divided into three groups, plus a sham-operated group. EEG was recorded for one week. Then, an HCB probe (120 nmol / kg) was injected via the tail vein. After 4 hours, the mice were anesthetized, the skin on their heads was cut open, and they were fixed to a lateral ventricle locator. A cranial drill was used to make a hole directly above the cerebellar region. We used a handheld Raman spectrometer (Ocean Optics QE65 Pro, excitation wavelength 785 nm, time 500 ms) to image the cerebral cortex. Raman spectra of 64 sites on the mouse cerebral cortex were acquired using Oceanview software, resulting in Raman maps, as shown below. Figure 3 As shown. Before drug treatment, elevated I levels were observed at the lesion site. 928 / I 482 and I 928 / I 590 The ratio, and the EEG data match, such as Figure 3 As shown.

[0163] Furthermore, three groups of epilepsy model mice were intraperitoneally injected with DMSO, TPM, and RTPM, respectively. During drug treatment, we examined the level of neuroinflammation and pH fluctuations in the brain using the HCB probe. After drug treatment, we again examined the level of neuroinflammation and pH fluctuations in the brain using the HCB probe. The procedure was as described above, and the results are as follows. Figure 3 As shown, we discovered that the HCB probe can monitor the level of neuroinflammation and pH fluctuations in the brain in real time. Probe monitoring results showed that in the brains of epilepsy model mice... 928 / I 482 and I 928 / I 590 The ratio map signal was significantly higher in the group compared to the sham-operated group, indicating a high level of neuroinflammation and a lower pH in the brain. During TPM treatment, I 928 / I 482 The ratio map signal showed little difference from the DMSO group, while I 928 / I 590 The ratio map signal increased, and surface TPM could control the pH decrease caused by epileptic seizures, but after discontinuation of medication, I... 928 / I 590 The ratio map signal increased again to near pre-treatment levels, indicating that pH remained at a low level characteristic of epileptic states, and TPM had virtually no effect on neuroinflammation. In contrast, the RTPM group showed improved I... 928 / I 482 and I 928 / I 590 The ratio map signal showed no significant difference compared to the DMSO group, verifying that RTPM can significantly restore neuroinflammation in mice and regulate pH to near-normal levels, and that the therapeutic effect does not diminish upon discontinuation of the drug. This result is consistent with the EEG results.

Claims

1. A tandem ratiometric response Raman probe characterized in that, A substance composed of gold nano substrate, dye molecules and polymers, the structure of which is shown in the following formula V: ； A is a gold nano substrate; X 1 The structure is shown as formula I a : ； wherein S is connected to the gold of said A surface via a gold-sulfur bond, R 1 and R 2 are independently hydrogen, halogen, C1-C4 alkyl, sulfonic acid group or nitro group; R 1 and R 2 are the same or different; X 2 for L 1 -L 2 , L 1 is a group connected to the gold of the A surface through a gold-sulfur bond, L 2 is a hydrophilic polymer group, and Int. indicates the connection of both, likewise below. X 3 for L 1 -L 3 -L 4 , L 1 is a group which is linked to the gold of the surface of A through a gold-sulfur bond, L 3 is a hydrophilic polymer group, L 4 is a targeting group; A as a Raman signal enhancing substrate, for providing electromagnetic field hot spots to enhance Raman reporter molecules X 1 Raman signal, improving the sensitivity of detection; X 1 as a reactive molecule with H2O2 and pH dual-ratio response, for providing a quantitative basis for the target marker; X 2 for increasing the biocompatibility of the probe; X 3 to endow the probe with the performance of high-efficiency targeting across the blood-brain barrier.

2. The tandem ratiometric Raman probe of claim 1, wherein, The particle size of A is 5nm-200nm.

3. The tandem ratiometric Raman probe of claim 1, wherein, The substance shown in formula V: X 1 molar ratio of 1 x 10 4 :1 to 5 x 10 4 :1; X 2 molar ratio of 5 x 10 4 :1 to 50 x 10 4 :1; X 3 molar ratio of 1 x 10 6 :1 to 3 x 10 6 :

1.

4. The tandem ratiometric Raman probe according to claim 1, wherein: The structure of X2 is: ， The X 3 The structural formula is: 。 5. The tandem ratiometric Raman probe according to claim 1, wherein: The hydrated particle size of the substance shown in formula V is 110±30nm; The surface charge of the substance shown in formula V is 9±5mV; The substance shown in formula V has Raman effect.

6. The tandem ratiometric Raman probe of claim 1, wherein, Raman spectrum of a substance represented by Formula V has one or more characteristic peaks at 303 cm -1 , 482 cm -1 , 520 cm -1 , 552 cm -1 , 590 cm -1 , 928 cm -1 , 790 cm -1 , 1169 cm -1 , and 1430 cm -1 .

7. Use of the tandem ratiometric Raman probe according to any one of claims 1-6 in detecting reactive oxygen species and pH, wherein the reactive oxygen species is superoxide anion, hydrogen peroxide, singlet oxygen, hydroxyl radical, lipid peroxidation radical, ozone, hypochlorite or hypobromite; The concentration of the reactive oxygen species is 5μmol / L-100μmol / L; The pH range in the environment of detecting reactive oxygen species is 2.0-10.

0.

8. Use according to claim 7, characterized in that, The tissue or cell containing reactive oxygen species and pH change is a neuron cell or brain tissue.

9. A method of making a tandem ratiometric response Raman probe as claimed in any one of claims 1 to 6, wherein, In a solvent, the gold nano substrate A, I a The substance X shown 1 , polymer X 2 and polymer X 3 react to obtain substance V; The solvent is an alcohol solvent and / or an aqueous solvent.

10. The method of claim 9, wherein, The alcohol solvent is methanol and / or ethanol; the aqueous solvent is deionized water, distilled water or pure water; When the solvent is a combination of alcohol solvent and aqueous solvent, the volume ratio of the alcohol solvent to the aqueous solvent is 1:10-1:500; The substance I a with A is 1 x 10 4 :1 ~ 5 x 10 4 :1; said polymer X 2 molar ratio to A is 1 x 10 3 :1 ~ 1 x 10 5 :1; The polymer X 3 molar ratio to A is 1 x 10 6 :1~5 x 10 6 :1; The polymer X 2 With polymer X 3 The molar ratio is 1:50 to 1:1000; The molar volume ratio of A to the solvent is 1:50-1:500; The substance I a a molar volume ratio to the solvent of 50:1 to 150:1; said polymer X 3 a molar volume ratio of 5000:1 to 15000:1 to said solvent.

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