Preparation method of near-infrared two-region ratiometric fluorescent probe for quantitatively detecting ROS (reactive oxygen species)

Abstract

The invention discloses a preparation method of a near-infrared two-region ratiometric fluorescent probe for quantitatively detecting ROS (reactive oxygen species). The preparation method comprises the following steps: 1) preparing NaYF4: 20% Yb and 2% Er DCNPs by coprecipitation of lanthanum acetate hydrate, ODE and OA; 2) based on NaYF4: 20% Yb, 2% Er DCNPs, preparing NaYF4: 20% Yb, 2% Er-coated NaYF4: 30% Nd core-shell DCNP through a seed-mediated core-shell growth method, and preparing NaYF4: 20% Yb, 2% Er-coated NaYF4: 30% Nd core-shell DCNP through a seed-mediated core-shell growth method; 3) azobenzene-4, 4 '-dicarboxylic acid, EDC, NHS and DSPE-PEG-2000 are dissolved in DMF for a reaction, after resuspension, a product is dissolved in DMF, EDC, NHS and cysteamine are added for a reaction, and after the product is resuspended again, modified DSPE-PEG is obtained; 4) dispersing the core-shell DCNP and a dye in dichloromethane, adding the modified DSPE-PEG for reaction, and performing rotary evaporation, water adding centrifugation and cleaning on a product to obtain a DCNP dye; (5) adding chloroauric acid into a mixed solution of tetrakis (hydroxymethyl) phosphonium chloride and NaOH under high-speed stirring, and stirring to obtain gold seeds; and reacting the gold seed with the DCNP dye. The ratiometric fluorescent probe prepared by the invention is provided with a reference, so that errors can be effectively eliminated, and accurate quantitative imaging of target molecules is realized.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescence imaging, and in particular to a method for preparing a near-infrared two-zone ratio fluorescence probe for quantitatively detecting ROS. Background Art

[0002] Radiotherapy is the main treatment for many cancers and kills cancer cells by producing reactive oxygen species (ROS). However, within the range of radiation doses tolerated by normal tissues, the current average cure rate for tumors is less than 40%. Reducing the radiation dose or increasing the radiation time will cause radiation resistance in tumor cells, and excessively high doses will increase the toxic side effects on normal cells. At the same time, due to the hypoxic microenvironment inside the tumor, tumor cells will resist radiation and be difficult to kill, eventually causing metastasis or recurrence of tumor cells, leading to radiotherapy failure. In order to solve the problem of radiotherapy resistance, gold nanoparticles of high atomic number elements are introduced as radiotherapy sensitizers. Since gold has a high photoelectric absorption cross-section and a strong absorption capacity for X-rays, it is introduced into tumor tissues. After being irradiated by high-energy rays, secondary electrons such as photoelectrons, Auger electrons, and Compton electrons are released, which can react with water to increase the production of ROS free radicals, causing indirect damage to DNA by attacking bases, ribose, oligonucleotides, etc., or direct damage to biomembranes and proteins by lipid peroxidation.

[0003] Real-time monitoring of ROS is an important prerequisite for in-depth elucidation of pathogenic mechanisms and early warning and diagnosis of diseases. There is an urgent need for in vivo detection and therapeutic effects of ROS. However, most of the current probes are single signal imaging and monitoring of ROS responses, which are easily interfered by non-target factors and mainly provide qualitative imaging information, making it difficult to accurately quantify ROS.

[0004] The advantages of fluorescence imaging, including non-invasiveness, real-time feedback capability, and high sensitivity, have been widely used for in vivo monitoring of bioactive molecules. Among them, the second near-infrared (NIR-II, 900-1700nm) FL imaging technology, especially the emission distribution in the tissue-transparent NIR-II spectral window (1000-1350nm and 1500-1700nm), shows a significantly higher signal-to-background ratio (SBR) compared with the near-infrared region I (NIR-I, 650-900nm).

[0005] Therefore, in order to address the problems that current in vivo fluorescence imaging is mainly qualitative and difficult to accurately quantify ROS in vivo, the present invention constructs a ROS-specific activated ratiometric NIR-II fluorescence imaging probe, and realizes the qualitative and quantification of ROS through NIR-II ratiometric fluorescence imaging. Summary of the invention

[0006] In view of the above problems, the purpose of the present invention is to provide a method for preparing a near-infrared II ratiometric fluorescent probe for quantitative detection of ROS. By connecting fluorescent small molecules as sensitizing antennas to down-conversion nanoparticles (DCNPs), and connecting AuNPs with ROS-responsive small molecules, a ROS-responsive NIR-II ratiometric fluorescent imaging diagnostic and therapeutic probe DCNP@IR-Au is designed for real-time in vivo imaging of ROS under radiosensitization therapy and early evaluation of radiotherapy effects.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] The present invention provides a method for preparing a near-infrared second-zone ratiometric fluorescent probe for quantitatively detecting ROS, comprising the following steps:

[0009] 1) NaYF4:20%Yb,2%Er DCNPs were prepared by co-precipitation of lanthanum acetate hydrate with ODE and OA;

[0010] 2) Based on NaYF4:20%Yb,2%Er DCNPs, NaYF4:20%Yb,2%Er@NaYF4:30%Nd core-shell DCNPs were prepared by a seed-mediated core-shell growth method;

[0011] 3) azobenzene-4,4'-dicarboxylic acid, EDC, NHS, and DSPE-PEG-2000 are dissolved in DMF for reaction, the reaction solution is resuspended, the product is dissolved in DMF, and EDC, NHS, and cysteamine are added for reaction, and the obtained reaction product is resuspended again to obtain the modified DSPE-PEG;

[0012] 4) dispersing the core-shell DCNP and the dye obtained in step 2) in dichloromethane, and then adding the modified DSPE-PEG to react, and the reaction product is subjected to rotary evaporation, centrifugation with water, and washing to obtain DCNP@dye;

[0013] 5) adding chloroauric acid to a mixed solution of tetrakis(hydroxymethyl)phosphonium chloride and NaOH under high-speed stirring, and then continuing to stir to obtain a gold seed; reacting the gold seed with the DCNP@dye, and dispersing the reaction product in deionized water after centrifugation and washing.

[0014] As a possible implementation, further, step 1) specifically includes the following steps:

[0015] 1.1) 0.78 mmol Y(AC)3, 0.2 mmol Yb(AC)3 and 0.02 mmol Er(AC)3 were added to a mixed solution containing 17 mmol OA and 42 mmol ODE;

[0016] 1.2) The mixed solution was slowly heated to 80° C. at 2° C. / min under continuous stirring, and maintained in vacuum for 20 minutes when the temperature reached 70° C., and then stirred continuously at 100° C. for 20 minutes under an argon atmosphere. Finally, the mixture was slowly heated to 150° C. and stirred for 40 minutes, and then cooled to room temperature to obtain a reaction solution I;

[0017] 1.3) 10 mL of 4 mmol / mL methanol solution of ammonium fluoride and 2.5 mL of 1 mmol / mL methanol solution of sodium hydroxide were mixed and added to the reaction solution I which was rapidly stirred at 700 rpm, and then the mixed solution was slowly heated at 0.6°C / min to 47.5°C and kept for 35 minutes, and when the temperature reached 70°C, vacuum was maintained for 20 minutes, and then slowly heated to 100°C and kept for 30 minutes, and then stirred at 298°C for 90 minutes and then cooled to room temperature;

[0018] 1.4) The reaction product was centrifuged at 6000 rpm for 15 minutes to collect the obtained nanoparticles and add ethanol, then 环己烷 :V 乙醇 The resulting mixture was washed three times with a mixed solution of cyclohexane and ethanol in a ratio of 3:1 and then dispersed in 10 mL of cyclohexane.

[0019] As a possible implementation, further, step 2) specifically includes the following steps:

[0020] 2.1) Add 0.7 mmol Y(AC)3 and 0.3 mmol Nd(AC)3 to a mixed solution containing 17 mmol OA and 42 mmol ODE;

[0021] 2.2) stirring the mixed solution at a constant speed of 700 rpm, and slowly heating the reaction system at 4°C / min, and keeping it under an argon atmosphere for 25 minutes when the temperature reaches 100°C, then slowly heating it to 150°C and keeping it under an argon atmosphere for 40 minutes, and then cooling it to room temperature to obtain a reaction solution II;

[0022] 2.3) The synthesized NaYF4:20% Yb, 2% Er DCNPs nanoparticles and 4mmol NH4F methanol solution were added to the reaction solution II, and then 1mmol NaOH was added. Then, the reaction system was slowly heated to 47.5℃ at 0.6℃ / min and kept for 35 minutes, and then gradually heated to 298℃ under argon atmosphere and kept for 90 minutes, and then cooled to room temperature;

[0023] 2.4) The reaction product cooled to room temperature was centrifuged to obtain core-shell DCNPs, which were then washed twice with cyclohexane and anhydrous ethanol and dispersed in 10 mL of cyclohexane.

[0024] As a possible implementation, further, step 3) specifically includes the following steps:

[0025] 3.1) Dissolve azobenzene-4,4'-dicarboxylic acid, EDC, NHS, and DSPE-PEG-2000 in DMF at a molar ratio of 1:1.2:1.2:1 and react overnight;

[0026] 3.2) The reaction solution was resuspended in 10-fold anhydrous ether and centrifuged twice at 2000 r / min, each time for 5 min;

[0027] 3.3) The obtained product was dissolved in 1 mL of DMF and EDC, NHS and 0.03 mol of cysteamine were added to react overnight; wherein the amount of EDC and NHS added was the same as in step 3.1);

[0028] 3.4) The reaction solution was resuspended in ten times anhydrous ether and centrifuged twice at 2000 rpm, each time for 5 min, to obtain the modified DSPE-PEG.

[0029] As a possible implementation, further, step 4) specifically includes the following steps:

[0030] 4.1) Disperse the core-shell DCNP and the dye in dichloromethane and stir evenly, then add the modified DSPE-PEG to react;

[0031] 4.2) The dichloromethane was dried by rotary evaporation at 100 rpm and 30° C., and then water was added and centrifuged at 7800 rpm for 7 minutes to collect the product, which was then washed twice with deionized water to obtain DCNP@dye.

[0032] As a possible implementation manner, further, the dye is selected from IR-808 or other organic or inorganic materials that are excited by 808nm laser and whose fluorescence emission peak is tailed to 980nm.

[0033] As a possible implementation manner, further, when the dye is IR-808, step 4) is specifically as follows:

[0034] 4.1) Disperse 100 μL 35 mg / mL core-shell DCNP and 655 μL 0.1 mg / mL IR808 in dichloromethane and stir evenly, then add 30 mg modified DSPE-PEG to react;

[0035] 4.2) The dichloromethane was dried by rotary evaporation at 100 rpm and 30° C., and then water was added and centrifuged at 7800 rpm for 7 minutes to collect the product, which was then washed twice with deionized water to obtain DCNP@IR.

[0036] As a possible implementation, further, step 5) specifically includes the following steps:

[0037] 5.1) 12 μL of tetrakis(hydroxymethyl)phosphonium chloride solution and 1M NaOH solution were mixed and stirred evenly, and then 1 mL of 20 mg / mL chloroauric acid was quickly added to the mixture solution under high-speed stirring at 400 rpm and continued to stir to obtain a gold seed, and the obtained gold seed was placed at room temperature;

[0038] 5.2) DCNP@IR and gold seed were mixed at a mass ratio of 1:10 and reacted for 6 hours. The product DCNP@IR-Au was collected by centrifugation at 7800 rpm for 7 minutes, then washed twice with deionized water and dispersed in deionized water.

[0039] The spatial distance between DCNP and Au in the near-infrared second-zone ratiometric fluorescent probe DCNP@IR-Au for quantitative detection of ROS prepared according to the above preparation method is less than 10 nm;

[0040] The probe emits fluorescence (FL) at 1550nm under 980nm laser excitation. 1550,980Ex ) emission is strong, under 808nm laser excitation, the fluorescence (FL 1550,808Ex ) emission is weaker.

[0041] The present invention innovatively combines the advantages of low background noise signal of NIR-II light in human tissue, non-invasive, rapid and real-time imaging of fluorescence imaging, and ratio probes that can eliminate the limitations of traditional optical probes. The reference signal and the response signal are designed to be the same wavelength, and both are located in NIR-II with high selectivity, high sensitivity, and accurate quantification of target molecules. Real-time imaging and absolute quantification of ROS in vivo are achieved, and early evaluation of radiotherapy efficacy is achieved by means of the correlation mechanism between in vivo imaging information and the amount of ROS.

[0042] Secondly, the present invention proposes for the first time a ROS-responsive NIR-II ratio fluorescence imaging method, which uses the changes in the NIR-II ratio fluorescence signal to monitor in real time the generation and distribution of ROS under tumor radiotherapy sensitization. By utilizing the advantages of NIR-II ratio fluorescence imaging, diagnostic and therapeutic drug molecules based on NIR-II ratio probes are designed, and a precise radiotherapy staging and efficacy evaluation system based on in vivo optical imaging is established, which shortens the detection time and enables early prediction of efficacy, so as to timely adjust treatment strategies, reduce side effects, and improve efficacy, making the new in vivo imaging technology more suitable for human application.

[0043] The beneficial effects of the present invention are as follows:

[0044] 1) Compared with traditional fluorescent probes, the ratio fluorescent probe prepared by the present invention can eliminate false positives of probes, errors caused by probe concentration per unit area and some instrument errors due to its own reference, thus achieving accurate quantitative imaging of target molecules. Compared with near-infrared imaging in the first region (NIR-I), NIR-II imaging has a higher signal-to-noise ratio and higher imaging resolution.

[0045] 2) The response molecules in the ratiometric fluorescent probe designed in the present invention can be flexibly replaced according to the target substance. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0047] Figure 1 TEM image of DCNPs;

[0048] Figure 2 TEM image of DCNP@shell;

[0049] Figure 3 TEM image of DCNP@IR;

[0050] Figure 4 TEM image of DCNP@IR-Au;

[0051] Figure 5 The fluorescence emission peaks of DCNP@IR and DCNP@IR-Au at 1550nm are excited by 980nm and 808nm lasers;

[0052] Figure 6 is the absorption range of DCNP, DCNP@IR, Au seed, and DCNP@IR-Au;

[0053] Figure 7 DCNP@IR-Au was treated with 100 μM Fe 2+ , 808nm laser and 980nm laser excited fluorescence spectra after 25μM H2O2 treatment;

[0054] Figure 8 The emission changes at 1550nm and FL under 808 excitation, 980 excitation and ·OH concentration range of 0-60μM Ex808,1550 / FL Ex980,1550 The ratio and ·OH concentration ranged from 0 to 60 μM. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] See attached Figure 1 As shown, this embodiment provides a method for preparing a near-infrared second-zone ratiometric fluorescent probe for quantitatively detecting ROS, comprising the following steps:

[0057] 1) Synthesis of NaYF4:20%Yb,2%Er DCNPs: NaYF4:20%Yb,2%Er DCNPs were prepared by co-precipitation of lanthanum acetate hydrate with ODE and OA, specifically comprising the following steps:

[0058] 0.78mmol Y(AC)3, 0.2mmol Yb(AC)3 and 0.02mmol Er(AC)3 were added to a flask (100mL) containing a mixed solution of OA (17mmol) and ODE (42mmol). The mixed solution was slowly heated to 80°C at 2°C / min under continuous stirring, and maintained in vacuum for 20 minutes when it reached 70°C, and then stirred continuously at 100°C for 20 minutes under an argon atmosphere. Finally, the mixture was slowly heated to 150°C and stirred for 40 minutes, and then cooled to room temperature to obtain a reaction solution I. Subsequently, 10mL of ammonium fluoride methanol solution (4mmol / mL) and 2.5mL of sodium hydroxide methanol solution (1mmol / mL) were mixed and added to the reaction solution I which was rapidly stirred at 700rpm. The mixed solution was then slowly heated at 0.6°C / min to 47.5°C for 35 minutes, maintained in vacuum for 20 minutes at 70°C, slowly heated to 100°C for 30 minutes, and then stirred at 298°C for 90 minutes before cooling to room temperature. The reaction product was centrifuged at 6000 rpm for 15 minutes to collect the resulting nanoparticles and add ethanol, followed by cyclohexane and ethanol (V 环己烷 :V 乙醇 =3:1) mixed solution was washed three times and then dispersed in 10 mL cyclohexane for later use. The TEM of the prepared DCNPs is shown in the attached figure. Figure 1 shown.

[0059] 2) Synthesis of NaYF4:20%Yb,2%Er@NaYF4:30%Nd core-shell DCNPs: Based on NaYF4:20%Yb,2%ErDCNPs, NaYF4:20%Yb,2%Er@NaYF4:30%Nd core-shell DCNPs were prepared by a seed-mediated core-shell growth method, which specifically includes the following steps:

[0060] 0.7mmol Y(AC)3 and 0.3mmol Nd(AC)3 were added to a flask (100mL) containing a mixed solution of OA (17mmol) and ODE (42mmol). The mixed solution was stirred at a constant speed of 700rpm throughout the reaction. The reaction system was slowly heated at 4℃ / min, and when it was heated to 100℃, it was kept in an argon atmosphere for 25 minutes, then slowly heated to 150℃ and kept in an argon atmosphere for 40 minutes, and then cooled to room temperature to obtain reaction solution II; the synthesized NaYF4:20%Yb,2%Er DCNPs nanoparticles were added to the reaction solution II together with a methanol solution (12.5mL) of NH4F (4mmol), and then NaOH (1mmol) was added, and then the reaction system was slowly heated to 47.5℃ at 0.6℃ / min and kept for 35 minutes, and then gradually heated to 298℃ in an argon atmosphere and kept for 90 minutes, and then cooled to room temperature. The reaction product was centrifuged to obtain core-shell DCNP (denoted as DCNP@shell), which was then washed twice with cyclohexane and anhydrous ethanol and dispersed in 10 mL of cyclohexane. TEM images of the obtained core-shell DCNP are shown in the attached figure. Figure 2 shown.

[0061] 3) Preparation of ROS response molecules: azobenzene-4,4'-dicarboxylic acid, EDC, NHS, and DSPE-PEG-2000 are dissolved in DMF for reaction, the reaction solution is resuspended, the product is dissolved in DMF, and EDC, NHS and cysteamine are added for reaction, and the obtained reaction product is resuspended again to obtain the modified DSPE-PEG, which specifically comprises the following steps:

[0062] 3.1) Dissolve azobenzene-4,4'-dicarboxylic acid, EDC, NHS, and DSPE-PEG-2000 in DMF at a molar ratio of 1:1.2:1.2:1 and react overnight;

[0063] 3.2) The reaction solution was resuspended in 10-fold anhydrous ether and centrifuged twice at 2000 r / min, each time for 5 min;

[0064] 3.3) The obtained product was dissolved in 1 mL of DMF and EDC, NHS and 0.03 mol of cysteamine were added to react overnight; wherein the amount of EDC and NHS added was the same as in step 3.1);

[0065] 3.4) The reaction solution was resuspended in ten times anhydrous ether and centrifuged twice at 2000 rpm, each time for 5 min, to obtain the modified DSPE-PEG.

[0066] 4) Synthesis of DCNP@dye: The core-shell DCNP and the dye prepared in step 2) are dispersed in dichloromethane, and then the modified DSPE-PEG is added to react. The reaction product is subjected to rotary evaporation, centrifugation with water, and washing to obtain DCNP@dye, which specifically comprises the following steps:

[0067] 4.1) Disperse the core-shell DCNP and the dye in dichloromethane and stir evenly, then add the modified DSPE-PEG to react;

[0068] 4.2) The dichloromethane was dried by rotary evaporation at 100 rpm and 30° C., and then water was added and centrifuged at 7800 rpm for 7 minutes to collect the product, which was then washed twice with deionized water to obtain DCNP@dye dispersion.

[0069] The dye may be IR-808, or other organic or inorganic materials excited by 808 nm laser, whose fluorescence emission peak can be tailed to 980 nm.

[0070] In this embodiment, the dye is IR-808, and the synthesis method of DCNP@IR specifically includes the following steps:

[0071] 100 μL of core-shell DCNP (35 mg / mL) and 655 μL of IR808 (0.1 mg / mL) were dispersed in dichloromethane and stirred evenly, and then 30 mg of modified DSPE-PEG was added to react for 12 h. The dichloromethane was dried by rotary evaporation (100 rpm, 30 ° C), and then water was added and centrifuged at 7800 rpm for 7 minutes to collect the product, which was then washed twice with deionized water to obtain DCNP@IR. The TEM of the prepared DCNP@IR is shown in the attached figure. Figure 3 shown.

[0072] 5) Synthesis of DCNP@IR-Au: HAuCl4 is added to a mixed solution of tetrakis(hydroxymethyl)phosphonium chloride (THPC) and NaOH under high-speed stirring, and then the mixture is stirred to obtain a gold seed; the gold seed is reacted with the DCNP@dye, and the reaction product is dispersed in deionized water after centrifugation and washing, specifically comprising the following steps:

[0073] 12 μL of tetrakis(hydroxymethyl)phosphonium chloride solution and 1M NaOH solution were mixed and stirred evenly, and then 1 mL of chloroauric acid (20 mg / mL) was quickly added to the mixture solution under high-speed stirring at 400 rpm, and stirring was continued for 12 hours to obtain a gold seed, and the obtained gold seed was placed at room temperature. Subsequently, DCNP@IR and the gold seed were mixed at a mass ratio of 1:10 and reacted for 6 hours. The product DCNP@IR-Au was collected by centrifugation at 7800 rpm for 7 minutes, and then washed twice with deionized water and dispersed in deionized water. The TEM of the prepared DCNP@IR-Au is shown in the attached figure. Figure 4 shown.

[0074] The probe DCNP@IR-Au is composed of a substrate that can be cut off in response to ROS molecules, connecting DCNP and Au. The spatial distance between DCNP and Au is less than 10nm, which satisfies the precursor conditions of the non-radiative energy resonance transfer mechanism. The probe has a fluorescence (FL) of 1550nm under 980nm laser excitation. 1550,980Ex ) emission is strong, under 808nm laser excitation, the fluorescence (FL 1550,808Ex ) emission is weaker.

[0075] See attached Figure 5 As shown, after DCNP@IR was connected to the gold seed, the fluorescence energy resonance transfer caused the fluorescence at 1550nm excited by 808nm to decrease, while 980 remained unchanged, which proved the successful synthesis of DCNP@IR-Au material.

[0076] See attached Figure 6 As shown, the absorption of DCNP@IR at around 800nm ​​disappears after the gold seed is attached, which proves the successful synthesis of DCNP@IR-Au material.

[0077] The substrate that responds to ROS molecules in the probe DCNP@IR-Au is an organic molecule that connects azobenzene-4,4'-dicarboxylic acid with DSPE-PEG2000 and cysteamine through an amidation reaction. Azobenzene-4,4'-dicarboxylic acid can be destroyed by hydroxyl radicals (OH·), resulting in the cleavage of the nitrogen-nitrogen double bond.

[0078] When the probe is in the initial state, due to the FRET effect, part of the energy of the fluorescent small molecule after being excited by the 808nm laser will be transferred to the AuNPs for quenching, making the fluorescence emission peak of DCNP@IR-Au at 1550nm under 808nm laser excitation (FL 1550,808Ex Once the solution is exposed to X-rays, the ROS generated by the irradiation will cause the ROS response small molecules on the probe to cleave, releasing AuNPs, and DCNPs will be sensitized by the fluorescent small molecules as sensitizing antennas again, making FL 1550,808ExAs a response signal, it will gradually increase. The fluorescence signal (FL 1550,980Ex ) does not change and can be used as a reference signal.

[0079] See attached Figure 7 As shown, after treatment with hydroxyl radicals, the fluorescence at 1550 nm excited by 808 nm was restored, and 980 remained unchanged as an internal standard, which proved that DCNP@IR-Au could be responded to by hydroxyl radicals, and ·OH could destroy the response bond azobenzene dicarboxylic acid, causing the gold seed to fall off.

[0080] The ratio probe DCNP@IR-Au satisfies the requirement that after responding to the target substance, the molecule connecting DCNP and Au breaks, and Au moves away from DCNP. At this time, the probe emits fluorescence (FL) at 1550nm under 808nm laser excitation. 1550,808Ex ) becomes stronger, and under 980nm laser excitation, the fluorescence (FL 1550,980Ex ) emission remains unchanged. The ratio of the two is FL 1550,980Ex / FL 1550,808Ex Become smaller.

[0081] The probe DCNP@IR-Au prepared by the present invention generates a large amount of ROS in cells under the induction of X-rays and the radiosensitization effect of AuNPs, thereby improving the radiotherapy efficacy. The quantitative relationship between the ratiometric NIR-II fluorescence imaging probe signal and ROS is studied by the ratio change and intensity distribution of the imaging signal in vivo (such as the attached Figure 8 Thus, the quantitative detection of ROS can be achieved.

[0082] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a near-infrared second-zone ratiometric fluorescent probe for quantitative detection of ROS, characterized in that: The steps include: 1) NaYF4:20%Yb,2%Er DCNPs were prepared by co-precipitation of lanthanum acetate hydrate with ODE and OA; 2) Based on NaYF4:20%Yb,2%Er DCNPs, NaYF4:20%Yb,2%Er@NaYF4:30%Nd core-shell DCNPs were prepared by a seed-mediated core-shell growth method; 3) azobenzene-4,4'-dicarboxylic acid, EDC, NHS, and DSPE-PEG-2000 are dissolved in DMF for reaction, the reaction solution is resuspended, the product is dissolved in DMF, and EDC, NHS, and cysteamine are added for reaction, and the obtained reaction product is resuspended again to obtain the modified DSPE-PEG; 4) dispersing the core-shell DCNP and the dye obtained in step 2) in dichloromethane, and then adding the modified DSPE-PEG to react, and the reaction product is subjected to rotary evaporation, centrifugation with water, and washing to obtain DCNP@dye; 5) adding chloroauric acid to a mixed solution of tetrakis(hydroxymethyl)phosphonium chloride and NaOH under high-speed stirring, and then continuing to stir to obtain a gold seed; reacting the gold seed with the DCNP@dye, and dispersing the reaction product in deionized water after centrifugation and washing.

2. The method for preparing a near-infrared second-region ratiometric fluorescent probe for quantitatively detecting ROS according to claim 1, characterized in that: Step 1) specifically includes the following steps: 1.1) 0.78 mmol Y(AC)3, 0.2 mmol Yb(AC)3 and 0.02 mmol Er(AC)3 were added to a mixed solution containing 17 mmol OA and 42 mmol ODE; 1.2) The mixed solution was slowly heated to 80° C. at 2° C. / min under continuous stirring, and maintained in vacuum for 20 minutes when the temperature reached 70° C., and then stirred continuously at 100° C. for 20 minutes under an argon atmosphere. Finally, the mixture was slowly heated to 150° C. and stirred for 40 minutes, and then cooled to room temperature to obtain a reaction solution I; 1.3) 10 mL of 4 mmol / mL methanol solution of ammonium fluoride and 2.5 mL of 1 mmol / mL methanol solution of sodium hydroxide were mixed and added to the reaction solution I which was rapidly stirred at 700 rpm, and then the mixed solution was slowly heated at 0.6°C / min to 47.5°C and kept for 35 minutes, and when the temperature reached 70°C, vacuum was maintained for 20 minutes, and then slowly heated to 100°C and kept for 30 minutes, and then stirred at 298°C for 90 minutes and then cooled to room temperature; 1.4) The reaction product was centrifuged at 6000 rpm for 15 minutes to collect the obtained nanoparticles and add ethanol, then 环己烷 :V 乙醇 The resulting mixture was washed three times with a mixed solution of cyclohexane and ethanol in a ratio of 3:1 and then dispersed in 10 mL of cyclohexane.

3. The method for preparing a near-infrared second-region ratiometric fluorescent probe for quantitatively detecting ROS according to claim 1, characterized in that: Step 2) specifically includes the following steps: 2.1) Add 0.7 mmol Y(AC)3 and 0.3 mmol Nd(AC)3 to a mixed solution containing 17 mmol OA and 42 mmol ODE; 2.2) stirring the mixed solution at a constant speed of 700 rpm, and slowly heating the reaction system at 4°C / min, and keeping it under an argon atmosphere for 25 minutes when the temperature reaches 100°C, then slowly heating it to 150°C and keeping it under an argon atmosphere for 40 minutes, and then cooling it to room temperature to obtain a reaction solution II; 2.3) The synthesized NaYF4:20% Yb, 2% Er DCNPs nanoparticles and 4mmol NH4F methanol solution were added to the reaction solution II, and then 1mmol NaOH was added. Then, the reaction system was slowly heated to 47.5℃ at 0.6℃ / min and kept for 35 minutes, and then gradually heated to 298℃ under argon atmosphere and kept for 90 minutes, and then cooled to room temperature; 2.4) The reaction product was cooled to room temperature and centrifuged to obtain core-shell DCNPs, which were then washed twice with cyclohexane and anhydrous ethanol and dispersed in 10 mL of cyclohexane.

4. The method for preparing a near-infrared second-region ratiometric fluorescent probe for quantitatively detecting ROS according to claim 1, characterized in that: Step 3) specifically includes the following steps: 3.1) Dissolve azobenzene-4,4'-dicarboxylic acid, EDC, NHS, and DSPE-PEG-2000 in DMF at a molar ratio of 1:1.2:1.2:1 and react overnight; 3.2) The reaction solution was resuspended in 10-fold anhydrous ether and centrifuged twice at 2000 r / min, each time for 5 min; 3.3) The obtained product was dissolved in 1 mL of DMF and EDC, NHS and 0.03 mol of cysteamine were added to react overnight; wherein the amount of EDC and NHS added was the same as in step 3.1); 3.4) The reaction solution was resuspended in ten times anhydrous ether and centrifuged twice at 2000 rpm, each time for 5 min, to obtain the modified DSPE-PEG.

5. The method for preparing a near-infrared second-region ratiometric fluorescent probe for quantitative detection of ROS according to claim 1, characterized in that: Step 4) specifically includes the following steps: 4.1) Disperse the core-shell DCNP and the dye in dichloromethane and stir evenly, then add the modified DSPE-PEG to react; 4.2) The dichloromethane was dried by rotary evaporation at 100 rpm and 30° C., and then water was added and centrifuged at 7800 rpm for 7 minutes to collect the product, which was then washed twice with deionized water to obtain DCNP@dye.

6. The method for preparing a near-infrared second-region ratiometric fluorescent probe for quantitatively detecting ROS according to claim 5, characterized in that: The dye is selected from IR-808 or other organic or inorganic materials which are excited by 808nm laser and whose fluorescence emission peak is tailed to 980nm.

7. The method for preparing a near-infrared second-region ratiometric fluorescent probe for quantitatively detecting ROS according to claim 6, characterized in that: When the dye is IR-808, step 4) is as follows: 4.1) Disperse 100 μL 35 mg / mL core-shell DCNP and 655 μL 0.1 mg / mL IR808 in dichloromethane and stir evenly, then add 30 mg modified DSPE-PEG to react; 4.2) The dichloromethane was dried by rotary evaporation at 100 rpm and 30° C., and then water was added and centrifuged at 7800 rpm for 7 minutes to collect the product, which was then washed twice with deionized water to obtain DCNP@IR.

8. The method for preparing a near-infrared second-region ratiometric fluorescent probe for quantitative detection of ROS according to claim 7, characterized in that: Step 5) specifically includes the following steps: 5.1) 12 μL of tetrakis(hydroxymethyl)phosphonium chloride solution and 1M NaOH solution were mixed and stirred evenly, and then 1 mL of 20 mg / mL chloroauric acid was quickly added to the mixture solution under high-speed stirring at 400 rpm and continued to stir to obtain a gold seed, and the obtained gold seed was placed at room temperature; 5.2) DCNP@IR and gold seed were mixed at a mass ratio of 1:10 and reacted for 6 hours. The product DCNP@IR-Au was collected by centrifugation at 7800 rpm for 7 minutes, then washed twice with deionized water and dispersed in deionized water.

9. A near-infrared second-zone ratiometric fluorescent probe DCNP@IR-Au for quantitatively detecting ROS prepared according to the preparation method of claim 7 or 8, characterized in that: The spatial distance between DCNP and Au in the DCNP@IR-Au is less than 10 nm; The probe emits fluorescence (FL) at 1550nm under 980nm laser excitation. 1550,980Ex ) emission is strong, under 808nm laser excitation, the fluorescence (FL 1550808Ex ) emission is weaker.

Images