Water-soluble near-infrared region stimuli-responsive chemiluminescent macromolecular fluorescent probe as well as preparation method and application of water-soluble near-infrared region stimuli-responsive chemiluminescent macromolecular fluorescent probe

Abstract

The invention discloses a water-soluble near-infrared region stimuli-responsive chemiluminescent macromolecular fluorescent probe and a preparation method thereof, the chemiluminescent macromolecular fluorescent probe is MCP 795 or NPMCP 795, the MCP 795 can transmit chemiluminescence to a near-infrared fluorescent dye through energy resonance transfer between molecules, and near-infrared region chemiluminescence responsive to active oxygen is realized; nPMCP 795 is subjected to in-situ oxidation of a chemiluminescence element by using active oxygen generated by a photosensitizer under 660 nm illumination, and near-infrared chemiluminescence triggered by low oxygen is realized through an intramolecular energy resonance transfer form; the two can be assembled together to form CP795, the CP795 has good water solubility and chemical stability and strong near-infrared region chemiluminescence, orthogonal detection of active oxygen and nitroreductase can be realized through a light gate control strategy, and the CP795 has good tissue penetration depth, high imaging signal-to-noise ratio and high detection sensitivity. The orthogonal detection of a plurality of markers in tumors can be realized.

Description

Technical Field

[0001] The invention belongs to the technical field of chemiluminescent macromolecular fluorescent probes, and specifically relates to a water-soluble near-infrared region stimulus-responsive chemiluminescent macromolecular fluorescent probe and a preparation method and application thereof. Background Art

[0002] The high heterogeneity and dynamic changes of tumor structure mean that the detection of multiple markers in tumors can not only better understand the functions in cancer biology, but also improve the early screening of tumor diseases (D. Lambrechts et al., Nature 2016, 537, 63-68). Among various imaging methods, fluorescence imaging technology can detect physiological and pathological processes at the molecular and cellular levels with high spatiotemporal resolution.

[0003] In the past few decades, trigger-activated fluorescent probes have been widely used in the detection of biomarkers. However, only a few systems can achieve bio-orthogonal detection, and these systems rely on the real-time excitation of multiple fluorescent reporters, which will cause a lot of light scattering and autofluorescence interference from biological tissues, and signal crosstalk due to spectral overlap between emission spectra. These will lead to poor resolution and sensitivity, signal-to-noise ratio and tissue penetration depth of biomarker detection.

[0004] Non-excitation luminescence imaging has developed into an ultra-sensitive means of detecting endogenous substances (N. Murthy et al., Nat. Mater. 2007, 6, 765-769). Due to the elimination of background interference of excitation light, it has a higher tissue penetration depth and imaging signal-to-noise ratio. Compared with traditional bioluminescence imaging performed by genetic means, the chemiluminescence imaging module can achieve high-sensitivity detection of over-expressed signal molecules in the microenvironment through molecular design.

[0005] Among the known types of chemiluminescence, the chemiluminescence system based on 1,4-dioxetane has attracted much attention. The phenol group in the molecular structure can be protected by various responsive trigger activation primitives, thereby achieving high sensitivity response to reactive oxygen / reactive nitrogen and various enzymes. However, due to the large spectral overlap of traditional chemiluminescent groups, it is difficult to achieve orthogonal detection of multiple markers in tumors by imaging. Summary of the invention

[0006] To solve the above technical problems, the present invention provides a water-soluble near-infrared region stimulus-responsive chemiluminescent macromolecular fluorescent probe and a preparation method thereof. The chemiluminescent macromolecular fluorescent probe is MCP 795 or NPMCP795. MCP 795 can transfer chemiluminescence to a near-infrared fluorescent dye through intermolecular energy resonance transfer to achieve near-infrared region chemiluminescence responsive to reactive oxygen species; NPMCP 795 in-situ oxidizes a chemiluminescent motif by reactive oxygen species generated by a photosensitizer under 660 nm light irradiation, and through an intramolecular energy resonance transfer form, thereby achieving near-infrared region chemiluminescence triggered by low oxygen.

[0007] The present invention also provides a macromolecular fluorescent probe CP795 for orthogonal detection of multiple substances under light-controlled conditions. The macromolecular fluorescent probe is co-assembled by MCP 795 and NPMCP 795, has good water solubility and chemical stability, has strong near-infrared region chemiluminescence, and can achieve orthogonal detection of reactive oxygen species and nitroreductase through a light gating control strategy. It not only has good tissue penetration depth, but also has a high signal-to-noise ratio for imaging, and can achieve orthogonal detection of multiple markers in tumors.

[0008] The present invention also provides an application of a macromolecular fluorescent probe for orthogonal detection of multiple substances under light-controlled conditions in orthogonal detection of tumor markers.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] A water-soluble near-infrared region stimulus-responsive chemiluminescent macromolecular fluorescent probe, and the structural formula of the fluorescent probe is:

[0011] Among them, the structural formula of R1 is structural formula A or structural formula B;

[0012] The structural formula A is:

[0013]

[0014] The structural formula B is:

[0015]

[0016] In structural formula A, x + y = 1, 0 < x < 1, 0 < y < 1, and n is a natural number greater than 1;

[0017] In structural formula B, a + b + c = 1, 0 < a < 1, 0 < b < 1, 0 < c < 1, and n is a natural number greater than 1;

[0018] Both R2 and R3 are One of a nitro group and a phosphate group, and R2 and R3 are different.

[0019] When the structural formula of R1 is structural formula A, the chemiluminescent macromolecular fluorescent probe is named MCP 795; when the structural formula of R1 is structural formula B, the chemiluminescent macromolecular fluorescent probe is named NPMCP 795.

[0020] The probe co-assembled from MCP 795 and NPMCP 795 can achieve orthogonal detection of multiple substances.

[0021] When R2 is When R3 is a nitro group, the probe co-assembled by MCP 795 and NPMCP 795 can achieve orthogonal detection of nitroreductase and reactive oxygen species.

[0022] The present invention also provides a macromolecular fluorescent probe CP795 for realizing orthogonal detection of substances under light-controlled conditions. The macromolecular fluorescent probe is formed by co-assembling MCP 795 and NPMCP 795.

[0023] Furthermore, the mass ratio of the MCP 795 to the NPMCP 795 is 1:1.

[0024] The present invention also provides a method for preparing the water-soluble near-infrared region stimulus-responsive chemiluminescent macromolecular fluorescent probe, the preparation method comprising the following steps:

[0025] (1) preparing an aza-fluoro-boron fluorescent substance, wherein the aza-fluoro-boron fluorescent substance has the structural formula:

[0026]

[0027] (2) oligoethylene glycol methacrylate and methacrylate azidoethanol ester, or oligoethylene glycol methacrylate, methacrylate azidoethanol ester and monoporphyrin monoacrylate, and azafluoroboron fluorescent substances are randomly copolymerized under the initiation of azobisisobutyronitrile to obtain a macromolecular fluorescent probe; the structural formula of the macromolecular fluorescent probe is: The structural formula of R4 is: or

[0028] (3) A click chemical reaction is carried out between the macromolecular fluorescent probe and the alkyne-functionalized chemiluminescent precursor to obtain a water-soluble near-infrared region stimulus-responsive chemiluminescent macromolecular fluorescent probe; the structural formula of the alkyne-functionalized chemiluminescent precursor is:

[0029]

[0030] Furthermore, in step (2), the molar ratio of the azafluoroboron fluorescent substance, azobisisobutyronitrile, oligoethylene glycol methacrylate, and azidoethanol methacrylate is 1:0.2-0.5:30-80:10-30.

[0031] In step (2), the molar ratio of the aza-fluoroboric fluoride substance, azobisisobutyronitrile, oligoethylene glycol methacrylate, azidoethanol methacrylate, and monoporphyrin monoacrylate is 1:0.2-0.5:30-80:10-30:1-10.

[0032] In step (3), the molar ratio of the macromolecular fluorescent probe to the alkyne-functionalized chemiluminescent precursor is 1:1.0-5.0.

[0033] The present invention also provides the use of the macromolecular fluorescent probe for realizing orthogonal detection of substances under light-controlled conditions in orthogonal detection of tumor markers.

[0034] The present invention provides two water-soluble near-infrared stimulus-responsive chemiluminescent macromolecular fluorescent probes, MCP795 and NPMCP795. MCP795 can transfer chemiluminescence to near-infrared fluorescent dyes through intermolecular energy resonance transfer to achieve near-infrared chemiluminescence in response to active oxygen; NPMCP795 utilizes active oxygen generated by a photosensitizer to in-situ oxidize chemiluminescent primitives under 660nm light, and achieves hypoxia-triggered near-infrared chemiluminescence through intramolecular energy resonance transfer. The macromolecular fluorescent probe co-assembled by MCP795 and NPMCP795 has good water solubility and chemical stability, strong near-infrared chemiluminescence, and can achieve orthogonal detection of active oxygen and nitroreductase through a light gate control strategy. It not only has a good tissue penetration depth, but also has a high signal-to-noise ratio for imaging, and can achieve orthogonal detection of multiple markers in tumors.

[0035] The specific process of orthogonal detection is as follows:

[0036] First, the first trigger element is added to the detection system containing the second trigger element to make the chemiluminescent signal decay to the baseline. Then, after near-infrared light irradiation, the chemiluminescent signal triggered is derived from the second trigger element, thereby achieving the purpose of orthogonal detection.

[0037] In the preparation method of the water-soluble near-infrared stimulus-responsive chemiluminescent macromolecular fluorescent probe provided by the present invention, firstly, an atom transfer radical polymerization initiator based on aza-fluoroboron fluorescence with near-infrared emission is synthesized, and then oligoethylene glycol monomethyl ether methacrylate (OEGMA) and methacrylate azidoethanol ester are initiated to obtain a water-soluble polymer; and a chemiluminescent moiety responsive to active oxygen is covalently modified to the polymer through a post-polymerization modification strategy to obtain MCP 795. A chemiluminescent moiety responsive to hypoxia and a photosensitizer capable of generating active oxygen are modified to the polymer chain through a post-polymerization modification strategy to obtain NPMCP 795.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The water-soluble near-infrared stimulus-responsive chemiluminescent macromolecular fluorescent probes MCP 795 and NPMCP 795 provided by the present invention have novel structures and good light stability and chemical stability in water. After the two are co-assembled and combined, the chemiluminescent precursor and the photosensitizer that can generate singlet oxygen are combined, and the active oxygen generated by the photosensitizer under light is used to in situ oxidize the chemiluminescent precursor, thereby realizing chemiluminescence orthogonal detection of multiple markers under light-controlled conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the H-NMR spectrum of BPSOCL;

[0041] Figure 2 is the NMR carbon spectrum of BPSOCL;

[0042] Figure 3 Matrix-assisted desorption ionization time-of-flight mass spectrometry of BPSOCL;

[0043] Figure 4 is the H-NMR spectrum of BPCL;

[0044] Figure 5 is the C NMR spectrum of BPCL;

[0045] Figure 6 is the H-NMR spectrum of CBDP-CTA;

[0046] Figure 7 is the NMR spectrum of CBDP-CTA;

[0047] Figure 8 It is matrix-assisted desorption ionization time-of-flight mass spectrometry of CBDP-CTA;

[0048] Fig. 9 is the H NMR spectrum of MP795-N3;

[0049] Fig.10is the H NMR spectrum of CP795;

[0050] Fig.11 is the H-NMR spectrum of NPSOCL;

[0051] Fig.12 is the NMR carbon spectrum of NPSOCL;

[0052] Fig.13 Matrix-assisted desorption ionization time-of-flight mass spectrometry for NPSOCL;

[0053] Fig.14 The scattered light intensity and particle size changes of CP795 nanoparticles before and after co-incubation with ONOO-, as well as the corresponding TEM characterization results;

[0054] Fig.15 CP795 nanoparticles and ONOO - Changes in absorption and chemiluminescence spectra after co-incubation, as well as the corresponding chemiluminescence half-life results;

[0055] Fig.16 For CP795 nanoparticles ONOO - Detection limit, selectivity of CP795 for different reactive oxygen species;

[0056] Fig.17 (a, b) are the changes in particle size measured by light scattering after incubation of NPMCP795 with nitroreductase and irradiation with 660nm light, and the corresponding transmission electron microscopy results; (c) is the difference in the ability of NPMCP795 nanoparticles to produce reactive oxygen species after incubation with different equivalents of nitroreductase;

[0057] Fig.18 (a) After NPMCP795 was incubated with nitroreductase and then irradiated with 660nm light, the intensity of chemiluminescence in the near infrared region changed with time; (b) NPMCP795 nanoparticles can only produce chemiluminescence when nitroreductase and light are present at the same time, and the chemiluminescence increases with the extension of the illumination time; (c) The intensity of chemiluminescence can be affected by regulating the oxygen content of the system in different ways;

[0058] Fig.19 (a) CP795 nanoparticles were prepared by coassembling MCP795 and NPMCP795; (b) CP795 nanoparticles were used for orthogonal detection of nitroreductase and ONOO in vitro - (c, d, e) the experiment is carried out according to the presence or absence of nitroreductase, ONOO - and illumination conditions were divided into eight groups, and the changes in chemiluminescence over time under each group were measured to demonstrate orthogonal detection;

[0059] Fig. 20 (a) Schematic diagram of orthogonal detection of CP795 nanoparticles in vivo, (b) CP795 nanoparticles were injected into the tumor, the chemiluminescence decayed to the baseline within 120 minutes, and then the chemiluminescence increased rapidly after 5 minutes of 660nm LED light, and dicoumarol was used as an enzyme inhibitor, (c) the change of chemiluminescence intensity of the corresponding tumor site over time;

[0060] Fig.21 This is a schematic diagram of the structure of a water-soluble near-infrared stimulus-responsive chemiluminescent macromolecular fluorescent probe provided by the present invention. DETAILED DESCRIPTION

[0061] The present invention is described in detail below with reference to the embodiments.

[0062] The sources of the raw materials used in each embodiment are as follows:

[0063] alkyne-SOCL was synthesized with reference to J.Am.Chem.Soc., 2017, 139(37): 13243-13248.

[0064] Alkyne-CBDP was synthesized with reference to Angew.Chem.Int.Ed., 2022, 134(5):e202114273.

[0065] N3-CTA was synthesized according to J. Am. Chem. Soc., 2012, 134(30): 12596-12603.

[0066] Example 1

[0067] A water-soluble near-infrared stimulus-responsive chemiluminescent macromolecular fluorescent probe MCP 795, whose structural formula is:

[0068] Among them, the structural formula of R1 is: Where R2 is

[0069] The synthetic route of described MCP 795 is as follows:

[0070]

[0071] The specific preparation process of the MCP 795 is as follows:

[0072] (1) Synthesis of BPSOCL: alkyne-SOCL (150 mg, 0.36 mmol, 1.0 equivalent), 4-bromomethylphenylboronic acid pinacol ester (128 mg, 0.432 mmol, 1.2 equivalent) and cesium carbonate (176 mg, 0.54 mmol, 1.5 equivalent) were added to a round-bottom flask. Under nitrogen stirring, 5 mL of anhydrous acetonitrile was added and reacted at 50°C for 4 h. After returning to room temperature, the acetonitrile was removed under reduced pressure and redispersed in 50 mL of dichloromethane. The mixture was washed twice with saturated brine (50 mL each time). The organic phase was collected and dried over anhydrous sodium sulfate. After concentration, it was subjected to column chromatography. The eluent for column chromatography was a mixed solution of dichloromethane and methanol in a volume ratio of 99:1 to obtain a bright yellow solid, BPSOCL (120 mg, yield: 51%). 1 H NMR(400MHz,Chloroform-d)δ7.88–7.81(m,2H),7.75–7.67(m,1H),7.52–7. 46(m,2H),7.38–7.34(m,1H),7.09–7.03(m,1H),6.54–6.43(m,1H),5.02(d,J =7.2Hz,2H),4.14(dt,J=5.3,2.1Hz,2H),3.34–3.30(m,3H),3.28(d,J=6.5H z,1H),2.21(s,1H),2.06(s,1H),1.96–1.67(m,12H),1.35(d,J=1.8Hz,12H). 13 C NMR(101MHz,Chloroform-d)δ165.85,154.26,139.95,139.75,138.15,136.38,135.56,132.88,130.40,130.17,128.83,128.28,1 26.62,123.28,79.88,74.40,72.43,72.01,57.78,39.30,37.24,33.18,29.95,29.65,28.55,19.52.MALDI-TOF-MS(m / z)calc.for C 37 H 43 BClNO5Na:650.9945[M+Na] + found:650.3135. Its H-NMR, C-NMR and mass spectra are as follows: Figure 1 , Figure 2 , Figure 3 shown.

[0073] (2) Synthesis of BPCL: Compound BPSOCL (30 mg, 0.046 mmol, 1.0 equivalent) and a catalytic amount of methylene blue were dissolved in 20 mL of dichloromethane and stirred in an ice-water bath at 660 nm and 200 mW / cm 2 The mixture was irradiated with LED light for 2 hours, concentrated and separated and purified by reverse phase HPLC, and a mixed solution of acetonitrile and water in a volume ratio of 1:1 was used as the mobile phase, and finally 20 mg of bright yellow solid powder was obtained with a yield of 65%. 1 H NMR(400MHz,Chloroform-d)δ7.82(dt,J=25.4,12.8Hz,4H),7.48(dd,J=16.9,8.0Hz,3H),6.52(t,J=15.2Hz,1H),5.05–4.89(m, 2H),4.13(dd,J=7.7,4.8Hz,2H),3.24(d,J=43.6Hz,3H),3.00(s,1H),2.29(d,J=15.1Hz,1H),2.06–1.55(m,13H),1.34(s,12H). 13 C NMR(101MHz,Chloroform-d)δ165.25,154.43,139.03,135.38,135.28,135.23 ,134.77,131.93,129.06,128.05,126.07,124.12,112.00,96.61,84.12,79.57 ,76.11,72.10,60.64,49.94,38.82,36.80,34.08,33.79,32.82,32.45,31.80, 31.71,29.62,26.38,25.99,25.10,21.29,14.42.MALDI-TOF-MS(m / z)calc.for C 37 H 43 BClNO7Na:682.9933[M+Na] + ,found:682.6616. Its H NMR and C NMR are as follows Figure 4 , Figure 5 shown.

[0074] (3) Synthesis of CBDP-CTA: Alkyne-CBDP (30 mg, 0.036 mmol, 1.0 equivalent) and N3-CTA (27.4 mg, 0.0786 mmol, 2.2 equivalent) were dissolved in 5 mL of dichloromethane, and then N,N-diisopropylethylamine (2.3 mg, 0.018 mmol, 0.5 equivalent), CH3COOH (1.0 mg, 0.018 mmol, 0.5 equivalent) and cuprous bromide (2.6 mg, 0.018 mmol, 0.5 equivalent) were added. The mixture was reacted at room temperature for 12 h, diluted with 20 mL of dichloromethane, washed twice with saturated brine, 20 mL each time, and the organic phase was dried over anhydrous sodium sulfate and then subjected to column chromatography. The eluent used for column chromatography was a mixed solution of dichloromethane and methanol in a volume ratio of 99:1 to obtain CBDP-CTA solid powder (42 mg, yield: 76.4%). 1 H NMR(400MHz,Chloroform-d)δ8.86(s,2H),8.29(d,J=8.4Hz,2H),8.09(d,J=8.2Hz,4H),7 .99(d,J=8.0Hz,2H),7.89–7.85(m,4H),7.72(s,2H),7.52(d,J=3.2Hz,2H),7.45–7.32(m, 10H),7.04(dd,J=15.3,7.4Hz,8H),5.23(s,4H),4.64–4.59(m,4H),4.52–4.47(m,4H),4. 27(q,J=6.9Hz,4H),2.67–2.52(m,8H),1.86(d,J=3.0Hz,6H),1.38(dd,J=7.3,4.4Hz,6H). 13 C NMR(101MHz,Chloroform-d)δ222.50,171.22,160.26,157.30,145.65,144.54,144.27 ,140.82,140.58,133.43,131.72,128.86,127.60,126.89,126.24,125.39,124.18,12 3.70,123.51,122.23,121.14,119.44,118.75,117.07,115.02,109.14,108.99,63.08 ,62.11,49.29,45.83,37.90,33.27,29.81,24.31,14.21.MALDI-TOF-MS(m / z)calc.for C 84 H 72 BF2N 13O6S4:1536.47[MH] - found:1536.35. Its H-NMR, C-NMR and mass spectra are as follows: Figure 6 , Figure 7 , Figure 8 shown.

[0075] (4) Synthesis of MP795-N3: CBDP-CTA (10 mg, 0.0065 mmol, 1.0 equivalent), azobisisobutyronitrile (0.2 mg, 0.0013 mmol), oligoethylene glycol monomethyl ether methacrylate (162.5 mg, 0.325 mmol, 50.0 equivalent), and methyl methacrylate azidoethanol (20.0 mg, 0.13 mmol, 20.0 equivalent) were added to a sealed tube containing a magnetic rod, and then 1,4-dioxane (350 mg) was added. After three cycles of freeze-degassing-thawing, the tube was sealed and reacted in an oil bath at 70°C for 4 h. The tube was frozen with liquid nitrogen, the sealed tube was opened, diluted with dichloromethane, and precipitated into excess ether. This process was repeated three times. Finally, 95 mg of an oily product was obtained after drying, with a yield of 52%. The molecular weight and molecular weight distribution measured by tetrahydrofuran GPC were 1.08×10 4 Da and M w / M n of 1.13, and the final structural formula was determined by NMR to be CBDP-P (OEGMA 0.66 -co-(N3) 0.34 ) 25 , named MP 795-N3. Its H NMR spectrum is as follows Fig. 9 shown.

[0076] (5) Synthesis of MCP795: MP795-N3 (50 mg, 0.04 mmol N3, 1.0 equivalent), BPCL (40 mg, 0.06 mmol, 1.5 equivalent), pentamethyldiethylenetriamine (1.4 mg, 0.008 mmol, 0.2 equivalent), and acetic acid (0.5 mg, 0.008 mmol, 0.2 equivalent) were added to a sealed tube, 2 mL of DMF was added thereto, and cuprous bromide (1.1 mg, 0.008 mmol, 0.2 equivalent) was added under nitrogen freezing. The reaction system was subjected to three cycles of freezing-degassing-thawing and then reacted at room temperature for 48 h. After the reaction was completed, the sealed tube was opened, 1.0 mL of DCM was added for dilution, and the solution was passed through an alkaline alumina column to remove the catalyst. After concentration, it was precipitated into excess ether. This process was repeated three times and the final product was obtained after drying. The molecular weight and molecular weight distribution were measured by tetrahydrofuran GPC and were 1.6×10 4 kDa and M w / M nof 1.25, the final molecular structure was characterized by NMR and named MCP795. Fig.10 shown.

[0077] Example 2

[0078] A water-soluble near-infrared stimulus-responsive chemiluminescent macromolecular fluorescent probe NPMCP 795, whose structural formula is:

[0079] Among them, the structural formula of R1 is: Wherein R3 is nitro.

[0080] The synthetic route of NPMCP 795 is as follows:

[0081]

[0082] The specific preparation process of NPMCP 795 is as follows:

[0083] (1) Synthesis of NPSOCL: alkyne-SOCL (150 mg, 0.36 mmol, 1.0 equivalent), p-nitrobenzyl bromide (93 mg, 0.432 mmol, 1.2 equivalent) and cesium carbonate (176 mg, 0.54 mmol, 1.5 equivalent) were added to a round-bottom flask, and 5 mL of acetonitrile was added thereto under a nitrogen environment. The mixture was stirred at 50°C for 4 h, and the acetonitrile was removed after returning to room temperature. The mixture was redissolved with dichloromethane and washed twice with saturated brine, each time with 50 mL. The organic phase was dried over anhydrous sodium sulfate, and then subjected to column chromatography. The eluent used in the column chromatography was a mixed solution of dichloromethane and methanol in a volume ratio of 99:1, to obtain a light yellow solid, NPSOCL. 1 H NMR(400MHz,Chloroform-d)δ8.21(dd,J=8.7,1.6Hz,2H),7.85(d,J=15.7Hz,1H), 7.67(dd,J=8.6,1.6Hz,2H),7.41(d,J=8.1Hz,1H),7.08(dd,J=8.0,1.3Hz,1H),6.4 8(dd,J=15.8,1.3Hz,1H),5.05(dd,J=11.0,4.5Hz,2H),4.13(dd,J=5.3,2.5Hz,2H) ,3.31(d,J=1.3Hz,3H),3.25(s,1H),2.25(s,1H),2.02(s,1H),1.95–1.69(m,12H). 13CNMR(101MHz,Chloroform-d)δ165.23,153.43,147.99,143.70,139.48,138.15,135.74,133.08,129.88,128.83,128.38,1 25.54,123.99,122.78,79.48,74.40,72.06,57.56,39.25,37.22,33.18,29.95,29.65,28.37.MALDI-TOF-MS(m / z)calc.for C 31 H 31 ClN2O5:547.0410[M] + ,found:546.6072. Its H-NMR, C-NMR and MS spectra are as follows: Fig.11 , Fig.12 , Fig.13 shown.

[0084] (2) Synthesis of PMP 795-N3: CBDP-CTA (5 mg, 0.00325 mmol, 1.0 equivalent), azobisisobutyronitrile (0.1 mg, 0.00065 mmol), oligoethylene glycol monomethyl ether methacrylate (81.3 mg, 0.1625 mmol, 50.0 equivalent), methyl methacrylate azidoethanol (10.0 mg, 0.065 mmol, 20.0 equivalent), porphyrin methyl methacrylate (4.5 mg, 0. 0065mmol, 2.0 equivalents) was added to a sealed tube containing a magnetic electron, and then 1,4-dioxane (350mg) was added. After freezing-degassing-thawing cycles for three times, the tube was sealed and reacted in an oil bath at 70°C for 4h. The tube was frozen with liquid nitrogen, the sealed tube was opened, diluted with dichloromethane, and precipitated into excess ether. This process was repeated three times. Finally, 51mg of an oily product was obtained after drying. The yield was 50.6%. The molecular weight and molecular weight distribution measured by tetrahydrofuran GPC were 1.4×10 4 Da and M w / M n of 1.27, and its final structural formula is CBDP-P(OEGMA 0.60 -co-TPP 0.08 -co-(N3) 0.32 ) 34 .

[0085] (3) Synthesis of NPMCP 795: PMP795-N3 (50 mg, 0.038 mmol N3, 1.0 equivalent), NPSOCL (32 mg, 0.058 mmol, 1.5 equivalent), pentamethyldiethylenetriamine (1.4 mg, 0.008 mmol, 0.2 equivalent), and acetic acid (0.5 mg, 0.008 mmol, 0.2 equivalent) were added to a sealed tube, 2 mL of DMF was added thereto, and cuprous bromide (1.1 mg, 0.008 mmol, 0.2 equivalent) was added under nitrogen freezing. The reaction system was subjected to three cycles of freeze-degassing-thawing and then reacted at room temperature for 48 h. After the reaction was completed, the sealed tube was opened, the catalyst was removed, and it was precipitated into excess ether. This process was repeated three times, and the final product was obtained after drying. The molecular weight and molecular weight distribution were measured by tetrahydrofuran GPC and were 2.1×10 4 kDa and M w / M n of1.32, and the final molecular structure was named NPMCP795.

[0086] Example 3

[0087] 2 mg of polymer probe MCP795 was dissolved in 0.2 mL of DMSO and quickly added to 9.8 mL of high-speed stirring NMR deionized water to obtain nanoparticles by flash precipitation. First, its responsiveness to reactive oxygen species was verified, such as Fig.14 As shown, the nanoparticles were mixed with a pH 7.4 PBS buffer solution at a volume ratio of 1:1, and 0.1 mM peroxynitrite (ONOO - ), the light scattering tracking results showed that the scattered light intensity decreased significantly, accompanied by a decrease in particle size, and the TEM results also proved that the nanoparticles had indeed degraded. The near-infrared chemiluminescence characteristics of this system were then verified, such as Fig.15 As shown, 0.1 mg / mL nanoparticles were mixed with 0.1 mM ONOO at pH 7.4. - After the reaction, the ultraviolet absorption spectrum around 400nm increased significantly, and the chemiluminescence at 795nm increased by 1066 times. The half-life of chemiluminescence at 795nm reached 21min, which further proved that the CP795 probe can transfer chemiluminescence to the near-infrared dye through the energy within the molecule to achieve chemiluminescence in the near-infrared region. The selectivity of the CP795 probe was further proved. Fig.16 As shown, different types of active oxygen (including H2O2, O2 - , ClO - OH . , the concentration of which was 0.1 mM) and incubated for 12 h, only ONOO -Significant chemiluminescence enhancement was achieved, and the detection limit of the CP795 probe reached 2.5 nM.

[0088] Example 4

[0089] 2 mg of polymer probe NPMCP 795 was dissolved in 0.2 mL of DMSO and then quickly added to 9.8 mL of deionized water under high-speed stirring to obtain nanoparticles by flash precipitation. First, the responsiveness of the nanoparticles to nitroreductase and the ability of the nanoparticles to produce singlet oxygen were verified. Nitroreductase (concentration of 15 μg / mL) / NADPH (1.0 mM) was added to 0.1 mg / mL nanoparticle solution (pH 7.4). After incubation for 12 h, the particle size change was tracked by dynamic light scattering, as shown in FIG. Fig.17 As shown in Figure a, the particle size decreased from the initial 125nm to 45nm, and after further irradiation with 660nm light, the particle size further decreased to 30nm. TEM results also further confirmed the process of nanoparticles becoming smaller, such as Fig.17 As shown in Figure b, it further illustrates that the nitroreductase and 660nm light irradiation promote the degradation of nanoparticles. After adding different concentrations of nitroreductase for 12 hours (nitroreductase concentrations are 0, 2, 5, 10, 15μg / mL, NADPH concentration is 1.0mM), 660nm light (power is 200mW / cm 2 ), using a singlet oxygen probe (9,10-anthracenediyl-bis(methylene)dicarboxylic acid) as an indicator, tracking the decrease in the 400nm absorption peak to evaluate the ability to produce singlet oxygen. It can be seen that with the increase in the concentration of nitroreductase, the ability to produce singlet oxygen gradually increases. Compared with the case without enzyme, its ability to produce singlet oxygen increases by 4.27, 6.14, 7.76, and 8.45 times, respectively. Fig.17 As shown in Figure c, after the enzyme promotes the micelle dissociation, the micelles partially dissociate and the aggregation quenching between the dyes is weakened, thereby enhancing the ability to produce singlet states.

[0090] Next, the ability of light-controlled near-infrared chemiluminescence was verified by incubating 0.1 mg / mL NPMCP 795 nanoparticles with nitroreductase (15 μg / mL) in a pH 7.4 buffer solution for 15 min and then irradiating with light at 660 nm (200 mW / cm 2 ) After 6 minutes, significant near-infrared chemiluminescence will be produced, and the luminescence time can last for more than 1 hour. Fig.18 As shown in Figure a. However, chemiluminescence cannot be produced by only nitroreductase or only light conditions, and the intensity of chemiluminescence is closely related to the illumination time. The longer the illumination time, the greater the luminescence intensity. Fig.18As shown in Figure b. In the sample solution, the nitrogen or oxygen is passed in advance to adjust the oxygen concentration in the solution, or the singlet oxygen quencher is added, which will significantly affect the intensity of chemiluminescence, such as Fig.18 As shown in Figure c.

[0091] Example 5

[0092] Preparation of CP795 nanoparticles and in vitro orthogonal detection. Take 1.0 mg of MCP795 and 1.0 mg of NPMCP795 probe and dissolve them in 0.2 mL of DMSO, quickly add them into 9.8 mL of high-speed stirring deionized water, and flash precipitate to obtain the CP795 probe. First, verify the ability of the CP795 probe for orthogonal detection. First, CP795 nanoparticles (final concentration of 0.1 mg / mL) were incubated with nitroreductase (NTR, 15 μg / mL) / coenzyme NADPH (1.0 mM) in a pH 7.4 buffer solution at 37 degrees for 12 hours, and then 0.1 mM ONOO was added. - , and then detect chemiluminescence. The chemiluminescence produced at this time is ONOO - When the chemiluminescence decays to the baseline, the 660nm light (200mW / cm 2 ) After 5 minutes, a strong chemiluminescent signal is generated again. This is because the nitroreductase (NTR) / coenzyme (NADPH) triggers the reaction. The 660nm light oxidizes the chemiluminescent precursor in situ, and the generated chemiluminescent product quickly rearranges and releases chemiluminescence. The results are as follows Fig.19 As shown in Figure ad. The whole system is divided into eight groups, namely Group 1: (-)NTR / (-)ONOO - / (-)hv; Group 2: (-)NTR / (-)ONOO - / (+)hv; Group 3: (-)NTR / (-)ONOO - / (+)hv; Group 4: (-)NTR / (+)ONOO - / (+)hv; Group 5: (+)NTR / (-)ONOO - / (-)hv; Group 6: (+)NTR / (-)ONOO - / (+)hv; Group 7: (+)NTR / (+)ONOO - / (-)hv; Group 8: (+)NTR / (+)ONOO - / (+)hv, where (+) means that such conditions exist, and (-) means that such conditions do not exist. Fig.19As can be seen from Figures e and f, in the first exposure, chemiluminescence can be triggered as long as ONOO- is present, regardless of the presence or absence of nitroreductase. When the first chemiluminescence ends, only the experimental group treated with nitroreductase in advance can continue to chemiluminesce after the second illumination. This is enough to show that under the action of light control, nitroreductase and ONOO- can be achieved. - Orthogonal detection.

[0093] Example 6

[0094] CP795 nanoparticles are used for orthogonal detection in vivo. Fig. 20 As shown, 50 μL of CP795 nanoparticles (2.0 mg / mL) were injected into the tumor, and the chemiluminescent signal at the tumor site increased rapidly, which was used to detect ONOO- in the tumor; it decayed to the baseline after 120 minutes, and then the 660nm (200mW / cm 2 ) After 5 minutes of illumination, the chemiluminescence continued to increase. At this time, the signal of nitroreductase was detected, and both showed a high signal-to-noise ratio, indicating that orthogonal detection of light-controlled ONOO- and nitroreductase in vivo can be achieved. At the same time, dicoumarol (1.0mM) was used in advance, and 20μL was injected intratumorally to inhibit the activity of nitroreductase, and then imaging was performed. At this time, the chemiluminescence signal decreased significantly.

[0095] The detailed description of a water-soluble near-infrared stimulus-responsive chemiluminescent macromolecular fluorescent probe and its preparation method and application by the above reference embodiments is illustrative rather than restrictive, and several embodiments can be listed according to the limited scope. Therefore, changes and modifications without departing from the overall concept of the present invention should fall within the scope of protection of the present invention.

Claims

1. A water-soluble near-infrared stimulus-responsive chemiluminescent macromolecular fluorescent probe, characterized in that: The structural formula of the fluorescent probe is as follows: Wherein the structural formula of R1 is structural formula A or structural formula B; The structural formula A is as follows: The structural formula B is as follows: In structural formula A, x + y = 1, 0 < x < 1, 0 < y < 1, and n is a natural number greater than 1; In structural formula B, a + b + c = 1, 0 < a < 1, 0 < b < 1, 0 < c < 1, and n is a natural number greater than 1; R2 and R3 are One of a nitro group and a phosphate group, and R2 and R3 are different.

2. The water-soluble near-infrared stimulus-responsive chemiluminescent macromolecular fluorescent probe according to claim 1, characterized in that: When the structural formula of R1 is structural formula A, the chemiluminescent macromolecular fluorescent probe is named MCP 795; when the structural formula of R1 is structural formula B, the chemiluminescent macromolecular fluorescent probe is named NPMCP 795.

3. The water-soluble near-infrared stimulus-responsive chemiluminescent macromolecular fluorescent probe according to claim 2, characterized in that: The probe formed by co-assembling MCP 795 and NPMCP 795 can achieve orthogonal detection of multiple substances.

4. The water-soluble near-infrared stimulus-responsive chemiluminescent macromolecular fluorescent probe according to claim 2, characterized in that: When R2 is When R3 is a nitro group, the probe co-assembled by MCP 795 and NPMCP 795 can achieve orthogonal detection of nitroreductase and reactive oxygen species.

5. A macromolecular fluorescent probe for orthogonal detection of multiple substances under light-controlled conditions, characterized in that: The macromolecular fluorescent probe is formed by co-assembling MCP 795 and NPMCP 795 in claim 2.

6. The macromolecular fluorescent probe according to claim 5, characterized in that: The mass ratio of MCP 795 and NPMCP 795 is 1:

1.

7. The method for preparing a water-soluble near-infrared stimulus-responsive chemiluminescent macromolecular fluorescent probe according to any one of claims 1 to 4, characterized in that: The preparation method includes the following steps: (1) Prepare aza-boron dipyrromethene compounds, and the structural formula of the aza-boron dipyrromethene compounds is (2) oligoethylene glycol methacrylate and methacrylate azidoethanol ester, or oligoethylene glycol methacrylate, methacrylate azidoethanol ester and monoporphyrin monoacrylate, and azafluoroboron fluorescent substances are randomly copolymerized under the initiation of azobisisobutyronitrile to obtain a macromolecular fluorescent probe; the structural formula of the macromolecular fluorescent probe is: The structural formula of R4 is: (3) A click chemical reaction is carried out between the macromolecular fluorescent probe and the alkyne-functionalized chemiluminescent precursor to obtain a water-soluble near-infrared region stimulus-responsive chemiluminescent macromolecular fluorescent probe; the structural formula of the alkyne-functionalized chemiluminescent precursor is:

8. The preparation method according to claim 7, characterized in that: In step (2), the molar ratio of the aza-boron dipyrromethene compounds, azobisisobutyronitrile, oligoethylene glycol methacrylate, and azidoethanol methacrylate is 1:0.2 - 0.5:30 - 80:10 - 30; or, the molar ratio of the aza-boron dipyrromethene compounds, azobisisobutyronitrile, oligoethylene glycol methacrylate, azidoethanol methacrylate, and monoporhyrin monoacrylate is 1:0.2 - 0.5:30 - 80:10 - 30:1 - 10.

9. The preparation method according to claim 4, characterized in that: In step (3), the molar ratio of the macromolecular fluorescent probe to the alkyne-functionalized chemiluminescent precursor is 1:1.0 - 5.

0.

10. The application of the macromolecular fluorescent probe according to claim 5 or 6 in the orthogonal detection of tumor markers.

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