Beta-lactoglobulin modified near-infrared fluorescent targeting probe as well as preparation method and application thereof

Abstract

The invention discloses a beta-lactoglobulin modified near-infrared fluorescent targeting probe as well as a preparation method and application thereof. The probe comprises indocyanine green (ICG) and beta-lactoglobulin (beta-LG) which are covalently bound. Compared with the prior art, the probe disclosed by the invention has the advantages that (1) the probe disclosed by the invention improves the stability of the near-infrared fluorescent dye in a body and prolongs the retention time of the near-infrared fluorescent dye, so that the observation window time is prolonged, imaging information is fully acquired in real time, and the effective circulation time in the body is prolonged; (2) the probe has the advantages of good biocompatibility, low toxicity and the like; and (3) the probe can specifically enrich in renal tubules and perform targeted imaging in kidneys, so that diagnosis and treatment of kidney injury, especially acute injury, are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of diagnostic reagents and relates to an improved fluorescent dye in a diagnostic reagent, specifically a beta-lactoglobulin-modified near-infrared fluorescent targeting probe, a preparation method and an application thereof. Background Art

[0002] Acute kidney injury (AKI) is asymptomatic and has no characteristic clinical manifestations before the extreme loss of renal function. Diagnosis usually occurs in the context of other acute diseases. The sudden decline in renal secretory function is the hallmark of acute kidney injury (AKI). Acute kidney disease (AKD), including AKI, is a group of diseases characterized by mild decline in renal function or persistent renal dysfunction and irreversible loss of renal cells and nephrons, which may develop into chronic kidney disease (CKD). At present, acute kidney injury (AKI) is monitored by urine, and one side effect of acute kidney injury is low urine, which leads to the inability to quickly and effectively diagnose. The kidney is the main organ for rapid elimination of endogenous excretion and exogenous drugs / toxins in the body. However, the exact elimination pathway used for solutes depends largely on its interaction with the renal compartment. After interacting with the renal compartment, molecules can take two different pathways, namely glomerular filtration and tubular secretion, to be cleared by the kidney. Clinically, it is necessary to use new biomarkers to detect renal damage before loss of renal function. The development of bioimaging probes with rapid distribution and long-term retention in the kidney is of great significance for the accurate diagnosis of kidney diseases.

[0003] Optical fluorescence imaging has been used to monitor biological activities, providing unprecedented insights into cellular processes and the diagnosis and treatment of human diseases. Since near-infrared (NIR) fluorescence is in the range of 780nm-1700nm, its tissue absorption, scattering and autofluorescence are reduced compared with ultraviolet visible light (200nm-700nm). Therefore, the higher tissue penetration and better signal-to-noise ratio of NIR fluorescent dyes make them valuable tools for complex imaging technologies in biomedicine. Currently, indocyanine green (ICG) is the only NIR dye approved by the FDA for clinical use. It is mainly used for tumor targeting and metastatic lymph node localization. It has a strong binding affinity with serum proteins and can be rapidly cleared from the body through the liver pathway. However, ICG is hindered by challenges such as extremely short blood half-life, poor photostability and solubility. Currently, commercial near-infrared dyes ICG and IRdye800CW have the disadvantage of poor bioconjugation effect.

[0004] By selecting proteins with different properties, the fluorescence properties of the probe can be adjusted. Cyanine dyes can interact with proteins through covalent conjugation and supramolecular encapsulation, which can change their metabolic behavior in the body. Covalently linked cyanine dyes to proteins can improve the stability of drugs, prolong the circulation time of drugs in the body, reduce the toxicity of drugs, and improve the biocompatibility of drugs. After ICG is completely bound to serum proteins and covalently linked to proteins, the probe dynamically binds to serum proteins. Summary of the invention

[0005] Technical problem to be solved: In order to overcome the deficiencies of the prior art, the existing ICG is modified or modified to improve its stability in serum and solubility in conventional solvents (such as water, phosphate buffer, fetal bovine serum, etc.), while ensuring that the modified or modified ICG has fluorescence properties and spectral absorption capacity close to or even better than the original, so that the imaging of the kidney can be better and in real time monitored, thereby realizing rapid diagnosis of acute kidney injury (AKI). On the other hand, the false positive of the diagnostic signal of the traditional fluorescent dye can be avoided, and the delivery and long-term accumulation of the fluorescent group to the kidney can be promoted. In view of this, the present invention provides a near-infrared fluorescent targeting probe modified with β-lactoglobulin, a preparation method and an application.

[0006] Technical solution: A β-lactoglobulin-modified near-infrared fluorescent targeting probe, wherein the probe comprises covalently bound indocyanine green (ICG) and β-lactoglobulin (β-LG).

[0007] Preferably, the β-lactoglobulin is a renal clear protein with a molecular weight of 18.4 kDa, which acts as a carrier protein for indocyanine green and can target renal tubular cells.

[0008] The method for preparing the beta-lactoglobulin modified near-infrared fluorescent targeting probe described above activates the -COOH of indocyanine green, reacts it with succinimide to obtain a -CO-NHS group, and then reacts it with -NH2 in beta-lactoglobulin through an ester substitution reaction to obtain the probe.

[0009] Preferably, the method comprises the following steps:

[0010] S1, activating the -COOH of indocyanine green and reacting it with succinimide to replace the hydrogen atom in succinimide to produce N-hydroxysuccinimide active ester (ICG-NHS);

[0011] S2. Add β-lactoglobulin to the N-hydroxysuccinimide active ester in a molar ratio of 10:1-1:10. The -CO-NHS group in N-hydroxysuccinimide reacts with the -NH2 in β-lactoglobulin to undergo an ester substitution reaction, and one molecule of N-hydroxysuccinimide is removed to obtain a probe.

[0012] Preferably, the relative molecular mass of ICG-NHS in S1 is 828.04, and the molecular structure is:

[0013]

[0014] Preferably, the molar ratio of N-hydroxysuccinimide active ester to β-lactoglobulin in S2 is 1:1.

[0015] Preferably, the reaction conditions of S2 are: in a PBS buffer with a pH of 7.0-8.0, stirring the reaction at 70°C in the dark; after the reaction is completed, dialyze to remove the organic solvent, pass through a Sephadex LH-20 column, collect the first elution phase solution, ultrafilter to remove the free dye, obtain the β-LG-ICG probe, and store at -4°C.

[0016] The application of the above-mentioned β-lactoglobulin modified near-infrared fluorescent targeting probe in renal tubule targeted fluorescence imaging.

[0017] The application of the above-mentioned β-lactoglobulin modified near-infrared fluorescent targeted probe in the detection of renal injury.

[0018] The use of the beta-lactoglobulin modified near-infrared fluorescent targeting probe described above in the preparation of a detection kit that can be rapidly distributed and retained in the kidney for a long time.

[0019] The idea or principle of designing the β-lactoglobulin modified near-infrared fluorescent targeting probe of the present invention is: using the near-infrared fluorescent dye ICG as the near-infrared fluorescent imaging functional group, utilizing the targeting ability of β-lactoglobulin to the renal tubules in the kidney, and using ICG modified by N-hydroxysuccinimide (-CO-NHS) ester group and -NH2 in β-lactoglobulin to covalently connect, synthesize the near-infrared fluorescent targeting probe β-LG-ICG. After injection, the targeting probe β-LG-ICG is reabsorbed and degraded in the renal tubule segment of the kidney, while the free dye injection will not be absorbed by the cells, so it is used to monitor renal injury.

[0020] Beneficial effects: (1) The probe of the present invention improves the stability of near-infrared fluorescent dyes in the body and prolongs their residence time, thereby extending the observation window time, fully acquiring imaging information in real time, and prolonging the effective circulation time in the body; (2) The probe has the advantages of good biocompatibility and low toxicity; (3) The probe can be specifically enriched in the renal tubules and targeted for imaging in the kidneys, thereby realizing the diagnosis and treatment of kidney damage, especially acute damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the gel electrophoresis diagram of three proteins BSA, HSA, and β-LG after covalently linking with ICG-NHS. The left picture is natural light, and the right picture is near-infrared light.

[0022] Figure 2 are the UV absorption spectra (A) and fluorescence emission spectra (B) of BSA-ICG, HSA-ICG, β-LG-ICG, and ICG-NHS;

[0023] Figure 3 The fluorescence intensity of BSA-ICG, HSA-ICG, β-LG-ICG, and ICG-NHS in PBS buffer, where the left picture is the fluorescence intensity observed under the small animal imaging device, and the right picture is the bar graph analysis;

[0024] Figure 4 The gel electrophoresis diagrams of the β-LG-ICG prepared in the examples with molar ratios of β-LG:ICG=10:1, β-LG:ICG=1:1, and β-LG:ICG=1:10 are shown, wherein the left diagram is natural light and the right diagram is near-infrared light;

[0025] Figure 5 The ultraviolet absorption spectra (A) and fluorescence emission spectra (B) of the β-LG-ICG prepared in the examples with molar ratios of β-LG:ICG=10:1, β-LG:ICG=1:1, and β-LG:ICG=1:10, respectively;

[0026] Figure 6 The gel electrophoresis (A), UV-visible spectrum (B) and fluorescence emission spectrum (C) of β-LG-ICG prepared in the example;

[0027] Figure 7 is the fluorescence emission spectrum of the β-LG-ICG prepared in the embodiment after being digested with 50% Trypsin by volume;

[0028] Figure 8 This is a serum protein binding test chart detected by agarose gel electrophoresis;

[0029] Fig. 9 The in vivo dorsal fluorescence images of renal injury and normal mice at different time points after tail vein injection of β-LG-ICG;

[0030] Fig.10 HE staining of normal mice and AKI mice after tail vein injection of β-LG-ICG;

[0031] Fig.11 Agarose gel electrophoresis of β-lactoglobulin (β-LG) and ICG non-specific adsorption (A) and front and back imaging of mice injected with tail vein and non-specifically adsorbed ICG by β-LG (B);

[0032] Fig.12The fluorescence intensity comparison of isolated organs of renal injury mice and normal mice 20 minutes after tail vein injection of β-LG-ICG (A) and kidney bar graph (B), the renal tubule fluorescence imaging comparison under 10x microscope in kidney slices (C), and the renal tubule fluorescence imaging comparison under 40x microscope in kidney slices (D);

[0033] Fig.13 This is the fluorescence co-localization image of β-LG-ICG cell uptake in human renal tubular epithelial cells (HK-2) after incubation for 2 hours. DETAILED DESCRIPTION

[0034] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0035] Example 1

[0036] The β-lactoglobulin modified near-infrared fluorescent targeting probe was prepared by the following method:

[0037] S1, activating the -COOH of indocyanine green and reacting it with succinimide to replace the hydrogen atom in succinimide to produce N-hydroxysuccinimide active ester (ICG-NHS);

[0038] S2. Add β-lactoglobulin to the N-hydroxysuccinimide active ester in a molar ratio of 10:1, 1:1, and 1:10, respectively. Stir in PBS at pH 7.0-8.0 at 70°C in the dark for 2 h. Remove the organic solvent by dialysis, pass through a Sephadex LH-20 column, collect the first elution phase solution, and remove the free dye by ultrafiltration. The -CO-NHS group in N-hydroxysuccinimide reacts with the -NH2 in β-lactoglobulin to undergo ester substitution reaction, remove one molecule of N-hydroxysuccinimide, and obtain the probe, which is stored at -4°C.

[0039] Comparative Example 1

[0040] The difference from Example 1 is that β-lactoglobulin is replaced by bovine serum albumin (BSA) and human serum albumin (HSA), and the products are recorded as BSA-ICG and HSA-ICG, respectively.

[0041] In conjunction with the instruction manual Figure 1-13 The results of Example 1 and Comparative Example 1 are analyzed as follows:

[0042] like Figure 1, the β-LG-ICG (molar ratio of 1:1) prepared in Example 1, BSA-ICG and HSA-ICG prepared in Comparative Example 1 were subjected to gel electrophoresis test, and ICG-NHS without protein modification was used as a blank control. The results were as follows: 20uBSA-ICG, HSA-ICG, β-LG-ICG and ICG-NHS solutions were added to the sample slot of the gel plate, and the wire was connected for electrophoresis. When it moved to 1-2cm away from the front edge, the electrophoresis was stopped. The test results are as follows Figure 1 As shown. Figure 1 It can be seen that since ICG-NHS and BSA-ICG, HSA-ICG, and β-LG-ICG are negatively charged in the solution, they move toward the positive electrode after power is applied. When the ICG-NHS fluorescent dye is coupled with BSA, HSA, and β-LG, it has the same mobility as ICG-NHS, but the moving distance is significantly longer than ICG-NHS; while ICG-NHS basically does not move, indicating that the fluorescent dye ICG-NHS is successfully coupled with BSA, HSA, and β-LG.

[0043] like Figure 2 The spectral properties of β-LG-ICG (molar ratio of 1:1) prepared in Example 1, BSA-ICG and HSA-ICG prepared in Comparative Example 1 were tested, including absorption spectral properties and emission spectral properties, and ICG-NHS without protein modification was used as a blank control. The results are as follows: Using a UV-2550 spectrometer, the spectral absorption intensity of ICG-NHS, BSA-ICG, HSA-ICG and β-LG-ICG was measured in the spectral range of 400nm-900nm, see Figure 2 As shown in A. Using the fluorescence spectrometer FS5, set the excitation light to 713nm, and test the relative fluorescence intensity of ICG-NHS, BSA-ICG, HSA-ICG, and β-LG-ICG in the near-infrared I region (740nm-900nm), see Figure 2 As shown in B. As shown in A, protein-modified BSA-ICG, HSA-ICG, and β-LG-ICG exhibit spectral absorption capabilities that are basically consistent with those of unmodified ICG-NHS. As shown in B, the fluorescence emission spectrum shows that the fluorescence quenching effect of the fluorescence spectrum after modification with lactoglobulin is the most obvious.

[0044] like Figure 3 ICG-NHS, BSA-ICG, HSA-ICG, and β-LG-ICG (molar ratio of 1:1) were dissolved in PBS, respectively, and their fluorescence intensity was observed under a small animal imaging instrument and a bar graph was performed. The test results are shown in FIG. Figure 3 As shown. Figure 3It can be seen that the fluorescence intensity of β-LG-ICG in PBS is the weakest after the three proteins are coupled.

[0045] like Figure 4 The β-LG-ICG prepared in three molar ratios in Example 1 was subjected to agarose gel electrophoresis. Figure 4 As shown, there is a difference in charge under the three different ratios.

[0046] like Figure 5 The β-LG-ICG prepared in three molar ratios in Example 1 was tested for its spectral properties, including absorption spectral properties and emission spectral properties, and ICG-NHS not modified by protein was used as a blank control. Figure 5 From A and B in FIG, it can be seen that the fluorescence quenching was basically the same at the molar ratio of 1:1 and 1:10, so the subsequent experiments were all based on the 1:1 ratio as an example.

[0047] like Figure 6 The β-LG-ICG prepared in Example 1 was compared with the ICG-NHS not modified by protein by agarose gel electrophoresis. Figure 6 As shown in A, both of them move toward the positive electrode, but the moving distance of β-LG-ICG is significantly longer than that of ICG-NHS, indicating the successful coupling of β-LG. Using a UV-2550 spectrometer, the spectral absorption intensity of ICG-NHS and β-LG-ICG was measured in the spectral range of 400nm-900nm, see Figure 6 As shown in B; using a fluorescence spectrometer Spectrofluorometer FS5, set the excitation light to 713nm, and test the relative fluorescence intensity of ICG-NHS and β-LG-ICG in the near-infrared I region (740nm-900nm), see Figure 6 As shown in C in FIG. 1 ; it can be seen from B and C that under similar ultraviolet absorption, the coupled β-LG-ICG has obvious fluorescence quenching, and the shoulder peak in the ultraviolet absorption spectrum moves up.

[0048] like Figure 7 , β-LG-ICG was hydrolyzed by trypsin at 37°C, and its fluorescence properties were tested. Figure 7 It can be seen that after adding trypsin, trypsin hydrolyzes protein and the quenching effect gradually weakens.

[0049] like Figure 8To test the protein binding of ICG after β-LG coupling, ICG, β-LG-ICG and FBS were incubated at 37°C for 30 minutes. In order to identify the colorless protein bands, ICG and β-LG-ICG incubated with FBS were stained with Coomassie Brilliant Blue 250 (CBB250). All samples were analyzed using agarose gel electrophoresis. 2% agarose gel was used for electrophoresis analysis, and 0.1X Britton-Robinson buffer (pH = 7.4) was selected as the electrolyte. A DYY-8C gel electrophoresis instrument was used for electrophoresis operation, and it was run at a current of 50mA for 1h at room temperature. Figure 8 As shown in the figure, fluorescent images were taken by the FOBI imaging system, and ICG and fetal bovine serum were basically completely bound. Although β-LG-ICG also bound to fetal bovine serum, the amount was less than that of ICG itself, which also verified that β-LG-ICG can be metabolized by both the liver and kidneys.

[0050] like Fig. 9 , test the fluorescence properties of β-LG-ICG, and use the FOBI in vivo imaging system to observe the distribution and metabolism of the dye in the body. Imaging conditions: excited by 808nm laser, 900nm long-wave filter FELH0900 separates the excitation light from the emission light, and the FOBI near-infrared camera is used for imaging and photography. Six healthy mice (about 20g) were divided into two groups, 3 mice in each group. The three mice in the experimental group were modeled with renal injury, and the control group was healthy mice. The two groups of mice were injected with 50μM β-LG-ICG through the tail vein. The two groups of mice were injected with the same concentration, and the dose was 125μL. By near-infrared I region fluorescence imaging, the back images of the two groups of mice were taken for up to 48h. The mouse imaging system was anesthetized by the animal anesthesia system SWD. After the tail vein injection of β-LG-ICG, the back of the mouse was imaged and photographed by near-infrared I region fluorescence imaging at the same time points of 20min, 1h, 6h, and 24h, respectively, and see the attached Fig. 9 The results are shown in the photo group. Fig. 9 As shown in the figure, after the tail vein injection of β-LG-ICG, a large amount of β-LG-ICG can be observed to accumulate in the kidney tissue area. The fluorescence signal of β-LG-ICG in the kidney still maintains a high fluorescence intensity for a long time after intravenous injection, and remains basically stable within a 24-hour period. This phenomenon is different from the observation results of organic dyes filtered by the glomerulus, such as ZW800-1, in which the fluorescence signal from the kidney reaches a peak soon after intravenous injection and then decays rapidly in the body. By comparing with the fluorescence imaging of renal injury mice, it can be seen that the fluorescence intensity of the kidneys of renal injury mice is significantly weaker, indicating that β-LG-ICG has a good application prospect in the application of renal disease imaging.

[0051] like Fig.1020 min after β-LG-ICG injection into normal mice and mice with renal injury, the kidneys of mice were dissected and collected, fixed, processed and embedded in paraffin. Two consecutive sections of the kidney were stained with hematoxylin and eosin for pathological analysis. Fig.10 It can be seen that the surface capsule of the kidney tissue of normal mice (Control) is composed of dense connective tissue of uniform thickness; the glomeruli are evenly distributed in the cortex, the number of cells and matrix in the glomeruli are uniform, the renal tubular epithelial cells are round and plump, and the brush borders are arranged neatly and regularly; the connective tissue between the urinary tubules is the renal interstitium, and there is no obvious hyperplasia of the interstitium; no obvious inflammatory cell infiltration is observed. In the kidneys of mice with renal injury (AKI), eosinophilic substances (black arrows) can be seen in a small number of renal capsules, and more renal tubules are filled with eosinophilic substances, forming casts (red arrows), and occasionally renal tubular epithelial cells fall off into the tubular lumen (yellow arrows), so the reabsorption function of the renal tubules is affected after modeling.

[0052] like Fig.11 ICG is easily non-specifically adsorbed to proteins. In order to explore the difference between the non-specific adsorption of ICG to β-lactoglobulin and the metabolism of ICG-NHS covalently linked β-lactoglobulin in mice, Fig.11 A in the figure is the agarose gel electrophoresis of free ICG and β-lactoglobulin incubated at 37°C for 2h. It can be seen that ICG will non-specifically adsorb to β-lactoglobulin. After adsorption, tail vein injection was performed and it was found that Fig.11 From B, we can see that after non-specific adsorption of ICG and β-lactoglobulin, it can only be metabolized by the liver, while ICG-NHS covalently linked to β-lactoglobulin can be metabolized by the kidneys.

[0053] like Fig.12 , a group of 6 BALB / c mice were selected, 3 normal mice (Control) and 3 mice with renal injury (AKI) were injected with 125μL 50μM LG-ICG through the tail vein and then killed 20 minutes later. The in vitro images of the heart (He), liver (Li), spleen (Sp), lung (Lu), and kidney (Kid) were collected and their fluorescence intensity was captured by near-infrared I zone imaging using a small animal in vivo fluorescence imager. Fig.12 As shown in A, the organ fluorescence imaging after 20 minutes shows that obvious fluorescence signals can be observed in the liver and kidneys of both normal mice and mice with renal injury. The fluorescence intensity of the liver of normal mice and mice with renal injury is basically the same, but the fluorescence of the kidneys of mice with renal injury is significantly weaker than that of normal healthy mice. Fig.12B in the figure is a bar chart comparison of the fluorescence intensity of the kidneys of normal mice and renal injury mice in the above group. It can be seen that the fluorescence intensity of normal mice is about twice that of renal injury mice, which can be used for the detection of early renal injury, indicating that β-LG-ICG has a good application prospect in the application of renal disease imaging. Normal mice and renal injury mice were killed 20 minutes after the tail vein injection of β-LG-ICG, and the kidneys of normal mice and renal injury mice were collected and immediately fixed in 10% neutral buffered formalin, followed by standard dehydration and paraffin embedding. The embedded tissue was then cut into 4 μm slices and stained with DAPI for nuclei. In order to identify the distribution of β-LG-ICG in the kidney, the final slices were observed under an Olympus IX-73 fluorescence microscope to observe the distribution and fluorescence intensity of β-LG-ICG in the renal tubules in the near-infrared region. It can be seen that after ICG is linked to β-lactoglobulin, β-LG-ICG can selectively target and detect the renal tubules, where β-lactoglobulin is reabsorbed and restores the fluorescence of quenched ICG. Fig.12 C shows a kidney section under a 10x microscope and Fig.12 D in the figure is the distribution and fluorescence intensity of β-LG-ICG in the renal tubules observed under a 40x microscope. It can be observed that β-LG-ICG is more widely distributed in the renal tubules of normal mice under both 10x and 40x microscopes, and the fluorescence intensity is stronger than that of mice with renal injury, with a significant distribution gap. Therefore, β-LG-ICG can be well used for early diagnosis of renal tubular secretion dysfunction and renal tubular injury.

[0054] like Fig.13 , the material β-LG-ICG was tested for cell co-localization, see Fig.13 HK2 cells were incubated with β-LG-ICG dissolved in PBS for 2 h, and lysosomes and cell nuclei were stained with Lyso Tracker Green and Hoechst 33342. The intracellular distribution of β-LG-ICG was observed. Fig.13 Fluorescence imaging shows that β-LG-ICG can be effectively absorbed by HK2 cells and is mainly distributed in the cytoplasm.

Claims

1. A near-infrared fluorescent targeting probe modified with β-lactoglobulin, characterized in that: The probes include covalently bound indocyanine green (ICG) and beta lactoglobulin (β-LG).

2. The β-lactoglobulin modified near-infrared fluorescent targeting probe according to claim 1, characterized in that: The beta lactoglobulin is a renal transparent protein with a molecular weight of 18.4 kDa, which acts as a carrier protein for indocyanine green and can target renal tubular cells.

3. The method for preparing the beta-lactoglobulin modified near-infrared fluorescent targeting probe according to claim 1 or 2, characterized in that: The method activates -COOH of indocyanine green, reacts with succinimide to obtain a -CO-NHS group, and then reacts with -NH2 in beta lactoglobulin through an ester substitution reaction to prepare a probe.

4. The method for preparing the beta-lactoglobulin modified near-infrared fluorescent targeting probe according to claim 3, characterized in that: The method comprises the following steps: S1, activating the -COOH of indocyanine green and reacting it with succinimide to replace the hydrogen atom in succinimide to produce N-hydroxysuccinimide active ester (ICG-NHS); S2. Add β-lactoglobulin to the N-hydroxysuccinimide active ester in a molar ratio of 10:1-1:

10. The -CO-NHS group in N-hydroxysuccinimide reacts with the -NH2 in β-lactoglobulin to undergo an ester substitution reaction, and one molecule of N-hydroxysuccinimide is removed to obtain a probe.

5. The method for preparing the beta-lactoglobulin modified near-infrared fluorescent targeting probe according to claim 4, characterized in that: The relative molecular mass of ICG-NHS in S1 is 828.04, and the molecular structure is:

6. The method for preparing the beta-lactoglobulin modified near-infrared fluorescent targeting probe according to claim 4, characterized in that: The molar ratio of N-hydroxysuccinimide active ester to β-lactoglobulin in S2 is 1:

1.

7. The method for preparing the beta-lactoglobulin modified near-infrared fluorescent targeting probe according to claim 4, characterized in that: The reaction conditions of S2 are: in PBS buffer with a pH of 7.0-8.0, react with stirring at 70°C in the dark; after the reaction, dialyze to remove the organic solvent, pass through a Sephadex LH-20 column, collect the first elution phase solution, and ultrafilter to remove the free dye to obtain the β-LG-ICG probe, which is stored at -4°C.

8. Use of the beta-lactoglobulin-modified near-infrared fluorescent targeting probe according to claim 1 or 2 in renal tubule targeted fluorescent imaging.

9. Use of the beta-lactoglobulin modified near-infrared fluorescent targeting probe according to claim 1 or 2 in the detection of renal injury.

10. Use of the beta-lactoglobulin-modified near-infrared fluorescent targeting probe according to claim 1 or 2 in the preparation of a detection kit capable of rapid distribution and long-term retention in the kidney.

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