Probes, their preparation methods, and applications
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- PEOPLES HOSPITAL OF ZHENGZHOU
- Filing Date
- 2026-05-09
- Publication Date
- 2026-07-03
Smart Images
Figure CN122321180A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of bionanotechnology and molecular imaging, specifically to probes, their preparation methods, and applications. Background Technology
[0002] In oncological surgery, positive surgical margins are an independent risk factor for postoperative recurrence and poor prognosis. Currently, intraoperative assessment of tumor boundaries mainly relies on the surgeon's palpation experience and intraoperative frozen sections: frozen section analysis takes 15-30 minutes, and sampling errors lead to a false negative rate as high as 10-25%; palpation cannot identify the boundaries of microinvasive lesions or carcinoma in situ. Fluorescent surgical navigation technology can achieve real-time visualization, but existing probes have significant limitations.
[0003] MMP-2-only responsive: Fluorescence recovery is achieved through enzymatic cleavage of matrix metalloproteinase-2 (MMP-2) specific substrate peptides (such as PLGVR and PLGLAG). Drawbacks: Positive results are observed in inflammatory tissues (where macrophages secrete MMP-2) and wound healing areas, resulting in a high false-positive rate.
[0004] pH-responsive only: Fluorescence recovery is achieved through the protonation and shedding of pH-sensitive polymers such as polyhistidine (pHis, pKa ~6.5) and poly-β-amino esters. Drawback: The central necrotic region of the tumor (acidic but without active invasion) shows a positive result, leading to misjudgment of the tumor boundary and an expansion of the resection area.
[0005] Recent literature reports "simultaneous-response" dual-lock probes, which respond simultaneously to MMP-2 and acidic pH at the same spatial location. Their fundamental flaw lies in failing to consider the biological timing characteristics of the tumor invasion front—extracellular matrix degradation (MMP-2 activation) occurs early in invasion, while extracellular acidification (lactate accumulation, proton pump upregulation) is a secondary event. Although the central necrotic zone shows acidification, MMP-2 activity is extremely low; while the inflammatory zone contains MMP-2, the pH is normal. The simultaneous-response logic cannot distinguish between the "invasive front" and "accompanying inflammation / necrosis," and the false-positive problem remains unresolved. Summary of the Invention
[0006] To address the shortcomings of existing technologies, this invention provides a probe, its preparation method, and its application, solving the technical problems of insufficient specificity and misjudgment of intraoperative boundaries in existing single-lock and simultaneous-response dual-lock probes.
[0007] To achieve the above objectives, the present invention provides the following technical solution: a time-response type dual-lock activation probe, comprising: (a) Near-infrared II fluorescence core; (b) A gold nanoshell coating on the surface of the fluorescent core, wherein the gold shell and the fluorescent core form a FRET quenching pair; (c) A first responsive layer covalently coupled to the surface of the gold shell by thiol groups, the first responsive layer comprising peptides having matrix metalloproteinase-specific cleavage sites; (d) A second responsive layer coupled to the end of the peptide, the second responsive layer comprising a pH-sensitive polymeric material; The fluorescence recovery of the probe occurs only after the following timing events: (i) The peptide is cleaved by matrix metalloproteinases, exposing the pH-sensitive polymer coupled to its end to the solvent environment; (ii) When exposed to an acidic environment with pH ≤ 6.8, the pH-sensitive polymer material undergoes a conformational change or detaches from the probe surface; Furthermore, when the peptide is not cleaved, the pH-sensitive polymer material is shielded by the physical barrier formed by the gold shell and the intact peptide, and does not come into contact with the solvent even in an acidic environment, thus preventing fluorescence recovery.
[0008] Preferably, the emission wavelength of the near-infrared II fluorescence core is 1000-1700nm, more preferably 1300-1400nm.
[0009] Preferably, the fluorescent core is selected from Ag2S quantum dots, PbS / CdS core-shell quantum dots, Ag2Se quantum dots, or nanoparticles coated with organic NIR-II fluorescent dyes.
[0010] Preferably, the thickness of the gold nanoshell is 5-20 nm, more preferably 8-12 nm, and its FRET quenching efficiency with the fluorescent core is ≥95%.
[0011] Preferably, the matrix metalloproteinase-responsive peptide is an MMP-2 and / or MMP-9 specific substrate peptide, which contains an amino acid sequence selected from SEQ ID NO:1 or SEQ ID NO:3; preferably, the peptide is the complete modified sequence shown in SEQ ID NO:2.
[0012] Preferably, the peptide is self-assembled onto the gold shell surface via Au-S covalent bonds through C-terminal or N-terminal cysteine residues, with a modification density of 0.2-2.0 nmol / cm², preferably 0.5-1.0 nmol / cm².
[0013] Preferably, the pH-sensitive polymer material is selected from polyhistidine, polyβ-amino ester, acetylated dextran or copolymers thereof, and its protonation response pH threshold is 5.5-6.8, preferably 6.0-6.5.
[0014] Preferably, the second response layer is coupled to the C-terminus of the peptide via click chemistry or biotin-avidin bridging.
[0015] Preferably, the probe also includes a targeting unit selected from RGD peptide, folic acid, EGFR antibody or its antigen-binding fragment, which is modified on the outermost layer of the probe.
[0016] A method for preparing the probe according to any one of claims 1-9, comprising the following steps: (1) Synthesize near-infrared II fluorescence cores with emission wavelengths of 1000-1700 nm; (2) A gold nanoshell is coated on the surface of the fluorescent core, with a thickness controlled at 5-20 nm; (3) Peptides containing matrix metalloproteinase-specific cleavage sites are modified onto the gold shell surface by thiol self-assembly; (4) Couple a pH-sensitive polymer material to the end of the peptide segment; (5) Purify to obtain a time-responsive double-lock activation probe.
[0017] Preferably, the peptide modification density in step (3) is controlled at 0.2-2.0 nmol / cm² by the feed ratio.
[0018] Preferably, the coupling method in step (4) is maleimide-thiol click reaction or biotin-avidin bridging.
[0019] Preferably, the purification in step (5) is performed by ultrafiltration centrifugation or size exclusion chromatography.
[0020] Application of a time-responsive dual-lock activation probe in the preparation of formulations for intraoperative navigation in tumor surgery.
[0021] Preferably, the tumor is a solid tumor, selected from breast cancer, colorectal cancer, gastric cancer, lung cancer, glioma, and head and neck squamous cell carcinoma.
[0022] Preferably, the intraoperative navigation is achieved through a near-infrared two-zone fluorescence imaging system with an imaging depth ≥5mm.
[0023] Preferably, the preparation is administered by intravenous injection or intraoperative local spraying.
[0024] Compared with the prior art, the present invention provides a probe, its preparation method and application, which have the following beneficial effects: Specificity: The fluorescence ratio (T / NT) at the invasion front to normal tissue is ≥5.0, which is more than twice that of existing single-lock probes (≤2.5); Sensitivity: Can detect minute infiltrates with a diameter ≤1mm; Imaging depth: Penetration depth ≥5mm under NIR-II window, covering common tissue thicknesses during surgery; Response speed: Peak response time is reached 4-6 hours after intravenous injection, which is consistent with the single-dose administration window during surgery. Attached Figure Description
[0025] Figure 1 is a schematic diagram of the preparation process of the dual-lock activation probe of the present invention; Figure 2 is a schematic diagram of the dual-lock activation probe structure of the present invention; Figure 3 is a flowchart of the dual-lock activation probe activation logic of the present invention. Detailed Implementation
[0026] To better understand the purpose, structure, and function of this invention, the probe, its preparation method, and its application are described in further detail below with reference to the accompanying drawings.
[0027] Please see Figure 1-3 This invention provides a time-responsive dual-lock activation probe, the core design logic of which is based on the microenvironmental characteristics of the tumor invasion front. For example... Figure 1 As shown, the probe consists of a near-infrared II (NIR-II) fluorescent core, a gold nanoshell, an MMP-responsive peptide layer, and a pH-sensitive polymer layer from the inside out.
[0028] 1. Structure and quenching mechanism In this embodiment, the near-infrared light emitted by the fluorescent core (such as Ag2S quantum dots) is efficiently quenched by a tightly coated gold nanoshell through the fluorescence resonance energy transfer (FRET) effect. The thickness of the gold shell is controlled at 5-20 nm (preferably 8-12 nm) to ensure a quenching efficiency of over 95%, thereby ensuring that the probe remains in a "silent" state in blood circulation and normal tissues, reducing background noise.
[0029] 2. Timing-based double-lock activation logic (core mechanism) The key to this invention lies in the spatial configuration relationship between the first responsive layer (peptide) and the second responsive layer (pH-sensitive polymer): Physical shielding phase: In normal tissue or a purely acidic environment, the peptide remains intact due to low MMP-2 / 9 enzyme levels. At this stage, due to the peptide modification density (0.2-2.0 nmol / cm²) and the steric hindrance effect of the gold shell surface, the pH-sensitive polymer at the ends is "suppressed" or "encased" in a microenvironment near the probe surface, preventing effective contact with solvents in the external environment. Even at low external pH, protons struggle to penetrate the physical barrier formed by the intact peptide to induce a conformational change in the polymer.
[0030] Step 1 Unlocking (Enzyme Digestion): When the probe reaches the tumor invasion front, the highly expressed MMP-2 / 9 cleaves the specific peptide (such as SEQ ID NO: 1), breaking the physical confinement.
[0031] The second step, unlocking (acid response): After peptide fragmentation, previously shielded pH-sensitive polymers (such as polyhistidine) are exposed to the solvent. Under the acidic environment of the tumor stroma (pH≤6.8), the polymers undergo protonation, and charge repulsion or conformational changes cause them to detach from the gold shell surface or loosen their structure. This increases the distance between the fluorescent core and the gold shell or alters the environment, allowing the fluorescence signal to be restored (ON).
[0032] 3. Preparation process route The preparation process strictly adheres to stoichiometry. First, a high-quality core is synthesized via thermal injection or hydrothermal methods, followed by gold shell deposition on the surface using a gold salt reduction method. Peptide modification leverages the high affinity of gold-thiol (Au-S) groups, with the modification density precisely adjusted by controlling the feed ratio. Finally, click chemistry (e.g., the reaction of maleimide with thiol groups) is used to couple pH materials to the peptide ends. Figure 1 Flowchart for activation logic: Example 1: Fabrication of a time-response dual-lock activation probe In this embodiment, a time-responsive dual-lock activation probe is prepared using near-infrared II fluorescent quantum dots as the fluorescent core, gold nanoshells as the FRET quenching layer, MMP-responsive peptides as the first response layer, and pH-sensitive polymers as the second response layer.
[0033] 1. Preparation of near-infrared II fluorescent cores Ag2S quantum dots are preferably used as the near-infrared II fluorescence core. Specifically, silver and sulfur precursors are thermally injected into a high-boiling-point solvent system, and the reaction temperature, time, and ligand ratio are controlled to obtain Ag2S quantum dots with uniform particle size and emission peaks around 1300 nm.
[0034] In a preferred embodiment, silver salt is dissolved in a ligand / solvent system containing oleylamine, octadecene, etc., and after heating to remove water and oxygen, it is rapidly injected into a sulfur source solution under inert gas protection to induce quantum dot nucleation and growth. After the reaction, Ag₂S quantum dots with stable ligands coated on the surface are obtained by ethanol precipitation, centrifugation, and redispersion. Their particle size distribution can be observed using transmission electron microscopy, and their emission is confirmed to be in the near-infrared II region of 1000-1700 nm by fluorescence spectroscopy.
[0035] 2. Coating with gold nanoshells After the surface of the Ag2S quantum dots was appropriately hydrophilized, a gold nanoshell was coated on the surface using a seed growth method.
[0036] Preferably, an active group or intermediate layer that can coordinate with the gold precursor is first introduced onto the surface of the quantum dots, and then a chloroauric acid solution is added. The mixture is then slowly reduced in the presence of a reducing agent to form a continuous gold shell. By adjusting the amount of gold salt added, the reduction rate, and the reaction time, the thickness of the gold shell can be controlled to be 5-20 nm, more preferably 8-12 nm.
[0037] After the gold shell is applied, an effective FRET quenching pair is formed between the fluorescent core and the gold layer, which significantly quenches the probe fluorescence in the initial state and reduces the fluorescence background to a near-minimum level, thereby providing a high signal-to-noise ratio for subsequent "restorative imaging".
[0038] 3. Construction of the first response layer A substrate peptide containing MMP-specific cleavage sites is introduced onto the surface of a gold nanoshell. The peptide preferably contains MMP-2 and / or MMP-9 recognizable sequences, such as PLGLAG, PLGVR, or their equivalent substrate sequences, and a cysteine residue is introduced at the peptide terminus to facilitate covalent self-assembly with the gold surface via an Au-S bond.
[0039] For example, peptides with the following structures can be used: Cys-substrate sequence-connector arm-active site Cys is used to form stable Au-S bonds with the gold shell surface, the substrate sequence is used to be specifically cleaved by MMP-2 or MMP-9, and the linker arm is used to improve spatial accessibility and avoid steric hindrance affecting enzyme digestion efficiency.
[0040] In a preferred embodiment, the peptide modification density is controlled at 0.2-2.0 nmol / cm², preferably 0.5-1.0 nmol / cm². This density ensures that the first response layer forms a continuous coverage on the gold shell surface, and also provides sufficient sites for subsequent enzymatic digestion, avoiding difficulties in enzymatic digestion due to excessively dense stacking.
[0041] 4. Construction of the second response layer A pH-sensitive polymer is further coupled to the peptide ends of the first response layer to form a second response layer. Preferred pH-sensitive polymers include polyhistidine, polyβ-amino esters, acetylated dextran and their copolymers, with polyhistidine being the most preferred because its protonation response threshold is within the acidic range commonly found in the tumor microenvironment.
[0042] In a preferred embodiment, reactive groups such as maleimide, active ester, or biotin are first reserved on the pH-sensitive polymer, and then coupled with the thiol or biotin group at the end of the peptide segment to form a stable link. The coupling method can be a maleimide-thiol click reaction or a biotin-avidin bridging method.
[0043] The protonation response pH threshold of the pH-sensitive polymer material is preferably set to 5.5-6.8, more preferably 6.0-6.5, to match the typical pH range of the tumor invasion front and the local acidified microenvironment. 5. Purification and Formulation After the above modifications are completed, the probe is purified by ultrafiltration centrifugation, dialysis or size exclusion chromatography to remove free peptides, free polymers and impurity particles without gold coating, and finally obtains a time-responsive dual-lock activation probe.
[0044] After purification, it can be resuspended in PBS buffer, Hepes buffer, or a physiologically compatible solution containing an appropriate amount of stabilizer to prepare an injectable probe formulation. Example 2: Probe structural characterization and timing response verification This embodiment verifies the structure, optical properties, and response mechanism of the probe prepared in Example 1.
[0045] 1. Structural characterization Transmission electron microscopy was used to observe the morphology of the probe, which showed that the fluorescent core was covered with a continuous gold shell, and the whole structure was a core-shell structure. Dynamic light scattering results showed that after modification with peptides and pH-sensitive polymers, the particle size was further increased compared with the gold shell intermediate, indicating that a bi-responsive layer was successfully constructed on the surface.
[0046] Zeta potential analysis can further verify the success of each modification step: The fluorescent core phase is typically characterized by an initial potential determined by surface ligands; The potential changes significantly after gold coating; After peptide modification, the potential shifts further due to the presence of charged groups. After pH-sensitive polymer coupling, the surface charge changes again.
[0047] This gradual change indicates that each functional layer was successfully constructed in sequence.
[0048] 2. Initial fluorescence quenching verification The luminescence intensity of the probe in the unactivated state was determined using fluorescence spectroscopy. Due to the formation of an efficient FRET quenching pair between the gold nanoshell and the fluorescent core, the probe exhibited only a very weak background signal under excitation conditions, indicating that fluorescence was effectively shut off in the initial state. 3. Timing Response Verification The probes were tested under the following conditions: Acidic conditions only: pH 6.0 or 6.5, but without MMP; Enzyme digestion conditions only: Add MMP-2 or MMP-9, but pH 7.4; Enzyme-first, acid-later conditions: First add MMP-2 for incubation, then transfer the system to an acidic environment; Simultaneous conditions: MMP and an acidic environment coexist; Control conditions: no enzymes, no acid.
[0049] The results show that: Under acidic conditions alone, the probe fluorescence did not recover significantly. This indicates that when the peptide fragment is intact, the pH-sensitive polymer layer remains shielded, and the acidic environment cannot fully act on the polymer layer.
[0050] Under enzyme digestion conditions alone, the probe showed little or no recovery, indicating that even if the peptide was cleaved, the polymer layer did not undergo significant conformational changes or detachment in the absence of an acidic environment.
[0051] Under the condition of enzyme followed by acid, the fluorescence intensity recovered significantly, and the recovery range was significantly higher than that of the control group, indicating that the probe does indeed follow the timing activation logic of "cleavage first, acidification later".
[0052] Fluorescence recovery can also be observed under the same conditions, but its essence is still that after the enzyme cleavage opens the barrier, the acidic microenvironment further triggers the second layer response.
[0053] The control group showed almost no luminescence, confirming that the system has good background suppression capabilities.
[0054] 4. Specificity verification The probe was incubated with different proteases, including MMP-2, MMP-9, trypsin, collagenase, and other unrelated proteases, and the fluorescence recovery was compared under the same acid-base conditions.
[0055] The results showed that the probe had a significant response to MMP-2 and / or MMP-9, but a weak response to unrelated proteases, indicating that the first response layer had good substrate specificity. This further demonstrated that probe activation depended on "specific enzymatic cleavage" rather than random degradation. Example 3: In vitro and in vivo evaluation of its use for intraoperative navigation in tumor surgery This embodiment illustrates the application effect of the probe in intraoperative tumor navigation.
[0056] 1. Validation of in vitro tissue models Constructing tissue models simulating the tumor invasion front, inflammatory tissue, and necrotic tissue: Invasion frontier model: possessing both high MMP activity and an acidic microenvironment; Inflammatory model: exhibits high MMP activity but near-neutral pH; Necrosis model: pH is acidic but MMP activity is low; Normal tissue model: low MMP activity and neutral pH.
[0057] After adding the probes to the aforementioned models, the results showed that the fluorescence recovery was most significant in the invasion front model, while the signals in the inflammation and necrosis models were significantly weaker, and normal tissue showed almost no signal. These results demonstrate that the temporal dual-locking mechanism of this invention can more accurately distinguish the true invasion boundary from inflammatory and necrotic areas, thereby reducing false positives.
[0058] 2. Validation of small animal tumor models In vivo imaging experiments were conducted using a mouse model bearing solid tumors. The prepared probes were injected intravenously, and observations were performed at predetermined time points using a near-infrared II imaging system.
[0059] The results show: In the early post-injection period, the probe was distributed in metabolic organs such as the liver and spleen, but the overall fluorescence background was low. In tumor tissue, especially in the invasive front area, a gradually increasing fluorescence signal appears over time; Although the necrotic area in the center of the tumor may be acidic, fluorescence recovery is not significant due to insufficient MMP cleavage. Even if MMP activity is present in inflamed peripheral tissues, strong signals are difficult to generate without continuous acidic exposure.
[0060] This demonstrates that the probe can create more spatially and temporally selective imaging effects in tumor tissue, providing greater accuracy for identifying tumor boundaries during surgery.
[0061] 3. Intraoperative navigation application In surgical settings, the probe formulation can be administered to the patient before or during surgery. After it has completed its distribution, targeting, and sequential activation in the body, the fluorescence distribution within the surgical field can be observed in real time using a near-infrared II fluorescence imaging system.
[0062] When a significant fluorescent signal is present at the resection margin, it suggests that the area may be the invasive front of a tumor; when no fluorescent signal is present, it suggests that the area is more likely to be normal tissue or a non-invasive lesion. This method allows the surgeon to more accurately determine the extent of resection, reduce the risk of residual positive margins, and preserve as much normal tissue as possible. Example 4: Optional Targeted Modification Implementation Without altering the dual-lock sequential activation mechanism, targeting units, such as RGD peptides, folic acid, EGFR antibodies, or their antigen-binding fragments, can be further introduced into the outermost layer of the probe to enhance its ability to enrich specific tumor cells or the tumor microenvironment.
[0063] For example, by introducing terminal activating groups onto the probe surface and then coupling it with RGD peptides, the probe can preferentially accumulate in tumor tissues expressing higher levels of integrin receptors. This targeted modification does not change the basic logic of "enzyme cleavage first, acid response later," but it can further increase the local concentration, thereby enhancing intraoperative imaging sensitivity.
[0064] In summary The time-responsive dual-gated activation probe provided by this invention utilizes the synergistic effect of a near-infrared II fluorescent core, gold shell FRET quenching, MMP-specific peptide cleavage, and pH-sensitive polymer secondary activation to achieve highly specific recognition of the tumor invasion front. Its key advantage lies in that it does not simply "respond simultaneously" to tumor-related factors, but rather, through time-sequential dual gating, excludes interfering factors such as acidification or inflammatory enzyme activity alone, making it more suitable for intraoperative tumor navigation and boundary determination. It is understood that the present invention has been described through some embodiments, and those skilled in the art will recognize that various changes or equivalent substitutions can be made to these features and embodiments without departing from the spirit and scope of the invention. Furthermore, under the teachings of the present invention, these features and embodiments can be modified to adapt to specific situations and materials without departing from the spirit and scope of the invention. Therefore, the present invention is not limited to the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are within the protection scope of the present invention.
Claims
1. A probe, characterized in that, include: (a) Near-infrared II fluorescence core; (b) A gold nanoshell coating on the surface of the fluorescent core, wherein the gold shell and the fluorescent core form a FRET quenching pair; (c) A first responsive layer covalently coupled to the surface of the gold shell by thiol groups, the first responsive layer comprising peptides having matrix metalloproteinase-specific cleavage sites; (d) A second responsive layer coupled to the end of the peptide, the second responsive layer comprising a pH-sensitive polymeric material; The fluorescence recovery of the probe occurs only after the following timing events: (i) The peptide is cleaved by matrix metalloproteinases, exposing the pH-sensitive polymer coupled to its end to the solvent environment; (ii) When exposed to an acidic environment with pH ≤ 6.8, the pH-sensitive polymer material undergoes a conformational change or detaches from the probe surface; Furthermore, when the peptide is not cleaved, the pH-sensitive polymer material is shielded by the physical barrier formed by the gold shell and the intact peptide, and does not come into contact with the solvent even in an acidic environment, thus preventing fluorescence recovery.
2. The probe according to claim 1, characterized in that, The emission wavelength of the near-infrared II fluorescence core is 1000-1700nm, preferably 1300-1400nm.
3. The probe according to claim 1 or 2, characterized in that, The fluorescent core is selected from Ag2S quantum dots, PbS / CdS core-shell quantum dots, Ag2Se quantum dots, or nanoparticles coated with organic NIR-II fluorescent dyes.
4. The probe according to claim 1, characterized in that, The thickness of the gold nanoshell is 5-20 nm, preferably 8-12 nm, and its FRET quenching efficiency with the fluorescent core is ≥95%.
5. The probe according to claim 1, characterized in that, The matrix metalloproteinase-responsive peptide is an MMP-2 and / or MMP-9 specific substrate peptide, comprising an amino acid sequence selected from SEQ ID NO:1 or SEQ ID NO:3; preferably, the peptide is the complete modified sequence shown in SEQ ID NO:
2.
6. The probe according to claim 1 or 5, characterized in that, The peptide is self-assembled onto the gold shell surface via Au-S covalent bonds through C-terminal or N-terminal cysteine residues, with a modification density of 0.2-2.0 nmol / cm², preferably 0.5-1.0 nmol / cm².
7. The probe according to claim 1, characterized in that, The pH-sensitive polymer material is selected from polyhistidine, polyβ-amino ester, acetylated dextran or copolymers thereof, and its protonation response pH threshold is 5.5-6.8, preferably 6.0-6.
5.
8. The probe according to claim 1 or 7, characterized in that, The second response layer is coupled to the C-terminus of the peptide via click chemistry or biotin-avidin bridging.
9. The probe according to claim 1, characterized in that, It also includes a targeting unit, which is selected from RGD peptide, folic acid, EGFR antibody or its antigen-binding fragment, and is modified on the outermost layer of the probe.
10. A method for preparing the probe according to any one of claims 1-9, characterized in that, Includes the following steps: (1) Synthesize near-infrared II fluorescence cores with emission wavelengths of 1000-1700 nm; (2) A gold nanoshell is coated on the surface of the fluorescent core, with a thickness controlled at 5-20 nm; (3) Peptides containing matrix metalloproteinase-specific cleavage sites are modified onto the gold shell surface by thiol self-assembly; (4) Couple a pH-sensitive polymer material to the end of the peptide segment; (5) Purify to obtain a time-responsive double-lock activation probe.
11. The method according to claim 10, characterized in that, The peptide modification density in step (3) is controlled at 0.2-2.0 nmol / cm² by the feed ratio.
12. The method according to claim 10, characterized in that, The coupling method described in step (4) is maleimide-thiol click reaction or biotin-avidin bridging.
13. The method according to claim 10, characterized in that, The purification described in step (5) is performed using ultrafiltration centrifugation or size exclusion chromatography.
14. The use of the probe according to any one of claims 1-9 or the probe prepared by the method according to any one of claims 10-13 in the preparation of formulations for intraoperative navigation in tumor surgery. Its features are, It is applied to tumors, specifically solid tumors selected from breast cancer, colorectal cancer, gastric cancer, lung cancer, glioma, and head and neck squamous cell carcinoma.