CN115029324B

CN115029324B - Fluorescent covalent labeling method of phage, phage with fluorescent label and application of phage

Info

Publication number: CN115029324B
Application number: CN202210798366.2A
Authority: CN
Inventors: 吴明雨; 陈实; 辜美佳; 万瑜
Original assignee: Southwest Jiaotong University
Current assignee: Southwest Jiaotong University
Priority date: 2022-07-08
Filing date: 2022-07-08
Publication date: 2023-06-06
Anticipated expiration: 2042-07-08
Also published as: CN115029324A

Abstract

The invention provides a fluorescence covalent labeling method of phage, a phage with fluorescence labeling and application thereof, wherein the labeling method can obtain fluorescence labeled AIE engineering phage through co-incubating the phage and a compound with aggregation-induced emission performance in water, and nucleophilic substitution/addition reaction between the compound with aggregation-induced emission performance and sulfhydryl on the phage. The AIE engineering phage can be used for rapidly identifying the pathogen of the sepsis so as to realize visual fluorescence detection of the sepsis, and can also carry out visual monitoring on the whole treatment process, thereby providing more accurate and practical information for us and optimizing and improving the treatment effect; effectively solves the problems that the existing phage cocktail therapy has low antibacterial activity and is difficult to realize rapid diagnosis and treatment of pathogenic bacteria.

Description

Fluorescent covalent labeling method of phage, phage with fluorescent label and application of phage

Technical Field

The invention belongs to the technical field of biochemistry, and particularly relates to a fluorescence covalent labeling method of phage, a phage with fluorescence labeling and application of phage.

Background

Sepsis is an acute systemic infectious disease caused by invasion of blood circulation by a variety of pathogenic bacteria and production of toxins by growth and reproduction in blood; multiple organ failure can occur in severe cases, which is life threatening. Identification and rapid diagnosis of the pathogen species of sepsis is critical, and blood culture and Next Generation Sequencing (NGS) are commonly used clinically. However, these methods have drawbacks of cumbersome steps, complicated operations, long time consumption, and false positive results. In addition, resistance problems caused by antibiotic abuse have increased the difficulty of sepsis treatment. Therefore, there is an urgent need to develop new methods for detecting and eliminating pathogenic bacteria in blood accurately, safely, and efficiently.

"phage cocktail therapy" now shows great potential in pathogen selective elimination as a new biocontrol method, however, low antibacterial activity restricts the use of phage in clinical therapies, especially acute infections. More importantly, although phage specifically target host bacteria, rapid diagnosis of pathogenic bacteria and real-time monitoring of therapeutic processes is difficult to achieve due to the lack of sensitive signaling radar.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a fluorescence covalent labeling method of phage, phage with fluorescence label and application thereof, wherein the fluorescence labeled phage not only can be used for rapidly identifying pathogenic bacteria of septicemia so as to realize visual fluorescence detection of septicemia, but also can carry out visual monitoring on the whole treatment process, thereby providing more accurate and practical information for us and optimizing and improving treatment effect; more importantly, the marked phage can kill bacteria through phage cocktail therapy, and can also kill bacteria through photodynamic therapy, so that the combined therapy of phage cocktail therapy and photodynamic therapy is realized, and the problems that the existing phage cocktail therapy is low in antibacterial activity and is difficult to realize rapid diagnosis and treatment of pathogenic bacteria are effectively solved.

In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a fluorescence covalent labeling method of phage comprises incubating phage with compound with aggregation-induced emission in water.

Further, the phage contains sulfhydryl groups, and during incubation, the aggregation-induced emission performance compound undergoes nucleophilic substitution/addition reaction with sulfhydryl groups in the phage structure.

Further, phages include E.coli, P.aeruginosa, methicillin-resistant Staphylococcus aureus, salmonella typhimurium, bacillus subtilis, staphylococcus aureus, staphylococcus epidermidis, enterococcus faecalis, proteus vulgaris, proteus mirabilis, enterococcus and Corynebacterium, klebsiella pneumoniae, bacteroides fragilis, candida albicans and Streptococcus pneumoniae.

Further, the chemical structural formula of the compound having aggregation-induced emission properties is as follows:

wherein ,

R ₁ ：

n＝0-7/>

r' is C1-C12 alkyl

/>

R ₂ ：

n＝0-10,A＝Cl,Br,I

X is O, S or Se;

Y ^- is Cl ^- 、Br ^- 、I ^- 、PF ₆ ^- 、PF ₄ ^- 、CH ₃ COO ^- Or CF (CF) ₃ COO ^- 。

Further, the compound having luminescence inducing property uses E-2, 3-bis (5-bromothiophene) acrylonitrile, E-2, 3-bis (5-bromofuran) acrylonitrile or E-2, 3-bis (5-bromoselenophene) acrylonitrile as a molecular structure skeleton by changing R on the molecular structure skeleton ₁ Electron donating group in position and R ₂ The group which can be covalently bound with the thiol reaction in the phage surface protein can be used for obtaining the compound with aggregation-induced emission performance.

Further, the specific preparation method of the compound with aggregation-induced emission performance comprises the following steps:

(1) Adding the compound A, the compound B and alkali into an alcohol solvent according to the molar ratio of (1-1.5) to (1-5), stirring at room temperature for reaction, and then filtering, washing and drying a solid product to obtain a compound C;

wherein the compound A is (5-bromo-2-thiophene) -acetonitrile, the compound B is 5-bromothiophene-2-formaldehyde, and the compound C is E-2, 3-bis (5-bromothiophene) acrylonitrile;

or, the compound A is (5-bromo-2-furan) -acetonitrile, the compound B is 5-bromo-furan-2-formaldehyde, and the compound C is E2, 3-bis (5-bromo-furan) acrylonitrile;

or, the compound A is (5-bromo-2-selenophen) -acetonitrile, the compound B is 5-bromoselenophen-2-formaldehyde, and the compound C is E2, 3-bis (5-bromoselenophen) acrylonitrile;

(2) Adding the compound C, the compound D, a palladium catalyst and inorganic alkali into a mixed solvent according to the molar ratio of 1 (1.3-3) (0.02-0.1) (3-30), and heating and refluxing until the reaction is complete; then extracting the reaction product by using an extractant to obtain an organic phase, washing the organic phase by using water and saturated saline sequentially, drying by using a drying agent, removing the organic solvent by reduced pressure distillation, and separating and purifying the obtained crude product by using a silica gel column chromatography to obtain a compound E;

(3) Adding a compound E, a compound F, a palladium catalyst and inorganic base into the mixed solvent according to the molar ratio of 1 (1.3-3) (0.02-0.1) (3-30), heating and refluxing until the reaction is complete, extracting a reaction product by using an extractant to obtain an organic phase, washing the organic phase by using water and saturated saline sequentially, drying by using a drying agent, distilling under reduced pressure to remove the organic solvent, and separating and purifying the obtained crude product by using a silica gel column chromatography to obtain a compound G;

(4) Adding a compound G and a compound H into acetonitrile or toluene according to the molar ratio of 1 (3-30), refluxing and stirring under the protection of argon until the reaction is complete, distilling under reduced pressure to remove an organic solvent, washing the obtained solid product with water, filtering under reduced pressure, drying, and separating and purifying by using a silica gel column chromatography to obtain a compound I, namely a compound with aggregation-induced emission performance;

wherein the structural formula of the compound A is

The structural formula of the compound B is as follows: />

The structural formula of the compound C is as follows: />

The compound D is 4-pyridine boric acid, and the structural formula of the compound E is as follows:

the compound F is arylboronic acid, and the structural formula of the compound F is R ₁ -B(OH) ₂ Or->

The structural formula of the compound G is as follows: />

The structural formula of the compound H is as follows: A-R ₂ A=cl, br, I, wherein X in compound a, compound B, compound C, compound E, and compound G is O, S or Se; r in Compound F and Compound G ₁ The method comprises the following steps:

R1：

n＝0-7 />

r' is C1-C12 alkyl->

/>

In the compound HR ₂ The method comprises the following steps:

n＝0-10，A＝CI，Br，I

further, in the step (1), the alcohol solvent is at least one of methanol, ethanol and isopropanol; the alkali is sodium hydroxide, potassium hydroxide, sodium methoxide, sodium ethoxide or sodium tert-butoxide; the palladium catalyst in the step (2) and the step (3) is tetra (triphenylphosphine) palladium, di (triphenylphosphine) palladium chloride, [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride, tris (dibenzylideneacetone) dipalladium or palladium acetate; the inorganic base in the step (2) and the step (3) is sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate or barium hydroxide.

Further, the extractant in the step (2) and the step (3) is dichloromethane, ethyl acetate or chloroform; the drying agent is anhydrous sodium sulfate, anhydrous calcium chloride, anhydrous magnesium chloride or anhydrous magnesium sulfate; the mixed solvent is a mixed solution composed of tetrahydrofuran and water with the volume ratio of (10-5) 1, or a mixed solution composed of toluene, ethanol and water with the volume ratio of (10-6) 1, or a mixed solution composed of dioxane and water with the volume ratio of (10-5) 1.

A bacteriophage with fluorescent label is prepared by the method.

The phage with fluorescent markers can be used for rapid diagnosis and treatment of pathogenic bacteria.

Further, it is useful for diagnosis and treatment of sepsis.

The beneficial effects of the invention are as follows:

the invention is based on E-2, 3-bis (5-bromothiophene) acrylonitrile, E-2, 3-bis (5-bromofuran) acrylonitrile or E-2, 3-bis (5-bromoselenol) acrylonitrile structure with aggregation-induced emission performance as molecular skeleton, R on the molecular skeleton is changed ₁ Electron donating group in position and R ₂ The group which can be covalently combined with sulfhydryl on phage is synthesized into a series of compounds with aggregation-induced emission performance, and the compounds have near infrared emission spectrum, high active oxygen yield and high light stabilitySex, etc.

The compounds of the invention emit no fluorescence emission in a single molecule state, emit strong fluorescence due to limited intramolecular movement after interaction with bacteria or cells, and show aggregation-induced emission (AIE) performance, which gives the compounds application potential in imaging; the compound can selectively and efficiently inactivate bacteria by generating a large amount of active oxygen under the irradiation of visible light or laser, has no toxicity to normal cells and tissues, and endows the compound with application potential as a photosensitizer for photodynamic therapy.

According to the method, the AIE engineering phage is constructed by the phage and the compound with aggregation-induced emission performance, the engineering phage has multiple functions of host specificity of the phage, fluorescence imaging of photosensitizer and photodynamic antibacterial, visual detection and treatment of septicemia pathogenic bacteria can be achieved, visual monitoring can be carried out on the whole treatment process, more accurate and practical information can be provided for us, treatment effects are optimized and improved, and visual detection and accurate treatment of septicemia are achieved. More importantly, the engineering phage not only can kill bacteria through phage cocktail therapy, but also can kill bacteria through photodynamic therapy, so that the combined treatment of phage cocktail therapy and photodynamic therapy is realized, and the treatment effect is improved.

Drawings

FIG. 1 is a synthetic route diagram of an aggregation-induced emission performance photosensitizer in an embodiment of the present invention;

FIG. 2 is a graph showing the UV-vis absorption spectrum of the photosensitizer prepared in example 1 in solvent DMSO;

FIG. 3 is a graph showing fluorescence spectra of the photosensitizer prepared in example 1 in solvents Toluene and DMSO;

FIG. 4 shows the determination of the AIE properties of the photosensitizers prepared in example 1;

fluorescence emission spectra of compounds in DMSO/Toluene solutions of different volume ratios and fluorescence emission intensity (I/I) ₀ ) A variation map;

FIG. 5 is a graph showing the dynamic light scattering particle size distribution of the photosensitizer in aqueous solution prepared in example 1;

FIG. 6 shows the fluorescence emission spectrum and the fluorescence intensity (I/I) at 525nm of the photosensitizer prepared in test example 1 for the indicator H2DCF-DA at different illumination times ₀ ) A variation map;

FIG. 7 shows the photosensitizer prepared in example 1 ¹ O ₂ A test chart;

the UV-vis absorption spectrum of ABDA alone under the irradiation of white light and the absorption spectrum and the decomposition rate constant of ABDA under the existence of photosensitizers TBTCP-PMB and RB are measured; wherein A is ₀ The initial absorbance value of the ABDA at 378nm, and A is the absorbance value of the ABDA at 378nm at different irradiation times;

FIG. 8 is a graph showing cytotoxicity assays of photosensitizer, phage, and "AIE engineered phage" prepared in CCK8 test example 1;

FIG. 9 is a chart showing a cytotoxicity test of "AIE engineered phage" formed by the photosensitizer and phage prepared in example 1, tested by erythrocyte hemolysis;

FIG. 10 is a chart showing cytotoxicity assays of "AIE engineered phage" mixtures of photosensitizers and phage prepared in example 1 using flow cytometry;

FIG. 11 is a graph showing fluorescence spectra measured after co-culturing the photosensitizer prepared in example 1 with phage;

FIG. 12 is a diagram showing the selective imaging (strategy demonstration+experiment) of photosensitizers and phage formation "" AIE engineered phage "prepared in example 1 for host bacteria;

FIG. 13 is a graph showing statistics of bacterial viability measured by plate coating;

the photosensitizers, phages and "AIE engineered phages" prepared in example 1 were used for in vitro photodynamic antibiosis of four pathogens in a common pathogen model for sepsis, including plate-coating and bacteria-counting;

FIG. 14 is a chart showing the statistics of bacterial viability measured by fluorescence imaging;

bacteriofluorescence imaging of four pathogens for sepsis, including imaging and fluorometric bacterial viability profiles, respectively, of phage and "AIE engineered phage";

FIG. 15 is a graph showing the morphology analysis of bacteria observed by a scanning electron microscope and a transmission electron microscope;

Wherein the photosensitizer prepared in example 1 and the AIE engineered phage are used for in vitro photodynamic inactivation of four pathogens in a sepsis model, respectively;

FIG. 16 is a strategy demonstration of phage cocktail therapy showing construction of a mouse sepsis model;

FIG. 17 is a photograph showing the treatment of a photosensitizer prepared in example 1 with phage to form an "AIE engineered phage" for sepsis model;

wherein, the method comprises the steps of carrying out bacterial count analysis graphs by a mouse form and a flat coating method after different treatment modes;

figure 18 shows statistical graphs of survival and body weight for the first eight days of differently treated mice;

FIG. 19 is a photograph showing histological analysis of mouse organs (heart, liver, spleen, lung, kidney, small intestine and large intestine) in a sepsis model;

FIG. 20 is a graph showing H & E staining analysis of mouse organs (heart, liver, spleen, lung, kidney, small intestine and large intestine) in a sepsis model; black is a scale (200 μm);

FIG. 21 is a graph showing the evaluation of tissue damage of organs (heart, liver, spleen, lung, kidney, small intestine and large intestine) in mice in a sepsis model;

FIG. 22 is a graph showing an analysis of immune response in mice with multifactorial sepsis;

including the determination of cytokines and inflammatory factors.

Detailed Description

The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The method takes a compound A and a compound B as raw materials, and reacts in an alcohol solution of strong alkali to obtain a compound C which is taken as a skeleton of a final compound I; then, the compound C and the compound D react in the presence of a palladium catalyst and inorganic base to obtain a compound E; reacting the compound E with a compound F to obtain a compound G; compound G reacted with compound H to give the final product: the specific reaction process of the compound I with aggregation-induced emission performance is shown in figure 1.

Example 1

A bacteriophage with a fluorescent label prepared by the method comprising: incubating phage and photosensitizer with aggregation-induced emission performance in water for 1 hr;

wherein, the preparation method of the compound with aggregation-induced emission performance comprises the following steps:

(1) 4.7g of 5-bromothiophene-2-carbaldehyde (Compound A) ₁ ) 5.0g of 2- (5-bromothiophene 2-) acetonitrile (Compound B) ₁ ) 1.3g of sodium methoxide and 50mL of methanol are added into a 100mL round bottom flask, the mixture is stirred at room temperature to react for 24 hours to precipitate a large amount of solids, the crude product obtained by filtration is further separated and purified by a silica gel chromatographic column, and 6.5g of yellow solid is obtained by using methylene dichloride as eluent: e-2, 3-bis (5-bromothiophene) acrylonitrile (Compound C) ₁ ) The yield is as follows: 69%;

the reaction formula of this step is as follows:

(2) 750.2mg of E-2, 3-bis (5-bromothiophene) acrylonitrile (2.0 mmol, compound C) ₁ ) 245.8mg of 4-pyridineboronic acid (2.0 mmol, compound D) ₁ ) And 2.76g of potassium carbonate (2.0 mmol) are added into a mixed solvent of 60mL of tetrahydrofuran and 10mL of water, the mixture is stirred and displaced for 30min under the protection of argon gas at room temperature, 46.2mg of tetraphenylpalladium phosphate (0.04 mmol) is added, the mixture is stirred for 5min under the protection of argon gas at room temperature, the temperature is raised to reflux reaction for 12h, 50mL of dichloromethane is added for extraction for three times after the reaction liquid is cooled to room temperature, the organic phase is combined, washed for three times with saturated saline solution, dried over anhydrous sodium sulfate, and the organic solvent is removed by reduced pressure distillation,the crude product is separated and purified by silica gel column chromatography, and the eluent is methylene dichloride: ethyl acetate=10:1 (volume ratio), giving 568mg of a yellowish brown solid as compound E ₁ Yield: 76%;

the reaction formula of this step is as follows:

(3) 90mg (E) -3- (5-bromothiophene-2-) -2- (5- (pyridine-4-) thiophene-2-) acrylonitrile (0.24 mmol, compound E) ₁ ) 89.6mg of 4-diphenylamino-phenylboronic acid (0.31 mmol, compound F) ₁ ) And 331.7mg of potassium carbonate (2.4 mmol) are added into a mixed solvent of 20mL of tetrahydrofuran and 3mL of water, the mixture is stirred and displaced for 30min under the protection of argon, 6mg of tetraphenylpalladium phosphate (0.005 mmol) is added, the mixture is stirred for 5min under the protection of argon, the temperature is raised to reflux reaction for 12h, 20mL of dichloromethane is added for extraction for three times after the reaction liquid is cooled to the room temperature, the organic phase is combined, the organic phase is washed for three times by saturated saline water, anhydrous sodium sulfate is dried, the organic solvent is distilled off under reduced pressure, the crude product is separated and purified by silica gel column chromatography, and the eluent is dichloromethane: ethyl acetate=10:1, giving 107.5mg of red solid as compound G ₁ Yield: 83.3%;

compound G ₁ Nuclear magnetic resonance spectrum and high resolution mass spectrum data of (c): ¹ H NMR(600MHz,DMSO-d ₆ )8.63(d,2H,J＝6.6Hz),8.03(s,1H),7.94(d,1H,J＝3.6Hz),7.81(d,1H,J＝3.6Hz),7.71(dd,2H,J＝1.8Hz,J＝4.8Hz),7.60(dd,2H,J＝1.8Hz,J＝6.6Hz),7.47(d,1H,J＝4.2Hz),7.41(d,1H,J＝3.6Hz,),7.36-7.34(m,4H),7.12-7.07(m,6H),6.98(dd,2H,J＝1.8Hz,J＝6.6Hz). ¹³ C NMR(150MHz,DMSO-d ₆ )170.6,167.6,166.7,164.7,164.1,158.6,156.1,155.8,151.8,149.7,149.2,148.3,147.3,146.6,146.3,144.7,144.5,143.9,143.8,142.4,141.8,139.7,136.7,122.2.HRMS(ESI):m/z[M] ⁺ calculated for C ₃₄ H ₂₃ N ₃ S ₂ :537.1333；found 537.1340.

the reaction formula of this step is as follows:

(4) Into a 50mL round bottom flask was charged 80.1mg (E) -3- {5- [4- (diphenylamino) phenyl ]]Thiophene-2- }2- [5- (pyridine-4-) thiophene-2-)]Acrylonitrile (0.149 mmol, compound G) ₁ ) 20mL of acetonitrile followed by 393.3mg of 1, 4-bis (bromomethyl) benzene (1.49 mmol, compound H) ₁ ) Adding the mixture into the solution, carrying out reflux reaction for 16h under the protection of argon, removing the organic solvent by reduced pressure distillation, and separating and purifying the crude product by silica gel column chromatography, wherein the eluent is dichloromethane: methanol=10:1, yielding 81.8mg of compound I as a dark red solid ₁ The yield was 68.5%.

Compound I ₁ Nuclear magnetic resonance spectrum and high resolution mass spectrum data of (c):

¹ H NMR(400MHz,DMSO-d ₆ )δ.9.11(t,2H,J＝4.2Hz),8.43(d,2H,J＝4.8Hz),8.33(d,1H,J＝3.2Hz),8.10(s,1H),7.90(d,1H,J＝2.8Hz),7.62–7.60(m,2H),7.55–7.48(m,6H),7.35(t,4H,J＝5.6Hz),7.14-7.10(m,6H),6.98(d,2H,J＝4.8Hz),5.81(s,2H),4.78(s,1H),4.72(s,1H). ¹³ C NMR(150MHz,DMSO-d ₆ )δ153.0,152.5,151.8,151.1,150.2,148.9,144.8,141.0,139.7,137.8,137.7,136.0,135.3,135.0,134.9,134.7,134.1,131.9,131.3,131.2,130.0,129.3,129.1,128.5,127.5,121.5,109.9,67.2,38.8.HRMS(ESI):m/z[M-Br ^- ] ⁺ calculated for C ₄₂ H ₃₁ B _r N ₃ S ₂ :720.1137；found:720.1138.

the reaction formula of this step is as follows:

example 2

Compound TBTCP (Compound I) with aggregation-induced emission performance ₂ ) The preparation process of (2) is similar to that of example 1, except that compound H in step (4) is reacted with ₁ 1, 4-bis (chloromethyl) benzene (Compound H) ₂ ) The experimental step (4) is operated as follows:

(4) Into a 50mL round bottom flask was charged 80.1mg (E) -3- {5- [4- (diphenylamino) phenyl ]]Thiophene-2- }2- [5- (pyridine-4-) thiophene-2-)]Acrylonitrile (0.149 mmol, compound G) ₁ ) 20mL of acetonitrile, then 260.8mg of 1, 4-bis (chloromethyl) benzene (1.49 mmol, compound H ₂ ) Adding the mixture into the solution, carrying out reflux reaction for 48 hours under the protection of argon, removing the organic solvent by reduced pressure distillation, and separating and purifying the crude product by silica gel column chromatography, wherein the eluent is dichloromethane: methanol=10:1, giving 64.6mg of compound I as a solid ₂ The yield was 60.8%.

Compound I ₂ The structural characterization data of (2) are: ¹ H NMR(400MHz,DMSO-d ₆ )δ9.26–8.84(m,2H),8.58–8.30(m,2H),7.89(s,1H),7.78(d,1H,J＝6.8Hz),7.57–7.34(m,9H),7.33–7.19(m,6H),7.16–7.08(m,4H),7.04(ddt,2H,J＝9.0,7.4,1.4Hz),6.11(d,2H,J＝1.0Hz),4.56(t,2H,J＝1.0Hz). ¹³ C NMR(150MHz,DMSO-d ₆ )δ146.8,146.6,145.1,142.3,142.0,142.0,138.6,136.4,136.3,134.8,134.7,134.5,129.6,128.9,128.8,128.7,127.7,127.5,126.7,126.1,124.7,124.3,123.6,121.4,115.0,105.7,64.2,46.1.HRMS(ESI):m/z[M-Cl ^- ] ⁺ calculated for C ₄₂ H ₃₁ ClN ₃ S ₂ :676.1642；found:676.1640.

the reaction formula of this step is as follows:

example 3

Compound TBTCP-PDI (compound I) with aggregation-induced emission performance ₃ ) The preparation process of (2) is similar to that of example 1, except that compound H in step (4) is reacted with ₁ 1, 3-diiodopropane (Compound H) ₃ ) The experimental step (4) is operated as follows:

(4) Into a 50mL round bottom flask was charged 80.0mg (E) -3- {5- [4- (diphenylamino) phenyl ]]Thiophene-2- }2- [5- (pyridine-4-) thiophene-2-)]Acrylonitrile (0.149 mmol, compound G) ₁ ) And 20mL of acetonitrile, then 440.9mg of 1, 3-diiodopropane (1.49 mmol, compound H) ₁ ) Adding the mixture into the solution, carrying out reflux reaction for 24 hours under the protection of argon, removing the organic solvent by reduced pressure distillation, and separating and purifying the crude product by silica gel column chromatography, wherein the eluent is dichloromethane: methanol=10:1, giving 69.2mg of solid as compound I ₃ The yield was 55.7%.

Compound I ₃ The structural characterization data of (2) are: ¹ H NMR(400MHz,DMSO-d ₆ )δ9.11–8.81(m,2H),8.50–8.27(m,2H),7.89(s,1H),7.78(d,1H,J＝6.8Hz),7.60–7.36(m,5H),7.34–7.19(m,6H),7.20–7.09(m,4H),7.04(ddt,2H,J＝9.0,7.4,1.4Hz),4.59(t,2H,J＝5.6Hz),3.30(t,2H,J＝6.4Hz),2.65–2.54(m,2H). ¹³ C NMR(150MHz,DMSO-d ₆ )δ147.0,147.0,146.8,146.1,139.4,139.1,139.1,136.9,135.5,133.5,129.5,128.8,128.8,127.8,127.6,125.1,124.8,124.7,124.5,123.8,123.0,121.1,115.2,105.7,60.5,34.0,3.2.HRMS(ESI):m/z[M-I ^- ] ⁺ calculated for C ₃₇ H ₂₉ IN ₃ S ₂ :706.0842；found:706.0844.

the reaction formula of this step is as follows:

example 4

Compound TBTCP-PMB (compound I) with aggregation-induced emission performance ₄ ) The preparation process of (2) is similar to that of example 1, except that compound F in step (3) ₁ Modification to 4-bis (4-methoxyphenyl) amino-phenylboronic acid (Compound F) ₂ ) The experimental step (3) is operated as follows:

(3) 90mg (E) -3- (5-bromothiophene-2-) -2- (5- (pyridine-4-) thiophene-2-) acrylonitrile (0.24 mmol, compound E) ₁ ) 108.3mg of 4-bis (4-methoxyphenyl) amino-phenylboronic acid (0.31 mmol, compound F) ₄ ) And 331.7mg of potassium carbonate (2.4 mmol) were added to a mixed solvent of 20mL of tetrahydrofuran and 3mL of water, and the mixture was stirred and displaced at room temperature under argon atmosphere for 30min. 6mg of tetraphenylpalladium phosphate (0) was added005 mmol) under argon, stirring at room temperature for 10min, and heating to reflux for 12h. The reaction mixture was cooled to room temperature, extracted three times with 20mL of dichloromethane, and the organic phases were combined, washed three times with saturated brine, and dried over anhydrous sodium sulfate. The organic solvent is removed by reduced pressure distillation, the crude product is separated and purified by silica gel column chromatography, and the eluent is methylene dichloride: ethyl acetate=10:1, giving 107.6mg of red solid as compound G ₂ Yield: 75%. The reaction formula of this step is as follows:

(4) 89.1mg of (E) -3- {5- [ 4-bis (4-methoxyphenylamino) -phenyl ] are placed in a 50mL round bottom flask]Thiophene-2- }2- [5- (pyridine-4-) thiophene-2-)]Acrylonitrile (0.149 mmol, compound G) ₂ ) 20mL of acetonitrile followed by 393.4mg of 1, 4-bis (bromomethyl) benzene (1.49 mmol, compound H) ₁ ) Adding the mixture into the solution, carrying out reflux reaction for 16h under the protection of argon, removing the organic solvent by reduced pressure distillation, and separating and purifying the crude product by silica gel column chromatography, wherein the eluent is dichloromethane: methanol=10:1, giving 85.2mg of solid compound as compound I ₄ The yield was 66.3%.

Compound I ₄ The structural characterization data of (2) are: ¹ H NMR(400MHz,DMSO-d ₆ )δ9.05–8.96(m,2H),8.48–8.38(m,2H),7.89(s,1H),7.78(d,1H,J＝6.8Hz),7.54(d,1H,J＝6.8Hz),7.53–7.47(m,2H),7.46(dd,2H,J＝6.8,2.2Hz),7.39(dt,2H,J＝8.0,1.0Hz),7.31–7.22(m,4H),7.18–7.11(m,4H),6.95–6.89(m,4H),6.11(t,2H,J＝1.0Hz),4.53(t,2H,J＝1.0Hz),3.78(s,6H). ¹³ C NMR(150MHz,DMSO-d ₆ )δ158.5,146.5,145.1,142.3,142.0,142.0,140.5,138.6,137.4,136.3,134.8,134.5,134.3,129.7,128.7,128.6,127.7,127.5,126.7,126.1,123.9,123.6,121.4,115.0,114.4,105.7,64.2,55.4,35.9.HRMS(ESI):m/z[M-Br ^- ] ⁺ calculated for C ₄₄ H ₃₅ B _r N ₃ O ₂ S ₂ :780.1349；found:780.1350.

the reaction formula of this step is as follows:

the test results of example 1 of the present invention are taken as examples to illustrate the compounds produced by the present invention, and other examples are not listed one by one since they react the same as the test results of example 1.

1. UV-visible absorption spectra in DMSO solutions

The compound TBTCP-PMB prepared in example 1 was tested for uv-vis absorption spectrum in solvent DMSO. As shown in FIG. 2, TBTCP-PMB has an absorption in DMSO ranging from 300nm to 700nm with a maximum absorption peak at 522nm.

2. Fluorescence spectra in both DMSO and Toluene solutions

The compound TBTCP-PMB prepared in example 1 was tested for fluorescence spectra in two solutions of DMSO and tolene, as shown in FIG. 3, the wavelength and intensity of the compound emission in different solvents were significantly different, and the compound showed strong emission at 725nm in the tolene solvent with a fluorescence Quantum Yield (QY) of 17.9%; whereas in DMSO solvents, very weak emission around 560nm was shown, fluorescence quantum yields were as low as 0.11%.

3. Fluorescence quantum yield determination

The compound TBTCP-PMB prepared in example 1 was assayed for fluorescence quantum yield in solvent (DMSO, tolutene). Measurement of fluorescence quantum yield of samples with cresyl violet (fluorescence quantum yield in ethanol Φ=0.53) as a reference, the fluorescence quantum yield was calculated by the following formula:

Φ _X ＝Φ _S (A _S ×F _X /A _X ×F _S )(n _x /n _s ) ²

in the formula ,Φ_X Fluorescence quantum yield as photosensitizer, A _X and A_S Respectively representing the absorbance of the sample to be tested and the standard substance at the excitation wavelength; f (F) _X and F_S Integrating fluorescence areas of the sample to be detected and the standard substance; n is the refractive index of the solvent; subscripts s and x represent reference and unknown samples, respectively.

4. Determination of relative fluorescence intensity in DMSO and Toluene Mixed System

The compound TBTCP-PMB prepared according to example 1 was dissolved in DMSO and slightly dissolved in tolene, and its AIE properties were measured using tolene and DMSO as poor solvents and good solvents, respectively. The Toluene and DMSO solvents were mixed with 3mL of each of the mixed solvents of different proportions from 0% to 98% of Toluene in an EP tube and 6. Mu.L of TBTCP-PMB stock solution was taken and the final concentration of the solution was 10. Mu.M by shaking, and the Duetta fluorescence spectrometer was used for the sequential fluorescence measurement. As shown in fig. 4, the excited state energy in DMSO solution is dissipated mainly in a non-radiative pathway without fluorescence due to intramolecular motion. As the tolene content in the mixed solution increased to 80%, the fluorescence intensity increased significantly and blue shift occurred due to the intra-molecular movement (RIM) mechanism limitation. The compound TBTCP-PMB has an emission intensity increased up to 172 times in tolene. This experiment demonstrates that the class of compounds has typical AIE properties.

5. Dynamic light scattering analysis

The particle size distribution of the compound TBTCP-PMB prepared in example 1 in an aqueous solution is shown in FIG. 5, the average particle size of the compound is 88.58nm, and the dispersion index Pdi is 0.173.

6. Target molecule active oxygen assay (indicator H2 DCF-DA)

Detecting probe with 2, 7-dichloro-dihydro-fluorescein diacetate (H2 DCF-DA), testing ROS yield of compound TBTCP-PMB in solution, to convert H2DCF-DA into 2, 7-dichloro-dihydro-fluorescein (H2 DCF), adding 0.25mL of H2DCF-DA ethanol solution (1 mM) into 1mL of NaOH (10 mM) aqueous solution, stirring at room temperature for 30min, regulating pH value with 5mL of PBS solution (pH 7.4), freezing and storing the solution for standby, adding DMSO solution of compound into the above solution to obtain final concentration of 2 μm, placing sample in fluorescence spectrometer, automatically testing fluorescence intensity of the solution every 10s (lambda) _ex 488 nm), as shown in FIG. 6, the fluorescence intensity at 525nm was increased with an increase in illumination time, and the fluorescence intensity was increased 51.2 times higher than the initial value at 150 s. The experiment proves that the compound can effectively and rapidly generate ROS under the irradiation of light.

7. Singlet oxygen yield determination (indicator ABDA)

The singlet oxygen production performance of photosensitizer TBTCP-PMB and commercial photosensitizer Bengal Rose Bengal (RB) under light irradiation was examined using ABDA (9, 10-anthryl-bis (methylene) di-malonic acid) as an indicator, when ABDA and ¹ O ₂ In the reaction, the ABDA is oxidized to form a peroxy bridge structure, so that the absorbance value of the ABDA at 378nm is reduced at a speed which can indirectly reflect the photosensitizer under light ¹ O ₂ Yield. The absorbance of the photosensitizers TBTCP-PMB and RB (5. Mu.M) was first set as blank. ABDA (50. Mu.M) was mixed with a solution of photosensitizer (5. Mu.M) under dark conditions and the absorbance values of the solutions were measured immediately, followed by a white light lamp (20 mW/cm ² ) The solution mixture was irradiated and immediately after each irradiation for 1min, the absorbance value of the solution was recorded until the absorbance value did not decrease. As shown in fig. 7, the absorbance value of the white light irradiation 5min ABDA solution is unchanged, the absorbance at 378nm is rapidly reduced to 1.19% after the photosensitizer TBTCP-PMB is added, and the absorbance of RB is only reduced to 47.2% under the same experimental conditions; the decomposition rate of ABDA in the presence of PS-PMB is 6.27 times higher than that of RB. The experiment proves that the compounds can be efficiently produced under illumination ¹ O ₂ And is far superior to RB.

Investigation of biocompatibility by CCK8 method

Cytotoxicity was determined using the standard CCK-8 method. 100. Mu.L of human embryonic lung fibroblasts (MRC-5) and human embryonic kidney cells (HEK-293) were inoculated into 96-well black microplates, respectively, incubated at 37℃until the cell density reached 70-80%, then the medium was replaced with 100. Mu.L of fresh medium (0, 0.2, 0.5, 1 and 2. Mu.M) containing phage, TBTCP-PMB at different concentrations, DMEM was used as a blank control, after which the cells were incubated in the dark or white light (80 mW/cm) ² ) After 20min of irradiation followed by incubation of the cells in a 37℃incubator for 24 hours, 10. Mu.L of CCK-8 mixture (CCK-8: PBS=1:9) was added to each well and incubated for 2h, and the absorbance OD at 450nm was measured with a microplate reader ₄₅₀ The relative cell viability was calculated as follows:

cell viability (%) = (OD _{450 sample} /OD _{450 control} )×100％

As can be seen from FIG. 8, the cell viability was nearly 100% when incubated with normal cells, either in the dark or in the light, in the presence of phage or TBTCP-PMB alone. A slight decrease in cell viability occurred with increasing concentration of "AIE engineered phage", with cell viability higher than 90% at a concentration of 2 μm in dark conditions; cell viability was still higher than 80% at a concentration of 2 μm under light conditions. The experiments prove that both TBTCP-PMB and the AIE engineering phage have better biocompatibility.

9. Haemolysis assay to explore biocompatibility

Fresh mouse blood was collected and heparin sodium was added to prevent blood clotting by 5×10 ³ The erythrocytes were collected by centrifugation at rpm for 10 min, then resuspended in PBS, 50. Mu.L of the erythrocyte suspension were mixed with 150. Mu.L of PBS solution containing varying concentrations of TBTCP-PMB (0.08, 0.5, 1 and 2. Mu.M), incubated at 37℃for 30 min and then incubated at 2X 10 ³ Centrifugal at rpm for 10 min, OD of the supernatant was measured by an enzyme-labeled instrument ₅₄₀ Untreated erythrocyte suspension served as negative control, sterile water served as positive control, PBS served as solvent control.

Hemolysis ratio (%) = (OD _{540 sample} /OD _{540 control} )×100％

As shown in fig. 9, the red blood cells of the positive control group are broken in a large amount, the solution is dark red, and the hemolysis rate is as high as 100%; the solution of the blank control PBS group is clear, the liquid in the 96-well plate is still clear when the concentration of the experimental group TBTCP-PMB is up to 2 mu M, the erythrocyte does not have obvious hemolysis phenomenon, the hemolysis rate is almost equal to that of the PBS group, and the hemolysis rate is obviously different from that of the positive control group.

10. Flow cytometry investigation of biocompatibility

This study used Annexin V-FITC/PI apoptosis detection kit (Vazyme Biotech, nanjin, china) human embryonic kidney cells (HEK-293) were seeded into six well plates and at 37℃with 5% CO ₂ Culturing overnight in incubator, removing culture medium, gently washing cells with PBS, adding serum-free culture medium containing TBTCP-PMB (2 μm) to the cells, incubating for 30min, and taking the serum-free culture medium as blank control, wherein both groups of cells are irradiated with dark or white light (80 mW/cm) ² ) Treating for 20min, and discarding cultureAdding appropriate amount of pancreatin digested cells without EDTA into 6-well plate, waiting a certain time, adding fresh culture medium to stop digestion, collecting cells, and collecting 1×10 cells ³ Centrifugation at rpm for 5min, discarding supernatant, resuspending the collected cells in PBS and counting, and resuspending 50,000-100,00 cells at 1X 10 ³ After centrifugation at rpm for 5 minutes, the supernatant was discarded, 195. Mu.L of Annexin V-FITC conjugate, 5. Mu.L of Annexin V-FITC and 10. Mu.L of Propidium Iodide (PI) stain were added sequentially, gently mixed, the cells were incubated at room temperature for 10-20 minutes, and then examined with CytoFLEX (Beckman-Coulter, USA) and FITC: lambda _ex ＝375nm,λ _em ＝400-500nm.PI:λ _ex ＝488nm,λ _em =590-630 nm. In the flow cytometry, the proportion of the cell population in different quadrants is analyzed according to the double-dyeing experimental result, the cells in the first quadrant belong to necrotic cells, the cells in the second quadrant belong to cells in late apoptosis, the cells in the third quadrant belong to cells in early apoptosis, and the cells in the fourth quadrant belong to living cells. As shown in fig. 10, the cell viability was greater than 90% in both the light-irradiated and dark conditions, the necrosis was less than 9%, and the apoptosis was negligible, both in the blank and in the treated groups, which demonstrated good biocompatibility of TBTCP-PMB.

"AIE engineered phage" emission spectroscopy study

Determination of the emission spectra the photosensitizers TBTCP-PMB were examined for success in constructing "AIE engineered phages" after one hour of co-incubation with phages P-E.coli (E.coli), P-P.aeroginosa (P.aeroginosa), P-MRSA (MRSA) and P-S.tyrphinium (S.tyrphinium), respectively, in water. As shown in FIG. 11, photosensitizer TBTCP-PMB and all phages present in solution PBS alone exhibited weak fluorescence emission; the phenomenon that the emission at 650nm was significantly enhanced and the emission peak blue shifted after one hour of co-culture of the photosensitizer with phage, suggests that "AIE engineered phage" has been successfully constructed. This experiment demonstrates that the photosensitizer can be successfully combined with four phages, respectively, to form an engineered phage.

12. Bacterial fluorescence imaging explores specific recognition of host bacteria by "AIE engineered phages

The ability of the "AIE engineered phage" to specifically recognize host bacteria was determined using a laser confocal microscope (CLSM). All bacteria were first stained green with a green nucleic acid dye NucGreen, then co-cultured with the corresponding "AIE-engineered phage" which was found to label the corresponding host bacterial membrane to show red fluorescence, in order to further explore the specificity of the "AIE-engineered phage" for host bacteria, s.mutans was stained green only by NucGreen and no red fluorescence against streptococcus mutans (s.mutans); when 4 bacteria (E.coli, P.aeromonas, MRSA and S.tyrsimurium) were co-cultured with control bacteria (S.mutans), respectively, and incubated with "AIE engineered phage" (TBTCP-PMB+P-E.Coli, TBTCP-PMB+P-S.tyrsimurium, TBTCP-PMB+P-P.aeromonas and TBTCP-PMB+P-MRSA), only the corresponding host bacteria E.coli, P.aerospor, MRSA and S.tyrsimurium showed bright red fluorescence, as shown in FIG. 12. Bacterial fluorescence imaging experiments have shown that "AIE engineered phage" can be used as a biological probe for fluorescent imaging identification of host bacteria.

13. Plate counting method to explore the in vitro antibacterial Activity of "AIE engineered phages

In view of the high ROS and ability of the "AIE engineered phage" to specifically recognize both the host bacterium and the photosensitizer TBTCP-PMB, the phage ¹ O ₂ We explored the photodynamic antibacterial activity of "AIE engineered phage" specific for killing host bacteria using plate counting. As shown in fig. 13, bacteria on the plates in the PBS group were not subjected to any treatment, and were set as a blank group, and the colony appearance was reduced after the corresponding phage was sequentially added in four experimental groups, wherein the survival rate of bacteria e.coll p.aeromonas, MRSA and s.tyrphinium was lower than 60%; meanwhile, the single photosensitizer TBTCP-PMB can be combined with bacteria through electrostatic action and hydrophobic action, the four bacteria can be killed, a large amount of active oxygen can be generated under light irradiation to enhance photodynamic antibacterial activity, and the survival rate of the bacteria is about 30% -50%. Incubating the four bacteria with the corresponding "AIE engineered phage" to obtain a plate with a significantly lower colony count than the photosensitizer-added panel, wherein S.tyrphiminum, P.aeromonasAnd bacterial viability in MRSA was less than 35%, the group was exposed to (80 mW/cm ² ) White light is irradiated for 20min, colonies of the four bacteria are hardly observed on the flat plate, and the bacterial survival rate is lower than 1%. The experiment shows that the AIE engineering phage has optimal in vitro photodynamic antibacterial activity.

14. The kit explores the in vitro synergistic antibacterial activity of AIE engineering phage

LIVE/DEAD is selected ^TM The kit further evaluates the synergistic antibacterial properties of the "AIE engineered phage" against bacteria. As shown in fig. 14, without photosensitizer TBTCP-PMB and phage, all bacteria showed strong green fluorescence, almost all bacteria exhibited red fluorescence after incubation with the corresponding "AIE engineered phage", with mortality of bacteria e.coll and MRSA up to 95%, while bacteria red fluorescence treated with phage only accounted for only 5% -20% of all bacteria. This experiment shows that the therapeutic effect of the "AIE engineered phage" is far superior to phage and photosensitizer TBTCP-PMB used alone.

15. Scanning Electron Microscope (SEM) investigation of bacterial morphology

mu.L of photosensitizer TBTCP-PMB (160. Mu.M) and 999. Mu.L of phages (P-S.tyrlimurum, P-MRSA, P-P.aeromonas and P-E.coli) were placed in a thermostatic waterbath at 37℃for 1h to construct "AIE-engineered phages", and these four "AIE-engineered phages" were then mixed with the corresponding host bacteria and placed in a thermostatic incubator at 37℃for 30 minutes, after which they were incubated using (80 mW/cm ² ) The white light is irradiated for 20 minutes, then the bacteria form change is observed after a series of operations such as fixing by 2.5% glutaraldehyde fixing solution, ethanol gradient dehydration, natural drying, ion sputtering metal spraying and the like, a control group and a blank group are arranged in the experiment, wherein the control group can not form AIE engineering phage without photosensitizer, the blank group only contains 4 host bacteria, and the two groups are consistent with the operation of the experiment group.

From FIG. 15, the host bacterial cell walls in the untreated blank were smooth and intact, and the control group was irradiated with light after adding the photosensitizer TBTCP-PMB and leaving depressions in some of the host bacterial cell walls, while all of the host bacterial cell walls were collapsed and surface roughness was irregular after adding the "AIE-engineered phage" and being irradiated with white light, and the cell membranes were severely damaged. This experiment demonstrates that the constructed "AIE engineered phage" can selectively act on host bacteria and inactivate them efficiently after light irradiation.

16. Investigation of bacterial morphology by Transmission Electron Microscopy (TEM)

A series of operations such as grouping and initial incubation illumination are consistent with SEM, after bacteria are fixed by 2.5% glutaraldehyde and 1% osmium tetroxide are fixed for 3 hours, each time the bacteria are washed 3 times by fixed PBS, then the bacteria are dehydrated by gradient of ethanol (30%, 50%,70%, 90%, 95% and 100%) with different concentrations for 15 minutes, then the bacteria are dehydrated by acetone twice and 15 minutes each time, finally the bacteria are embedded and ultrathin sections are embedded in graded Spur 812 epoxy resin (30%, 50%,70% and 100%), and the bacteria are respectively subjected to double staining by 2% uranyl acetate and lead citrate for 15 minutes and then observed by a transmission electron microscope, and as shown in figure 15, after the AIE engineering phage is incubated with host bacteria and irradiated by white light, the outer surfaces of the host bacteria are obviously damaged, cell walls are blurred and vacuoles appear.

Antibacterial Activity of "AIE engineered phage" in vivo

The experiment was divided into a control group, a treatment group and a no-treatment group. Control group: the sepsis model is not constructed and has a treatment process; treatment-free group: constructing a sepsis model alone without a therapeutic course; treatment group: both a sepsis model and a treatment course are constructed, wherein the sepsis model is constructed: 100. Mu.L of the bacterial mixture (OD) ₆₀₀ =0.6, e.coll, p.aerobacking, mrsa and s.tyrphiminuium each 25 μl) into mouse blood.

Wherein, the treatment course is white light irradiation (three times a day) after the mice blood is incubated with AIE engineering phage in vitro, as shown in FIG. 17, compared with the control group, the untreated group mice have a great number of typical sepsis symptoms such as ecchymosis, erythema and the like, and die completely on the 4 th day; the symptoms of ecchymosis and erythema of mice in the treatment group are obviously reduced along with the extension of time, most mice still survive on the 8 th day, meanwhile, the bacterial quantity in the blood of the mice is monitored in real time by adopting a plate counting method, a sepsis model is not constructed in a control group, and no colony is observed on a plate; mice in the experimental group were injected with bacteria (e.coll, p.aerobosa, MRSA and s.tyrphiminuium) and a large number of bacteria appeared on the plates on day 1 both in the treated group and in the untreated group, with an increased number of bacteria in the blood on

days

2 and 4; bacteria numbers in the blood of the treatment group decreased sharply at day 4, with all disappearing at day 8.

The survival rate and weight change of the three groups of mice were examined, and as shown in fig. 18, the control group mice survived all, and the weight was steadily increased; untreated mice continued to lose weight and all died on day 4, treated mice did not die until day 8, the survival rate was higher than 60% on day 8, and the mice had a tendency to lose weight three days before the group, and body weight gradually increased from day 4 to day 8 of treatment.

18. Histological analysis of organs

By histological analysis of organs such as heart, liver, spleen, lung, kidney, small intestine and large intestine of three groups of mice, as shown in fig. 19, untreated groups of mice appeared multi-organ failure, heart, liver, spleen, lung, kidney, small intestine organs became dark in color, even dark red, blood vessel embolism was found in large intestine, ischemia, whitening was caused, and treated groups of only heart, spleen two organs were slightly darker in color than normal groups of mice.

19. Hematoxylin-eosin staining

In addition, hematoxylin-eosin (H & E) staining was also used to evaluate the pathological status of the different organs. As shown in fig. 20, a large number of inflammatory cells were seen in the cardiac interstitium, severe vacuolation was seen in liver tissue, and a large number of extramedullary hematopoiesis was seen in the spleen parenchyma in untreated mice. In addition, the lung tissue structure is disordered, erythrocytes are diffused, and the kidney tissue ischemia causes Acute Tubular Necrosis (ATN), and the intestinal tissue skin falls off. The visceral lesions of the treatment group are all improved, for example, inflammatory cells are obviously reduced, hollowness is obviously improved, extramedullary hematopoiesis of spleen disappears, lung tissue structure is normal, glomerular degeneration is improved, intestinal tissue mucous layer goblet cell structure is normal, and histological characteristics are complete.

20. Blood routine analysis of septicemic mice

To verify the results of the in vivo antibacterial activity of "AIE engineered phage", blood routine analysis was performed on septic mice. As shown in tables 1-3 below and fig. 21, the concentration of hemoglobin in the blood of untreated mice was significantly higher than the normal range, and increased hemoglobin tended to result in increased blood volume and blood viscosity, which increased the probability of vascular embolism and atherosclerotic plaque. In addition, the number of leukocytes and neutrophils in the blood of untreated mice was also significantly higher than normal, and these increases in index were often associated with the phenomena of Disseminated Intravascular Coagulation (DIC), central nervous system vascular congestion, and septic shock. The study shows that the tissue appearance of each organ of the mice in the treatment group is close to that of the mice in the control group, the blood routine index is in a normal range, and the result of the blood routine analysis is consistent with the experimental results of histological analysis, plate bacteria count and the like.

Table 1，Control

Table 2，WithoutTreatment

Table 3.With Treatment

21. Multi-factor detection of immune response in septic mice

Exogenous pathogens invade and bind to receptors on the surface of immune cells, which activate the immune response of the body. This process releases a variety of cytokines and inflammatory factors that accelerate the inflammatory process in the body, thereby exacerbating the sepsis condition. To understand the role of "AIE engineered phage" in related inflammation, we performed an inflammatory factor analysis. Tumor necrosis factor (TNF- α), interleukin (IL-1 β) and interleukin-6 (IL-6) are key cytokines that cause the inflammatory response of sepsis myocardial injury. Wherein IL-1 beta, IL-6 exacerbate tissue damage by neutrophil infiltration. In addition, IL-9 is also closely related to injury and death of the intestinal mucosal barrier in septic patients. As shown in FIG. 22, the expression of IL-6 was increased in the treated group compared with the control group, and the expression results of the remaining inflammatory factors were similar, whereas the overexpression occurred in the inflammatory factors IL-17A, IL-1β, IL-10, IL-17A, TFN- α and TFN- γ in the untreated group. The experiment shows that the AIE engineering phage can effectively control inflammatory factors.

All animal experiments above show that the photosensitizer can rapidly identify host bacteria after entering blood with the "AIE engineered phage" constructed by incubating the photosensitizer with four phages. The engineering phage combines the targeting and killing effects of phage on host bacteria and multiple functions of efficiently generating ROS (reactive oxygen species) for efficient inactivation of bacteria under illumination, so that the engineering phage has good biocompatibility and realizes rapid identification and efficient inactivation of host bacteria; it is also possible to further inhibit inflammatory factors, improve tissue fibrosis, and facilitate cell proliferation, angiogenesis and tissue repair, thereby promoting healing and improvement of sepsis in mice.

In summary, a series of compounds with aggregation-induced emission properties were designed and successfully synthesized in the present application, and nucleophilic substitution/addition reactions of these compounds with thiol groups in phage structures were performed to obtain fluorescently labeled phage, i.e. "AIE engineered phage". The engineering phage can be used for rapidly identifying the pathogen of the sepsis so as to realize visual fluorescence detection of the sepsis, and can also carry out visual monitoring on the whole treatment process, thereby providing more accurate and practical information for us and optimizing and improving the treatment effect; more importantly, the marked phage can kill bacteria through phage cocktail therapy, and can also kill bacteria through photodynamic therapy, so that the combination therapy of phage cocktail therapy and photodynamic therapy is realized, a better therapeutic effect is achieved, and the problems that the existing phage cocktail therapy is low in antibacterial activity and is difficult to realize rapid diagnosis and treatment of pathogenic bacteria are effectively solved.

Based on the information contained herein, it will be apparent to those skilled in the art that various changes may be made in the precise description of the invention without departing from the spirit or scope of the following claims. The subject matter of the present invention is not limited to the steps, properties and compositions defined herein, as these preferred embodiments and other descriptions are intended to illustrate various specific aspects of the invention. Indeed, various modifications of the described examples which fall within the scope of the claims may be made by those skilled in the chemical and biochemical arts.

Claims

1. A fluorescence covalent labeling method of phage is characterized in that phage and a compound with aggregation-induced emission performance are incubated together in water; the phage contains sulfhydryl, and in the incubation process, the compound with aggregation-induced emission performance and sulfhydryl in the phage structure undergo nucleophilic substitution/addition reaction; the chemical structural general formula of the compound with aggregation-induced emission performance is as follows:

wherein ,

/>

R ₂ ：

；

x is O, S or Se;

2. The fluorescent covalent labeling method of phage of claim 1, wherein the compound with aggregation-induced emission properties has E-2, 3-bis (5-bromothiophene) acrylonitrile, E-2, 3-bis (5-bromofuran) acrylonitrile or E-2, 3-bis (5-bromoselenophene) acrylonitrile as a molecular structure backbone by changing R on the molecular structure backbone ₁ Electron donating group in position and R ₂ The group which can be covalently bound with the thiol reaction in the phage surface protein can be used for obtaining the compound with aggregation-induced emission performance.

3. The fluorescent covalent labeling method of phage of claim 2, wherein the specific preparation method of the compound with aggregation-induced emission properties is:

(2) Adding the compound C, the compound D, a palladium catalyst and an inorganic base into a mixed solvent according to the molar ratio of 1 (1.3-3) (0.02-0.1) (3-30), and heating and refluxing until the reaction is complete; then extracting the reaction product by using an extractant to obtain an organic phase, washing the organic phase by using water and saturated saline sequentially, drying by using a drying agent, removing the organic solvent by reduced pressure distillation, and separating and purifying the obtained crude product by using a silica gel column chromatography to obtain a compound E;

(4) Adding a compound G and a compound H into acetonitrile or toluene according to a molar ratio of 1 (3-30), refluxing and stirring under the protection of argon until the reaction is complete, distilling under reduced pressure to remove an organic solvent, washing the obtained solid product with water, filtering under reduced pressure, drying, and separating and purifying by using a silica gel column chromatography to obtain a compound I, namely a compound with aggregation-induced emission performance;

wherein the structural formula of the compound A is

The structural formula of the compound B is as follows: />

The structural formula of the compound C is as follows: />

The structural formula of the compound G is as follows: />

The structural formula of the compound H is as follows: />

Wherein X in the compound A, the compound B, the compound C, the compound E and the compound G is O, S or Se; r in Compound F and Compound G ₁ The method comprises the following steps:

/>

r in Compound H ₂ The method comprises the following steps:

。

4. the fluorescent covalent labeling method of phage of claim 3, wherein the alcoholic solvent in step (1) is at least one of methanol, ethanol and isopropanol; the alkali is sodium hydroxide, potassium hydroxide, sodium methoxide, sodium ethoxide or sodium tert-butoxide; the palladium catalyst in the step (2) and the step (3) is tetra (triphenylphosphine) palladium, di (triphenylphosphine) palladium chloride, [1, 1' -bis (diphenylphosphino) ferrocene ] palladium dichloride, tri (dibenzylideneacetone) dipalladium or palladium acetate; the inorganic base in the step (2) and the step (3) is sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate or barium hydroxide.

5. The fluorescent covalent labeling method of phage of claim 3, wherein the extractant in step (2) and step (3) is dichloromethane, ethyl acetate or chloroform; the drying agent is anhydrous sodium sulfate, anhydrous calcium chloride, anhydrous magnesium chloride or anhydrous magnesium sulfate; the mixed solvent is a mixed solution composed of tetrahydrofuran and water in a volume ratio of (10-5), or a mixed solution composed of toluene, ethanol and water in a volume ratio of (10-6) 1:1, or a mixed solution composed of dioxane and water in a volume ratio of (10-5) 1.

6. Phage with fluorescent markers, characterized in that it is produced by the method according to any one of claims 1 to 5.

7. Use of a bacteriophage with a fluorescent label according to claim 6 for the preparation of a rapid diagnostic kit for pathogenic bacteria.