Self-luminous molecular probe as well as preparation method, application and load test paper thereof

Abstract

The invention relates to a self-luminous molecular probe as well as a preparation method and application thereof and load test paper, and belongs to the technical field of biological detection. The self-luminous molecular probe provided by the invention is simple in preparation method, cheap and easily available in raw materials, simple and convenient in synthesis process, easy to separate and purify, high in yield and suitable for large-scale production, popularization and application, has a specific enzyme trigger response chemical self-luminous effect, and can be used for preparing the self-luminous molecular probe. The interference of self-absorption and biological sample background fluorescence can be effectively avoided, and the method can be used for rapid and sensitive optical detection and visual portable test paper detection of ALP.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a self-luminous molecular probe, a preparation method thereof, an application thereof, and a loading test strip. Background Art

[0002] Alkaline phosphatase (ALP), as a key isoenzyme in many biological tissues, has the functions of catalyzing the hydrolysis of phosphate esters and phosphorylation, and plays an important role in the phosphate group transfer and metabolism processes of organs such as the liver, kidney, intestine, bone, and placenta. The activity level of ALP in serum is one of the important indicators in routine blood tests. Its abnormal increase is related to various diseases, such as bone diseases (osteogenic osteosarcoma, Paget's disease, osteomalacia), liver diseases (cancer, hepatitis, obstructive jaundice), breast cancer, prostate cancer, and diabetes, etc.; at the same time, the ALP level may also decrease due to metabolic disorders (such as Wilson's disease) or blood system diseases (such as aplastic anemia and chronic myeloid leukemia). Currently, the serum ALP level has been listed as a core biomarker for liver function and bone metabolism in the "Clinical Laboratory Operating Procedures" (WHO standard: 40 - 129 U / L).

[0003] However, the existing ALP detection technologies have many defects. Traditional colorimetric methods (such as p-nitrophenyl phosphate method) rely on enzyme-catalyzed color reactions, with low sensitivity, a detection limit greater than 5 U / L, and weak anti-interference ability. The inhibition rate of bilirubin / hemoglobin can reach more than 30%; fluorescence probe methods require an external excitation light source, are easily interfered by biological autofluorescence, resulting in a 40% - 60% decrease in the signal-to-noise ratio, and there is also a problem of photobleaching. The signal decays by more than 50% after continuous irradiation for 10 minutes; electrochemical sensors are greatly affected by electrode surface contamination and have poor reusability. The sensitivity decreases by 80% after 3 cycles. Therefore, it is of great practical significance to develop an ALP detection technology with strong anti-interference ability, high sensitivity, and fast and effective.

[0004] Chemiluminescence is a self-luminous process that uses a chemical reaction as an excitation source. Without external light excitation, it can directly excite the luminescent body using the chemical energy released by the ALP-catalyzed substrate decomposition reaction, avoiding the interference of endogenous fluorescent substances (NADH, bilirubin) in biological samples (such as blood, tissue fluid), having a higher detection signal-to-noise ratio and stronger anti-interference ability, and chemiluminescence also has the advantages of ultra-high sensitivity, wide linear range, and long luminescence lifetime.

[0005] In view of this, the present invention is specifically proposed to solve the above problems. Summary of the Invention

[0006] Aiming at the inherent defect problem of current traditional fluorescent molecular probes for ALP detection, the purpose of the present invention is to provide a self-luminescent molecular probe capable of rapidly detecting alkaline phosphatase, and disclose the preparation method and application to achieve rapid and ultrasensitive detection of ALP in vitro.

[0007] To achieve the above object, the present invention provides the following technical solutions: A self-luminescent molecular probe, characterized in that it has the following molecular formula:

[0008] Furthermore, the preparation method of the above self-luminescent molecular probe includes the following steps: Step 1: Dissolve 3-hydroxybenzaldehyde in methanol, add trimethyl orthoformate and tert-butylammonium tribromide, stir at room temperature, monitor the reaction by TLC. After the reaction is completed, dilute with ethyl acetate, wash with 0.01M sodium bicarbonate, dry the organic phase, concentrate, and purify by silica gel column chromatography to obtain Compound 1; Step 2: Dissolve Compound 1 and imidazole in dichloromethane, add tert-butyldimethylsilyl chloride, stir at room temperature, monitor the reaction by TLC, filter the precipitate, evaporate the solvent, and purify by silica gel column chromatography to obtain Compound 2; Step 3: Dissolve Compound 2 and trimethyl phosphite in dichloromethane, cool to 0 °C, dropwise add titanium tetrachloride, monitor the reaction by TLC. After the reaction, pour into saturated sodium bicarbonate aqueous solution, stir, extract, dry, concentrate, and purify by silica gel column chromatography to obtain Compound 3; Step 4: Under nitrogen protection, dissolve Compound 3 in anhydrous tetrahydrofuran, cool to -78 °C, add lithium diisopropylamide and stir, then add an anhydrous tetrahydrofuran solution of 2-adamantanone, react at -78 °C and then warm up to room temperature to continue the reaction, monitor the reaction by TLC. After the reaction, dilute with ethyl acetate, wash with saturated brine, dry, concentrate, and purify by silica gel column chromatography to obtain Compound 4; Step 5: Dissolve Compound 4 in tetrahydrofuran, add tetrabutylammonium fluoride, stir at room temperature, monitor the reaction by TLC. After the reaction, dilute with ethyl acetate, wash with dilute hydrochloric acid, dry, concentrate, and purify by silica gel column chromatography to obtain Compound 5; Step 6: Dissolve Compound 5 in toluene, cool to 0 °C, add N-iodosuccinimide in portions, stir, monitor the reaction by TLC. After the reaction, quench with saturated sodium thiosulfate, dilute with ethyl acetate, wash with brine, dry, concentrate, and purify by silica gel column chromatography to obtain Compound 6; Step 7: Dissolve compound 6 in anhydrous DMF, add copper(I) iodide, triphenylphosphine, palladium(II) acetate and benzothiazole, stir at 100 °C for 12 h, monitor the reaction by TLC. After the reaction, dilute with ethyl acetate, wash with brine, dry, concentrate and purify by silica gel column chromatography to obtain compound 7 with X = S; Step 8: Dissolve compound 7 in dry DMF, cool to 0 °C under argon, add sodium hydride, heat to room temperature and stir, then add diethyl (4-(bromomethyl)phenyl)phosphonate. Monitor the reaction by TLC. After the reaction, concentrate by evaporation under reduced pressure, and purify the crude product by HPLC to obtain compound 8 with X = S; Step 9: Take compound 8 and a catalytic amount of methylene blue and dissolve them in DCM, stir in an ice bath and react under white light irradiation with oxygen bubbling. Monitor the reaction by TLC. After the reaction, purify by HPLC to obtain compound CL–ALP with X = S, namely the self-luminescent molecular probe.

[0009] The present invention also provides an application of the above self-luminescent molecular probe. The self-luminescent molecular probe realizes the rapid detection of ALP by chemically reacting with ALP to generate fluorescence; Further, it includes the following steps: S1. Immerse the paper test strip in a THF solution containing CL-ALP for 0.5 min, then air dry; S2. Immerse the test strip obtained in step S1 in an aqueous ALP solution for 5 min, then air dry; S3. Take chemiluminescence imaging and fluorescence images of the test strip.

[0010] Further, the chemiluminescence imaging is obtained by an IVIS imaging system in the bioluminescence mode, using an open filter, and the exposure time is 5 s; the fluorescence image is obtained by exciting with an ultraviolet lamp at a wavelength of 365 nm.

[0011] The present invention also discloses a loaded test strip, which is loaded with the above self-luminescent molecular probe.

[0012] The beneficial effects of the present invention are as follows: 1. The preparation method of the chemiluminescent molecular probe for sensitive detection of ALP provided by the present invention is simple, the raw materials are cheap and easily available, the synthesis process is simple, the separation and purification are easy, the yield is high, and it is suitable for large-scale production and popularization.

[0013] 2. This molecular probe has a specific enzyme-triggered response chemiluminescence effect, can effectively avoid the interference of self-absorption and background fluorescence of biological samples, and can be used for rapid and sensitive optical detection of ALP and visual portable test strip detection.

[0014] The above description is only an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and implement it according to the content of the specification, the following describes the preferred embodiments of the present invention in detail with reference to the accompanying drawings. Brief Description of the Drawings

[0015] Figure 1: Ultraviolet absorption, fluorescence emission and chemiluminescence response spectra of compound CL - ALP in Example 2, where: Figure 1(a): Ultraviolet absorption spectra and fluorescence emission spectra of probe CL - ALP before and after responding to ALP; Figure 1(b): Chemiluminescence spectra of probe CL - ALP before and after responding to ALP. The inset is a picture taken before and after the response using an IVIS imaging system in the bioluminescence mode; Figure 2: Chemiluminescence response behavior of compound CL - ALP to pH value in Example 3, where: Figure 2(a): Chemiluminescence intensities of probe CL - ALP before and after responding at different pH values. The inset is a picture taken using an IVIS imaging system in the bioluminescence mode at different pH values; Figure 2(b): Signal - to - noise ratios of probe CL - ALP responding to ALP at different pH values. The values are mean ± SD (n = 3); Figure 3: Investigation of the response kinetics of compound CL - ALP in Example 4, specifically the chemiluminescence kinetics of the probe responding to ALP. The concentration of the probe is 10 μM, the concentration of ALP is 0 or 10 U / L, and the chemiluminescence intensity at a recording wavelength of 480 nm is recorded; Figure 4: Investigation of the response of compound CL - ALP to different concentrations of ALP in Example 5, where: Figure 4(a): Chemiluminescence pictures taken in the bioluminescence mode of an IVIS imaging system after different concentrations of ALP respond to probe CL - ALP (10 μM); Figure 4(b): Changes in chemiluminescence intensity at a wavelength of 480 nm after probe CL - ALP (10 μM) is incubated with different concentrations of ALP (0 - 80 U / L); Figure 5: Investigation of the response specificity of compound CL - ALP in Example 6, chemiluminescence intensity diagrams before and after adding the probe, interferent (L) and ALP; Figure 6: Chemiluminescence and fluorescence emission dual - mode visualization detection of ALP by the CL - ALP test strip in Example 7, where: Figure 6(a): Schematic diagram of the operation of the test strip for detecting ALP; Figure 6(b): The pictures are chemiluminescence pictures taken by the IVIS imaging system in the bioluminescence mode and fluorescence pictures taken under a portable ultraviolet lamp; Figure 6(c): The corresponding intensity values in the chemiluminescence pictures. The values are mean ± SD (n = 3). Detailed implementation manners

[0016] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0017] In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0018] Example 1 A self-luminous molecular probe for rapid detection of alkaline phosphatase shown in a preferred embodiment of the present application has the following structural formula:

[0019] When X = S, the synthesis steps are as follows:

[0020] Specifically, when X = S, the preparation method of the self-luminous molecular probe includes the following steps: Step 1: Synthesis of Compound 1 Dissolve 3-hydroxybenzaldehyde (2000 mg, 12.77 mmol) in methanol, add trimethyl orthoformate (2.24 mg, 20.44 mmol) and tert-butylammonium tribromide (308 mg, 0.64 mmol), and stir the solution at room temperature. Monitor the reaction progress by TLC method. After the reaction is completed, dilute the reaction mixture with ethyl acetate and wash it with 0.01 M sodium bicarbonate. Dry the organic phase over anhydrous sodium sulfate and concentrate it under reduced pressure. Purify by silica gel column chromatography to obtain colorless oily compound 1 (2460 mg, yield 95%).

[0021] Step 2: Synthesis of Compound 2 Compound 1 (336.4 mg, 2 mmol) and imidazole (272.7 mg, 4 mmol) were dissolved in dichloromethane. tert-Butyldimethylsilyl chloride (360.8 mg, 2.4 mmol) was added, and the solution was stirred at room temperature. The reaction progress was monitored by TLC. After the reaction was completed, the white precipitate was filtered off, and the solvent was evaporated under reduced pressure. Purification by silica gel column chromatography gave compound 2 as a colorless oil (595.1 mg, yield 94%).

[0022] Step 3: Synthesis of compound 3 Compound 2 (350 mg, 1.1 mmol) and trimethyl phosphite (170 μL, 1.4 mmol) were dissolved in dichloromethane. Then the reaction mixture was cooled to 0 °C, and titanium tetrachloride (150 μL, 1.3 mmol) was slowly added dropwise. The reaction progress was monitored by TLC. After the reaction was complete, the solution was poured into saturated aqueous sodium bicarbonate at 0 °C. After stirring for 10 minutes, dichloromethane was added for extraction. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purification by silica gel column chromatography gave compound 3 as a colorless oil (401 mg, yield 92%). Step 4: Synthesis of compound 4 Under nitrogen protection, compound 3 (395 mg, 1.0 mmol) was dissolved in anhydrous tetrahydrofuran. Then the system was cooled to -78 °C and stirred for 30 minutes. Then lithium diisopropylamide (2.0 mol / L, 0.6 mL, 1.2 mmol) was slowly added, and stirring was continued for 30 minutes. Then a solution of 2-adamantanone (225 mg, 1.5 mmol) dissolved in 10 mL of anhydrous tetrahydrofuran was slowly added to the system. The reaction was continued at -78 °C for 30 minutes, and then the reaction system was allowed to warm to room temperature and react for another 2 hours. The reaction progress was monitored by TLC. After the reaction was complete, the reaction mixture was diluted with ethyl acetate and washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purification by silica gel column chromatography gave compound 4 as a white solid (356 mg, yield 85%).

[0023] Step 5: Synthesis of compound 5 Compound 4 (419.2 mg, 1 mmol) was dissolved in tetrahydrofuran. Tetrabutylammonium fluoride (1.0 M, 1.1 mL, 1.1 mmol) was added, and the reaction system was stirred at room temperature for 1 hour. The reaction progress was monitored by TLC. After the reaction was complete, it was diluted with ethyl acetate and washed with 1 mol / L dilute hydrochloric acid. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. It was purified by silica gel column chromatography to obtain white solid compound 5 (290 mg, yield 95%).

[0024] Step 6: Synthesis of Compound 6 Compound 5 (269.6 mg, 1 mmol) was dissolved in toluene and cooled to 0 °C. N-Iodosuccinimide (224.9 mg, 1 mmol) was added portionwise, and the reaction was stirred for 1 hour. The reaction progress was monitored by TLC. After the reaction was complete, the reaction system was quenched with saturated sodium thiosulfate, then diluted with ethyl acetate and washed with brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. It was purified by silica gel column chromatography (PE:EA = 85:15) to obtain 182.2 mg of a white solid (yield 45%).

[0025] Step 7: Synthesis of Compound 7 Compound 6 (600 mg, 1.5 mmol) was dissolved in anhydrous DMF, then copper(I) iodide (catalytic amount), triphenylphosphine (390 mg, 1.5 mmol), palladium(II) acetate (10 mg, 0.05 mmol) and benzothiazole (270 mg, 2 mmol) were added. The reaction was stirred at 100 °C for 12 hours. The reaction progress was monitored by TLC. After the reaction was complete, it was diluted with ethyl acetate and washed with brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. It was purified by silica gel column chromatography to obtain 189 mg of a white solid product (yield 30%).

[0026] Step 8: Synthesis of Compound 8 Compound 7 (403.5 mg, 1 mmol) was taken and dissolved in dry DMF. It was cooled to 0 °C under argon, and sodium hydride (88 mg, 2.20 mmol) was added. It was heated to room temperature. After stirring for 15 minutes, diethyl 4-(bromomethyl)phenylphosphonate (387.8 mg, 1.2 mmol) was added. The reaction progress was monitored by TLC. After the reaction was complete, the reaction mixture was evaporated and concentrated under reduced pressure. The crude product was purified by HPLC to obtain white solid compound 8 (290.1 mg, yield: 45%).

[0027] Step 9: Synthesis of Compound CL-ALP Compound 8 (258.3 mg, 0.4 mmol) and a catalytic amount of methylene blue (16 mg) were dissolved in 10 mL of DCM. The reaction system was stirred under an ice bath and oxygen was introduced into the reaction system under white light irradiation. After reacting for 0.5 h, the reaction progress was monitored by TLC. After the reaction was completed, it was purified by HPLC to obtain white solid CL-ALP (154.2 mg, yield: 62%), which is the self-luminous molecular probe shown in the present invention. Example 2

[0028] Chemiluminescence Spectrum Study of Compound CL-ALP 10 μL of the CL-ALP mother liquor (1 mM) was added to a 2 mL EP tube, 980 μL of PBS buffer (20 mM, pH = 11) was added, and then 10 μL of ALP (1 mM) was added to form the post-response system. 10 μL of the CL-ALP mother liquor (1 mM) was added to a 2 mL EP tube, and 990 μL of PBS buffer (20 mM, pH = 11) was added as the pre-response system. The pre- and post-response systems were incubated in a 37 °C water bath for 30 minutes. Immediately afterwards, the ultraviolet-visible absorption spectra of the probe before and after the response were measured using an ultraviolet-visible spectrophotometer. The fluorescence spectra of the probe before and after the response were measured using a fluorescence spectrophotometer. 10 μL of the CL-ALP mother liquor (1 mM) was added to a 2 mL EP tube, 980 μL of PBS buffer (20 mM, pH = 11) was added, and then 10 μL of ALP (1 mM) was added. After incubation in a 37 °C water bath, the chemiluminescence spectrum was immediately read using a microplate reader. Each test data was measured three times repeatedly.

[0029] Please refer to Figure 1 , there are obvious differences in the spectra of CL-ALP before and after the reaction with ALP, which proves that this compound can specifically react with ALP, produce detectable spectral changes, and has the basic characteristics of a detection probe. Example 3

[0030] pH Investigation of Compound CL-ALP on ALP Prepare PBS buffers with different pH values in advance (pH = 4.0, 5.0, 6.0, 7.0, 8.0, 9.0). Take 10 μL of the CL-Hg stock solution and add it to a 2 mL EP tube. Then, add 980 μL of PBS buffers with different pH values respectively. Finally, add 10 μL of mercury chloride stock solution (1 mM). Make the volume of each system 1 mL. Pipette 300 μL of the above system into a white opaque 96-well plate, and place it in an enzyme-linked immunosorbent assay (ELISA) reader to measure the chemiluminescence intensity at different times at 37 °C. Take the maximum chemiluminescence value at 480 nm as the chemiluminescence intensity, and repeat each test data three times.

[0031] Please refer to Figure 2 , under different pH conditions, the chemiluminescence intensity and signal-to-noise ratio of CL-ALP before and after responding to ALP are different, indicating that pH is an important factor affecting the detection effect of CL-ALP for ALP. In actual detection, it is necessary to select an appropriate pH environment to optimize the detection performance. Example 4

[0032] Investigation of the response kinetics of compound CL-ALP to ALP Take 10 μL of the CL-ALP stock solution (1 mM) and add it to a 2 mL EP tube. Add 890 μL of PBS buffer (20 mM, pH = 7.4), and then add 10 μL of ALP (1 mM). Then add the system to a white opaque 96-well plate and place it in an ELISA reader to measure the change in chemiluminescence intensity at 480 nm (40 minutes) under the condition of 37 °C. Repeat each test data three times. Example 5

[0033] Investigation of the response of compound CL-ALP to different concentrations of ALP To evaluate the sensitivity of CL-ALP to ALP, CL-ALP (10 μM, 300 μL, PBS solution containing 1% DMSO, pH = 11.0) was incubated with different concentrations of ALP (0, 0.1, 0.5, 1, 2, 5, 10, 20, 40, 60, and 80 μM). After adding to a white opaque 96-well plate, it was measured on a microplate reader. In addition, the fluorescence intensity (excitation wavelength: 320 nm) after incubation of the above concentrations of ALP with CL-ALP (10 μM, 1000 μL, PBS solution containing 1% DMSO, pH = 11.0) was determined by a fluorescence spectrophotometer. According to the formula LOD = 3δ / k, the LOD values for chemiluminescence mode and fluorescence mode detection were calculated.

[0034] The results showed that CL-ALP had different degrees of response to different concentrations of ALP. By calculating the LOD values for chemiluminescence mode and fluorescence mode detection, its detection sensitivity was further quantified, demonstrating that CL-ALP had high sensitivity and accuracy in detecting different concentrations of ALP. Example 6

[0035] Investigation of the selectivity of compound CL-ALP for ALP To study the specificity of CL-ALP for ALP, CL-ALP (10 μM, 300 μL, PBS solution containing 1% DMSO, pH = 11.0) was co-incubated with five-fold amounts of different interferents, including cellulase (Cel), lysozyme (Lyso), β-galactosidase (β-Gal), β-glucuronidase (β-Glu), bovine serum albumin (BSA), ovalbumin (OVA), human serum albumin (HSA), cysteine (Cys), homocysteine (Hcy), glutathione (GSH), vitamin C (Vc), hydrogen peroxide (H2O2), hydrogen sulfide (H2S), CaCl2, MgCl2, and fetal bovine serum (FBS). The change in chemiluminescence intensity was measured using a microplate reader for 0 - 12 hours.

[0036] The results showed that after co-incubation with various interferents, CL-ALP was less affected by the interferents, and the change in chemiluminescence intensity mainly depended on the presence of ALP, indicating that this compound had high specificity for ALP and could effectively detect ALP in complex biological samples, reducing the influence of interference factors. Example 7

[0037] Test strip test of compound CL-ALP for ALP The paper test strips were immersed in a THF solution containing 50 μM CL-ALP for 0.5 minutes and then air-dried. The test strips loaded with CL-ALP were immersed in aqueous solutions of ALP with different concentrations (0, 2, 5, 10, 20, and 30 μM) for 5 minutes. After air-drying, chemiluminescence and fluorescence images of the strips were taken. Chemiluminescence imaging was obtained by the IVIS imaging system in the bioluminescence mode using an open filter with an exposure time of 5 seconds. The fluorescence images were obtained by excitation with a handheld ultraviolet lamp at a wavelength of 365 nm.

[0038] The results showed that the test strips loaded with CL-ALP could visually detect different concentrations of ALP through dual-mode chemiluminescence and fluorescence emission. This provided a new approach for the development of a convenient and visual ALP detection method, with potential practical application value.

[0039] In summary, through various tests, it can be seen that the compound CL-ALP probe shown in this application is faster, more sensitive in ALP detection, has a wider linear range and anti-interference ability, and does not require complex instrument equipment in practical applications. The test strip detection method is faster and more portable, with significant advantages compared to the prior art.

[0040] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0041] The above-described embodiments merely represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims.

Claims

1. A self-luminescent molecular probe, characterized in that, Has the following molecular formula:

2. A preparation method of the self-luminous molecular probe as described in claim 1, characterized in that, Including the following steps: Step 1: Dissolve 3-hydroxybenzaldehyde in methanol, add trimethyl orthoformate and tert-butylammonium tribromide, stir at room temperature, monitor the reaction by TLC, after the reaction is completed, dilute with ethyl acetate, wash with 0.01M sodium bicarbonate, dry, concentrate and purify by silica gel column chromatography to obtain Compound 1; Step 2: Dissolve Compound 1 and imidazole in dichloromethane, add tert-butyldimethylsilyl chloride, stir at room temperature, monitor the reaction by TLC, filter the precipitate, evaporate the solvent, and purify by silica gel column chromatography to obtain Compound 2; Step 3: Dissolve Compound 2 and trimethyl phosphite in dichloromethane, cool to 0 °C, dropwise add titanium tetrachloride, monitor the reaction by TLC, pour the reaction mixture into saturated sodium bicarbonate aqueous solution after the reaction, stir, extract, dry, concentrate and purify by silica gel column chromatography to obtain Compound 3; Step 4: Under nitrogen protection, dissolve Compound 3 in anhydrous tetrahydrofuran, cool to -78 °C, add lithium diisopropylamide and stir, then add an anhydrous tetrahydrofuran solution of 2-adamantanone, react at -78 °C and then warm up to room temperature to continue the reaction, monitor the reaction by TLC, after the reaction, dilute with ethyl acetate, wash with saturated brine, dry, concentrate and purify by silica gel column chromatography to obtain Compound 4; Step 5: Dissolve Compound 4 in tetrahydrofuran, add tetrabutylammonium fluoride, stir at room temperature, monitor the reaction by TLC, after the reaction, dilute with ethyl acetate, wash with dilute hydrochloric acid, dry, concentrate and purify by silica gel column chromatography to obtain Compound 5; Step 6: Dissolve Compound 5 in toluene, cool to 0 °C, add N-iodosuccinimide in portions, stir, monitor the reaction by TLC, quench the reaction with saturated sodium thiosulfate after the reaction, dilute with ethyl acetate, wash with brine, dry, concentrate and purify by silica gel column chromatography to obtain Compound 6; Step 7: Dissolve Compound 6 in anhydrous DMF, add copper(I) iodide, triphenylphosphine, palladium(II) acetate and benzothiazole, stir at 100 °C for 12 hours, monitor the reaction by TLC, after the reaction, dilute with ethyl acetate, wash with brine, dry, concentrate and purify by silica gel column chromatography to obtain Compound 7 with X = S; Step 8: Dissolve Compound 7 in dry DMF, cool to 0 °C under argon, add sodium hydride, heat to room temperature and stir, then add diethyl (4-(bromomethyl)phenyl)phosphonate, monitor the reaction by TLC, after the reaction, evaporate and concentrate under reduced pressure, and purify the crude product by HPLC to obtain Compound 8 with X = S; Step 9: Take Compound 8 and a catalytic amount of methylene blue and dissolve them in DCM, stir in an ice bath and react under white light irradiation with oxygen, monitor the reaction by TLC, and purify the reaction product by HPLC to obtain the self-luminescent molecular probe Compound CL–ALP with X = S; 3. Use of a self-luminescent molecular probe as described in claim 1, characterized in that, Through the chemical reaction between the self-luminescent molecular probe and ALP to produce fluorescence, the rapid detection of ALP is realized.

4. The application of the self-luminescent molecular probe according to claim 3, characterized in that, Including the following steps: S1. Immerse the paper test strip in a THF solution containing CL-ALP for 0.5 minutes, and then air dry it. S2. Immerse the test strip obtained in step S1 in an aqueous ALP solution for 5 minutes, and then air dry it. S3. Take the chemiluminescence imaging and fluorescence images of the test strip.

5. The application of the self-luminescent molecular probe according to claim 4, wherein The chemiluminescence imaging is obtained by an IVIS imaging system in the bioluminescence mode, using an open filter, and the exposure time is 5 seconds; the fluorescence image is obtained by excitation with an ultraviolet lamp at a wavelength of 365 nm.

6. A load test strip, characterized in that, The loaded test strip is loaded with the self-luminescent molecular probe as described in claim 1.

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