Neutrophil elastase-activatable near-infrared second-zone fluorescent carbon dots and their application in the diagnosis of inflammatory bowel disease

Abstract

The present invention discloses neutrophil elastase-activatable near-infrared II fluorescent carbon dots and their application in the diagnosis of inflammatory bowel disease. The carbon dots synthesized by a solvent thermal method are covalently bonded to pentafluoropropionic anhydride to obtain the desired result. The carbon dots of the present invention can respond specifically and sensitively to neutrophil elastase in cells and in vivo with high biocompatibility, displaying a strong NIR-II fluorescence signal. They can be used for real-time imaging of noninvasive diagnostic assessments of early inflammatory bowel disease, providing a promising approach for the development of clinical noninvasive diagnostic techniques for early inflammatory bowel disease.

Description

Technical Field

[0001] The present invention belongs to the field of nanomaterial preparation and biomedicine, and specifically relates to a neutrophil elastase-activated near-infrared second-zone fluorescent carbon dot and its application in the diagnosis of inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a common chronic inflammatory disease of the gastrointestinal tract, including Crohn's disease (CD) and ulcerative colitis (UC). Its etiology is complex and is actually a complex disease caused by genetic, environmental, microbial and immune factors. Over the past few decades, the incidence of inflammatory bowel disease has gradually increased. Early diagnosis and treatment are very important for patients with inflammatory bowel disease (IBD). Recent studies have shown that the diagnosis of IBD is often delayed, and this delay increases the risk of intestinal surgery. Currently, clinical diagnosis is mainly performed through endoscopic surgery. Compared with traditional examination methods, endoscopy has the advantage of being more intuitive, but endoscopy is invasive and may cause serious side effects. Therefore, for accurate clinical diagnosis and drug treatment, there is a great need for rapid and effective methods for detecting relevant biomarkers for non-invasive diagnosis of early IBD.

[0003] Neutrophil elastase (NE) is a serine protease normally found in monocytes, T cells, and mast cells. It plays an important role in chronic inflammation and acute responses to infection and injury. It is a well-known inflammatory perpetuator and a promising prognostic biomarker. Excessive activation and release of NE not only degrade the extracellular matrix, increase tissue permeability, and promote neutrophil infiltration and chemotaxis to sites of inflammation, but also release inflammatory factors that promote inflammatory responses, thereby triggering a variety of inflammation-related diseases. Neutrophil elastase expression is directly correlated with IBD, with NE being highly expressed at inflammatory sites in IBD patients. Therefore, in situ monitoring of NE activity at inflammatory sites in IBD patients could aid in the clinical diagnosis or treatment of early-stage IBD.

[0004] Carbon dots (CDs) are an emerging class of optical materials with unique applications in optoelectronic devices, catalysis, detection, and bioimaging. Due to their convenient synthesis procedures and effectively tunable chemical structures, utilizing CDs with bright NIR-II emission as standard imaging agents in the near-infrared second (NIR-II) imaging window may usher in a new era in disease detection and surgical visualization. Summary of the Invention

[0005] Inspired by the advantages of near-infrared fluorescence bioimaging of IBD-related biomarkers NE and CDs, the purpose of the present invention is to develop a neutrophil elastase-activated NIR-II fluorescent carbon dot (OPDG-PFA@PEI) that specifically responds to changes in NE content through near-infrared fluorescence imaging, thereby achieving non-invasive diagnosis of early IBD.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The method for synthesizing neutrophil elastase-activated near-infrared second-zone fluorescent carbon dots provided by the present invention comprises the following steps:

[0008] Step 1: o-phenylenediamine and glutathione are dissolved in formamide and dispersed uniformly by ultrasonication. The mixed solution is transferred to a polytetrafluoroethylene-lined autoclave for a solvothermal reaction. The resulting reaction product is then dialyzed and dried to obtain carbon dots, designated as OPDG.

[0009] Step 2: The N,N-dimethylformamide solution of OPDG obtained in Step 1 was mixed with pentafluoropropionic anhydride to obtain neutrophil elastase-activatable near-infrared region II fluorescent carbon dots, designated as OPDG-PFA. The resulting OPDG-PFA solution in dichloromethane was thoroughly mixed with an aqueous solution of PEI using ultrasonic probe technology to improve the stability of OPDG-PFA in the aqueous solution, and then dried by rotary evaporation to obtain OPDG-PFA@PEI.

[0010] Preferably, in step 1, the mass ratio of o-phenylenediamine to glutathione is 10.8-108 mg:1.5-15 mg.

[0011] Preferably, in step 1, the solvent thermal reaction is carried out at 140-180° C. for 8-12 hours.

[0012] Preferably, in step 1, the dialysis is performed by diluting the resulting reaction product 3-5 times with pure water, pouring it into a 500-1000 Da dialysis bag, and dialyzing it at room temperature for 24-48 hours, with the water in the dialysis system being changed every 2-4 hours. The formamide solvent and unreacted molecules are removed by dialysis, and OPDG is obtained by rotary evaporation after dialysis. The purpose of rotary evaporation is to remove water molecules to prevent them from hydrolyzing pentafluoropropionic anhydride in the next reaction.

[0013] Preferably, the preparation method of OPDG-PFA in step 2 is as follows: OPDG is dissolved in N,N-dimethylformamide and ultrasonically dispersed uniformly, triethylamine is added at room temperature, and then pentafluoropropionic anhydride is added in an ice bath. The resulting mixture is shaken at 30-40°C for 1-4 hours, and then rotary evaporated to obtain OPDG-PFA. The mass ratio of OPDG, triethylamine, and pentafluoropropionic anhydride is 3.73-37.3 mg: 2.02-20.2 mg: 6.2-62 mg. The purpose of rotary evaporation is to remove unreacted pentafluoropropionic anhydride and DMF solvent.

[0014] Preferably, in step 2, the mass ratio of OPDG-PFA to PEI is 3-30 mg:9-90 mg, the emulsification time using probe ultrasound is 10-30 min, and OPDG-PFA@PEI is obtained after rotary evaporation and drying. The purpose of rotary evaporation and drying is to remove the dichloromethane solvent.

[0015] The neutrophil elastase-activatable near-infrared zone II fluorescent carbon dots prepared according to the above method have the following characteristics: they are designed and synthesized through a two-step solvent thermal / surface covalent bond modification reaction, the procedure is simple, and the cost of the reaction precursors is low; their radial size is 100 to 120 nm, and they have good water solubility and stability; they have good biosafety and bioavailability at the cellular and animal levels; they can maintain good stability under continuous excitation at 808 nm; and they can specifically identify neutrophil elastase and release a strong NIR-II fluorescence signal.

[0016] The neutrophil elastase-activated near-infrared zone II fluorescent carbon dots prepared in the present invention have an emission wavelength of up to 1000-1100 nm. After oral administration, OPDG-PFA@PEI will be specifically activated by neutrophil elastase highly expressed in the inflammatory site, showing a strong NIR-II fluorescence signal. By monitoring the NIR-II fluorescence imaging of neutrophil elastase activity, non-invasive diagnosis and evaluation of early IBD can be achieved.

[0017] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0018] 1. The present invention adopts a two-step solvent thermal / surface covalent bond modification reaction to design and synthesize OPDG-PFA@PEI. The emission center of the obtained water-soluble carbon dots is formed by covalent bonding of conjugated carbon cores. The synthesis procedure is simple and the reaction precursor cost is low.

[0019] 2. The OPDG-PFA@PEI prepared in the present invention has good biocompatibility and bioavailability.

[0020] 3. The emission wavelength of the OPDG-PFA@PEI prepared in the present invention can reach 1091 nm, which can effectively penetrate superficial tissues and reach deeper tissue layers. In addition, the light scattering phenomenon in the tissue is weak, thereby reducing image blur caused by light scattering.

[0021] 4. The OPDG-PFA@PEI prepared in the present invention is specifically activated by neutrophil elastase, which is highly expressed in inflammatory sites, and displays a strong NIR-II fluorescence signal. By monitoring the NIR-II fluorescence imaging of neutrophil elastase activity, non-invasive diagnosis and evaluation of early IBD can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the synthesis of neutrophil elastase-activated near-infrared zone II fluorescent carbon dots of the present invention.

[0023] Figure 2 Transmission electron microscopy images of OPDG ((a) in the figure) and OPDG-PFA@PEI ((b) in the figure) nanoparticles prepared in Example 1.

[0024] Figure 3 These are NIR-II fluorescence imaging images of OPDG and OPDG-PFA@PEI prepared in Example 1.

[0025] Figure 4 This is the fluorescence spectrum (808nm excitation) of OPDG and OPDG-PFA@PEI prepared in Example 1.

[0026] Figure 5 This is a diagram showing the cytotoxicity experiment of OPDG-PFA@PEI prepared in Example 1.

[0027] Figure 6 This is the experimental diagram of cell NIR-II fluorescence imaging of OPDG-PFA@PEI prepared in Example 1.

[0028] Figure 7 This is a comparison chart of the results of quantitative analysis of the cell NIR-II fluorescence signal intensity of OPDG-PFA@PEI prepared in Example 1.

[0029] Figure 8 This is a statistical chart of the laser stability of OPDG and OPDG-PFA@PEI prepared in Example 1.

[0030] Figure 9 Statistical graph of the fluorescence response of OPDG-PFA@PEI prepared in Example 1 to different enzymes and related species.

[0031] Figure 10This is a NIR-II fluorescence imaging experiment diagram of a mouse in supine position for in vivo drug delivery imaging of OPDG-PFA@PEI prepared in Example 1.

[0032] Figure 11 This is a comparison chart of the results of quantitative analysis of the NIR-II fluorescence signal intensity of the abdomen of mice in the supine position for in vivo drug administration imaging of OPDG-PFA@PEI prepared in Example 1.

[0033] Figure 12 This is an experimental diagram of NIR-II fluorescence imaging of mouse colon tissue administered with OPDG-PFA@PEI prepared in Example 1.

[0034] Figure 13 These are statistical graphs of the routine IBD assessment experiment in mice using OPDG-PFA@PEI drug administration imaging prepared in Example 1. Figure (a) shows the weight change of mice, Figure (b) shows the length of the mouse colon, and Figures (c to f) are comparison graphs of the levels of MPO, IL-1β, IL-6, and TNF-α in the colon tissue homogenate, respectively. DETAILED DESCRIPTION

[0035] To make the above-mentioned objects, features, and advantages of the present invention more clearly understood, the following detailed description of the specific embodiments of the present invention is provided in conjunction with the following embodiments. The following is merely an example and illustration of the concept of the present invention. Any modification, supplement, or substitution of the described specific embodiments by a person skilled in the art, as long as it does not deviate from the concept of the invention or exceed the scope defined by the claims, shall fall within the scope of protection of the present invention.

[0036] Example 1

[0037] In this example, neutrophil elastase-activated near-infrared zone II fluorescent carbon dots were prepared as follows:

[0038] (1) Preparation of carbon dot OPDG: 108 mg of o-phenylenediamine and 15 mg of glutathione were dissolved in 10 mL of formamide and ultrasonicated for 10 min until the solution was clear and transparent. The mixed solution was transferred to a polytetrafluoroethylene-lined autoclave for solvent thermal reaction at a reaction temperature of 180 ° C and a reaction time of 12 h. After cooling to room temperature, three times the volume of pure water was added for dilution, and the product was washed in 1.0 L of deionized water using a dialysis bag (500 Da) for 24 h. The water in the dialysis system was changed every 2 h. Finally, the OPDG aqueous solution was completely evaporated using a rotary evaporator, and then placed in a vacuum drying barrel under vacuum overnight until the water was completely evaporated, and further dispersed in DMF (concentration of 18.65 mg / mL) and stored at 4 ° C.

[0039] (2) Preparation of neutrophil elastase-activated near-infrared second-zone fluorescent carbon dots OPDG-PFA@PEI: Take a DMF solution containing 37.3 mg of OPDG, add 20 mg of triethylamine at room temperature, ultrasonicate for 1 minute to fully mix the system, and slowly add 62 mg of pentafluoropropionic anhydride in an ice bath. The reaction mixture is shaken at 37°C for 3-4 hours. The DMF and unreacted pentafluoropropionic anhydride in the OPDG-PFA solution are completely evaporated using a rotary evaporator to obtain OPDG-PFA. Take 30 mg of OPDG-PFA and dissolve it in 6 mL of dichloromethane. Add 27 mL of an aqueous solution containing 90 mg of PEI. Use a probe ultrasonic emulsification, ultrasonicate for 10 minutes, and ultrasonic power of 50%. Evaporate the solvent of the mixture using a rotary evaporator at room temperature to obtain OPDG-PFA@PEI. Disperse OPDG-PFA@PEI in deionized water to obtain a dispersion with a concentration of 5 mg / mL, and store in the dark.

[0040] Figure 2 Transmission electron micrographs of OPDG and OPDG-PFA@PEI obtained in Example 1 were obtained. The OPDG and OPDG-PFA@PEI aqueous dispersions were dripped onto a copper transmission electron microscope grid, allowed to dry, and then observed under a transmission electron microscope. The images show spherical structures, with OPDG having a diameter of approximately 10 nm and OPDG-PFA@PEI having a diameter of 100-120 nm.

[0041] Figure 3 This is the NIR-II fluorescence imaging of 1 mg / mL aqueous solutions of OPDG and OPDG-PFA@PEI obtained in Example 1 under 808 nm excitation. The image shows that OPDG exhibits a strong NIR-II fluorescence signal, while the fluorescence signal of OPDG-PFA@PEI modified with the elastase-specific recognition group is significantly weakened. This indicates that pentafluoropropionic anhydride modification can reduce the NIR-II fluorescence intensity of OPDG.

[0042] Figure 4 Fluorescence spectra of 1 mg / mL aqueous solutions of OPDG and OPDG-PFA@PEI obtained in Example 1 under 808 nm excitation light. The figure shows that the emission wavelength of the prepared OPDG and OPDG-PFA@PEI reaches 1091 nm. The NIR-II fluorescence signal of OPDG-PFA@PEI is significantly lower than that of OPDG.

[0043] Figure 5 The cytotoxicity test diagram of OPDG-PFA@PEI obtained in Example 1 is as follows: 100 μL (1×10 5 ) of NCM460 cells and incubated at 37°C for 24 h. Figure 5 Different concentrations of OPDG-PFA@PEI were added and incubated at 37°C for 24 hours. Following incubation, 20 μL of MTT (5 mg / mL) was added to each well and incubated in a humidified incubator for 4 hours. Finally, the supernatant was aspirated and 150 μL of DMSO was added to each well to dissolve the formazan. The cells were incubated at 37°C in the dark for 10 minutes, and the absorbance at 490 nm was measured. As shown in the figure, when the OPDG-PFA@PEI concentration reached 100 μg / mL, the NCM460 cell survival rate remained above 80%, demonstrating the material's good biosafety and bioavailability.

[0044] Figure 6 The cell imaging experiment of OPDG-PFA@PEI obtained in Example 1 is shown in Figure 1. The characterization method is as follows: 100 μL (1×10 5 ) RAW264.7 cells were incubated at 37°C for 12 hours before the experiment began. RAW264.7 cells were divided into 7 groups (n=3 per group), and the experimental method for each group was as follows:

[0045] (1) Blank group: no treatment, OPDG-PFA@PEI (0.5 mg / mL) was added; (2) + Sivelestat group: Sivelestat (10 μM) was added, cultured at 37°C for 3 h, and then OPDG-PFA@PEI (0.5 mg / mL) was added; (3) - Sivelestat group: no Sivelestat was added, and OPDG-PFA@PEI (0.5 mg / mL) was added directly; (4) Sivelestat group: LPS (20 μg / mL) and Sivelestat (10 μM) were added, cultured at 37°C for 3 h, and then OPDG-PFA@PEI (0.5 mg / mL) was added; (5) 1 h group: addition LPS (20 μg / mL), cultured at 37℃ for 1 h, then sivelestat (10 μM) was added, cultured at 37℃ for 3 h, then OPDG-PFA@PEI (0.5 mg / mL) was added; (5) 5 h group: LPS (20 μg / mL) was added, cultured at 37℃ for 5 h, then sivelestat (10 μM) was added, cultured at 37℃ for 3 h, then OPDG-PFA@PEI (0.5 mg / mL) was added; (5) 8 h group: LPS (20 μg / mL) was added, cultured at 37℃ for 8 h, then sivelestat (10 μM) was added, cultured at 37℃ for 3 h, then OPDG-PFA@PEI (0.5 mg / mL) was added;

[0046] After the addition of the material, the cells were incubated at 37°C for 30 minutes. Near-infrared images were acquired using an InGaAs camera with a 900nm long-pass filter under 808nm excitation. The image shows a significant increase in the fluorescence intensity of inflammatory cells after incubation of RAW264.7 cells with OPDG-PFA@PEI.

[0047] Figure 7 for Figure 6 The experimental graph shows the quantification of fluorescence intensity, as measured using Image J. The graph shows that the fluorescence intensity of Raw264.7 cells inhibited by sivelestat is almost identical to that of the blank control group. The fluorescence intensity of Raw264.7 cells stimulated by LPS for 5 hours is the highest, indicating that OPDG-PFA@PEI can specifically recognize NE and release NIR-II fluorescence signals by cleaving the amide bond.

[0048] Figure 8 Figure 1 shows the laser stability of OPDG and OPDG-PFA@PEI obtained in Example 1. Characterization was performed by adding 100 μL of an aqueous dispersion of OPDG or OPDG-PFA@PEI (0.5 mg / mL) to a 96-well plate. The plate was excited with 808 nm light for 10 minutes. Near-infrared images were captured using an InGaAs camera with a 900 nm longpass filter at 0, 2, 4, 6, 8, and 10 minutes. The figure shows that after 10 minutes of continuous 808 nm excitation, the fluorescence intensity of OPDG and OPDG-PFA@PEINIR-II did not significantly decrease, demonstrating good laser stability.

[0049] Figure 9 The fluorescence response experimental diagram of OPDG-PFA@PEI obtained in Example 1 to different enzymes and related species was characterized as follows: 500 μg / mL OPDG-PFA@PEI was added to PBS buffer (pH 7.4) with various related species (200 μM) and enzymes (750 pg / mL). Twenty groups (n=3 per group) were set up: (1) blank; (2) vitamin C; (3) cysteine; (4) glutathione; (5) glucose; (6) glycine; (7) histidine; (8) leucine; (9) H2S; (10) Fe 3+ ; (11) Cu 2+ ; (12) Mg 2+ ; (13) Zn 2+ ; (14) HClO; (15) H2O2; (16) bovine albumin; (17) γ-glutamyltransferase; (18) β-galactosidase; (19) esterase; (20) NE. The cells were incubated at 37°C in the dark for 30 min. Near-infrared images were acquired using an InGaAs camera with a 900 nm long-pass filter under 808 nm excitation. The image shows that OPDG-PFA@PEI exhibits high specificity and sensitivity for NE.

[0050] Figure 10The figure shows the experimental image of near-infrared fluorescence imaging of OPDG-PFA@PEI in Balb / C female mice obtained in Example 1. The characterization method is as follows: 3.5g DSS was dissolved in 100mL deionized water to prepare a 3.5% DSS solution, which was used to construct an IBD mouse model. Balb / C female mice were randomly divided into three groups (n=3 per group). The experimental method for each group was as follows: (1) Control: fed with normal water for 7 days; (2) DSSDay4: fed with 3.5% DSS solution instead of normal water for 4 days, as an early IBD mouse model; (2) DSSDay7: fed with 3.5% DSS solution instead of normal water for 7 days, as a late IBD mouse model. All mice used for in vivo imaging were fed jelly 24 hours before image acquisition to reduce the interference of mouse feed on gastrointestinal imaging. After the modeling was completed, the three groups of mice were administered OPDG-PFA@PEI (100mg / kg) by gavage. Four hours after oral administration, NIR-II images were captured using an InGaAs camera with a 900nm longpass filter under 808nm excitation. The images show strong NIR-II fluorescence signals detected in the abdomens of mice on DSS Day 4 and DSSDay7, while relatively low fluorescence signals were detected in the abdomens of mice in the control group.

[0051] Figure 11 This figure shows the fluorescence intensity quantification of OPDG-PFA@PEI obtained in Example 1 in Balb / C female mice using NIR-II fluorescence imaging. The intensity of the mouse abdomen was read using Image J. The figure shows that the NIR-II fluorescence signal in the abdomen of DSSDay4 and DSSDay7 mice is significantly higher than that in the control group. This demonstrates that OPDG-PFA@PEI can highly specifically recognize NE and monitor early-stage IBD through NIR-II fluorescence imaging.

[0052] Figure 12The figure shows the experimental image of OPDG-PFA@PEI obtained in Example 1 in the colon tissue of Balb / C female mice. The characterization method is as follows: 3.5g DSS was dissolved in 100mL deionized water to make a 3.5% DSS solution, which was used to construct an IBD mouse model. Balb / C female mice were randomly divided into three groups (n=3 per group). The experimental method for each group was as follows: (1) Control: fed with normal water for 7 days; (2) DSSDay4: fed with 3.5% DSS solution instead of normal water for 4 days, as an early IBD mouse model; (3) DSSDay7: fed with 3.5% DSS solution instead of normal water for 7 days, as a late IBD mouse model. All mice used for in vivo imaging were fed jelly 24 hours before image acquisition to reduce the interference of mouse feed on gastrointestinal imaging. After the modeling was completed, the three groups of mice were administered OPDG-PFA@PEI (100mg / kg) by gavage. Four hours after oral administration, mice were sacrificed, and their colons were removed and NIR-II images were captured using an InGaAs camera with 808nm excitation and a 900nm longpass filter. The images show a strong NIR-II fluorescence signal detected in the colon tissues of DSSDay4 and DSSDay7 mice, while a relatively low signal was detected in the colon tissues of control mice. This demonstrates that OPDG-PFA@PEI can highly specifically identify NE in inflammatory sites of both early and late-stage IBD in mice, making it a promising candidate for noninvasive diagnosis and evaluation of early-stage IBD.

[0053] Figure 13 This is a routine assessment of IBD in Balb / C female mice. Figure (a) shows weight change, and Figure (b) shows colon length. Figures (c-f) show the pro-inflammatory status of colon homogenates. The following method was used: 0.5 g of colon was weighed, added to 10 mL of 7.4 PBS, and maintained at 2-8°C. The sample was thoroughly homogenized manually. Centrifuged at 2500 rpm for 20 minutes, the supernatant collected, and the tissue homogenate was obtained. MPO activity and levels of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The figures show a significant decrease in weight and shortened colon length in mice treated with DSS for 4 and 7 days. MPO, TNF-α, IL-1β, and IL-6 levels in colon homogenates were higher than those in the control group, indicating the presence of IBD symptoms in the mice.

[0054] In summary, the carbon dots of the present invention can respond specifically and sensitively to neutrophil elastase with high biocompatibility in cells and in vivo, showing a strong NIR-II fluorescence signal, and can be used for real-time imaging of noninvasive diagnosis and evaluation of early inflammatory bowel disease, providing a potential method for the development of clinical noninvasive diagnosis technology for early inflammatory bowel disease.

[0055] The above description is merely an exemplary embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A method for synthesizing neutrophil elastase-activated near-infrared second-zone fluorescent carbon dots, characterized in that: The following steps are involved: Step 1: o-phenylenediamine and glutathione are dissolved in formamide and dispersed uniformly by ultrasonication. The mixed solution is transferred to an autoclave for a solvothermal reaction. The resulting reaction product is then dialyzed and dried to obtain carbon dots, which are designated as OPDG. Step 2: The N,N-dimethylformamide solution of OPDG obtained in step 1 is mixed with pentafluoropropionic anhydride to obtain neutrophil elastase-activated near-infrared second-zone fluorescent carbon dots, which are designated as OPDG-PFA. The obtained OPDG-PFA dichloromethane solution and PEI aqueous solution were fully mixed by probe ultrasound to improve the stability of OPDG-PFA in the aqueous solution, and then rotary evaporated to obtain OPDG-PFA@PEI.

2. The method for synthesizing neutrophil elastase-activatable near-infrared second-zone fluorescent carbon dots according to claim 1, characterized in that: In step 1, the mass ratio of o-phenylenediamine to glutathione is 10.8-108 mg:1.5-15 mg.

3. The method for synthesizing neutrophil elastase-activatable near-infrared second-zone fluorescent carbon dots according to claim 1, characterized in that: In step 1, the solvent thermal reaction conditions are: reaction at 140-180° C. for 8-12 hours.

4. The method for synthesizing neutrophil elastase-activatable near-infrared second-zone fluorescent carbon dots according to claim 1, characterized in that: In step 1, the dialysis is performed by diluting the obtained reaction product 3 to 5 times with pure water, pouring it into a 500 to 1000 Da dialysis bag, and dialyzing it at room temperature for 24 to 48 hours, with the water in the dialysis system being changed every 2 to 4 hours.

5. The method for synthesizing neutrophil elastase-activatable near-infrared second-zone fluorescent carbon dots according to claim 1, characterized in that: The preparation method of OPDG-PFA in step 2 is as follows: OPDG is dissolved in N,N-dimethylformamide and ultrasonically dispersed uniformly, triethylamine is added at room temperature, and then pentafluoropropionic anhydride is added under ice bath conditions. The resulting mixture is shaken at 30-40°C for 1-4 hours, and then rotary evaporated to dryness to obtain OPDG-PFA.

6. The method for synthesizing neutrophil elastase-activatable near-infrared second-zone fluorescent carbon dots according to claim 5, characterized in that: The mass ratio of OPDG, triethylamine and pentafluoropropionic anhydride is 3.73~37.3 mg:2.02~20.2 mg:6.2~62 mg.

7. The method for synthesizing neutrophil elastase-activatable near-infrared second-zone fluorescent carbon dots according to claim 1, characterized in that: In step 2, the mass ratio of OPDG-PFA to PEI is 3-30 mg:9-90 mg. 8 . Neutrophil elastase-activatable near-infrared zone II fluorescent carbon dots prepared by the synthesis method according to any one of claims 1 to 7 .

9. The neutrophil elastase-activatable near-infrared second-zone fluorescent carbon dots according to claim 8, characterized in that: The emission wavelength of the fluorescent carbon dots can reach 1000-1100 nm.

10. A use of the neutrophil elastase-activatable near-infrared second-zone fluorescent carbon dots according to claim 8 or 9, characterized in that: Used to prepare preparations for specifically identifying neutrophil elastase for diagnosing inflammatory bowel disease.

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