A luminescent controllable afterglow type chemiluminescence molecule, a nanoprober and a preparation method and application thereof

Abstract

The application provides a luminescent controllable afterglow type chemiluminescence molecule, which outputs stable and persistent chemiluminescence signals under the control of a 'double key' of singlet oxygen activation and H2O2 response triggering, and the molecular structure is shown in formula (I): (I), wherein the borate group and the adamantyl alkene bond serve as a 'double lock', and the singlet oxygen and H2O2 serve as a 'double key'; when the afterglow type chemiluminescence molecule or the nanometer probe prepared from the afterglow type chemiluminescence molecule reacts with the 'double key' (the singlet oxygen activation and the H2O2 response triggering) to open the 'double lock', the persistent and stable chemiluminescence signals are output, and the problems of uncontrollable signal output, unstable signals and low sensitivity of the existing chemiluminescence substrate are effectively solved. The application further provides a luminescent controllable afterglow type chemiluminescence nanometer probe and a preparation method and application thereof.

Description

Technical Field

[0001] This invention relates to the field of chemiluminescence analysis and detection technology, specifically to a "dual-lock-key" luminescent controllable afterglow chemiluminescent molecule, nanoprobe, its preparation method, and analytical applications. Background Technology

[0002] Chemiluminescence is the light emitted when molecules in a system absorb energy and transition to an excited state due to the chemical energy generated by a chemical reaction, without the need for external electric fields, light, or heat excitation, and then return to the ground state. Because it does not require an external light source, it avoids background light and other stray light interference, thus reducing noise and improving the signal-to-noise ratio. Furthermore, its advantages, such as high sensitivity, simple instrumentation, fast analysis speed, and ease of automation, have led to its widespread application in molecular biology, environmental chemistry, and clinical testing.

[0003] Currently, there are two main categories of widely used chemiluminescent substrates. The first category is flash-type, represented by luminol. These substrates typically have high luminescence intensity, but short luminescence time, leading to low reliability, poor repeatability, and a limited range of detectable substances. The second category is glow-type, represented by adamantane-dioxane chemiluminescent substrates. These substrates contain high-energy peroxy bonds, and their wide detection range and high sensitivity have led to their rapid development in fields such as biosensing. Both types of chemiluminescent substrates release photons and output chemiluminescent signals during analyte recognition. In the analyte recognition and photon release processes, the analyte recognition and photon release processes are inseparable, resulting in uncontrollable luminescence, incomplete signal collection, and low sensitivity—a very challenging problem.

[0004] Therefore, it is necessary to provide a new chemiluminescent material to solve the above-mentioned technical problems. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the existing technology and provide a "dual lock-key" controllable afterglow chemiluminescent molecule and nanoprobe, which effectively solves the problems of uncontrollable signal output, unstable signal and low sensitivity of existing chemiluminescent substrates.

[0006] The first aspect of this invention is to provide a chemiluminescent molecule with controllable afterglow, which outputs a stable and persistent chemiluminescent signal under the "dual-key" control of singlet oxygen activation and H2O2 response triggering, and its molecular structure is shown in formula (I):

[0007] (I).

[0008] A second aspect of the present invention is to provide a method for synthesizing the afterglow-type chemiluminescent molecule with controllable luminescence as described in the first aspect of the present invention, comprising the following steps:

[0009] (1) Compound 1 was prepared from 2-chloro-3-hydroxybenzaldehyde and trimethyl orthoformate.

[0010] (2) Compound 2 was prepared using compound 1 and tert-butyldimethylchlorosilane as raw materials;

[0011] (3) Compound 3 was prepared from compound 2 and trimethyl phosphite;

[0012] (4) Compound 4 was prepared by using compound 3 and 2-adamantanone as raw materials;

[0013] (5) Compound 5 was prepared by using compound 4 as a raw material and tetrabutylammonium fluoride as a desilylation reagent;

[0014] (6) Compound 6 was prepared by reacting compound 5 and paraformaldehyde under nitrogen protection.

[0015] (7) Compound 7 was prepared by using compound 6 and (triphenylphosphine)acetonitrile as raw materials;

[0016] (8) Compound 8 was prepared using 4-(hydroxymethyl)phenylboronic acid pinacol ester, NaI and trimethylchlorosilane as raw materials;

[0017] (9) Using compounds 7 and 8 as raw materials, the luminescent afterglow type chemiluminescent molecule with controllable luminescence was prepared.

[0018] The third aspect of the present invention is to provide a luminescence-controllable afterglow chemiluminescent nanoprobe, which is prepared by loading the luminescence-controllable afterglow chemiluminescent molecules described in the first aspect of the present invention onto polystyrene nanospheres as a carrier. The luminescence-controllable afterglow chemiluminescent nanoprobe outputs a stable and persistent chemiluminescent signal under the "dual key" control of singlet oxygen activation and H2O2 response triggering.

[0019] Preferably, the polystyrene nanospheres have a particle size of 50~1000 nm.

[0020] Preferably, the polystyrene nanospheres are surface-carboxylated polystyrene nanospheres.

[0021] A fourth aspect of the present invention is to provide a method for preparing the luminescence-controllable afterglow chemiluminescent nanoprobe described in the third aspect of the present invention, comprising the following steps:

[0022] Step S1: Disperse polystyrene nanospheres in isopropanol and sonicate to obtain a stable polystyrene nanosphere dispersion.

[0023] Step S2: Dissolve the chemiluminescent molecules described in the first aspect in dichloromethane, then add them to the polystyrene nanosphere dispersion in step S1, mix well, and shake.

[0024] Step S3: The solution obtained in step S2 is rotary evaporated and washed with isopropanol and distilled water to obtain a afterglow-type chemiluminescent nanoprobe.

[0025] Preferably, in step S1, the concentration of the polystyrene nanosphere dispersion is 2 mg / mL.

[0026] Preferably, in step S2, the volume ratio of the solution containing chemiluminescent molecules to the polystyrene nanosphere dispersion is 1:5 to 1:2; specifically, the volume ratio can be 1:5, 1:4, 1:3 or 1:2, or other values ​​within this range.

[0027] Preferably, in step S2, the ratio of polystyrene nanospheres to chemiluminescent molecules is 1:0.5 to 1:1; specifically, the ratio can be 1:0.5, 1:0.8, or 1:1, or other values ​​within this range.

[0028] The fifth aspect of the present invention is to provide the application of the luminescence-controlled afterglow chemiluminescent molecule described in the first aspect of the present invention or the luminescence-controlled afterglow chemiluminescent nanoprobe described in the third aspect of the present invention in the analysis and detection of singlet oxygen.

[0029] The sixth aspect of the present invention is to provide the application of the luminescence-controllable afterglow chemiluminescent molecule described in the first aspect of the present invention or the luminescence-controllable afterglow chemiluminescent nanoprobe described in the third aspect of the present invention in the analysis and detection of H2O2.

[0030] The seventh aspect of the present invention is to provide the application of the luminescence-controlled afterglow chemiluminescent molecule described in the first aspect of the present invention or the luminescence-controlled afterglow chemiluminescent nanoprobe described in the third aspect of the present invention in the analysis and detection of Gram-negative bacteria.

[0031] When the luminescence-controllable afterglow chemiluminescent molecule described in the first aspect of this invention or the luminescence-controllable afterglow chemiluminescent nanoprobe described in the third aspect is applied to analyte detection, the operation steps for controlling luminescence using a "double lock-key" mechanism of singlet oxygen and H2O2 are as follows:

[0032] (1) The afterglow-type chemiluminescent nanoprobe is mixed with a photosensitizer and then irradiated; wherein the irradiation time is 3~8 min and the light source is 50~100mW / cm². 2The white light;

[0033] (2) Add H2O2 to the above mixed solution, mix well, and perform chemiluminescence detection; the concentration of the added H2O2 is 1 mM~10 mM.

[0034] Compared with existing technologies, the luminescence-controllable afterglow chemiluminescent molecules, nanoprobes, their preparation methods, and applications provided by this invention have the following advantages:

[0035] I. The luminescently controllable afterglow chemiluminescent molecule or nanoprobe provided by this invention possesses "double-lock-key" controllable luminescence performance, wherein the borate ester group and the adamantane bond act as the "double lock," while singlet oxygen and H2O2 act as the "double key," with singlet oxygen used to open the adamantane bond and H2O2 used to open the borate ester group. When the afterglow chemiluminescent molecule or nanoprobe reacts with the "double key" (singlet oxygen activation and H2O2 response triggering) to open the "double lock," a persistent and stable chemiluminescent signal is output. The principle is as follows: Figure 1 As shown. The "dual-lock-key" controllable luminescence afterglow-type chemiluminescent molecular or nanoprobe provided by this invention separates the analyte recognition process from the photon release process. By accumulating the signal of the intermediate first and then releasing the photons in a concentrated manner, it efficiently solves the problems of uncontrollable substrate signal output, unstable signal, low sensitivity, and unreliable results in existing chemiluminescent substrates. It has great application value in the fields of cold light source, biological detection, and biosensors.

[0036] Second, the luminescence-controllable afterglow chemiluminescent molecules or nanoprobes provided by this invention have the advantage of long-lasting luminescence. Compared with most known chemiluminescent systems with very short luminescence intensity peak decay time, the luminescence-controllable afterglow chemiluminescent molecules or nanoprobes provided by this invention can improve the stability and accuracy of detection results in analytical detection applications.

[0037] Third, the luminescence-controllable afterglow type chemiluminescent nanoprobe provided by the present invention selects surface carboxylated polystyrene nanospheres as a carrier to load chemiluminescent molecules. On the one hand, the carboxyl groups on the surface of polystyrene nanospheres are easy for biomolecules to be labeled and enhance the dispersion stability of nanospheres in aqueous solution. On the other hand, the hydrophobic environment inside polystyrene nanospheres can improve the stability of chemiluminescent molecules in the present invention and avoid the problem of chemiluminescent molecules being unstable and decomposing during storage. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 This is a schematic diagram illustrating the principle of chemiluminescence of controllable afterglow chemiluminescent molecules or nanoprobes in this invention.

[0040] Figure 2 This is a synthetic route diagram for the afterglow-type chemiluminescent molecule with controllable luminescence in this invention;

[0041] Figure 3 This is a chemiluminescence imaging detection image of the controllable afterglow type chemiluminescence nanoprobe in this invention changing over time;

[0042] Figure 4 This invention verifies the dual-control performance of singlet oxygen and H2O2 on the luminescence-controllable afterglow chemiluminescent nanoprobe.

[0043] Figure 5 This invention relates to the application of a luminescent, afterglow-type chemiluminescent nanoprobe in singlet oxygen analysis and detection.

[0044] Figure 6 This invention relates to the application of the controllable afterglow chemiluminescent nanoprobe in the analysis and detection of H2O2.

[0045] Figure 7 This invention demonstrates the feasibility of using the luminescent, afterglow-type chemiluminescent nanoprobe with controllable luminescence in the analysis and detection of Gram-negative bacteria.

[0046] Figure 8 This invention relates to the quantitative detection of Gram-negative bacteria using a luminescent, afterglow-type chemiluminescent nanoprobe with controllable luminescence. Detailed Implementation

[0047] 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 objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be further described below.

[0048] The endpoints and any values ​​of the ranges disclosed herein 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 the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0049] Example 1: Synthesis of a controllable afterglow chemiluminescent molecule

[0050] In this invention, the luminescent, afterglow-type chemiluminescent molecular structure is shown in formula (Ⅰ):

[0051] (I).

[0052] Please see Figure 2 This is a synthetic route diagram for the controllable afterglow chemiluminescent molecule of the present invention. A method for synthesizing a controllable afterglow chemiluminescent molecule includes the following steps:

[0053] (1) 2-Chloro-3-hydroxybenzaldehyde (3.52 g, 22.5 mmol), trimethyl orthoformate (4.8 g, 45 mmol) and tetrabutylammonium tribromide (616 mg, 1.28 mmol) were dissolved in 50 mL of methanol and stirred at room temperature for 12 hours. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was complete, the solution was diluted with ethyl acetate, washed with sodium bicarbonate aqueous solution and extracted three times with ethyl acetate. Finally, the product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (4 / 1) as eluent to obtain compound 1, a colorless oily liquid.

[0054] (2) Compound 1 (2.1 g, 10.4 mmol) was dissolved in 25 mL of anhydrous dichloromethane, and then imidazole (1.4 g, 20.8 mmol) and tert-butyldimethylchlorosilane (1.9 g, 12.3 mmol) were added in sequence. The mixture was stirred at room temperature for 2 h. The reaction was monitored by thin-layer chromatography (TLC). When the reaction was complete, the solvent was removed by rotary evaporation. Finally, the product was purified by silica gel column chromatography using petroleum ether / dichloromethane (4 / 1) as the eluent to obtain compound 2 as a colorless liquid.

[0055] (3) Compound 2 (3g, 9.6 mmol) and trimethyl phosphite (1.5mL, 12.3 mmol) were dissolved in 40mL of dichloromethane. The reaction mixture was cooled to 0℃, and titanium tetrachloride (1.4mL, 12.7mmol) was slowly added dropwise. The reaction was monitored by thin-layer chromatography. After the reaction was completed, saturated NaHCO3 aqueous solution was slowly added dropwise to the reaction solution at 0℃. When no more bubbles appeared, the addition of saturated NaHCO3 was stopped. After stirring for 30 minutes, the mixture was extracted with dichloromethane. The combined organic phases were dried with anhydrous Na2SO4 and concentrated under reduced pressure. The product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (1 / 4) as eluent to obtain colorless liquid compound 3.

[0056] (4) Compound 3 (3.3 g, 8.4 mmol) was placed in a 100 mL Schlenk flask. After replacing the air in the flask with nitrogen, 45 mL of anhydrous and oxygen-free tetrahydrofuran was added. The solution was cooled to -78 °C and then diisopropylaminolithium solution (2.0 M, 5 mL, 10 mmol, tetrahydrofuran) was slowly added dropwise. The mixture was stirred at low temperature for 30 min. 2-Adamantane (1.9 g, 12.6 mmol) was dissolved in 20 mL of anhydrous and oxygen-free tetrahydrofuran and added dropwise to the above reaction solution. The mixture was stirred at -78 °C for half an hour, and then the reaction was heated to room temperature and stirred. The reaction progress was monitored by thin-layer chromatography. When the reaction was complete, the reaction mixture was diluted with ethyl acetate, washed with brine, and then extracted with ethyl acetate. The combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (5 / 95) as the eluent to obtain a white solid compound 4.

[0057] (5) Compound 4 (3.3 g, 7.8 mmol) was dissolved in 50 mL of tetrahydrofuran, and a tetrahydrofuran solution of tetrabutylammonium fluoride (1 M, 10 mL, 10 mmol) was added. The mixture was stirred at room temperature. The reaction was monitored by thin-layer chromatography. After the reaction was completed, ethyl acetate was added to dilute the solution and the mixture was washed with dilute hydrochloric acid. The product was then extracted with ethyl acetate, and the combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (15 / 85) as the eluent to obtain compound 5 as a white solid.

[0058] (6) Compound 5 (2.3 g, 7.4 mmol) and MgCl2 (1.1 g, 11.1 mmol) were added to a 120 mL high-pressure flask. After replacing the air in the flask with nitrogen, 45 mL of anhydrous and oxygen-free tetrahydrofuran and triethylamine (4.1 mL, 29.1 mmol) were added. After stirring for 20 min, paraformaldehyde (5.2 g, 58.2 mmol) was added under nitrogen protection. The reaction mixture was heated and refluxed at 80 °C for 12 hours. The reaction was monitored by thin-layer chromatography. After the reaction was completed, the reaction was cooled to room temperature. The paraformaldehyde solid was dissolved with dilute hydrochloric acid and extracted with ethyl acetate. The combined organic phases were dried with anhydrous Na2SO4 and concentrated under reduced pressure. The product was purified by silica gel column chromatography using dichloromethane / petroleum ether (1 / 2) as eluent to obtain compound 6 as a white solid.

[0059] (7) 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 in anhydrous dichloride. The mixture was stirred at room temperature for 30 min. After the reaction was completed, the solvent was removed under vacuum. The residue was dissolved in CH2Cl2 (20 mL) and purified by column chromatography (ethyl acetate / petroleum ether = 1 / 6) through a diatomaceous earth pad. The combined eluent was concentrated under vacuum to obtain compound 7.

[0060] (8) 4-(hydroxymethyl)phenylboronic acid pinacol ester (1 g, 4.27 mmol) was dissolved in acetonitrile (30 mL) and cooled to 0 °C. Then, NaI (1.92 g, 12.8 mmol) and trimethylchlorosilane (1.63 mL, 12.9 mmol) were added. The mixture was stirred for 30 min at room temperature, diluted with ethyl acetate, washed with saturated Na2S2O3 solution, washed with water and saturated NaCl solution, and the organic layer was dried on Na2SO4. The mixture was concentrated by evaporation and purified by column chromatography (EA:PE = 5:95) to give a white solid compound 8.

[0061] (9) Compound 7 (500 mg, 1.41 mmol) was dissolved in DMF (15 mL), and the solution was cooled to 0 °C. K2CO3 (252 mg, 1.83 mmol) was then added, and the mixture was stirred at room temperature. Finally, compound 8 (629 mg, 1.83 mmol) was added, and the mixture was stirred at room temperature for 3 h. After the reaction was complete, the mixture was cooled to 0 °C, diluted with ether (15 mL), and saturated NH4Cl solution (15 mL) was added. Extraction and separation were performed. The aqueous phase was washed twice with ether, the organic layers were combined, dried with anhydrous MgSO4, and purified by column chromatography (EA:PE = 5:95) to obtain yellow solid compound 9, which is the target product with controllable afterglow chemiluminescence.

[0062] Example 2: Preparation method of luminescence-controllable afterglow chemiluminescent nanoprobes

[0063] A luminescence-controllable afterglow chemiluminescent nanoprobe is prepared from surface-carboxylated polystyrene nanospheres (smaller than 200 nm) and the aforementioned luminescence-controllable afterglow chemiluminescent molecule. The specific steps include:

[0064] (1) The purchased polystyrene nanospheres with a particle size of 174 nm and surface modified with carboxyl groups were dispersed in isopropanol (2 mg / mL, 1 mL), and then 400 μL of chemiluminescent molecular solution (1 mg dissolved in dichloromethane) was added. The mixture was mixed, protected from light, and shaken at room temperature for 3 h.

[0065] (2) Remove dichloromethane from the solution obtained in step (1) using a vacuum rotary evaporator, then centrifuge (12000 rpm, 30 min), wash three times with isopropanol and ultrapure water to obtain chemiluminescent polystyrene nanospheres, and finally disperse them in ultrapure water and store them in a refrigerator at 4°C in the dark.

[0066] Example 3: Verification of the luminescence persistence of a controllable afterglow chemiluminescent nanoprobe

[0067] The luminescence-controllable afterglow chemiluminescent nanoprobe (50 μL, 1 pmol) prepared in Example 2 was mixed uniformly with a photosensitizer or photosensitizer-loaded nanoparticles (50 μL, 1 pmol). In this example, the photosensitizer particles were prepared by loading methylene blue photosensitizer onto polystyrene nanospheres (174 nm), which was commercially available.

[0068] The method for preparing photosensitive particles by loading the photosensitizer methylene blue onto polystyrene nanospheres (174 nm) is the same as the operation process of loading chemiluminescent molecules onto polystyrene nanospheres (see Example 2).

[0069] Add 50 μL of the above mixture to a 384-well plate and use 50 mW / cm² water. 2 The sample was irradiated with white light for 5 min, then 10 μL of H2O2 was added. Chemiluminescence detection was performed using the chemiluminescence mode of the ChemiDoc XRS+ imaging system at 0 h, 1 h, 6 h, 12 h, 24 h, 48 h, and 60 h, and quantitative analysis was performed using the instrument's built-in software. The results are as follows: Figure 3 As shown.

[0070] Depend on Figure 3 As can be seen, the luminescence-controllable afterglow type chemiluminescent nanoprobe provided by the present invention has a long luminescence time, and the chemiluminescent signal can still be detected even after 60 hours.

[0071] Example 4: Verification of the dual-control performance of singlet oxygen and H2O2 on the luminescence-controllable afterglow chemiluminescent nanoprobes

[0072] The photosensitive particles used in this embodiment are the same as those in Example 3.

[0073] Four control experiments were set up:

[0074] Group 1: 50 μL of luminescent afterglow chemiluminescent nanoprobe (8 fmol) + illumination (5 min) + 10 μL H2O2;

[0075] Group 2: 50 μL of a mixture of luminescent afterglow chemiluminescent nanoprobes (8 fmol) and photosensitive particles (8 fmol) + light irradiation (5 min);

[0076] Group 3: 50 μL of a mixture of luminescent afterglow chemiluminescent nanoprobes (8 fmol) and photosensitive particles (8 fmol) + 10 μL of H2O2;

[0077] Group 4: 50 μL of a mixture of luminescent afterglow-type chemiluminescent nanoprobes (8 fmol) and photosensitive particles (8 fmol) + light irradiation (5 min) + 10 μL H2O2.

[0078] Chemiluminescence detection was performed using the chemiluminescence mode of the ChemiDoc XRS+ imaging system, and quantitative analysis was conducted using the instrument's built-in software. The results are as follows: Figure 4 As shown.

[0079] Depend on Figure 4 It can be seen that the chemiluminescent nanoprobe provided by this invention detects a relatively weak chemiluminescent signal in the presence of only singlet oxygen or H2O2. However, when both singlet oxygen activation and H2O2 response triggering are present, the "double lock" of the chemiluminescent molecules in the chemiluminescent nanoprobe can be unlocked, thereby detecting a stronger chemiluminescent signal.

[0080] Example 5: Application of controllable afterglow chemiluminescent nanoprobes in singlet oxygen analysis and detection

[0081] The photosensitive particles used in this embodiment are the same as those in Example 3.

[0082] The luminescence-controllable afterglow chemiluminescent nanoprobe (2.72 × 10⁻⁶) prepared in Example 2 was used. 10 (pcs / mL, 30μL) was mixed with 30μL containing different amounts of photosensitizer particles (1.08×10 pcs / mL, 30μL). 6 2.15×10 5 4.3×10 4 8.6×10 31.72×10 3 3.4×10 2 (60, 10, 1, 0). The above mixture was added to a 384 well plate and heated with 50 mW / cm² water. 2 The sample was irradiated with white light for 5 min, followed by the addition of 10 μL of H₂O₂. Chemiluminescence detection was performed using the chemiluminescence mode of the ChemiDoc XRS+ imaging system, and quantitative analysis was conducted using the instrument's built-in software. The results are as follows: Figure 5 As shown.

[0083] Depend on Figure 5 It can be seen that when the number of photosensitizer particles in the mixture is reduced to 10-100, the generated singlet oxygen still enables the chemiluminescent nanoprobe to release a chemiluminescent signal significantly higher than the background. This indicates that the prepared luminescently controllable afterglow chemiluminescent nanoprobe has excellent response performance to singlet oxygen.

[0084] Example 6: Application of controllable afterglow chemiluminescent nanoprobes in H2O2 analysis and detection

[0085] The photosensitive particles used in this embodiment are the same as those in Example 3.

[0086] The luminescence-controllable afterglow chemiluminescent nanoprobe (2.72 × 10⁻⁶) prepared in Example 2 was used. 8 (pcs / mL, 30μL) and photosensitizer particles (2.72×10 pcs / mL, 30μL) 8 Mix (pcs / mL, 30μL), add the above mixture to a 384 multi-well plate, and spray with 50mW / cm 2 The sample was irradiated with white light for 5 min. Then, 10 μL of different concentrations of H2O2 (0, 0.1, 1, 10, 100, 1000, 10000 μM) were added. Chemiluminescence detection was performed using the chemiluminescence mode of the ChemiDoc XRS+ imaging system, and quantitative analysis was performed using the instrument's built-in software. The results are as follows: Figure 6 As shown.

[0087] Depend on Figure 6 It can be seen that the chemiluminescence signal intensity of the prepared afterglow-type chemiluminescence nanoprobe increases with the increase of H2O2 concentration, and the chemiluminescence nanoprobe can still release a strong chemiluminescence signal when the H2O2 concentration is 10-100μM, indicating that the chemiluminescence nanoprobe has good response performance to H2O2 and has the potential for application in the analysis and detection of H2O2.

[0088] Example 7: Application of controllable afterglow chemiluminescent nanoprobes in the analysis and detection of Gram-negative bacteria

[0089] The photosensitive particle used in this embodiment is PCN-224 (zirconium-based MOF).

[0090] The detection of Gram-negative bacteria using a chemiluminescent nanoprobe with controllable afterglow is described in the following steps:

[0091] (1) Take 2mg H2TCPP and 6mg ZrOCl 2· 8H2O and 56 mg of benzoic acid were dissolved in a 10 mL round-bottom flask and stirred at 90 °C and 300 rpm for 5 h. Then, the mixture was centrifuged (12500 rpm, 40 min) and washed three times with DMF to obtain photosensitive nanoparticles PCN-224. Finally, the nanoparticles were dispersed in ultrapure water and stored in a refrigerator at 4 °C in the dark.

[0092] (2) After mixing PCN-224 (20 uL, 20 mg / mL) with EDC / NHS (200 uL, 5 / 6 mg / mL) for 1 h, the mixture was centrifuged and antimicrobial peptide (AMP, 50 uL, 1 mg / mL) was added and reacted overnight. Then, the mixture was centrifuged (14000 rpm, 8 min) and washed three times with PBS (10 mM, pH 7.4) to obtain the photosensitive nanoprobe PCN-224@AMP. Finally, the nanoprobe was dispersed in PBS (10 mM, pH 7.4) and stored in a refrigerator at 4 °C in the dark.

[0093] (3) Synthesize polystyrene nanospheres loaded with chemiluminescent molecules to form chemiluminescent nanoparticles CL@PS (see Example 2 for details);

[0094] (4) After mixing CL@PS (20 μL, 10 mg / mL) with EDC / NHS (100 μL, 5 / 6 mg / mL) and reacting for 1 hour, centrifuge and add... The mixture was reacted overnight with polymyxin B (PMB, 100 μL, 1 mg / mL). Then it was centrifuged (14000 rpm, 8 min) and rinsed with PBS (10 mM). The chemiluminescent nanoprobe CL@PS@PMB was obtained by washing three times with PBS (pH 6.0), and finally dispersed in PBS (10 mM, pH 6.0) and placed in a container. Store in a refrigerator at 4°C, away from light.

[0095] (5) Taking *Escherichia coli* as an example, the feasibility of the luminescent, controllable afterglow chemiluminescent nanoprobes provided in this invention in the analysis and detection of Gram-negative bacteria was investigated. Five control experiments were set up. Group 1: chemiluminescent nanoprobe CL@PS@PMB (50 μL, 1 μg / mL) and photosensitive nanoprobe PCN-224@AMP (50 μL, 1 μg / mL); Group 2: chemiluminescent nanoparticles CL@PS (50 μL, 1 μg / mL), photosensitive nanoparticles PCN-224 (50 μL, 1 μg / mL), and *Escherichia coli* (100 μL, 10 μg / mL). 4 Group 3: Chemiluminescent nanoparticles CL@PS (50 μL, 1 μg / mL), photosensitive nanoprobe PCN-224@AMP (50 μL, 1 μg / mL), and Escherichia coli (100 μL, 10 μg / mL). 4Group 4: Chemiluminescent nanoprobe CL@PS@PMB (50 μL, 1 μg / mL), photosensitive nanoparticles PCN-224 (50 μL, 1 μg / mL), and Escherichia coli (100 μL, 10 μg / mL). 4 Group 5: Chemiluminescent nanoprobe CL@PS@PMB (50 μL, 1 μg / mL), photosensitive nanoprobe PCN-224@AMP (50 μL, 1 μg / mL), and Escherichia coli (100 μL, 10 μg / mL). 4 CFU / mL). Mix and incubate for 5 min, then add the above mixed bacterial solution to a 384-well plate and spray with 50 mW / cm² water. 2 The sample was irradiated with white light for 4 min, followed by the addition of 10 μL of H₂O₂. Chemiluminescence detection was performed using the chemiluminescence mode of the ChemiDoc XRS+ imaging system, and quantitative analysis was conducted using the instrument's built-in software. The results are as follows: Figure 7 As shown.

[0096] Depend on Figure 7 It can be seen that when neither the chemiluminescent nanoparticles CL@PS nor the photosensitive nanoparticles PCN-224 are modified with the target molecule (PMB or AMP), or when only one of them is modified with the target molecule, almost no chemiluminescent signal can be detected. However, when both the chemiluminescent nanoparticles CL@PS@PMB and the photosensitive nanoparticles PCN-224 are modified with the target molecule, the chemiluminescent nanoprobes CL@PS@PMB and PCN-224@AMP simultaneously target the surface of E. coli, bringing the two probes closer together, a stronger chemiluminescent signal can be detected, demonstrating the feasibility of this chemiluminescent strategy for detecting Gram-negative bacteria.

[0097] (6) Taking *Escherichia coli* as an example, the quantitative detection of Gram-negative bacteria by the luminescently controlled afterglow chemiluminescent nanoprobe provided in this invention was investigated. The prepared luminescently controlled chemiluminescent nanoprobe CL@PS@PMB (50 μL, 2.4 ug / mL) was mixed evenly with the photosensitive nanoprobe PCN-224@AMP (50 μL, 0.2 ug / mL) and then mixed with 100 μL of *E. coli* at different concentrations (0, 10, 50, 10...). 2 5×10 2 10 3 5×10 3 10 4 Incubate the bacterial mixture (CFU / mL) for 10 minutes. Add the above mixed bacterial solution to a 384-well plate and incubate with 50 mW / cm² water. 2 The sample was irradiated with white light for 4 min, followed by the addition of 10 μL of H₂O₂. Chemiluminescence detection was performed using the chemiluminescence mode of the ChemiDoc XRS+ imaging system, and quantitative analysis was conducted using the instrument's built-in software. The results are as follows: Figure 8 As shown.

[0098] Depend on Figure 8 It can be seen that the chemiluminescence signal intensity of the luminescently controllable afterglow type chemiluminescent nanoprobe of the present invention increases with the increase of Escherichia coli concentration, indicating that the chemiluminescent nanoprobe has good response performance to Escherichia coli; and the concentration of Escherichia coli Log[C E.coli The signal intensity change (ΔSignal) is linearly related to the signal intensity change (CFU / mL), and the linear equation is Y = 691.9522X - 656.2668 (R0). 2 =0.9789), the detection limit for Escherichia coli concentration is 10 CFU / mL.

[0099] The embodiments of the present invention have been 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 still fall within the protection scope of the present invention.

Claims

1. A chemiluminescent molecule with controllable afterglow emission, characterized in that, It outputs a stable and persistent chemiluminescent signal under the "dual-key" controlled initiation of singlet oxygen activation and H2O2 response, and its molecular structure is shown in formula (Ⅰ): （Ⅰ）。 2. A method for synthesizing a luminescent, afterglow-type chemiluminescent molecule with controllable luminescence as described in claim 1, characterized in that, Includes the following steps: (1) Compound 1 was prepared from 2-chloro-3-hydroxybenzaldehyde and trimethyl orthoformate. (2) Compound 2 was prepared using compound 1 and tert-butyldimethylchlorosilane as raw materials; (3) Compound 3 was prepared from compound 2 and trimethyl phosphite; (4) Compound 4 was prepared by using compound 3 and 2-adamantanone as raw materials; (5) Compound 5 was prepared by using compound 4 as a raw material and tetrabutylammonium fluoride as a desilylation reagent; (6) Compound 6 was prepared by reacting compound 5 and paraformaldehyde under nitrogen protection. (7) Compound 7 was prepared by using compound 6 and (triphenylphosphine)acetonitrile as raw materials; (8) Compound 8 was prepared using 4-(hydroxymethyl)phenylboronic acid pinacol ester, NaI and trimethylchlorosilane as raw materials; (9) Using compounds 7 and 8 as raw materials, the afterglow chemiluminescent molecule with controllable luminescence as described in claim 1 is prepared.

3. A chemiluminescent nanoprobe with controllable afterglow, characterized in that, The afterglow chemiluminescent nanoprobe is prepared by loading the afterglow chemiluminescent molecule described in claim 1 onto polystyrene nanospheres as a carrier. The afterglow chemiluminescent nanoprobe outputs a stable and persistent chemiluminescent signal under the "dual key" control of singlet oxygen activation and H2O2 response triggering.

4. The luminescence-controllable afterglow chemiluminescent nanoprobe according to claim 3, characterized in that, The polystyrene nanospheres are surface-carboxylated polystyrene nanospheres with a particle size of 50~1000 nm.

5. A method for preparing a luminescent, afterglow-type chemiluminescent nanoprobe as described in claim 3, characterized in that, Includes the following steps: Step S1: Disperse polystyrene nanospheres in isopropanol and sonicate to obtain a stable polystyrene nanosphere dispersion. Step S2: Dissolve the chemiluminescent molecule described in claim 1 in dichloromethane, then add it to the polystyrene nanosphere dispersion in step S1, mix well, and shake. Step S3: The solution obtained in step S2 is rotary evaporated and washed with isopropanol and distilled water to obtain a luminescent afterglow-type chemiluminescent nanoprobe with controllable luminescence.

6. The method for preparing the luminescently controllable afterglow chemiluminescent nanoprobe according to claim 5, characterized in that, In step S1, the concentration of the polystyrene nanosphere dispersion is 2 mg / mL.

7. The method for preparing the luminescently controllable afterglow chemiluminescent nanoprobe according to claim 5, characterized in that, In step S2, the volume ratio of the solution containing chemiluminescent molecules to the polystyrene nanosphere dispersion is 1:5 to 1:2; the feed ratio of polystyrene nanospheres to chemiluminescent molecules is 1:0.5 to 1:

1.

8. The application of a luminescent afterglow chemiluminescent molecule as described in claim 1 or a luminescent afterglow chemiluminescent nanoprobe as described in claim 3 in singlet oxygen analysis and detection, for purposes other than disease diagnosis and treatment.

9. The application of a luminescent afterglow chemiluminescent molecule as described in claim 1 or a luminescent afterglow chemiluminescent nanoprobe as described in claim 3 in the analysis and detection of H2O2, for purposes other than disease diagnosis and treatment.

10. The application of a luminescently controlled afterglow chemiluminescent molecule as described in claim 1 or a luminescently controlled afterglow chemiluminescent nanoprobe as described in claim 3 in the analysis and detection of Gram-negative bacteria, for non-disease diagnosis and treatment purposes.

Images