BODIPY light-caged chemiluminescent molecules, nano-microspheres, and synthesis methods and applications thereof

Abstract

The present invention provides a BODIPY photocaged chemiluminescent molecule, the structure of which is shown in formula (I): (I); wherein:. The BODIPY photocaged chemiluminescent molecule of the present invention can generate a chemiluminescent signal under illumination conditions, the luminescence process is simple to operate, and has the characteristics of high luminescence brightness and good chemical stability. Based on the BODIPY photocaged chemiluminescent molecule, the present invention also provides a method for synthesizing the BODIPY photocaged chemiluminescent molecule, and its application in chemiluminescent immunoassay. The present invention also provides a BODIPY photocaged chemiluminescent nanosphere, which uses a swelling method to load the BODIPY photocaged chemiluminescent molecule Bsa‑CPMs into a polystyrene nanosphere PS. The present invention also provides an application of a BODIPY photocaged chemiluminescent nanosphere in chemiluminescent immunoassay.

Description

Technical Field

[0001] The present invention relates to the technical field of chemiluminescence analysis and detection, and in particular to a BODIPY light-caged chemiluminescent molecule, a nano-microsphere, and a synthesis method and application thereof. Background Art

[0002] Chemiluminescent immunoassay is an immunoassay method that combines highly sensitive chemiluminescent assay technology with highly specific immune reactions and directly labels antigens or antibodies with chemiluminescent agents. It is commonly used to detect various antigens, antibodies, fatty acids, vitamins, and drugs. Currently, the main immunoassay method used in chemiluminescent technology is acridinium ester chemiluminescent reagents. The acridinium ester luminescent system mainly uses the double antibody sandwich principle and acridinium ester as the dye. The luminescence principle is that acridinium ester emits light in alkaline H 2 O 2 The solution is attacked by hydrogen peroxide ions, generating unstable ethylene dioxide, which can quickly decompose into CO 2 The electronically excited N-methylacridone releases photons in the process of returning to the ground state, generating corresponding optical signals.

[0003] However, the luminescence system based on acridinium ester requires the addition of hydrogen peroxide to initiate the luminescence process, which is easily affected by environmental and external factors. For example, changes in temperature, pH value, etc. will affect the intensity and stability of chemiluminescence. Acridinium ester itself is also unstable in the buffer solution and easily hydrolyzed. In addition, the selectivity of the acridinium ester luminescence reaction is relatively low. In complex samples, other components may react non-specifically with acridinium ester, resulting in errors in the detection results of the target analyte. In addition, direct chemiluminescence of acridinium ester has high requirements for instruments and requires the addition of excitation solution in situ.

[0004] In view of this, it is necessary to provide a new luminescent material to solve the above technical problems. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a BODIPY light-caged chemiluminescent molecule that can generate chemiluminescent signals only under light conditions, has a simple luminescence process, and has the characteristics of high luminescence brightness and good chemical stability.

[0006] The first aspect of the present invention is to provide a BODIPY light-caged chemiluminescent molecule, the structure of which is shown in formula (I):

[0007] ;

[0008] (I)

[0009] in:

[0010] .

[0011] The second aspect of the present invention is to provide a method for synthesizing the BODIPY photocaged chemiluminescent molecule as described in the first aspect, comprising the following steps:

[0012] (1) Compound 1 was synthesized from 2-chloro-3-hydroxybenzaldehyde / m-hydroxybenzaldehyde / 2-bromo-3-hydroxybenzaldehyde, trimethyl orthoformate and tert-butylammonium tribromide;

[0013] (2) Compound 2 was synthesized using compound 1, imidazole and tert-butyldimethylsilyl chloride as raw materials;

[0014] (3) Compound 3 was synthesized using compound 2, trimethyl phosphite and titanium chloride as raw materials;

[0015] (4) Compound 4 is synthesized using compound 3, lithium diisopropylamide and compound A as raw materials; wherein compound A is one of 2-adamantanone, aldehyde-modified β-cyclodextrin, cyclobutanone, 3-oxetanone, 3-carbonyl-cyclobutanecarboxylic acid methyl ester and tetrahydrothioran-4-one 1,1-dihydrogenated;

[0016] (5) Compound 5 was synthesized using compound 4 and tetrabutylammonium fluoride as raw materials;

[0017] (6) Compound 6 was synthesized using compound 5, magnesium chloride and paraformaldehyde as raw materials;

[0018] (7) Compound 7 is synthesized from compound 6 and compound B as raw materials; wherein compound B is one of (triphenylphosphine)acetonitrile, 2-(triphenylphosphoranylidene)acetic acid, and methoxyformylmethylenetriphenylphosphine; when R 1 When it is an aldehyde group, it is compound 6;

[0019] (8) Compound 8 was synthesized using 2,4-dimethylpyrrole and acetoxyacetyl chloride as raw materials;

[0020] (9) Compound 9 was synthesized using compound 8 and lithium hydroxide monohydrate as raw materials;

[0021] (10) Compound 10 was synthesized from compound 9, triphosgene and N,N-diisopropylethylamine;

[0022] (11) Compound 7 and compound 10 were used as raw materials to synthesize BODIPY light-caged chemiluminescent molecules, namely compound 11;

[0023] The synthesis route is as follows:

[0024] ;

[0025] in:

[0026] ;

[0027] A = 2-adamantanone; cyclobutanone; 3-oxetanone; 3-carbonyl-cyclobutanecarboxylic acid methyl ester; tetrahydrothiopyran-4-one 1,1-dioxide;

[0028] B = (triphenylphosphine)acetonitrile; 2-(triphenylphosphoranylidene)acetic acid; ethoxycarbonylmethylenetriphenylphosphine.

[0029] The third aspect of the present invention is to provide a use of the BODIPY photocaged chemiluminescent molecule as described in the first aspect in chemiluminescent immunoassay.

[0030] The fourth aspect of the present invention is to provide a BODIPY photocaged chemiluminescent nanosphere, which is prepared by loading the BODIPY photocaged chemiluminescent molecules Bsa-CPMs described in the first aspect onto polystyrene nanospheres PS as carriers, and the prepared nanospheres are activated to emit light by light.

[0031] Furthermore, the method for preparing the BODIPY light-caged chemiluminescent nanospheres comprises the following steps:

[0032] Step S1, dispersing carboxylated polystyrene nanospheres PS in isopropanol;

[0033] Step S2, dissolving the BODIPY light-caged chemiluminescent molecule Bsa-CPMs in dichloromethane;

[0034] Step S3, mixing the solutions of step S1 and step S2 uniformly;

[0035] Step S4, rotary evaporating the sample in step S3, centrifuging and washing to obtain BODIPY light-caged chemiluminescent nanoparticles Bsa-CPM@PS.

[0036] Furthermore, the particle size of the carboxylated polystyrene nanospheres is 20-5000 nm.

[0037] Furthermore, in step S1, the concentration of the dispersed polystyrene nanospheres is 1-2 mg / ml; specifically, the concentration of the dispersed polystyrene nanospheres can be 1 mg / ml, 1.5 mg / ml or 2 mg / ml, or other values ​​within the range.

[0038] Furthermore, in step S2, the concentration of the BODIPY photocaged chemiluminescent molecule is 3.3-6.6 mg / ml; specifically, the concentration of the BODIPY photocaged chemiluminescent molecule can be 3.3 mg / ml, 4 mg / ml, 4.5 mg / ml, 5 mg / ml, 5.5 mg / ml, 6 mg / ml or 6.6 mg / ml, or other values ​​within this range.

[0039] The fifth aspect of the present invention is to provide a use of the BODIPY photocaged chemiluminescent nanospheres as described in the third aspect in chemiluminescent immunoassay.

[0040] The sixth aspect of the present invention is to provide a use of the BODIPY light-caged chemiluminescent nanospheres as described in the third aspect in the chemiluminescent immunoassay of the prostate cancer marker PSA. The specific method is as follows:

[0041] Step S1, BODIPY photocaged chemiluminescent nanoparticles (Bsa-CPMs@PS) were mixed with EDC / NHS solution (MES, 100 mM, pH 5.5), reacted at room temperature for 1 h, and centrifuged at 14000 rpm for 8 min to remove EDC / NHS;

[0042] Step S2, covalently linking rabbit anti-human prostate protein (PSA) monoclonal antibody (Rabbit MAb) to Bsa-CPMs@PS via an amide bond: adding Rabbit MAb to the activated Bsa-CPMs@PS solution in step S1, shaking the solution overnight, and then centrifuging and washing the solution twice at 14,000 rpm to remove excess Rabbit MAb, thereby obtaining rabbit antibody-modified chemiluminescent nanoparticles (Bsa-CPMs@PS-RMAb), which were dispersed in PBS;

[0043] Step S3, the carboxylated magnetic beads (MB-COOH) and the EDC / NHS mixed solution (MES, 100 mM, pH 5.5) were reacted at room temperature for 2 h, and then the supernatant was discarded by magnetic separation, washed three times with PBS buffer (pH 7.4) to remove excess EDC / NHS, and dispersed in PBS;

[0044] Step S4, adding mouse anti-human PSA monoclonal antibody (Mouse MAb) to the MB solution activated in step S3, shaking overnight and washing four times with PBS magnetic separation to obtain mouse antibody-modified magnetic beads (MB-MMAb), which are dispersed in PBS;

[0045] Step S5, taking different concentrations of PSA, incubating with MB-MMAb and Bsa-CPMs@PS-RMAb for 20 min, washing three times with PBS magnetic separation, dispersing in PBS, and performing chemiluminescence detection immediately after illumination.

[0046] Further, in step S1, the concentration of Bsa-CPMs@PS was 1-20 mg / ml, and the contents of EDC and NHS were 5 mg / ml and 6 mg / ml, respectively;

[0047] Furthermore, in steps S2 and S4, the concentration of Rabbit MAb and Mouse MAb is 1 mg / ml, and the added volume is 0.5-10 μL, which can be any value within this range.

[0048] Furthermore, the MB (magnetic beads) used in step S3 were modified with carboxyl groups at a concentration of 10 mg / ml.

[0049] Furthermore, the concentration ratio of PSA to MAb ranges from 1:250 to 1:1000.

[0050] Compared with the prior art, the BODIPY light-caged chemiluminescent molecules, nano-microspheres, and their synthesis methods and applications provided by the present invention have the following beneficial effects:

[0051] 1. The BODIPY light-caged chemiluminescent molecules provided by the present invention can be activated to generate chemiluminescent signals only under light conditions, without the need to add an excitation solution, and are easier to operate.

[0052] 2. Compared with most chemiluminescent molecules, the BODIPY photocaged chemiluminescent molecules provided by the present invention have the advantages of high luminescence brightness and good chemical stability (such as relatively slow self-decomposition rate). When used in immunoassay detection, they are more suitable for the detection of targets in complex environments and have great application value.

[0053] 3. The BODIPY photocaged chemiluminescent nanospheres provided by the present invention use a swelling method to load BODIPY photocaged chemiluminescent molecules (Bsa-CPMs) into polystyrene nanospheres (PS spheres). On the one hand, the PS spheres with carboxyl groups have strong chemical reactivity and can be used as the starting site for fixing certain molecules or compounds, or as active functional groups for coupling reactions of other functional groups, providing convenience for coupling specific biological molecules such as antibodies; on the other hand, the polystyrene nanospheres have good thermal stability and mechanical properties, which can maintain the stability and durability of Bsa-CPMs and avoid the instability and decomposition of chemiluminescent molecules during storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0055] Figure 1 It is a schematic diagram of the chemiluminescence principle of the BODIPY light-caged chemiluminescent molecule provided by the present invention;

[0056] Figure 2 is the ultraviolet absorption spectrum of the BODIPY light-caged chemiluminescent molecule synthesized in Example 1;

[0057] Figure 3 is a TEM image of Bsa-CPM@PS synthesized in Example 2;

[0058] Figure 4 It is the verification of the photoluminescence performance of the BODIPY photocaged chemiluminescent molecule synthesized in Example 1;

[0059] Figure 5 It is the verification of the photoluminescence performance of Bsa-CPM@PS synthesized in Example 2;

[0060] Figure 6 This is a diagram showing the chemiluminescent immunoassay effect of the prostate cancer marker PSA in Example 4. DETAILED DESCRIPTION

[0061] In order to enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention and to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention are further described below.

[0062] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0063] The BODIPY light-caged chemiluminescent molecule of the present invention is denoted as Bsa-CPMs, and its structure is shown in formula (I):

[0064] ;

[0065] (I)

[0066] in:

[0067] .

[0068] The BODIPY light-caged chemiluminescent molecules of the present invention can be activated under light conditions to output chemiluminescent signals. The specific luminescence mechanism is as follows: Figure 1 shown.

[0069] Under light conditions, the BODIPY photocaged chemiluminescent molecule generates singlet oxygen ( 1 O 2 ) oxidizes the C=C in the chemiluminescent molecule to form peroxobutane. At the same time, the CO bond connecting BODIPY to the chemiluminescent molecule is broken, and the unstable carbonate is converted to CO 2 The hydroxyl protecting group leaves and becomes an unstable intermediate II, which self-degrades to generate an excited state III, which then transitions to the ground state and releases photons. Unlike the acridinium ester luminescence system, the BODIPY photocaged chemiluminescence does not require oxidants such as hydrogen peroxide. The chemiluminescence process can be triggered after illumination, making it easier to operate and more suitable for the detection of targets in complex environments.

[0070] Example 1: Synthesis of BODIPY light-caged chemiluminescent molecules Bsa-CPMs

[0071] The synthesis route of BODIPY light-caged chemiluminescent molecules Bsa-CPMs is as follows:

[0072] ;

[0073] in:

[0074] ;

[0075] A = 2-adamantanone; cyclobutanone; 3-oxetanone; 3-carbonyl-cyclobutanecarboxylic acid methyl ester; tetrahydrothiopyran-4-one 1,1-dioxide;

[0076] B = (triphenylphosphine)acetonitrile; 2-(triphenylphosphoranylidene)acetic acid; ethoxycarbonylmethylenetriphenylphosphine.

[0077] This example takes BODIPY photocaged 1,2-dioxacycloadamantane chemiluminescent molecule as an example to describe its synthesis method in detail. Its structural formula is as follows:

[0078] ;

[0079] Specifically, the synthesis method comprises the following steps:

[0080] (1) Synthesis of compound 1

[0081] 2-Chloro-3-hydroxybenzaldehyde (2000 mg, 12.77 mmol) was dissolved in 20 mL of methanol, trimethyl orthoformate (2.24 mL, 20.44 mmol) and tert-butylammonium tribromide (308 mg, 0.64 mmol) were added to the above solution, and stirred at room temperature overnight. The reaction was monitored by thin layer chromatography. When the reaction was completed, ethyl acetate was added to the reaction mixture for dilution, and then washed with a 0.01 M aqueous solution of sodium bicarbonate and extracted with ethyl acetate. After the organic phases were combined and dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure. The product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (v / v=1 / 4) as an eluent to obtain compound 1 as a colorless oil.

[0082] The NMR data of compound 1 are: 1 H NMR (400 MHz, Chloroform- d ) δ 7.24 (dd, J=4.3,1.7 Hz, 2H), 7.06 (dd, J=5.7, 4.0 Hz, 1H), 5.80 (d, J=2.3 Hz, 1H), 5.67-5.57(m, 1H), 3.42 (d, J=1.3 Hz, 6H).

[0083] (2) Synthesis of compound 2

[0084] Compound 1 (2450 mg, 12.09 mmol) was dissolved in 15 mL of anhydrous dichloromethane, and then imidazole (1650 mg, 24.24 mmol) and tert-butyldimethylsilyl chloride (2180 mg, 14.46 mmol) were added in sequence, and stirred at room temperature for three hours. The reaction was monitored by thin layer chromatography. When the reaction was completed, the white precipitate was filtered, the solvent was evaporated under reduced pressure, and the product was purified by silica gel column chromatography using dichloromethane / petroleum ether (v / v=1 / 4) as an eluent to obtain a colorless oily compound 2.

[0085] The NMR data of compound 2 are: 1 H NMR (400 MHz, Chloroform- d) δ 7.31 (dt, J=8.2,2.1 Hz, 1H), 7.23 (t, J=7.9 Hz, 1H), 6.92 (dt, J=8.0, 1.7 Hz, 1H), 5.23 (d, J=15.7 Hz, 1H), 3.82 (d, J=10.6 Hz, 3H), 3.68 (d, J=10.4 Hz, 3H), 3.39 (s, 3H), 1.06 (s, 9H), 0.27-0.25 (m, 6H).

[0086] (3) Synthesis of compound 3

[0087] Compound 2 (3500 mg, 11.04 mmol) was dissolved in anhydrous dichloromethane, and trimethyl phosphite (1.7 mL, 14.41 mmol) was added. After cooling to 0°C, titanium chloride (1.45 mL, 13.22 mmol) was added dropwise to the reaction mixture. The reaction was monitored by thin layer chromatography. When the reaction was completed, a saturated aqueous solution of NaHCO3 was added to the reaction solution at 0°C. After stirring for 20 minutes, dichloromethane was added for extraction and the phases were separated. After the organic phases were combined and dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure. The product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (v / v=1 / 4) as the eluent to obtain a colorless oily compound 3.

[0088] The NMR data of compound 3 are: 1 H NMR (400 MHz, Chloroform- d ) δ 7.34-7.28 (m,1H), 7.22 (td, J=7.9, 2.5 Hz, 1H), 6.97-6.85 (m, 1H), 5.22 (dd, J=15.7, 2.6Hz, 1H), 3.82 (dd, J=10.6, 2.5 Hz, 3H), 3.67 (dd, J=10.5, 2.5 Hz, 3H), 3.38 (d, J=2.6 Hz, 3H), 1.06 (d, J=2.6 Hz, 9H), 0.25 (d, J=2.6 Hz, 6H).

[0089] (4) Synthesis of compound 4

[0090] Compound 3 (3950 mg, 10.0 mmol) was dissolved in 25 mL of anhydrous tetrahydrofuran, and the reaction was carried out under a nitrogen atmosphere at -78°C. Diisopropylamine lithium solution (2.0 M tetrahydrofuran, 6 mL, 12 mmol) was added and stirred at low temperature for 20 min. 2-Adamantane ketone (2250 mg, 14.98 mmol) was dissolved in tetrahydrofuran, and then the tetrahydrofuran solution of 2-adamantanone was slowly added dropwise to the above reaction solution, and continued to stir at -78°C for half an hour, and then the reaction was returned to room temperature for stirring. The reaction was monitored by thin layer chromatography. When the reaction was completed, ethyl acetate was added to the reaction mixture for dilution, washed with brine, and then extracted with ethyl acetate. After the organic phases were combined and dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure. The product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (v / v=5 / 95) as the eluent to obtain white solid compound 4.

[0091] The NMR data of compound 4 are: 1 H NMR (400 MHz, Chloroform- d ) δ 7.13 (t, J=7.8Hz, 1H), 6.90 (dq, J=7.6, 1.7 Hz, 2H), 3.34 (s, 3H), 3.30 (s, 1H), 2.09 (s,1H), 1.97-1.67 (m, 12H), 1.07 (s, 9H), 0.26 (s, 6H).

[0092] (5) Synthesis of compound 5

[0093] Compound 4 (3500 mg, 8.35 mmol) was dissolved in 30 mL of tetrahydrofuran, and tetrabutylammonium fluoride (1.0 M tetrahydrofuran, 9.2 mL, 9.2 mmol) was added, and the reaction was stirred at room temperature. The reaction was monitored by thin layer chromatography. When the reaction was completed, ethyl acetate was added to the reaction mixture for dilution, washed with a small amount of hydrochloric acid, and then extracted with ethyl acetate. After the organic phases were combined and dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure. The product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (v / v=15 / 85) as the eluent to obtain white solid compound 5.

[0094] The NMR data of compound 5 are: 1 H NMR (400 MHz, Chloroform- d) δ 7.25-7.10 (m,1H), 7.03 (dt, J=8.1, 1.6 Hz, 1H), 6.86 (dt, J=7.5, 1.5 Hz, 1H), 5.74 (s,1H), 3.34 (d, J=1.7 Hz, 3H), 3.31 (s, 1H), 2.14 (s, 1H), 1.99-1.73 (m, 12H).

[0095] (6) Synthesis of compound 6

[0096] Compound 5 (500 mg, 1.64 mmol) was dissolved in acetonitrile, MgCl2 (344 mg, 3.61 mmol) and Et3N (500 μL, 3.61 mmol) were added, and after stirring at room temperature for 10 min, paraformaldehyde (395 mg, 13.1 mmol) was added to the reaction mixture under nitrogen atmosphere. The reaction mixture was refluxed at 80 °C overnight. The reaction was monitored by thin layer chromatography. When the reaction was completed, it was cooled to room temperature, the paraformaldehyde solid was dissolved with dilute hydrochloric acid, and then extracted with ethyl acetate. The organic phases were combined and dried with anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was purified by silica gel column chromatography using dichloromethane / petroleum ether (v / v=1 / 2) as eluent to obtain white solid compound 6.

[0097] The NMR data of compound 6 are: 1 H NMR (400 MHz, Chloroform- d ) δ 11.66 (s, 1H),9.93 (s, 1H), 7.52 (d, J=7.9 Hz, 1H), 7.02 (d, J=7.8 Hz, 1H), 3.37 (s, 3H),3.32 (s, 1H), 2.11 (s, 1H), 2.01-1.78 (m, 12H).

[0098] (7) Synthesis of compound 7

[0099] Compound 6 (500 mg, 1.5 mmol) and (triphenylphosphine)acetonitrile (500 mg, 1.65 mmol) were added to a 50 mL round-bottom flask and dissolved with anhydrous dichloromethane, stirred at room temperature for 30 min, and after the reaction was completed, the solvent was removed in vacuo, the residue was dissolved in dichloromethane (20 mL), and passed through a celite pad, purified by column chromatography (ethyl acetate / petroleum ether = 1 / 6), and the combined eluent was concentrated in vacuo to obtain compound 7.

[0100] The NMR data of compound 7 are:1 H NMR (400 MHz, Chloroform- d ) δ 7.62 (d, J =16.8 Hz, 1H), δ 7.31 (d, 1H), 6.93 (d, 1.2 Hz, 1H), 6.36 (s, 1H), 6.22 (d,1.2 Hz, 1H), 3.35 (s, 3H), 3.31 (s, 1H), 2.13 – 1.83 (m, 13H).

[0101] (8) Synthesis of compound 8

[0102] 2,4-Dimethylpyrrole (0.2 g, 2.1 mmol, 2 eq) was dissolved in dry dichloromethane. Acetoxyacetyl chloride (0.14 mL, 1.3 mmol, 1.2 eq) was added to the solution and the reaction was stirred under argon at 40 °C. After 2 hours, it was cooled to room temperature and diisopropylethylamine (0.73 mL, 4.2 mmol, 4 eq) was added, and then boron trifluoride diethanolamine complex (0.53 mL, 4.2 mmol, 3 eq) was added dropwise after 15 minutes. During the addition of boron trifluoride diethanolamine complex, the color changed from light yellow to dark red. The reaction was stopped after 15 minutes, the solvent was evaporated under reduced pressure, and the crude reaction mixture was loaded on a silica flash column and eluted with 50% ethyl acetate / hexane to obtain orange-green crystals (0.25 g, 75% yield).

[0103] The NMR data of compound 8 are: 1 H NMR (400 MHz, Chloroform- d ) δ 6.09 (s, 2H), 5.30 (s, 2H), 2.53 (s, 6H), 2.36 (s, 6H), 2.13 (s, 3H).

[0104] (9) Synthesis of compound 9

[0105] Compound 8 (320 mg, 1 mmol, 1 eq) was dissolved in dry tetrahydrofuran under argon to give a 0.05 M solution. Lithium hydroxide monohydrate (115 mg, 5 mmol, 5 eq) was dissolved in an equal volume of water. The aqueous lithium hydroxide solution and the bulk solution in tetrahydrofuran were combined. The reaction mixture was stirred at room temperature under argon for 4 hours and then extracted with ethyl acetate (3×100 mL). The combined organic layers were washed with saturated aqueous ammonium chloride solution (3×100 mL) and brine (1×100 mL). The organic phase was collected and concentrated to give a crude product, which was loaded onto a silica flash column and eluted with 50% ethyl acetate / hexane to give compound 9 as an orange powder.

[0106] The NMR data of compound 9 are: 1 H NMR (400 MHz, d 6-DMSO) δ 6.21 (s, 2H), 5.52 (t, 1H), 4.70 (d, J=5.1 Hz, 2H), 2.47 (s, 6H), 2.39 (s, 6H).

[0107] (10) Synthesis of compound 10

[0108] Compound 9 (100 mg, 0.36 mmol, 1 equivalent) was dissolved in anhydrous dichloromethane, and N,N-diisopropylethylamine (190 μL, 1.08 mmol, 3 equivalents) was added at 0 °C. After stirring for 5 minutes, a solution of triphosgene (160 mg, 0.54 mmol, 1.5 equivalents) dissolved in anhydrous dichloromethane was slowly added dropwise. Stir at room temperature for 1 h, and the reaction was monitored by thin layer chromatography. When the reaction was completed, the mixture was washed with water and saturated brine, and the organic phase was collected and concentrated to obtain the crude product of compound 10, which was immediately used for the next step reaction without further purification.

[0109] (11) Synthesis of compound 11

[0110] Compound 7 (100 mg, 0.28 mool, 1 equivalent) was dissolved in anhydrous tetrahydrofuran, N,N-diisopropylethylamine (150 μL, 0.84 mmol, 3 equivalents) was added at 0 °C, and the crude product of compound 10 was added after stirring for 5 minutes, and the mixture was stirred at room temperature for 1 h. The reaction was monitored by thin layer chromatography. When the reaction was completed, the mixture was washed with water and saturated brine, the organic phase was collected and concentrated to obtain the crude product of compound 11, and the product was purified by silica gel column chromatography using ethyl acetate / hexane (v / v=1 / 5) as the eluent to obtain dark solid compound 11.

[0111] Mass spectrometry analysis data of compound 11: MALDI-TOF: m / z [M]+ calcd forC 36 H 37 BClF 2 N 3 O 4 :659.25; found: 659.43.

[0112] The ultraviolet absorption spectrum of the BODIPY light-caged chemiluminescent molecule Bsa-CPMs synthesized in this example is shown in FIG. Figure 2 shown.

[0113] It should be noted that in the synthesis process of this embodiment, when the raw material compound A used in the synthesis step of compound 4 is selected from 2-adamantanone, cyclobutanone, 3-oxetanone, 3-carbonyl-cyclobutanecarboxylic acid methyl ester, or tetrahydrothioran-4-one 1,1-dihydrogenate, the R 2 respectively correspond to adamantane group, cyclobutane group, 3-oxetane group, methyl cyclobutanecarboxylate group, or tetrahydrothioran-1,1-dihydrogenate group); similarly, when compound 7 (Note: when R 1 When R is H atom, it is compound 5. 1 When the raw material compound B used in the synthesis step is (triphenylphosphine) acetonitrile, ethoxyformylmethylenetriphenylphosphine, 2-(triphenylphosphoranylidene)acetic acid, or other electron-withdrawing groups, R in compound 7 1 They correspond to acrylonitrile groups, methyl acrylate groups, acrylic acid groups, and other electron-withdrawing groups respectively.

[0114] Example 2

[0115] A BODIPY light-caged chemiluminescent nanosphere is prepared by using polystyrene nanospheres as carriers and loading BODIPY light-caged chemiluminescent molecules on polystyrene nanospheres. The specific preparation method comprises the following steps:

[0116] Step S1, dispersing 20 μl of carboxylated polystyrene nanospheres (PS) (100 mg / ml) in 980 μl of isopropanol and mixing;

[0117] Step S2, dissolving 2 mg of the BODIPY light-caged chemiluminescent molecules (Bsa-CPMs) prepared in Example 1 in 300 μl of dichloromethane;

[0118] Step S3, mixing the solutions in step S1 and step S2 uniformly, and shaking at 1200 rpm for 3 hours;

[0119] Step S4, the reaction mixture in step S3 was rotary evaporated to remove dichloromethane, and then centrifuged at 12000 rpm and 15°C for 30 min, and then washed three times with isopropanol, ethanol, and deionized water respectively to remove excess CPMs to obtain the product Bsa-CPM@PS.

[0120] The TEM image of Bsa-CPM@PS prepared in this example is shown in Figure 3 shown.

[0121] Example 3: Verification of the luminescent properties of chemiluminescent molecules and chemiluminescent nanospheres

[0122] The BODIPY photocage chemiluminescent molecules (100 μM) prepared in Example 1 and the BODIPY photocage chemiluminescent nanospheres prepared in Example 2 were placed under a white light source, respectively. After irradiation with white light for 1 min, 30 μL of the solution was taken out and added to the wells of a 96-well plate, and then the images were quickly taken using the chemiluminescence mode of the ChemiDoc XRS+ imaging system. Figure 4 and Figure 5 As shown, the prepared chemiluminescent molecules and nano-microspheres are activated under light conditions to generate chemiluminescent signals with high luminescence brightness.

[0123] Example 4: Application of BODIPY photocaged chemiluminescent nanospheres in chemiluminescent immunoassay of prostate cancer marker PSA

[0124] The specific method is as follows:

[0125] Step S1, mixing the chemiluminescent nanoparticles (Bsa-CPMs@PS) prepared in Example 2 with an EDC / NHS solution (MES, 100 mM, pH 5.5), reacting at room temperature for 1 h, and centrifuging at 14000 rpm for 8 min to remove EDC / NHS; wherein the concentration of Bsa-CPMs@PS is 1-20 mg / ml, and the contents of EDC and NHS are 5 mg / ml and 6 mg / ml, respectively;

[0126] Step S2, covalently linking rabbit anti-human prostate protein (PSA) monoclonal antibody (Rabbit MAb) to Bsa-CPMs@PS via an amide bond: adding Rabbit MAb to the activated Bsa-CPMs@PS solution in step S1, shaking the reaction overnight, and then centrifuging and washing twice at 14000 rpm to remove excess Rabbit MAb, thereby obtaining rabbit antibody-modified chemiluminescent nanoparticles (Bsa-CPMs@PS-RMAb), which were dispersed in PBS; wherein the Rabbit MAb concentration was 1 mg / ml, and the added volume was 0.5-10 μL;

[0127] Step S3, reacting carboxylated magnetic beads (MB-COOH) with EDC / NHS mixed solution (MES, 100 mM, pH 5.5) at room temperature for 2 h, then magnetically separating and discarding the supernatant, washing three times with PBS buffer (pH 7.4) to remove excess EDC / NHS, and dispersing in PBS; wherein the MB (magnetic beads) used are modified with carboxyl groups at a concentration of 10 mg / ml;

[0128] Step S4, adding mouse anti-human PSA monoclonal antibody (Mouse MAb) to the MB solution activated in step S3, shaking overnight and washing four times with PBS magnetic separation to obtain mouse antibody-modified magnetic beads (MB-MMAb), which are dispersed in PBS; wherein the concentration of Mouse MAb is 1 mg / ml, and the added volume is 0.5-10 μL;

[0129] Step S5, take different concentrations of PSA, incubate with MB-MMAb and Bsa-CPMs@PS-RMAb for 20 min, wash three times with PBS magnetic separation, disperse in PBS (pH 7.4) solution, place under white light source for 1 min, take out 30 μL of solution and add it to the wells of 96-well plate, and quickly perform imaging detection using the chemiluminescence mode of ChemiDoc XRS+ imaging system.

[0130] like Figure 6 As shown, when PSA is present, the reaction system can emit a strong chemiluminescent signal, indicating that the chemiluminescent nanoparticles (Bsa-CPMs@PS) of the present invention can be used for chemiluminescent immunoassay of prostate cancer marker PSA.

[0131] Therefore, the BODIPY photocaged chemiluminescent molecules and nanoparticles Bsa-CPM@PS of the present invention can be applied in chemiluminescent immunoassays, have the characteristics of simple operation, high luminescence intensity, and good stability, and can improve the detection sensitivity.

[0132] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations made to these embodiments without departing from the principles and spirit of the present invention are still within the protection scope of the present invention.

Claims

1. A BODIPY light-caged chemiluminescent molecule, characterized in that: Its structure is shown in formula (I): ； （Ⅰ） in: 。 2. A method for synthesizing the BODIPY light-caged chemiluminescent molecule as claimed in claim 1, characterized in that: The steps include: (1) Compound 1 was synthesized from 2-chloro-3-hydroxybenzaldehyde / m-hydroxybenzaldehyde / 2-bromo-3-hydroxybenzaldehyde, trimethyl orthoformate and tert-butylammonium tribromide; (2) Compound 2 was synthesized using compound 1, imidazole and tert-butyldimethylsilyl chloride as raw materials; (3) Compound 3 was synthesized using compound 2, trimethyl phosphite and titanium chloride as raw materials; (4) Compound 4 is synthesized using compound 3, lithium diisopropylamide and compound A as raw materials; wherein compound A is one of 2-adamantanone, aldehyde-modified β-cyclodextrin, cyclobutanone, 3-oxetanone, 3-carbonyl-cyclobutanecarboxylic acid methyl ester and tetrahydrothiopyran-4-one 1,1-dioxide; (5) Compound 5 was synthesized using compound 4 and tetrabutylammonium fluoride as raw materials; (6) Compound 6 was synthesized using compound 5, magnesium chloride and paraformaldehyde as raw materials; (7) Compound 7 is synthesized using compound 6 and compound B as raw materials; wherein compound B is one of (triphenylphosphine)acetonitrile, 2-(triphenylphosphoranylidene)acetic acid, and methoxyformylmethylenetriphenylphosphine; (8) Compound 8 was synthesized using 2,4-dimethylpyrrole and acetoxyacetyl chloride as raw materials; (9) Compound 9 was synthesized using compound 8 and lithium hydroxide monohydrate as raw materials; (10) Compound 10 was synthesized from compound 9, triphosgene and N,N-diisopropylethylamine; (11) Compound 7 and compound 10 were used as raw materials to synthesize BODIPY light-caged chemiluminescent molecules, namely compound 11; The synthesis route is as follows: ； in: ； A = 2-adamantanone; cyclobutanone; 3-oxetanone; 3-carbonyl-cyclobutanecarboxylic acid methyl ester; tetrahydrothiopyran-4-one 1,1-dioxide; B = (triphenylphosphine)acetonitrile; 2-(triphenylphosphoranylidene)acetic acid; ethoxycarbonylmethylenetriphenylphosphine.

3. Use of the BODIPY photocaged chemiluminescent molecule as claimed in claim 1 in the preparation of chemiluminescent immunoassay products.

4. A BODIPY light-caged chemiluminescent nanosphere, characterized in that: The BODIPY light-caged chemiluminescent molecules Bsa-CPMs described in claim 1 are loaded on polystyrene nanospheres PS as carriers to prepare the nanospheres, and the prepared nanospheres are activated to emit light by light.

5. The BODIPY light-caged chemiluminescent nanosphere according to claim 4, characterized in that: The preparation method thereof comprises the following steps: Step S1, dispersing carboxylated polystyrene nanospheres PS in isopropanol; Step S2, dissolving the BODIPY light-caged chemiluminescent molecule Bsa-CPMs in dichloromethane; Step S3, mixing the solutions of step S1 and step S2 uniformly; Step S4, rotary evaporating the sample in step S3, centrifuging and washing to obtain BODIPY light-caged chemiluminescent nanoparticles Bsa-CPM@PS.

6. The BODIPY light-caged chemiluminescent nanosphere according to claim 5, characterized in that: The particle size of the carboxylated polystyrene nanospheres is 20-5000 nm.

7. The BODIPY light-caged chemiluminescent nanosphere according to claim 5, characterized in that: In step S1, the concentration of the dispersed polystyrene nanospheres is 1-2 mg / ml.

8. The BODIPY light-caged chemiluminescent nanosphere according to claim 5, characterized in that: In step S2, the concentration of BODIPY photocaged chemiluminescent molecules is 3.3-6.6 mg / ml.

9. Use of the BODIPY photocaged chemiluminescent nanospheres as claimed in claim 4 in the preparation of chemiluminescent immunoassay products.

10. Use of the BODIPY photocaged chemiluminescent nanospheres as claimed in claim 4 in preparing a chemiluminescent immunoassay product for a prostate cancer marker, PSA.

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