Tetraarylethene modified rhodamine-like derivative as well as preparation method and application thereof

Abstract

The invention discloses a tetraarylethene modified rhodamine-like derivative as well as a preparation method and application thereof. The rhodamine-like derivative has a structure as shown in a formula I, rhodamine is taken as a core skeleton, a tetraarylethene (TAE) structure with AIE characteristics is introduced into a meso site of the rhodamine, RL-TAE series molecules are obtained, the defect of aggregation-induced quenching (ACQ) is greatly improved through the strategy, the molar absorption coefficient is obviously increased, and the rhodamine-like derivative has a good application prospect. Therefore, a new scheme is provided for developing high-brightness aggregation-induced emission dye. The rhodamine-like derivative disclosed by the invention is simple in preparation method, mild in reaction condition and high in yield, has the characteristics of AIE effect, high molar absorption coefficient and brightness, photobleaching resistance and diamagnetism, is an excellent concentration-brightness correspondence dye, and can realize efficient coding application.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent materials and in vitro diagnostic detection, and specifically relates to a tetraarylethene-modified rhodamine-like derivative, a preparation method and an application thereof. Background Art

[0002] Long-wavelength dyes with maximum absorption wavelengths in the near-infrared spectrum have long been a hot research topic in dye chemistry. The main infrared fluorescent dyes in this field include borofluorinated pyrrole (BODIPY) dyes, squaric acid dyes, cyanine dyes, and rhodamine derivative dyes. Various near-infrared dyes have been widely used in infrared detection, anti-counterfeiting, disease diagnosis, optoelectronic materials and other application fields.

[0003] Rhodamine is a type of rhodamine derivative compound with the advantages of good photostability, high fluorescence quantum yield, and simple chemical modification. It is a good fluorescent small molecule dye probe and is widely used in the staining and fluorescent labeling of biological samples.

[0004] However, rhodamine has many shortcomings in its application as a fluorescent probe, especially when applied to biological samples. For example, the UV-visible light absorption and fluorescence emission of ordinary rhodamine are generally between 550-620nm, and the excitation wavelength of rhodamine B dye is in the UV-visible light region, while biological samples will produce serious signal interference in the UV-visible light region. On the other hand, ultraviolet visible light has poor penetration into tissues and has high phototoxicity to biological samples. These factors greatly affect the sensitivity and accuracy of rhodamine dye probes in fluorescence analysis of biological samples, greatly limiting their further application in fluorescence imaging of biological samples. Therefore, its application in the field of biological imaging has been greatly limited.

[0005] In 2008, Qian Xuhong innovatively used silicon to replace the bridging oxygen atom to obtain the first red-light-emitting silanthracene fluorophore: TMDHS. Compared with the rhodamine skeleton (Pyronine Y, PY), it has strong absorption and emission red shifted by 90nm, and the molar absorption coefficient is 10 5 L·mol -1 cm -1It is twice that of PY. The incorporation of Si has expanded the application and potential of rhodamine fluorophores. However, with the continuous development of technology, in order to meet the requirements of more biological sample analysis and imaging, researchers are driven to pursue a larger red shift while retaining the advantages of rhodamine compounds as much as possible. Researchers have proposed many modification methods. The commonly used methods are to (1) fix the nitrogen end to expand the conjugated system or enhance the rigidity of the molecule; (2) increase the aromatic ring to expand the conjugation. However, when the wavelength is greater than 800nm, there are generally deficiencies in photostability and fluorescence brightness. This is due to the increase in the rigidity of the molecular structure, which leads to a decrease in the hydrophilicity of the molecule, a decrease in the molar absorption coefficient, and easy π-π stacking. Serious aggregation leads to luminescence quenching (ACQ). As a result, even if the purpose of red shift can be achieved, it seriously affects the application effect.

[0006] In view of the above-mentioned defects of the prior art, there is an urgent need to provide a new type of rhodamine dye molecule that does not cause aggregation-induced luminescence quenching (ACQ) while maintaining a high molar absorption coefficient. Summary of the invention

[0007] The present invention aims to provide a tetraarylethene-modified rhodamine-like derivative and a preparation method and application thereof. The rhodamine-like derivative has a high molar absorption coefficient and aggregation-induced emission (AIE) properties.

[0008] The technical solution of the present invention is as follows:

[0009] The present invention provides a tetraarylethene-modified rhodamine-like derivative, the structural formula of which is as follows:

[0010]

[0011] Among them, the dashed line indicates bonding or non-bonding;

[0012] R1 is selected from H, halogen substituted or unsubstituted C 1-4 Alkyl, halogen substituted or unsubstituted C 1-4 Alkoxy and halogen substituted or unsubstituted C 2-4 or the C to which R1 is connected, the N to which R2 is connected, the C connecting the first two, R1 and R2 together form a 5-7 membered nitrogen-containing heterocyclic ring substituted with 0-4 substituents, each of which is independently selected from halogen-substituted or unsubstituted C 1-4 alkyl;

[0013] R 10 is selected from H, halogen substituted or unsubstituted C 1-4 Alkyl, halogen substituted or unsubstituted C 1-4 Alkoxy and halogen substituted or unsubstituted C 2-4 alkenyl; or R 10The connected C, R9 connected to N, the connected C, R9 and R 10 Together they form a 5-7 membered nitrogen-containing heterocyclic ring substituted by 0-4 substituents, each of which is independently selected from halogen-substituted or unsubstituted C 1-4 alkyl;

[0014] R2 and R3 are each independently selected from H, halogen substituted or unsubstituted C 1-6 Alkyl, halogen substituted or unsubstituted C 6-14 Aryl and halogen substituted or unsubstituted C 2-4 or the C to which R3 is connected, the N to which R2 is connected, R2 and R3 together form a 3-8 membered heterocyclic group or a 4-15 membered heterobridged ring group substituted with 0-5 substituents, each of which is independently selected from C 1-6 Alkyl, halogen and oxo (=O); or the C to which R3 is connected, the N to which R2 is connected, R2 and R3 together form a 3-8-membered cycloalkyl spiro 4-15-membered heterobridged ring group or a 3-8-membered heterocyclyl spiro 4-15-membered heterobridged ring group; the heterocyclyl and heterobridged ring groups have 1-5 heteroatoms independently selected from N, O and S;

[0015] R8, R9 are each independently selected from H, halogen substituted or unsubstituted C 1-6 Alkyl, halogen substituted or unsubstituted C 6-14 Aryl and halogen substituted or unsubstituted C 2-4 or C to which R8 is connected, N to which R9 is connected, R8 and R9 together form a 3-8 membered heterocyclic group or a 4-15 membered heterobridged ring group substituted with 0-5 substituents, each of which is independently selected from C 1-6 Alkyl, halogen and oxo; or the C to which R8 is connected, the N to which R9 is connected, R8 and R9 together form a 3-8-membered cycloalkyl spiro 4-15-membered heterobridged ring group or a 3-8-membered heterocyclyl spiro 4-15-membered heterobridged ring group; the heterocyclyl and heterobridged ring groups have 1-5 heteroatoms independently selected from N, O and S;

[0016] R4 is selected from hydrogen, amino, hydroxy, nitro, cyano, carboxyl, halogen, aryl, C 1-4 Alkoxy, C 2-4 Alkenyl, -SO3 - , -SO2X, -SO2NH2, X is halogen; or selected from C substituted by 0-2 substituents 1-4 Alkyl, substituents are each independently selected from C 1-4 or the C to which R3 is connected, the C to which R4 is connected, R3 and R4 together form a benzene ring substituted with 0-3 substituents, each of which is independently selected from halogen, C 1-4 Alkyl and C 1-4 Alkoxy;

[0017] R7 is selected from hydrogen, amino, hydroxy, nitro, cyano, carboxyl, halogen, aryl, C 1-4 Alkoxy, C 2-4 Alkenyl, -SO3 - , -SO2X, -SO2NH2, X is halogen; or selected from C substituted by 0-2 substituents 1-4 Alkyl, substituents are each independently selected from C 1-4 or the C to which R8 is connected, the C to which R7 is connected, R7 and R8 together form a benzene ring substituted with 0-3 substituents, each of which is independently selected from halogen, C 1-4 Alkyl and C 1-4 Alkoxy;

[0018] R5 and R6 are each independently selected from hydrogen, amino, hydroxy, nitro, cyano, carboxyl, halogen, aryl, C 1-4 Alkoxy, C 2-4 Alkenyl, -SO3 - , -SO2X, -SO2NH2, X is halogen; or selected from C substituted by 0-2 substituents 1-4 Alkyl, substituents are each independently selected from C 1-4 Alkoxy, halogen, azido, amino, and thiol;

[0019] R 11 , R 12 Each is independently selected from hydrogen, amino, hydroxy, nitro, cyano, carboxyl, halogen, aryl, C 1-4 Alkoxy, C 2-4 Alkenyl, -SO 3- , -SO2X, -SO2NH2, X is halogen; or selected from C substituted by 1-5 substituents 1-4 Alkoxy, the substituents are each independently selected from halogen, C 1-4 Alkoxy and azido; or selected from C 1-4 Alkyl, substituents are each independently selected from C 1-4 Alkoxy, halogen, azido, amino and mercapto; or R 11 , R 12 Together with the X1 to which it is connected, it forms a 3-8 membered heterocyclic group substituted by 0-5 substituents, each of which is independently selected from C 1-6 Alkyl, halogen and oxo; or R 11 , R 12 Together with the X1 to which it is connected, it forms an aromatic group substituted by one or more substituents, each of which is independently selected from -F, -Cl, -Br, -I, -OH, -NH2, -NO2, -CN, -N3, -COOH, C 1-6Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl or C 1-6 Alkylamino;

[0020] Ar1, Ar2, Ar3, Ar4 are each independently selected from aromatic groups substituted by one or more substituents, and the substituents are each independently selected from -F, -Cl, -Br, -I, -OH, -NH2, -NO2, -CN, -N3, -COOH, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl and C 1-6 Alkylamino group; wherein Ar1 plays a role in linking the rhodamine-like structure and the tetraaryl olefin;

[0021] X1 is selected from one of C, O, Si, P, Ge, Sn, and Pb;

[0022] X2 and X3 are each independently selected from one of H, C, S, O, and N; when X2 and X3 are not H elements, they can form a 4-6 membered heterocyclic group substituted by 0-5 substituents with the adjacent N and the connected C, and the substituents are each independently selected from C 1-6 Alkyl, halogen, and oxo;

[0023] A is an anion;

[0024] n represents The number of the intermediate benzene rings, n, is independently an integer of 1 to 10.

[0025] Preferably, R 11 , R 12 The aromatic group structural formula formed by the X1 to which it is connected and the aromatic group substituted by one or more substituents is as follows:

[0026]

[0027] “—*” indicates the attachment site of the group;

[0028] Preferably, the anion is an acid radical ion deprotonated by an acid, and the acid is selected from one or more of HCl, trifluoroacetic acid, methanesulfonic acid, toluenesulfonic acid, acetic acid, and sulfuric acid;

[0029] Preferably, when n=1, 2 or 3, it represents a benzene ring, a naphthalene ring or an anthracene ring, respectively, and each unit may be the same or different. For example, when n=2, two R5 may be the same or different.

[0030] Preferably, halogen is fluorine, chlorine, bromine or iodine.

[0031] Preferably, the tetraarylethene-modified rhodamine-like derivative has the following structural formula:

[0032]

[0033]

[0034] The present invention provides a method for preparing the above-mentioned tetraarylethene-modified rhodamine-like derivative, comprising the following steps:

[0035] (1) When X1=Si and n=1, the compound of formula II and the compound of formula III are subjected to activation reaction in the presence of an alkyl lithium reagent using THF as a solvent, and after the activation reaction, a silicon-containing compound R is added 11 R 12 SiCl2, stirred at room temperature for reaction, added water to quench the reaction, extracted, evaporated to remove the solvent, separated, to obtain compound IV;

[0036] (2) The compound of formula IV and the compound of formula V are reacted in the presence of p-toluenesulfonic acid monohydrate, and the product obtained after the reaction is mixed with M + A - Reaction and separation to obtain a tetraarylethene-modified rhodamine-like derivative compound of formula I;

[0037] M + represents a positively charged cation;

[0038] The reaction formula is as follows:

[0039]

[0040] Among them, R 1- R 12 ,Ar 1- Ar4, X 2- X3 and A have the same defined ranges as in Formula I above.

[0041] Preferably, the alkyl lithium reagent includes n-butyl lithium and / or tert-butyl lithium; more preferably n-butyl lithium.

[0042] Preferably, the molar ratio of the compound of formula II to the compound of formula III is 1:(1-4);

[0043] Preferably, the molar ratio of the alkyl lithium reagent to the total amount of the compound of formula II and the compound of formula III is (1-2):1; further preferably, the molar ratio of the alkyl lithium reagent to the total amount of the compound of formula II and the compound of formula III is (1-1.8):1.

[0044] Preferably, the silicon-containing compound R 11 R 12 The molar ratio of SiCl2 to the total amount of compound II and compound III is 1:(1-2);

[0045] Preferably, the temperature of the activation reaction in step (1) is -80 to -70°C;

[0046] Preferably, the activation reaction time in step (1) is 1-2 hours; the stirring reaction time is 2-12 hours;

[0047] Preferably, the separation in step (1) is silica gel column chromatography separation;

[0048] Preferably, the eluent for the silica gel column chromatography separation in step (1) is a mixed solvent of petroleum ether and ethyl acetate, and the volume ratio of ethyl acetate to petroleum ether is 1:1 to 1:100, more preferably 1:60.

[0049] Preferably, the molar ratio of the compound of formula IV to the compound of formula V is 1:(1-1.5);

[0050] Preferably, the temperature for reacting the compound of formula IV with the compound of formula V in step (2) is 50-150° C.;

[0051] Preferably, the reaction time of the compound of formula IV and the compound of formula V in step (2) is 2-24 hours;

[0052] Preferably, the separation in step (2) is silica gel column chromatography separation;

[0053] Preferably, the eluent for the silica gel column chromatography separation in step (2) is a mixed system of dichloromethane and methanol or a mixed system of ethyl acetate, petroleum ether and triethylamine;

[0054] Preferably, the M + Selected from Na + , K + or NH4 + ;

[0055] Preferably, the product obtained after the reaction is + A - The molar ratio is 1:(1.5-2.5);

[0056] Preferably, the product obtained after the reaction is+ A - The reaction temperature is 15-40°C;

[0057] Preferably, the product obtained after the reaction is + A - The reaction time is 0.5-3h.

[0058] The invention provides a fluorescent dye composition, which contains the above-mentioned tetraarylethene-modified rhodamine-like derivative.

[0059] The invention provides a kit containing the above-mentioned tetraarylethene-modified rhodamine-like derivatives.

[0060] Preferably, the fluorescent dye composition or kit may further include an optional solvent;

[0061] Preferably, the solvent is selected from one or more of MeOH, EtOH, PrOH, iPrOH, BuOH, water, acetonitrile, acetone, DMF, DMSO, pyridine, dichloromethane, chloroform, dichloroethane, benzene, toluene, p-xylene, chlorobenzene, nitrobenzene, 1,4-dioxane, THF, ethyl acetate, AcOH and buffer;

[0062] Further preferably, the buffer is PBS buffer.

[0063] The present invention provides the use of the tetraarylethene-modified rhodamine-like derivatives in the preparation of electroluminescent devices, metal complexes, fluorescent microspheres, near-infrared fluorescence imaging, photothermal therapy and / or photodynamic therapy agents.

[0064] The present invention provides an AIE magnetic encoding microsphere, comprising an AIE polymer microsphere and magnetic particles, wherein the magnetic particles are dispersed inside the AIE polymer microsphere or deposited on the surface of the AIE polymer microsphere; the AIE polymer microsphere comprises an AIE molecule and a blank polymer microsphere, wherein the AIE molecule is dispersed inside the blank polymer microsphere, and the AIE molecule comprises the above-mentioned tetraarylethene-modified rhodamine-like derivative.

[0065] Preferably, the blank polymer microspheres are selected from at least one of polystyrene microspheres, polymethyl methacrylate microspheres and polyglycidyl acrylate microspheres.

[0066] Preferably, the magnetic particles are Fe3O4.

[0067] Preferably, the encoding capacity of the AIE magnetically encoded microspheres is ≥ 8.

[0068] Preferably, the AIE polymer microspheres are obtained by embedding AIE molecules inside blank polymer microspheres, and the magnetic particles are deposited on the surface of the AIE polymer microspheres;

[0069] Preferably, the AIE molecules are embedded in the blank polymer microspheres by an active swelling method, and the amount of the AIE molecules in the active swelling method is 0.01-10 wt % of the amount of the blank polymer microspheres;

[0070] More preferably, the active swelling method is to allow the AIE molecules to swell with the blank polymer microspheres in a swelling agent so that the AIE molecules are embedded inside the blank polymer microspheres;

[0071] More preferably, in the active swelling method, the swelling agent is selected from at least one of dichloromethane, tetrahydrofuran, chloroform, anisole, benzyl alcohol and toluene, and the particle size of the blank polymer microspheres ranges from 0.03 μm to 50 μm;

[0072] Preferably, the magnetic particles are deposited on the surface of the AIE polymer microspheres by an in-situ precipitation method of iron salts, wherein the amount of iron salts used in the in-situ precipitation method is 0.1-25 wt % of the amount of blank polymer microspheres;

[0073] More preferably, the in-situ precipitation method is to add an alkaline solution to react with the iron salt chelated on the surface of the AIE polymer microspheres to generate magnetic particles which are deposited on the surface of the AIE polymer microspheres;

[0074] More preferably, in the in-situ precipitation method, the surface of the AIE polymer microspheres is modified by sulfonation, the alkaline solution is selected from at least one of sodium hydroxide solution, ammonia water, potassium hydroxide solution and sodium bicarbonate solution; the iron salt is selected from at least one of trivalent iron salt and divalent iron salt; the trivalent iron salt is selected from at least one of ferric chloride hexahydrate and anhydrous ferric chloride; the divalent iron salt is selected from at least one of ferrous chloride tetrahydrate, ferrous chloride dihydrate, ferrous sulfate and ferrous sulfate heptahydrate.

[0075] The present invention provides an AIE magnetically encoded microsphere immunoassay reagent, comprising the above-mentioned AIE magnetically encoded microspheres marked with antibodies.

[0076] Preferably, the antibody-labeled AIE magnetically encoded microspheres include AIE magnetically encoded microspheres with different encoding intensities respectively labeled with antibodies to multiple different antigens;

[0077] Preferably, the antibody is selected from at least one of a CRP monoclonal antibody, a PCT monoclonal antibody, an IL-6 monoclonal antibody and a SAA monoclonal antibody. More preferably, the antibody is a mouse antibody.

[0078] The present invention provides a method for preparing the above-mentioned AIE magnetically encoded microsphere immunoassay reagent, comprising the following steps:

[0079] The AIE magnetically encoded microspheres are carboxyl modified, carboxyl activated, and antibody coupled to obtain antibody-labeled AIE magnetically encoded microspheres, that is, AIE magnetically encoded microsphere immunoassay reagents.

[0080] Preferably, the carboxyl modification comprises: mixing and reacting the AIE magnetically encoded microspheres, a base, TEOS and a silane coupling agent, magnetically separating, and adding a monomer containing a double bond and a carboxyl group to react to obtain the carboxyl-modified AIE magnetically encoded microspheres;

[0081] Preferably, the activating carboxyl groups comprises: dispersing the carboxyl-modified AIE magnetically encoded microspheres in an acidic buffer, adding a carbodiimide condensation reagent, and stirring the reaction to activate the carboxyl groups, thereby obtaining the AIE magnetically encoded microspheres with activated carboxyl groups;

[0082] Preferably, the antibody coupling comprises: dispersing the AIE magnetically encoded microspheres with activated carboxyl groups in an alkaline buffer, adding antibodies, stirring and incubating, magnetically separating, adding blocking solution and preservation solution, and obtaining antibody-labeled AIE magnetically encoded microspheres.

[0083] The present invention provides an application of the aforementioned AIE magnetically encoded microsphere immunoassay reagent in antigen immunoassay, comprising the following steps:

[0084] The AIE magnetically encoded microsphere immunoassay reagent is added to the test solution containing the corresponding antigen. After the reaction, it is separated by magnetic absorption and the encoding peak and fluorescence qualitative detection antigen type are obtained by flow cytometry.

[0085] The present invention utilizes rhodamine-derivative AIE materials as high-brightness fluorescent dyes to prepare multiple AIE magnetically encoded microspheres, whose high discrimination can achieve high-sensitivity and long linear range detection in complex samples of multiple markers, providing a new detection strategy for multiple marker immunoassays.

[0086] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0087] (1) The present invention innovatively combines the TPE structure with the rhodamine derivative dye. On the one hand, the introduction of the TPE structure improves the defects of the rhodamine derivative dye ACQ. On the other hand, the novel rhodamine-like derivatives after the combination of the TPE and rhodamine structure of the present invention have extremely high molar absorption coefficients. For example, the SiR-TPE molecule not only greatly improves the ACQ defect, but also significantly improves the molar absorption coefficient, up to 1.9 times.

[0088] (2) The preparation method of the rhodamine-like derivatives of the present invention is simple, the reaction conditions are mild, and the yield is high. The fluorescent dye has the characteristics of AIE, high molar absorption coefficient, high brightness and photobleaching resistance, and is an excellent coded microsphere material.

[0089] (3) Compared with the fluorescent materials of traditional magnetic encoded microspheres, the AIE aggregation-induced luminescence magnetic encoded microspheres of the present invention have high molar absorption coefficient AIE materials that improve the discrimination of magnetic encoded microspheres and solve the aggregation-induced quenching effect of traditional fluorescent materials inside the microspheres. At the same time, the AIE materials have high photostability, so that there is no obvious fluorescence attenuation after repeated irradiation.

[0090] (4) The present invention provides a simple method, which utilizes the high specificity of AIE encoded microspheres to obtain an immunoassay technology based on AIE magnetic encoded microspheres, providing a new detection strategy for multiple marker immunoassays. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 The UV absorption spectra of SiR-TPE in Example 1 in different solvent solutions;

[0092] Figure 2 The fluorescence emission spectra of SiR-TPE in different solvent solutions in Example 1 are shown;

[0093] Figure 3 This is the AIE curve of SiR-TPE in n-hexane and ethanol system in Example 1;

[0094] Figure 4 Encoding for 8-fold fluorescent microspheres prepared using SiR-TPE. DETAILED DESCRIPTION

[0095] The technical solution of the present invention is described in detail below with reference to examples and drawings. The technical solution of the present invention is illustrated by the following examples but is not limited thereto.

[0096] Example 1

[0097] Step 1:

[0098]

[0099] In a 500mL long-necked reaction bottle equipped with a magnetic stirrer, add 27.376g (120.0mmol) of N,N-diethyl-m-bromoaniline. Under nitrogen protection, add 120mL of THF and stir magnetically at -78°C for 5min. Slowly drop n-BuLi 52.5mL (2.4Minn-hexane, 126.0mmol) into the reaction solution. After the addition is complete, continue the reaction at -78°C for 2h. Dissolve 8.8m (72.0mmol) of dimethyldichlorosilane in 30mL of THF and slowly drop it into the above reaction solution. After the addition is complete, slowly warm the reaction to room temperature and stir overnight. 150 mL of water was added to the reaction solution, and the organic layer was separated by a separatory funnel. The aqueous layer was extracted with ether (50 mL × 3). The organic layers (including the organic layer separated by the separatory funnel and the extracted organic layer) were combined, washed once with 150 mL of water, washed once with 150 mL of saturated NaCl aqueous solution, dried with anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The product was purified by silica gel column chromatography, and the eluent was petroleum ether: ethyl acetate = 80:1 by volume to obtain yellow oily SiN, 17.507 g, with a yield of 95%. HRMS (ESI) calcd. for C 10 H 14 BrN[M] + :227.03,found227.0354.

[0100] Step 2:

[0101]

[0102] In a 15mL polymerization tube equipped with a magnetic stirrer, add 106mg (0.3mmol) of raw material SiN, 540mg (1.5mmol) of 1,1,2-triphenyl-2-(4-formaldehydephenyl)ethylene, and 57mg (0.3mnol) of p-toluenesulfonic acid monohydrate, react in an oil bath at 140°C for 12 hours, cool naturally to room temperature, add 2mL of dichloromethane and methanol to dissolve, purify by silica gel column chromatography, the eluent is dichloromethane:methanol = 40:1 by volume, and the target compound SiR-TPE is obtained with a yield of 60%. HRMS (ESI) calcd.forC 49 H 51 N2Si + [M] + :695.38,found 695.3816.

[0103] Example 2

[0104] Step 1:

[0105]

[0106] In a 250mL long-necked reaction bottle equipped with a magnetic stirrer, add 4.070g (18.0mmol) of 1-ethyl-6-bromoindoline. Under nitrogen protection, add 50mL of THF and stir magnetically at 0℃ for 5min. Slowly add 11.8mL of n-BuLi (1.6Min n-hexane, 18.9mmol) to the reaction solution. After the addition is complete, continue the reaction at 0℃ for 2h. Dissolve 1.3mL (10.8mmol) of dimethyldichlorosilane in 10mL of THF and slowly add it to the above reaction solution. After the addition is complete, the reaction is slowly warmed to room temperature and stirred overnight. 50 mL of water was added to the reaction solution, the organic layer was separated by a separatory funnel, the aqueous layer was extracted with ether (30 mL × 3), the organic layers (including the organic layer separated by the separatory funnel and the extracted organic layer) were combined, washed once with 50 mL of water, washed once with 50 mL of saturated NaCl aqueous solution, dried over anhydrous Na2SO4, the solvent was removed by rotary evaporation, and purified by silica gel column chromatography, the eluent was petroleum ether: ethyl acetate = 40:1 by volume, and yellow oily SiN2 was obtained, 2.423 g, with a yield of 77%. HRMS (ESI) calcd.ForC 22 H 32 N2Si

[0107] [M] + :352.23, found 352.2365.

[0108] Step 2:

[0109]

[0110] The method is the same as Example 1, except that SiN in the raw material is replaced by SN2, and the target compound SiR2-TPE is obtained with a yield of 34%. HRMS (ESI) calcd.For C 49 H 47 N2Si + [M] + :691.35,found 691.3522.

[0111] Example 3

[0112]

[0113] The method is the same as Example 1, except that the N, N-diethyl-m-bromoaniline in the raw material of step 1 is replaced with 2-bromo-10-ethyl-phenothiazine to obtain the target compound SiR3-TPE with a yield of 50%. HRMS (ESI) calcd.For C 57 H 47 N2S2Si + [M] + :851.29, found 851.2934.

[0114] Determination of compound spectra

[0115] 1. Determination of sample absorption spectrum

[0116] The prepared sample SiR-TPE solution (10 -5 mol / L) was measured with a SHIMADZU UV-2600 spectrophotometer (quartz dish, optical path length 1 cm) in the range of 450nm to 750nm. The maximum absorption wavelength was measured and the molar absorption coefficient of the sample was calculated. The absorption spectrum is shown in Figure 1 .

[0117] 2. Determination of sample emission spectrum

[0118] The prepared sample SiR-TPE solution (10 -5 mol / L) was measured with a HORIBA Fluoromax-4 spectrometer, with an excitation wavelength of 610nm and a measurement range of 620nm to 750nm to measure its maximum emission wavelength. Figure 2 .

[0119] The fluorescence data results are shown in Table 1 below.

[0120] Table 1 SiR-TPE spectral data

[0121]

[0122] The existing SiRB molecule Comparative test with SiR-TPE in acetic acid solvent (10 -5 mol / L). The comparison results are shown in Table 2

[0123] Table 2 Comparison of spectral data of SiR-TPE and SiRB

[0124]

[0125] From Tables 1 and 2, we can see that although the introduction of TPE has little effect on the luminescence peak position, its maximum molar absorption coefficient can reach 1.9×10 5 L / mol cm -1 .

[0126] As shown in Table 2, the molar absorption coefficient of SiR-TPE is about 1.9×10 5 L / mol cm -1 , the molar absorption coefficient of SiRB is about 10×10 5 L / mol cm -1 , roughly increased by 1.9 times.

[0127] The introduction of TPE can not only improve the molar absorption coefficient, but also endow silicon-based rhodamine with certain AIE properties. The AIE curve of SiR-TPE in n-hexane and ethanol system is shown in Figure 2. Figure 3 ,As shown in the figure, as the n-hexane component increases, that is, the aggregation degree increases, the fluorescence intensity of SiR-TPE molecules increases linearly, and the fluorescence intensity is the maximum when the volume components of n-hexane and ethanol each account for 50%.

[0128] In summary, the present invention provides a strategy for improving the ACQ properties of silicon-based rhodamine derivatives and increasing the molar absorption coefficient, which greatly promotes the application prospects of silicon-based rhodamine and obtains a tetraarylethylene-modified rhodamine-like derivative with AIE properties and high molar absorption coefficient.

[0129] Application Example 1

[0130] 1. Carboxyl-modified SiR-TPE magnetic encoding microspheres

[0131] Weigh 1 g of blank polymer microspheres and disperse them in 100 g of water to obtain a microsphere dispersion;

[0132] 5 mg of SiR-TPE molecules were dissolved in 10 mL of tetrahydrofuran to obtain a swelling agent solution; the two solutions were mixed together (the mass of SiR-TPE molecules accounted for 0.5 wt% of the mass of blank polymer microspheres), and ultrasonic treatment was performed at a power of 100 W for 1 min. After the closed swelling was performed for 2 h, the solvent was evaporated for 4 h, and the supernatant was removed by centrifugation to obtain SiR-TPE polymer microspheres. The obtained SiR-TPE polymer microspheres were added to 50 mL of concentrated sulfuric acid (98.3 wt%), the temperature was raised to 80 ° C, and the reaction was performed for 8 h; the microspheres were centrifuged at a speed of 5000 rpm, and then resuspended in water, and repeated three times to obtain SiR-TPE polymer microspheres with sulfonated surfaces.

[0133] Ferrous sulfate heptahydrate and ferric chloride hexahydrate were mixed with 100g water in a mass ratio of 2:1 (the total mass of iron salt accounted for 5wt% of the mass of blank polymer microspheres), and the SiR-TPE polymer microspheres with sulfonation modified as above were added. After soaking and reacting for 1h, the microspheres were centrifuged at 5000rpm and resuspended in water. The process was repeated three times to obtain SiR-TPE polymer microspheres chelated with iron salts. 50mL of 1mol / L sodium hydroxide solution was added, the temperature was raised to 80℃, and the reaction was carried out for 2h. The iron salts reacted in situ to generate magnetic particles that were deposited on the surface of SiR-TPE polymer microspheres. The microspheres were centrifuged at 5000rpm and resuspended in water. The process was repeated three times to obtain aggregation-induced luminescence magnetically encoded microspheres. 3 mL of ammonia water (28%), 1.5 mL of LTEOS and 0.6 mL of 3-vinyltrimethoxysilane were added to the above-mentioned aggregation-induced emission magnetically encoded microspheres (0.1 g). After reacting for 6 hours and magnetic separation, 0.5 g of acrylic acid monomer was added, the temperature was raised to 80°C, and after reacting for 8 hours and magnetic separation, carboxyl-modified SiR-TPE magnetically encoded microspheres were obtained.

[0134] The amount of SiR-TPE molecules was changed to 0.05 mg, 0.1 mg, and 0.5 mg respectively to obtain carboxyl-modified SiR-TPE magnetic encoding microspheres with different encoding intensities (other parameters remained unchanged).

[0135] 2. Antibody-labeled SiR-TPE magnetically encoded microspheres

[0136] The carboxyl-modified SiR-TPE magnetically encoded microspheres prepared above (SiR-TPE molecule dosage is 5 mg) are added to 10 g MES buffer (pH = 6.5), and after ultrasonic dispersion, 0.0095 g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added, and the mixture is placed in a mixer to activate the carboxyl group at a speed of 300 rpm for 5 min. The supernatant is removed by magnetic suction to obtain activated SiR-TPE magnetically encoded microspheres, 3 g boric acid buffer (pH = 8) and 10 μg mouse antibody of the inflammatory marker SAA are added, and the mixture is placed in a mixer for incubation for 30 min, the supernatant is removed by magnetic suction, 0.5 mL of blocking solution BSA (0.5%) and Tris (0.1%) microsphere storage solution are added, and ultrasonic dispersion is performed to obtain a SAA antibody-labeled SiR-TPE magnetically encoded microsphere solution (0.2 g / mL).

[0137] The preparation method of the SiR-TPE magnetically encoded microsphere solution labeled with the antibody corresponding to the inflammatory marker CRP is the same as above, except that the mouse antibody of the inflammatory marker SAA is replaced with the mouse antibody of the inflammatory marker CRP, and the microspheres are replaced with SiR-TPE magnetically encoded microspheres with different encoding strengths (the amount of SiR-TPE molecules used is 0.05 mg);

[0138] The preparation method of the SiR-TPE magnetically encoded microsphere solution labeled with the antibody corresponding to the inflammatory marker IL-6 is the same as above, except that the mouse antibody of the inflammatory marker SAA is replaced with the mouse antibody of the inflammatory marker IL-6, and the microspheres are replaced with SiR-TPE magnetically encoded microspheres with different coding strengths (the amount of SiR-TPE molecules used is 0.1 mg);

[0139] The preparation method of SiR-TPE magnetically encoded microsphere solution labeled with the antibody corresponding to the inflammatory marker PCT is the same as above, the only difference is that the mouse antibody of the inflammatory marker SAA is replaced by the mouse antibody of the inflammatory marker PCT, and the microspheres are replaced with SiR-TPE magnetically encoded microspheres with different coding strengths (the amount of SiR-TPE molecules used is 0.5 mg).

[0140] 3. Determination of multiple marker immunoassays

[0141] 10 μL of each SiR-TPE magnetically encoded microsphere solution labeled with four different types of antibodies was added to the flow tube, and then 5 μL of the sample to be tested (a mixed solution of four inflammatory markers SAA, CRP, IL-6, and PCT) was added. After magnetic separation, the coding peak was obtained through the coding channel of the flow cytometer, and the type of inflammatory markers in the test solution was distinguished by the coding channel and intensity.

[0142] like Figure 4 As shown, SIR-TPE magnetically encoded microspheres have been successfully made. As the mass of molecules encapsulated in the prepared microspheres increases, not only can the particle size be kept uniform, but the luminescence intensity of the fluorescent microspheres also increases linearly. The figure shows eight-fold magnetic single fluorescent microsphere encoding.

[0143] The multiple encoding properties of this fluorescent microsphere can be applied to the determination of multiple marker immunoassays. The present invention adds antibody-labeled SIR-TPE magnetic encoding microspheres to a test solution containing the corresponding antigen, and after the reaction, the SIR-TPE magnetic encoding microspheres are separated by magnetic attraction, and the encoding peaks and the types of antigens detected by fluorescence qualitative detection are obtained by flow cytometry. Long linear range detection is achieved in complex samples with multiple markers, and a new detection strategy is provided for multiple marker immunoassays.

[0144] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A tetraarylethene-modified rhodamine-like derivative, characterized in that: The structural formula is as follows: Among them, the dashed line indicates bonding or non-bonding; R1 is selected from H, halogen substituted or unsubstituted C 1-4 Alkyl, halogen substituted or unsubstituted C 1-4 Alkoxy and halogen substituted or unsubstituted C 2-4 or the C to which R1 is connected, the N to which R2 is connected, the C connecting the first two, R1 and R2 together form a 5-7 membered nitrogen-containing heterocyclic ring substituted with 0-4 substituents, each of which is independently selected from halogen-substituted or unsubstituted C 1-4 alkyl; R 10 is selected from H, halogen substituted or unsubstituted C 1-4 Alkyl, halogen substituted or unsubstituted C 1-4 Alkoxy and halogen substituted or unsubstituted C 2-4 alkenyl; or R 10 The connected C, R9 connected to N, the connected C, R9 and R 10 Together they form a 5-7 membered nitrogen-containing heterocyclic ring substituted by 0-4 substituents, each of which is independently selected from halogen-substituted or unsubstituted C 1-4 alkyl; R2 and R3 are each independently selected from H, halogen substituted or unsubstituted C 1-6 Alkyl, halogen substituted or unsubstituted C 6-14 Aryl and halogen substituted or unsubstituted C 2-4 or the C to which R3 is connected, the N to which R2 is connected, R2 and R3 together form a 3-8 membered heterocyclic group or a 4-15 membered heterobridged ring group substituted with 0-5 substituents, each of which is independently selected from C 1-6 Alkyl, halogen and oxo; or the C to which R3 is connected, the N to which R2 is connected, R2 and R3 together form a 3-8-membered cycloalkyl spiro 4-15-membered heterobridged ring group or a 3-8-membered heterocyclyl spiro 4-15-membered heterobridged ring group; the heterocyclyl and heterobridged ring groups have 1-5 heteroatoms independently selected from N, O and S; R8, R9 are each independently selected from H, halogen substituted or unsubstituted C 1-6 Alkyl, halogen substituted or unsubstituted C 6-14 Aryl and halogen substituted or unsubstituted C 2-4 or C to which R8 is connected, N to which R9 is connected, R8 and R9 together form a 3-8 membered heterocyclic group or a 4-15 membered heterobridged ring group substituted with 0-5 substituents, each of which is independently selected from C 1-6 Alkyl, halogen and oxo; or the C to which R8 is connected, the N to which R9 is connected, R8 and R9 together form a 3-8-membered cycloalkyl spiro 4-15-membered heterobridged ring group or a 3-8-membered heterocyclyl spiro 4-15-membered heterobridged ring group; the heterocyclyl and heterobridged ring groups have 1-5 heteroatoms independently selected from N, O and S; R4 is selected from hydrogen, amino, hydroxy, nitro, cyano, carboxyl, halogen, aryl, C 1-4 Alkoxy, C 2-4 Alkenyl, -SO3 - , -SO2X, -SO2NH2, X is halogen; or selected from C substituted by 0-2 substituents 1-4 Alkyl, substituents are each independently selected from C 1-4 or the C to which R3 is connected, the C to which R4 is connected, R3 and R4 together form a benzene ring substituted with 0-3 substituents, each of which is independently selected from halogen, C 1-4 Alkyl and C 1-4 Alkoxy; R7 is selected from hydrogen, amino, hydroxy, nitro, cyano, carboxyl, halogen, aryl, C 1-4 Alkoxy, C 2-4 Alkenyl, -SO3 - , -SO2X, -SO2NH2, X is halogen; or selected from C substituted by 0-2 substituents 1-4 Alkyl, substituents are each independently selected from C 1-4 or the C to which R8 is connected, the C to which R7 is connected, R7 and R8 together form a benzene ring substituted with 0-3 substituents, each of which is independently selected from halogen, C 1-4 Alkyl and C 1-4 Alkoxy; R5 and R6 are each independently selected from hydrogen, amino, hydroxy, nitro, cyano, carboxyl, halogen, aryl, C 1-4 Alkoxy, C 2-4 Alkenyl, -SO3 - , -SO2X, -SO2NH2, X is halogen; or selected from C substituted by 0-2 substituents 1-4 Alkyl, substituents are each independently selected from C 1-4 Alkoxy, halogen, azido, amino, and thiol; R 11 , R 12 Each is independently selected from hydrogen, amino, hydroxy, nitro, cyano, carboxyl, halogen, aryl, C 1-4 Alkoxy, C 2-4 Alkenyl, -SO 3- , -SO2X, -SO2NH2, X is halogen; or selected from C substituted by 1-5 substituents 1-4 Alkoxy, the substituents are each independently selected from halogen, C 1-4 Alkoxy and azido; or selected from C 1-4 Alkyl, substituents are each independently selected from C 1-4 Alkoxy, halogen, azido, amino and mercapto; or R 11 , R 12 Together with the X1 to which it is connected, it forms a 3-8 membered heterocyclic group substituted by 0-5 substituents, each of which is independently selected from C 1-6 Alkyl, halogen and oxo; or R 11 , R 12 Together with the X1 to which it is connected, it forms an aromatic group substituted by one or more substituents, each of which is independently selected from -F, -Cl, -Br, -I, -OH, -NH2, -NO2, -CN, -N3, -COOH, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl or C 1-6 Alkylamino; Ar1, Ar2, Ar3, Ar4 are each independently selected from aromatic groups substituted by one or more substituents, and the substituents are each independently selected from -F, -Cl, -Br, -I, -OH, -NH2, -NO2, -CN, -N3, -COOH, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl and C 1-6 Alkylamino; X1 is selected from one of C, O, Si, P, Ge, Sn, and Pb; X2 and X3 are each independently selected from one of H, C, S, O, and N; when X2 and X3 are not H elements, they can form a 4-6 membered heterocyclic group substituted with 0-5 substituents with the adjacent N and the connected C, and the substituents are each independently selected from C 1-6 Alkyl, halogen, and oxo; A is an anion; n represents The number of the intermediate benzene rings, n, is independently an integer of 1 to 10.

2. The tetraarylethene-modified rhodamine-like derivative according to claim 1, characterized in that: R 11 , R 12 The aromatic group structural formula formed by the X1 to which it is connected and the aromatic group substituted by one or more substituents is as follows: The anion is an acid radical ion deprotonated by an acid, and the acid is selected from one or more of HCl, trifluoroacetic acid, methanesulfonic acid, toluenesulfonic acid, acetic acid, and sulfuric acid; When n=1, 2 or 3, it represents a benzene ring, a naphthalene ring or an anthracene ring, respectively, and each unit may be the same or different.

3. The tetraarylethene-modified rhodamine-like derivative according to claim 1, characterized in that: The structural formula is as follows:

4. The method for preparing the tetraarylethene-modified rhodamine-like derivative according to any one of claims 1 to 3, characterized in that: The steps include: (1) When X1=Si and n=1, the compound of formula II and the compound of formula III are subjected to activation reaction in the presence of an alkyl lithium reagent using THF as a solvent, and after the activation reaction, a silicon-containing compound R is added 11 R 12 SiCl2, stirred at room temperature for reaction, added water to quench the reaction, extracted, evaporated to remove the solvent, separated, to obtain compound IV; (2) The compound of formula IV and the compound of formula V are reacted in the presence of p-toluenesulfonic acid monohydrate, and the product obtained after the reaction is mixed with M + A - Reaction and separation to obtain a tetraarylethene-modified rhodamine-like derivative compound of formula I; M + represents a positively charged cation; The reaction formula is as follows:

5. The preparation method according to claim 4, characterized in that: The alkyl lithium reagent includes n-butyl lithium and / or tert-butyl lithium; The molar ratio of the compound of formula II to the compound of formula III is 1:(1-4); The molar ratio of the alkyl lithium reagent to the total amount of the compound of formula II and the compound of formula III is (1-2): 1; The silicon-containing compound R 11 R 12 The molar ratio of SiCl2 to the total amount of compound II and compound III is 1:(1-2); The temperature of the activation reaction in step (1) is -80 to -70°C; The activation reaction time in step (1) is 1-2 hours; the stirring reaction time is 2-12 hours; The separation in step (1) is separation by silica gel column chromatography; The eluent for the silica gel column chromatography separation in step (1) is a mixed solvent of petroleum ether and ethyl acetate, with the volume ratio of ethyl acetate to petroleum ether being 1:1 to 1:100; The molar ratio of the compound of formula IV to the compound of formula V is 1:(1-5); In step (2), the temperature for reacting the compound of formula IV with the compound of formula V is 50-150° C.; Step (2) The reaction time of the compound of formula IV and the compound of formula V is 2-24 hours; The separation in step (2) is silica gel column chromatography separation; The eluent for the silica gel column chromatography separation in step (2) is a mixed system of dichloromethane and methanol or a mixed system of ethyl acetate, petroleum ether and triethylamine; The M + Selected from Na + , K + or NH4 + ; The product obtained after the reaction is + A - The molar ratio is 1:(1.5-2.5); The product obtained after the reaction is + A - The reaction temperature is 15-40°C; The product obtained after the reaction is + A - The reaction time is 0.5-3h.

6. A fluorescent dye composition or kit, characterized in that: A rhodamine-like derivative modified with tetraarylethene as described in any one of claims 1 to 3.

7. Use of the tetraarylethene-modified rhodamine-like derivative according to any one of claims 1 to 3 in the preparation of electroluminescent devices, metal complexes, fluorescent microspheres, near-infrared fluorescence imaging, photothermal therapy and / or photodynamic therapy agents.

8. An AIE magnetically encoded microsphere, characterized in that: The invention comprises AIE polymer microspheres and magnetic particles, wherein the magnetic particles are dispersed inside the AIE polymer microspheres or deposited on the surface of the AIE polymer microspheres; the AIE polymer microspheres comprise AIE molecules and blank polymer microspheres, wherein the AIE molecules are dispersed inside the blank polymer microspheres, and the AIE molecules comprise the tetraarylethene-modified rhodamine-like derivatives according to any one of claims 1 to 3.

9. An AIE magnetically encoded microsphere immunoassay reagent, characterized in that: The AIE magnetically encoded microspheres of claim 8 comprising antibody labels.

10. The method for preparing the AIE magnetically encoded microsphere immunoassay reagent according to claim 9, characterized in that: The following steps are involved: The AIE magnetically encoded microspheres are carboxyl modified, carboxyl activated, and antibody coupled to obtain antibody-labeled AIE magnetically encoded microspheres, that is, AIE magnetically encoded microsphere immunoassay reagents.

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