Near-infrared two-region ratiometric fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of fluorescence imaging, and discloses a near-infrared two-region ratiometric fluorescent probe as well as a preparation method and application thereof. The preparation method comprises the following steps: performing coprecipitation on yttrium acetate, ytterbium acetate and erbium acetate in a mixed solution of 1-octadecene and oleic acid to prepare NaYF4: 20% Yb, 2% Er down-conversion nanoparticles which are marked as DCNP-core; preparing NaYF4: 20% Yb, 2% Er at NaYF4: 30% Nd from DCNP at core through a seed mediation method, and recording the NaYF4: 20% Yb, 2% Er at NaYF4: 30% Nd as a core-shell structure DCNP; the preparation method comprises the following steps: dispersing DCNP with a core-shell structure in cyclohexane, and adding NOBF4 for reaction to obtain DCNP (at) NOBF4; the preparation method comprises the following steps: reacting DCNP (at) NOBF4 and peptide in DMF (Dimethyl Formamide) to obtain DCNP (at) PEP; the DCNP (at) PEP and PEG are subjected to a reaction in DMF, and DCNP (at) PEG is obtained; dCNP-coated PEG and IR808 are subjected to a reaction in DMF, a reaction solution is centrifuged, a solid phase is collected, DCNP-coated IR808 is obtained, and the near-infrared two-region ratiometric fluorescent probe is obtained. The near-infrared two-region ratiometric fluorescent probe can eliminate errors and instrument errors caused by different attenuation coefficients in tissues, accurately quantifies the content of the HNE enzyme in lung tissues, can be used for detecting the HNE enzyme, and evaluates the severity of lung inflammation through the expression quantity of the HNE enzyme.

Description

Technical Field

[0001] The present application relates to the field of fluorescence imaging technology, and in particular to a near-infrared second-region ratiometric fluorescence probe and a preparation method and application thereof. Background Art

[0002] Pneumonia is a widespread disease caused by viral and bacterial infections, affecting approximately 200 million people annually and resulting in millions of deaths, posing a serious threat to human health and social stability. It is generally believed that pneumonia or uncontrolled systemic inflammation is the primary pathogenesis of acute lung injury (ALI) / acute respiratory distress syndrome (ARDS). When stimulated, most inflammatory cells (including macrophages, neutrophils, and lymphocytes) release large amounts of proinflammatory cytokines, such as IL-1, IL-6, and TNF-α. Neutrophils are the first cells recruited to the site of injury during an inflammatory response, exacerbating tissue damage by releasing human neutrophil elastase (HNE). When the balance between anti-HNE proteins and HNE is disrupted, excessive HNE release can lead to the development of related diseases. Therefore, quantitative detection of HNE can monitor the extent of lung inflammation in real time. However, due to the complexity of lung tissue and the inflammatory response, accurate quantification of HNE has been a challenging task.

[0003] Optical imaging utilizes the interaction of light with matter to obtain information about the interior or surface of an object. Near-infrared second-region fluorescence imaging (NIR-II imaging) is a fluorescence imaging technique operating in the 1000-1700 nm wavelength range. Compared to conventional visible light (400-700 nm) and near-infrared first region (NIR-I, 700-900 nm) imaging, NIR-II imaging offers deeper tissue penetration, higher spatial resolution, and lower background noise, thus showing great potential in biomedical research and clinical applications. Currently, lung disease-related markers are qualitatively imaged using conventional probes in an on / off mode for the qualitative and quantitative detection of bioactive molecules in vivo. Although responsive "turn-on" and "turn-off" probes have been widely developed, most NIR-II fluorescence probes, similar to these, rely on single-channel imaging and detection and are subject to autofluorescence, which can lead to detection bias and hinder the accurate detection and quantification of these active molecules. Real-time and accurate concentration quantification remains a significant challenge. The ratiometric FL probe has a built-in self-calibration signal, which can effectively overcome the interference of instrument and environmental errors, making it a more sensitive and reliable detection method.

[0004] Based on this, it is of great significance to develop a new near-infrared second region (NIR-II) ratiometric fluorescence (FL) probe to achieve accurate quantitative detection of HNE enzyme. Summary of the Invention

[0005] The present application provides a near-infrared second-region ratiometric fluorescent probe and its preparation method and application, aiming to solve the technical problem that the existing technology is difficult to accurately and quantitatively detect HNE enzyme.

[0006] In order to achieve the above objectives, this application adopts the following technical solutions.

[0007] In a first aspect of the present application, a method for preparing a near-infrared second-region ratiometric fluorescent probe is provided, comprising:

[0008] S1, NaYF4:20%Yb,2%Er down-conversion nanoparticles were prepared by coprecipitation of yttrium acetate, ytterbium acetate and erbium acetate in a mixed solution of 1-octadecene and oleic acid, denoted as DCNP@core;

[0009] S2, DCNP@core was prepared by seed-mediated method to prepare NaYF4:20% Yb, 2% Er@NaYF4:30% Nd, which was recorded as core-shell DCNP;

[0010] S3, dispersing the core-shell structure DCNP in cyclohexane, adding NOBF4 to react, and obtaining DCNP@NOBF4;

[0011] S4, reacting DCNP@NOBF4 and peptide in DMF to obtain DCNP@PEP, wherein the peptide is a neutrophil elastase-specific peptide;

[0012] S5, reacting DCNP@PEP and PEG in DMF to obtain DCNP@PEG;

[0013] S6, DCNP@PEG and IR808 are reacted in DMF, the reaction solution is centrifuged to collect the solid phase, and DCNP@IR808, a near-infrared second region ratiometric fluorescent probe, is obtained.

[0014] Preferably, in step S3, the core-shell structure DCNP and NOBF4 are ultrasonically treated in cyclohexane, and the supernatant is removed after standing and stratification; then a mixed coagulant is added, and the precipitate is collected by centrifugation to obtain DCNP@NOBF4.

[0015] Further preferably, the mass ratio of the core-shell structure DCNP to NOBF4 is 1:(2-4).

[0016] Further preferably, the mixed coagulant is a mixture of toluene and cyclohexane in a volume ratio of 1:1.

[0017] Preferably, in step S4, DCNP@NOBF4 and peptide are reacted in DMF at 25° C. in the dark with stirring for 10 to 15 h at a stirring rate of 300 to 500 rpm;

[0018] In step S5, DCNP@PEP and PEG are reacted in DMF at 25° C. in the dark with stirring for 5 to 8 h at a stirring rate of 300 to 500 rpm;

[0019] In step S6, DCNP@PEG and IR808 were reacted in DMF at 25° C. in the dark with stirring for 12 to 15 h at a stirring rate of 300 to 500 rpm.

[0020] Preferably, the molar ratio of DCNP@NOBF4, peptide, PEG and IR808 is 1:(1500-2500):(19-21):(17-19).

[0021] Preferably, in step S1, yttrium acetate, ytterbium acetate, and erbium acetate are mixed in a mixed solution of 1-octadecene and oleic acid, and then heated to 70-80° C. under vacuum and kept warm. Then, the mixture is first heated to 100° C. and kept warm under an argon atmosphere, and then heated to 150° C. and kept warm, and then cooled to room temperature to obtain a reaction solution.

[0022] The precipitant is added to the reaction solution, and the temperature is slowly raised to 45-50° C. and maintained, then heated to 65-70° C., evacuated, and maintained; then heated to 100° C. and maintained, then heated to 300-310° C. and maintained, and then cooled to room temperature; solid particles are collected by centrifugation and washed to obtain NaYF4:20% Yb, 2% Er down-conversion nanoparticles;

[0023] The precipitant is a mixture of ammonium fluoride methanol solution and sodium hydroxide methanol solution; wherein the molar ratio of ammonium fluoride to sodium hydroxide is 16:1.

[0024] Preferably, in step S2, yttrium acetate and neodymium acetate are mixed in a mixed solution of 1-octadecene and oleic acid, and then gradually heated to 150° C. under an argon atmosphere, and then cooled to room temperature to obtain a reaction solution;

[0025] The DCNP@core prepared in step S1, a methanol solution of NH4F and a methanol solution of sodium hydroxide were added to the reaction solution, and the mixture was heated to 300-310°C for insulation, and then cooled to room temperature, centrifuged and washed to obtain a core-shell structure DCNP.

[0026] In a second aspect of the present application, a near-infrared second-region ratiometric fluorescent probe prepared by the above preparation method is provided.

[0027] The third aspect of the present application provides the use of the above-mentioned near-infrared second-region ratiometric fluorescent probe in detecting HNE enzyme.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] The near-infrared second-zone ratio fluorescent probe prepared in this application reaches the site of lung inflammation by inhalation administration. The amide bond will be cut off by the HNE enzyme overexpressed by neutrophils. The substrate peptide, IR808 will be detached from the probe, losing the sensitization effect, and under 808nm laser excitation, the NIR-II fluorescence signal (1550nm) is weakened. At the same time, the NIR-II fluorescence signal (1550nm, 980nm excitation) remains unchanged as a reference signal. The ratio of the two signals and the HNE enzyme concentration within a certain range will be linearly related. The content of the HNE enzyme can be accurately quantified by the ratio reaction signal (ratio = F1550Em, 808Ex / F1550Em, 980Ex).

[0030] The proposed near-infrared second-zone ratiometric fluorescent probe eliminates errors caused by varying attenuation coefficients within tissues and instrumental errors, accurately quantifying the amount of HNE enzyme in lung tissue. It can be used to detect HNE enzyme and assess the severity of lung inflammation based on its expression, thereby enabling real-time, accurate lung inflammation grading and guiding treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] To more clearly illustrate the technical solutions of the embodiments of this application, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some of the embodiments described in this application. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0032] Figure 1 TEM image of DCNP@core of Example 1;

[0033] Figure 2 TEM image of the core-shell DCNP of Example 1;

[0034] Figure 3 TEM image of DCNP@IR808 of Example 1;

[0035] Figure 4 Zeta potential diagrams of DCNP@NOBF4, DCNP@PEP, DCNP@PEG and DCNP@IR808 in Example 1;

[0036] Figure 5 DLS images of DCNP@NOBF4, DCNP@PEP, DCNP@PEG and DCNP@IR808 in Example 1;

[0037] Figure 6 The fluorescence spectra of DCNP@NOBF4, DCNP@PEP, DCNP@PEG and DCNP@IR808 in Example 1 at 1550 nm under 808 nm excitation;

[0038] Figure 7 The fluorescence spectra of DCNP@NOBF4, DCNP@PEP, DCNP@PEG and DCNP@IR808 in Example 1 at 1550 nm under 980 nm excitation;

[0039] Figure 8 Statistical graph of fluorescence imaging ratio of DCNP@NOBF4, DCNP@PEP, DCNP@PEG and DCNP@IR808;

[0040] Figure 9 Fluorescence imaging ratio diagram of DCNP@NOBF4, DCNP@PEP, DCNP@PEG and DCNP@IR808;

[0041] Figure 10 This is a time-linear curve of the ratio signal value generated by the reaction of DCNP@IR808 with different concentrations of HNE enzyme. DETAILED DESCRIPTION

[0042] The following will be combined with the accompanying drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0043] In the following description of this embodiment, the terms "include", "comprising", "having" and "containing" are open-ended terms, meaning including but not limited to.

[0044] In the following description of this embodiment, the term "and / or" is used to describe the association relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, B exists alone, and both A and B exist. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0045] In the following description of this embodiment, the term "at least one" refers to one or more, and "plurality" refers to two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0046] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0047] Those skilled in the art should understand that in the following description of the embodiments of the present application, the order of serial numbers does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0048] It will be understood by those skilled in the art that the numerical ranges in the examples of the present application are to be understood as specifically disclosing each intermediate value between the upper and lower limits of the range. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also included in the present application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

[0049] Unless otherwise indicated, the technical / scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application belongs. Although this application describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0050] In a first aspect, the present application provides a method for preparing a near-infrared second-region ratiometric fluorescent probe, comprising:

[0051] S1, NaYF4:20%Yb,2%Er down-conversion nanoparticles were prepared by coprecipitation of yttrium acetate, ytterbium acetate and erbium acetate in a mixed solution of 1-octadecene and oleic acid, denoted as DCNP@core;

[0052] S2, DCNP@core was prepared by seed-mediated method to prepare NaYF4:20% Yb, 2% Er@NaYF4:30% Nd, which was recorded as core-shell DCNP;

[0053] S3, dispersing the core-shell structure DCNP in cyclohexane, adding NOBF4 to react, and obtaining DCNP@NOBF4;

[0054] In this application, core-shell DCNP and NOBF4 were ultrasonically treated in cyclohexane, where the mass ratio of the core-shell DCNP to NOBF4 was 1:(2-4). After standing and stratification, the supernatant cyclohexane was removed. A mixed coagulant was then added, and the residual solvent was removed by centrifugation at 12,000 x g. The precipitate was collected to obtain DCNP@NOBF4. The mixed coagulant was a mixture of toluene and cyclohexane in a volume ratio of 1:1.

[0055] S4, reacting DCNP@NOBF4 and peptide in DMF to obtain DCNP@PEP;

[0056] In the present application, the peptide is a neutrophil elastase-specific peptide, preferably a HNE enzyme-linked peptide.

[0057] Specifically, the DCNP@NOBF4 obtained in S3 and the peptide were reacted in DMF at 25° C. in the dark with stirring for 10 to 15 h at a stirring rate of 300 to 500 rpm.

[0058] S5, reacting DCNP@PEP and PEG in DMF to obtain DCNP@PEG;

[0059] Specifically, the DCNP@PEP prepared in S4 and PEG were reacted in DMF at 25° C. in the dark with stirring for 5 to 8 h at a stirring rate of 300 to 500 rpm.

[0060] After the polyethylene glycol (PEG)-carboxyl group is activated, the amino group of each elastase-specific peptide is effectively conjugated to the PEG-carboxyl group by forming a covalent amide bond.

[0061] S6, DCNP@PEG and IR808 are reacted in DMF, the reaction solution is centrifuged to collect the solid phase, and DCNP@IR808, a near-infrared second region ratiometric fluorescent probe, is obtained.

[0062] Specifically, the DCNP@PEG prepared in S5 and IR808 were reacted in DMF at 25° C. in the dark with stirring for 12 to 15 h at a stirring rate of 300 to 500 rpm.

[0063] In the present application, the molar ratio of DCNP@NOBF4, peptide, PEG and IR808 is 1:(1500-2500):(19-21):(17-19), and more preferably the molar ratio of DCNP@NOBF4, peptide, PEG and IR808 is 1:2000:20:18.

[0064] In this application, NaYF4:20% Yb, 2% Er down-conversion nanoparticles (i.e. DCNP@core) were prepared by co-precipitation. The specific method includes:

[0065] Yttrium acetate, ytterbium acetate, and erbium acetate are mixed in a mixed solution of 1-octadecene and oleic acid, and the mixture is heated to 70-80° C. under vacuum and kept warm. Then, the mixture is heated to 100° C. under an argon atmosphere and kept warm, and then heated to 150° C. and kept warm, and then cooled to room temperature to obtain a reaction solution.

[0066] A precipitant is added to a reaction solution, and the temperature is slowly raised to 45-50° C. and maintained, then heated to 65-70° C., vacuumed, and maintained; then heated to 100° C. and maintained, then heated to 300-310° C. and maintained, and then cooled to room temperature; solid particles are collected by centrifugation and washed to obtain NaYF4:20% Yb, 2% Er down-conversion nanoparticles; the precipitant is a mixture of ammonium fluoride methanol solution and sodium hydroxide methanol solution; wherein the molar ratio of ammonium fluoride to sodium hydroxide is 16:1.

[0067] In this application, DCNP@core was prepared by a seed-mediated method to prepare NaYF4:20% Yb, 2% Er@NaYF4:30% Nd (i.e., core-shell structure DCNP), specifically comprising:

[0068] Yttrium acetate and neodymium acetate are mixed in a mixed solution of 1-octadecene and oleic acid, and the mixture is gradually heated to 150° C. under an argon atmosphere, and then cooled to room temperature to obtain a reaction solution;

[0069] The DCNP@core prepared in step S1, a methanol solution of NH4F and a methanol solution of sodium hydroxide were added to the reaction solution, and the mixture was heated to 300-310°C for insulation, and then cooled to room temperature, centrifuged and washed to obtain a core-shell structure DCNP.

[0070] The near-infrared second-zone ratio fluorescent probe prepared in this application reaches the site of lung inflammation by inhalation administration. The substrate peptide will be cut off by the HNE enzyme overexpressed by neutrophils, and IR808 will detach from the probe, losing the sensitization effect. Under 808nm laser excitation, the NIR-II fluorescence signal (1550nm) is weakened. At the same time, the NIR-II fluorescence signal (1550nm, 980nm excitation) remains unchanged as a reference signal. The content of HNE enzyme can be accurately quantified by the ratio reaction signal (ratio = F1550Em, 808Ex / F1550Em, 980Ex).

[0071] The proposed near-infrared second-zone ratiometric fluorescent probe eliminates errors caused by varying attenuation coefficients within tissues and instrumental errors, accurately quantifying the amount of HNE enzyme in lung tissue. The proposed near-infrared second-zone ratiometric fluorescent probe can be used to detect HNE enzyme and assess the severity of lung inflammation based on its expression, thereby enabling real-time, accurate grading of lung inflammation and guiding treatment.

[0072] The present application is further described below through specific examples.

[0073] The peptide in the examples was purchased from Wuhan Dangang Biotechnology Co., Ltd. with the serial number QPMAVVQSVPQK(Ac) and the serial number S-8861.

[0074] Example 1

[0075] S1, 0.78mmol of yttrium acetate, 0.2mmol of ytterbium acetate and 0.02mmol of erbium acetate were added to a flask (100mL) containing a mixed solution of 17mmol of OA and 4mmol of ODE, and the temperature was slowly raised to 80℃ under continuous stirring. When the temperature reached 70℃, vacuum was maintained for 20 minutes, and then the temperature was raised to 100℃ under argon atmosphere and stirred for 20 minutes. Then, the temperature was slowly heated to 150℃ and stirred for 40 minutes, and then cooled to room temperature to obtain a reaction solution. 10mL of 4mmol mL –1 2.5 mL of ammonium fluoride methanol solution with a concentration of 1 mmol mL –1 The mixture was mixed with a methanol solution of sodium hydroxide and added to the rapidly stirred reaction solution. The mixture was slowly heated to 47.5°C and kept warm for 35 minutes. The mixture was then heated to 70°C and kept in vacuum for 20 minutes. The mixture was then slowly heated to 100°C and kept warm for 30 minutes. The mixture was then heated to 305°C and stirred for 90 minutes, and then cooled to room temperature. The reaction solution was centrifuged at 8000 rpm for 10 minutes to collect the solid phase, and NaYF4:20% Yb, 2% Er down-conversion nanoparticles were obtained, which were designated as DCNP@core. The reaction mixture was purified by cyclohexane and ethanol (V 环己烷 :V 乙醇=1:3) for three times, and then dispersed in 10 mL of cyclohexane for later use.

[0076] S2, 0.7 mmol of yttrium acetate and 0.3 mmol of neodymium acetate were added to a flask containing a mixed solution of 17 mmol of OA and 42 mmol of ODE, and the mixed solution was stirred at a constant speed for reaction.

[0077] The reaction system was slowly heated to 100°C and held at 100°C for 25 minutes under an argon atmosphere. The temperature was then slowly increased to 150°C and held under argon for 40 minutes. The reaction mixture was then cooled to room temperature. The DCNP@core from step S1, 12.5 mL of a 4 mmol methanolic solution of NH4F, and 1 mmol of NaOH were then added. The reaction system was then slowly heated to 47.5°C and held for 35 minutes. Furthermore, the reaction mixture was gradually heated to 306°C under argon and held for 90 minutes before being cooled to room temperature. The core-shell DCNPs were obtained by centrifugation and washed twice with cyclohexane and anhydrous ethanol. The resulting mixture was then dispersed in 10 mL of cyclohexane to a quantitative concentration of 20 mg / mL.

[0078] In step S3, the core-shell DCNPs were mixed with NOBF4 at a mass ratio of 1:3 and sonicated for 10 minutes. After stratification, the cyclohexane in the supernatant was removed. A coagulant mixture of toluene and cyclohexane at a volume ratio of 1:1 was then added. The mixture was centrifuged twice at 12,000 x g to remove residual solvent, yielding a precipitate, DCNP@NOBF4.

[0079] S4, 2 mg of DCNP@NOBF4 and 0.18 mg of peptide were dissolved in 2 mL of DMF and stirred at 300 rpm at 25 °C for 12 h in the dark to obtain a DCNP@PEP solution;

[0080] In step S5, 1 mg of PEG was dissolved in 2 mL of DMF to prepare a solution. 53.3 μL of this solution was added to the DCNP@PEP solution obtained in step S4. The mixture was stirred at 300 rpm at 25°C in the dark for 6 h to obtain a DCNP@PEG solution.

[0081] S6. Weigh 2 mg of IR808 and dissolve it in 6 mL of DMF to prepare a solution. Take 2.7 μL of this solution and add it to the DCNP@PEG solution obtained in S5. Stir in the dark at 300 rpm at 25°C for 12 h to obtain a DCNP@IR808 solution. Then centrifuge at 20,000 x g for 18 min and disperse in 100 μL of UP water to obtain DCNP@IR808.

[0082] Example 2

[0083] The difference between Example 2 and Example 1 is that the amount of peptide used is 0.36 mg, and the rest is the same as Example 1.

[0084] Example 3

[0085] The difference between Example 3 and Example 1 is that in step S5, the amount of PEG added is 0.0534 mg, and the rest is the same as Example 1.

[0086] Example 4

[0087] The difference between Example 4 and Example 1 is that in step S6, the amount of IR808 added is 0.0018 mg, and the rest is the same as Example 1.

[0088] The product and intermediate product of Example 1 were tested by TEM images for morphology evaluation.

[0089] Figure 1 TEM image of DCNP@core of Example 1, Figure 1 It can be seen that the nanoparticles are in a uniform state, proving that the synthesis is successful.

[0090] Figure 2 TEM image of the core-shell structure DCNP of Example 1, Figure 2 It can be seen that the prepared core-shell DCNP is in a uniform state, proving that the synthesis is successful.

[0091] Figure 3 TEM image of DCNP@IR808 of Example 1. Figure 3 It can be seen that IR808 is evenly coated on the surface of the nanoparticles, indicating that DCNP@IR808 is successfully prepared.

[0092] The Zeta potential diagrams of DCNP@NOBF4, DCNP@PEP, DCNP@PEG and DCNP@IR808 in Example 1 were tested. The test results are shown in FIG. Figure 4 As shown. Figure 4 It can be seen that the neutrophil elastase-specific peptide was successfully grafted onto DCNP.

[0093] The DLS images of DCNP@NOBF4, DCNP@PEP, DCNP@PEG and DCNP@IR808 in Example 1 are shown in FIG. Figure 5 As shown. Figure 5 It can be seen that the hydrodynamic diameter of the nanoparticles measured by DLS increases accordingly, and the hydrodynamic diameter of the prepared probe nanoparticles DCNP@IR808 is about 100 nm, which is conducive to cellular uptake and inhalation administration.

[0094] The fluorescence spectra of DCNP@NOBF4, DCNP@PEP, DCNP@PEG and DCNP@IR808 in Example 1 are shown in the figure below: Figure 6 As shown in the figure, the fluorescence spectrum at 1550nm under 980nm excitation is as follows Figure 7 shown.

[0095] from Figure 6 and Figure 7 It can be seen that when irradiated with 808nm laser, the FL signal of the nanoprobe DCNP@IR808 at 1550nm increased significantly compared with the control DCNP@PEG. This is due to the successful sensitization of DCNP by IR808 as a dye antenna molecule. However, when irradiated with 980nm laser, the NIR-II FL signal at 1550nm showed no change.

[0096] The fluorescence imaging ratio statistics and fluorescence imaging ratio diagrams of DCNP@NOBF4, DCNP@PEP, DCNP@PEG and DCNP@IR808 are shown in Figure 2 . Figure 8 and Figure 9 shown.

[0097] from Figure 8 It can be clearly seen that the fluorescence ratio value increased significantly after the IR808 was injected. Figure 9 It can be seen that under irradiation with 808 nm laser, the NIR-II FL signal at 1550 nm gradually becomes brighter, while after irradiation with 980 nm laser, the NIR-II FL signal at 1550 nm remains basically unchanged.

[0098] The near-infrared second-region ratiometric fluorescent probe DCNP@IR808 prepared in Example 1 was reacted with different concentrations of HNE enzyme for 240 min, and the generated ratiometric signal value was detected; the linear curve was as follows: Figure 10 shown.

[0099] from Figure 10 DCNP@IR808 exhibited a ratiometric response to HNE enzyme in the range of 0.2 to 1.2 μg / mL, with a coefficient of determination of 0.91. The ratio values ​​corresponding to different HNE enzyme concentrations at 240 minutes were selected to demonstrate the feasibility of quantitative HNE detection using a standard curve.

[0100] Although this specification has been used to fully describe the present application using general descriptions and specific embodiments, it is obvious to those skilled in the art that modifications or improvements may be made based on the present application. Therefore, such modifications or improvements made without departing from the spirit of the present application are within the scope of protection claimed in this application.

Claims

1. A method for preparing a near-infrared second-region ratiometric fluorescent probe, characterized in that: include: S1, NaYF4:20%Yb,2%Er down-conversion nanoparticles were prepared by coprecipitation of yttrium acetate, ytterbium acetate and erbium acetate in a mixed solution of 1-octadecene and oleic acid, denoted as DCNP@core; S2, DCNP@core was prepared by seed-mediated method to prepare NaYF4:20% Yb, 2% Er@NaYF4:30% Nd, which was recorded as core-shell DCNP; S3, dispersing the core-shell structure DCNP in cyclohexane, adding NOBF4 to react, and obtaining DCNP@NOBF4; S4, reacting DCNP@NOBF4 and peptide in DMF to obtain DCNP@PEP, wherein the peptide is a neutrophil elastase-specific peptide; S5, reacting DCNP@PEP and PEG in DMF to obtain DCNP@PEG; S6, DCNP@PEG and IR808 are reacted in DMF, the reaction solution is centrifuged to collect the solid phase, and DCNP@IR808, a near-infrared second region ratiometric fluorescent probe, is obtained.

2. The preparation method according to claim 1, characterized in that In step S3, the core-shell structure DCNP and NOBF4 are ultrasonically treated in cyclohexane, and the supernatant is removed after standing and stratification; Then, a mixed coagulant is added and the precipitate is collected by centrifugation to obtain DCNP@NOBF4.

3. The preparation method according to claim 2, characterized in that The mass ratio of the core-shell structure DCNP to NOBF4 is 1:(2-4).

4. The preparation method according to claim 2, characterized in that The mixed coagulant is a mixture of toluene and cyclohexane in a volume ratio of 1:

1.

5. The preparation method according to claim 1, characterized in that In step S4, DCNP@NOBF4 and peptide are reacted in DMF at 25°C in the dark with stirring for 10-15 h at a stirring rate of 300-500 rpm; In step S5, DCNP@PEP and PEG are reacted in DMF at 25° C. in the dark with stirring for 5 to 8 h at a stirring rate of 300 to 500 rpm; In step S6, DCNP@PEG and IR808 were reacted in DMF at 25° C. in the dark with stirring for 12 to 15 h at a stirring rate of 300 to 500 rpm.

6. The preparation method according to claim 1, characterized in that The molar ratio of the DCNP@NOBF4, peptide, PEG and IR808 is 1:(1500-2500):(19-21):(17-19).

7. The preparation method according to claim 1, characterized in that In step S1, yttrium acetate, ytterbium acetate, and erbium acetate are mixed in a mixed solution of 1-octadecene and oleic acid, and then heated to 70-80° C. under vacuum and kept warm. Then, the mixture is heated to 100° C. and kept warm under an argon atmosphere, and then heated to 150° C. and kept warm, and then cooled to room temperature to obtain a reaction solution. The precipitant is added to the reaction solution, and the temperature is slowly raised to 45-50° C. and maintained, then heated to 65-70° C., evacuated, and maintained; then heated to 100° C. and maintained, then heated to 300-310° C. and maintained, and then cooled to room temperature; solid particles are collected by centrifugation and washed to obtain NaYF4:20% Yb, 2% Er down-conversion nanoparticles; The precipitant is a mixture of ammonium fluoride methanol solution and sodium hydroxide methanol solution; wherein the molar ratio of ammonium fluoride to sodium hydroxide is 16:

1.

8. The preparation method according to claim 1, characterized in that In step S2, yttrium acetate and neodymium acetate are mixed in a mixed solution of 1-octadecene and oleic acid, and then gradually heated to 150° C. under an argon atmosphere, and then cooled to room temperature to obtain a reaction solution; The DCNP@core prepared in step S1, a methanol solution of NH4F and a methanol solution of sodium hydroxide were added to the reaction solution, and the mixture was heated to 300-310°C for insulation, and then cooled to room temperature, centrifuged and washed to obtain a core-shell structure DCNP.

9. A near-infrared second-region ratiometric fluorescent probe prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the near-infrared second-region ratiometric fluorescent probe according to claim 9 in detecting HNE enzyme.

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