BPNs chemiluminescence probe, BPNs chemiluminescence probe-loaded scopoletin composite material and chemiluminescence detection method of BPNs chemiluminescence probe-loaded scopoletin composite material

Abstract

The invention discloses a BPNs chemiluminescence probe, a BPNs chemiluminescence probe loaded scopoletin composite material and a chemiluminescence detection method of the BPNs chemiluminescence probe loaded scopoletin composite material. The BPNs chemical light-emitting probe synthesized through water bath ultrasonic wave loads scopoletin through hydrogen bond interaction to obtain the composite material, and the composite material has efficient active oxygen scavenging capacity and reacts with potassium permanganate to generate a strong chemical light-emitting signal. On the basis, a chemiluminescence detection platform of the BPNs / scopoletin is constructed. The method has the advantages of high sensitivity, low detection cost, simplicity and convenience in operation and the like.

Description

Technical Field

[0001] The present invention relates to the interdisciplinary field of materials science and analytical chemistry, and in particular to a BPNs chemiluminescent probe and a composite material of the BPNs chemiluminescent probe loaded with scopoletin and a chemiluminescent detection method thereof. Background Art

[0002] Scopolamine (SP) is a natural coumarin compound found in a wide variety of plants. Due to its unique chemical structure, it possesses a variety of pharmacological and biological activities, including antidiabetic, antistress, anticancer, antimicrobial, anti-inflammatory, and antioxidant activities. In particular, SP has been shown to act as a potent antioxidant in reactive oxygen species (ROS)-mediated diseases, mitigating the effects of oxidants on the body by scavenging ROS. Furthermore, in cell and animal studies, SP has been shown to reduce oxidative stress and enhance antioxidant enzyme activity, thereby protecting against oxidative damage. However, the antioxidant mechanism of SP remains unclear. Therefore, studying the mechanism of action of SP with ROS is crucial for further understanding its pharmacological activities.

[0003] Chemiluminescence (CL) is the phenomenon in which excited products of a chemical reaction release photons upon returning to their ground state, accompanied by the conversion of ROS. This method offers advantages such as high sensitivity, rapidity, and real-time monitoring capabilities, and has been widely used in bioanalysis and chemical reaction mechanism research. Therefore, we believe CL will be an effective method for studying the antioxidant mechanism of SP. However, the weak luminescence signal of conventional CL systems hinders mechanistic investigations.

[0004] In recent years, black phosphorus (BP) nanomaterials, due to their unique structure and excellent optoelectronic properties, have been demonstrated to be useful as reducing agents, emitters, and catalysts for amplifying CL signals, injecting new vitality into CL analysis techniques. Furthermore, two-dimensional BP, with its unique layered structure, excellent biocompatibility, and high biodegradability, has been shown to possess stable drug-carrying properties in the medical field. Therefore, we aim to leverage BP's excellent drug-carrying capabilities and CL performance to investigate the antioxidant mechanisms of SP through CL analysis, thereby enhancing our understanding of the pharmacological activities of SP.

[0005] Currently, no existing patents have explored the antioxidant mechanism of SP through chemiluminescence technology, allowing for the simultaneous detection of SP in cigarettes and cigarette smoke. This invention uses bulk BP as a raw material and synthesizes oxygen-rich BP nanosheets (BPNs) via an ultrasonic exfoliation strategy. This nanomaterial possesses stable drug-carrying properties, can load SP through hydrogen bonding interactions, exhibits excellent reactive oxygen species scavenging ability, and displays excellent chemiluminescence properties in response to potassium permanganate. Based on this, a highly selective chemiluminescent method for the detection of SP has been successfully developed. Summary of the Invention

[0006] Purpose of the invention: The present invention provides a BPNs chemiluminescent probe and a composite material of a BPNs chemiluminescent probe loaded with scopoletin that efficiently scavenges reactive oxygen species, as well as a simple, low-cost and highly selective chemiluminescent detection method for scopoletin.

[0007] The technical solution is as follows: A BPNs chemiluminescent probe is synthesized using bulk black phosphorus as raw material through water bath ultrasonic exfoliation. The specific preparation steps are as follows:

[0008] 15-25 mg of bulk black phosphorus was placed in a round-bottom flask, 30-50 mL of distilled water was added, and the flask was sealed. The flask was then placed in an ultrasonic cleaner with an operating frequency of 30-50 kHz for ultrasonic stripping for 7-9 hours. The water bath temperature in the ultrasonic cleaner was 10-30°C to obtain a black mixture. The black mixture was collected and centrifuged at a speed of 10,000-15,000 rpm for 5-15 minutes. 85-95% of the suspension was collected to obtain BPNs rich in oxygen functional groups, i.e., BPNs chemical photoluminescence probes.

[0009] The aforementioned BPNs chemical photoluminescence probe is synthesized from bulk black phosphorus by water bath ultrasonic exfoliation. The specific preparation steps are as follows:

[0010] 15-20 mg of bulk black phosphorus was placed in a round-bottom flask, 30-40 mL of distilled water was added, and the flask was sealed. The flask was then placed in an ultrasonic cleaner with an operating frequency of 35-45 kHz for ultrasonic stripping for 7.5-8.5 hours. The water bath temperature in the ultrasonic cleaner was 16-25°C to obtain a black mixture. The black mixture was collected and centrifuged at 12000 rpm for 8-12 minutes. 90% of the suspension was collected to obtain a BPNs suspension rich in oxygen functional groups.

[0011] Specifically, the aforementioned BPNs chemiluminescent probe is characterized in that: the BPNs chemiluminescent probe is synthesized using bulk black phosphorus as raw material through water bath ultrasonic exfoliation, and the specific preparation steps are as follows:

[0012] 15 mg of bulk black phosphorus was placed in a 100 mL round-bottom flask, 30 mL of distilled water was added, and the flask was sealed. The flask was then placed in an ultrasonic cleaner operating at a frequency of 40 kHz for ultrasonic stripping for 8 h. The water bath temperature in the ultrasonic cleaner was 16-25°C to obtain a black mixture. The black mixture was collected and centrifuged at 12,000 rpm for 10 min. 90% of the suspension was collected to obtain a BPNs suspension rich in oxygen functional groups.

[0013] A composite material of a BPNs chemiluminescent probe loaded with scopoletin as claimed in claim 1, wherein the composite material is a BPNs chemiluminescent probe loaded on scopoletin via hydrogen bonding to obtain a BPNs-loaded scopoletin composite material, wherein the composite material has a highly efficient reactive oxygen species scavenging ability and can react with potassium permanganate to produce a strong chemiluminescent signal;

[0014] The specific preparation method of the composite material is to place 500-700 μL of BPNs chemical light emitting probe in a quartz reaction cell, and add 150-250 μL of 5-15 μg / mL scopoletin solution to obtain the composite material.

[0015] The specific preparation method of the composite material is to place 600 μL of BPNs suspension in a quartz reaction cell, and add 200 μL of 10 μg / mL scopoletin solution to obtain a BPNs-loaded scopoletin composite material.

[0016] A chemiluminescent detection method comprises placing 800 μL of the composite material of the BPNs chemiluminescent probe loaded with scopoletin according to claim 4 or 5 in a chemiluminescent dish, using a peristaltic pump static injection method, externally introducing 200 μL of potassium permanganate to generate a chemiluminescent signal, using a chemiluminescent signal detection instrument to collect the light signal, and quantitatively analyzing the concentration of scopoletin based on the signal intensity.

[0017] The aforementioned chemiluminescence signal detection instrument sets the operating voltage of the photomultiplier tube to -1000 V, and the data integration time of the BPCL ultra-weak luminescence analyzer to 0.1 s.

[0018] A method for detecting scopoletin in cigarettes or cigarette smoke using the aforementioned BPNs chemical luminescence probe.

[0019] The method for detecting scopoletin in the aforementioned cigarettes is carried out according to the following steps:

[0020] (1) Scopoletin standard solutions with concentrations of 0.1, 0.3, 0.5, 1, 3, 5, 8, and 10 μg / mL were prepared, and then mixed with 600 μL of BPNs chemiluminescent probe to obtain 8 BPNs-loaded scopoletin composites with different concentrations;

[0021] (2) The eight BPNs-loaded scopoletin composite materials with different concentrations were placed in the luminescent dishes of the reaction system, and then 0.01 mol / L potassium permanganate was introduced externally to react with them. At the same time, a BPCL ultra-weak luminescence analyzer equipped with a photomultiplier tube was used to monitor the chemiluminescence signal, construct a chemiluminescence platform, draw a standard curve, and confirm the regression equation;

[0022] (3) 5 g of cigarette smoke was mixed with 10 mL of n-hexane and ultrasonicated at 18-25°C for 15 min at a frequency of 40 kHz and a power of 500 W. The mixture was then centrifuged at 4500 rpm for 4 min to remove triglycerides. This step was repeated twice to obtain a crude cigarette extract.

[0023] (4) adding 50 mL of methanol to the crude cigarette extract, ultrasonicating the solution at 18-25°C for 30 min at a frequency of 40 kHz and a power of 500 W, then filtering the solution through a Buchner funnel and finally filtering the solution through a 0.22 μm filter membrane to obtain the cigarette extract;

[0024] (5) The cigarette extract was mixed with the BPNs chemiluminescent probe and placed in a luminescent dish of the reaction system. A PBS=11 buffer solution was added, and then 0.01 mol / L potassium permanganate was introduced. At the same time, an ultra-weak luminescence analyzer was used to record the chemiluminescent signal generated by the reaction. The obtained chemiluminescent signal intensity was substituted into the regression equation to calculate the scopoletin content in the cigarette extract.

[0025] The aforementioned method for detecting scopoletin in cigarette smoke is carried out according to the following steps:

[0026] (1) Scopoletin standard solutions with concentrations of 0.1, 0.3, 0.5, 1, 3, 5, 8, and 10 μg / mL were prepared, and then mixed with 600 μL of BPNs chemiluminescent probe to obtain 8 BPNs-loaded scopoletin composites with different concentrations;

[0027] (2) The eight BPNs-loaded scopoletin composite materials with different concentrations were placed in the luminescent dishes of the reaction system, and then 0.01 mol / L potassium permanganate was introduced externally to react with them. At the same time, a BPCL ultra-weak luminescence analyzer equipped with a photomultiplier tube was used to monitor the chemiluminescence signal, construct a chemiluminescence platform, draw a standard curve, and confirm the regression equation;

[0028] (3) 0.9 g of cigarette smoke was burned in a natural environment, and smoke was pumped into a closed round-bottom flask containing 50 mL of methanol for 3 minutes to simulate the smoking process; after the smoke was completely dissolved, a cigarette smoke sample was obtained;

[0029] (4) A cigarette smoke sample was mixed with the BPNs chemiluminescent probe and placed in a luminescent dish of the reaction system. A PBS=11 buffer solution was added, and then 0.01 mol / L potassium permanganate was introduced. At the same time, an ultra-weak luminescence analyzer was used to record the chemiluminescent signal generated by the reaction. The obtained chemiluminescent signal intensity was substituted into the regression equation to calculate the scopoletin content in the cigarette smoke.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The synthesized BPNs, rich in oxygen functional groups on their surfaces, loaded scopoletin via hydrogen bonding. This loaded complex exhibited reactive oxygen species scavenging capabilities. The BPNs-loaded scopoletin efficiently scavenged singlet oxygen, hydroxyl radicals, and superoxide anions, and exhibited excellent chemiluminescence properties in response to potassium permanganate. Consequently, a chemiluminescent detection platform for scopoletin in cigarettes and cigarette smoke was established. This method offers advantages such as good selectivity, low detection cost, and ease of use.

[0032] Scopoletin showed good linearity over the concentration range of 0.1 μg / mL to 10.0 μg / mL, with a limit of detection of 0.002 μg / mL, demonstrating the high sensitivity of this method for detecting scopoletin. Furthermore, the method was used to detect and recover scopoletin in cigarettes and cigarette smoke, with recoveries ranging from 92.6% to 101.6%, demonstrating its reliability. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : TEM image of BPNs;

[0034] Figure 2 : The method of BPNs loading scopoletin (A is FT-IR spectrum; B is Zeta value);

[0035] Figure 3 : CL performance test (A is the CL response of BPNs / SP and potassium permanganate; B is the selectivity experiment);

[0036] Figure 4 : ROS scavenging mechanism of BPNs loaded with scopoletin (A is ROS scavenging experiment; B is EPR experiment of singlet oxygen; C is EPR experiment of hydroxyl radical; D is chemical probe experiment of superoxide anion);

[0037] Figure 5 : Linear relationship diagram between SP concentration and relative chemiluminescence intensity (A is the CL intensity-time curve of BPNs / SP-KMnO4 system under different SP concentrations; B is the linear relationship diagram between SP concentration and chemiluminescence intensity). DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the examples, but they are not intended to limit the present invention.

[0039] Example 1:

[0040] Preparation of the BPNs chemical luminescence probe: Place 15 mg of bulk black phosphorus in a 100 mL round-bottom flask and add 30 mL of distilled water. The flask is then sealed and ultrasonically exfoliated in a 40 kHz ultrasonic cleaner for 8 hours (water bath temperature 16-25°C) to produce a black mixture. The black mixture is collected and centrifuged at 12,000 rpm for 10 minutes. 90% of the suspension is collected to obtain the oxygen-rich BPNs, the resulting BPNs chemical luminescence probe.

[0041] Example 2

[0042] Preparation of the BPNs chemical luminescence probe: Place 20 mg of bulk black phosphorus in a 100 mL round-bottom flask and add 40 mL of distilled water. The flask is then sealed and ultrasonically exfoliated for 7.5 hours in a 45 kHz ultrasonic cleaner (water bath temperature 16-25°C) to produce a black mixture. The mixture is then collected and centrifuged at 12,000 rpm for 12 minutes. 90% of the suspension is collected to obtain the oxygen-rich BPNs, the resulting BPNs chemical luminescence probe.

[0043] Example 3:

[0044] Preparation of the BPNs chemical luminescence probe: 25 mg of bulk black phosphorus was placed in a 100 mL round-bottom flask and 50 mL of distilled water was added. The round-bottom flask was then sealed and subjected to ultrasonic exfoliation in a 35 kHz ultrasonic cleaner for 8.5 hours (water bath temperature 16-25°C), yielding a black mixture. The mixture was then collected and centrifuged at 12,000 rpm for 8 minutes. 90% of the suspension was collected to obtain the oxygen-rich BPNs, the resulting BPNs chemical luminescence probe.

[0045] Example 4

[0046] Preparation of BPNs chemiluminescent probe-loaded scopoletin composite material: 600 μL of BPNs chemiluminescent probe (prepared in Example 1) was placed in a quartz cell, and 200 μL of 10 μg / mL scopoletin solution was added to obtain a BPNs-loaded scopoletin composite material.

[0047] Example 5

[0048] Preparation of BPNs chemiluminescent probe-loaded scopoletin composite material: 600 μL of BPNs chemiluminescent probe (prepared in Example 2) was placed in a quartz cell, and 200 μL of 10 μg / mL scopoletin solution was added to obtain a BPNs-loaded scopoletin composite material.

[0049] Example 6

[0050] Preparation of BPNs chemiluminescent probe-loaded scopoletin composite material: 600 μL of BPNs chemiluminescent probe (prepared in Example 3) was placed in a quartz cell, and 200 μL of 10 μg / mL scopoletin solution was added to obtain a BPNs-loaded scopoletin composite material.

[0051] Example 7

[0052] Preparation of BPNs chemiluminescent probe-loaded scopoletin composite material: 500 μL of BPNs chemiluminescent probe (prepared in Example 1) was placed in a quartz cell, and 150 μL of 5 μg / mL scopoletin solution was added to obtain a BPNs-loaded scopoletin composite material.

[0053] Example 8

[0054] Preparation of BPNs chemiluminescent probe-loaded scopoletin composite material: 700 μL of BPNs chemiluminescent probe (prepared in Example 1) was placed in a quartz cell, and 250 μL of 15 μg / mL scopoletin solution was added to obtain a BPNs-loaded scopoletin composite material.

[0055] Example 9:

[0056] Chemiluminescence detection

[0057] (1) The chemical reaction was carried out using the peristaltic pump static injection method. The luminescence signal generated by the reaction was monitored by a BPCL ultra-weak luminescence analyzer equipped with a photomultiplier tube. The operating voltage of the photomultiplier tube was set to -1000 V, and the data integration time of the BPCL ultra-weak luminescence analyzer was set to 0.1 s.

[0058] (2) 800 μL of the BPNs chemiluminescent probe-loaded scopoletin composite (prepared in Example 4) was placed in a dedicated luminescent dish. 200 μL of potassium permanganate solution was placed in a plastic tube. A peristaltic pump was then activated to rapidly inject the potassium permanganate solution from the disposable plastic tube into the luminescent dish. Simultaneously, a chemiluminescent signal detector was turned on to collect the light signal.

[0059] Example 10:

[0060] Chemiluminescence detection

[0061] (1) The chemical reaction was carried out using the peristaltic pump static injection method. The luminescence signal generated by the reaction was monitored by a BPCL ultra-weak luminescence analyzer equipped with a photomultiplier tube. The operating voltage of the photomultiplier tube was set to -1000 V, and the data integration time of the BPCL ultra-weak luminescence analyzer was set to 0.1 s.

[0062] (2) Place 800 μL of the BPNs chemiluminescent probe-loaded scopoletin composite (prepared in Example 5) in a dedicated luminescent dish. Place 200 μL of potassium permanganate solution in a plastic tube. Then, activate a peristaltic pump to rapidly inject the potassium permanganate solution from the disposable plastic tube into the luminescent dish. Simultaneously, turn on a chemiluminescent signal detector to collect the light signal.

[0063] Example 11:

[0064] Chemiluminescence detection

[0065] (1) The chemical reaction was carried out using the peristaltic pump static injection method. The luminescence signal generated by the reaction was monitored by a BPCL ultra-weak luminescence analyzer equipped with a photomultiplier tube. The operating voltage of the photomultiplier tube was set to -1000 V, and the data integration time of the BPCL ultra-weak luminescence analyzer was set to 0.1 s.

[0066] (2) 800 μL of the BPNs chemiluminescent probe-loaded scopoletin composite (prepared in Example 6) was placed in a dedicated luminescent dish. 200 μL of potassium permanganate solution was placed in a plastic tube. A peristaltic pump was then activated to rapidly inject the potassium permanganate solution from the disposable plastic tube into the luminescent dish. Simultaneously, a chemiluminescent signal detector was turned on to collect the light signal.

[0067] Example 12: Detection of Cigarettes

[0068] 1. Construction of chemiluminescence platform

[0069] (1) Scopoletin standard solutions with concentrations of 0.1, 0.3, 0.5, 1, 3, 5, 8, and 10 μg / mL were prepared, and then mixed with 600 μL of BPNs solution (prepared in Example 1) to obtain 8 BPNs-loaded scopoletin composite materials with different concentrations;

[0070] (2) The eight BPNs-loaded scopoletin composite materials of different concentrations obtained in (1) were placed in luminescent dishes of the reaction system, and then 0.01 mol / L potassium permanganate was introduced externally to react with them, and the chemiluminescence signal was monitored by a BPCL ultra-weak luminescence analyzer equipped with a photomultiplier tube;

[0071] (3) Finally, a standard curve was drawn with a range of 0.1 μg / mL-10 μg / mL, and the detection limit was determined to be 0.002 μg / mL, resulting in the regression equation I = 1208.59 [SP] + 362.74.

[0072] 2. Testing process

[0073] (1) 5 g of cigarette smoke was added to 10 mL of n-hexane and ultrasonicated for 15 minutes (ultrasonic frequency: 40 kHz, temperature: 20°C, ultrasonic power: 500 W). The mixture was then centrifuged at 4500 rpm for 4 minutes to remove triglycerides (the previous step was repeated twice) to obtain a crude cigarette extract. 50 mL of methanol was then added and ultrasonicated for 30 minutes (ultrasonic frequency: 40 kHz, temperature: 20°C, ultrasonic power: 500 W). The mixture was then filtered using a Buchner funnel and finally filtered through a 0.22 μm filter membrane to obtain a cigarette extract.

[0074] (2) The cigarette extract was mixed with the BPNs of Example 1 and placed in a luminescent dish of the reaction system. A buffer solution of PBS = 1:1 was added, and then 0.01 mol / L potassium permanganate was introduced. At the same time, the chemiluminescence signal generated by the reaction was recorded using an ultra-weak luminescence analyzer. The obtained chemiluminescence (CL) signal intensity was substituted into the regression equation I = 1208.59 [SP] + 362.74, and the scopoletin content in the cigarette extract was calculated to be 0.62 μg / mL.

[0075] Example 13: Detection of cigarette smoke

[0076] 1. Construction of chemiluminescence platform

[0077] (1) Scopoletin standard solutions with concentrations of 0.1, 0.3, 0.5, 1, 3, 5, 8, and 10 μg / mL were prepared, and then mixed with 600 μL of BPNs solution (prepared in Example 1) to obtain 8 BPNs-loaded scopoletin composite materials with different concentrations;

[0078] (2) The eight BPNs-loaded scopoletin composite materials of different concentrations obtained in (1) were placed in luminescent dishes of the reaction system, and then 0.01 mol / L potassium permanganate was introduced externally to react with them, and the chemiluminescence signal was monitored by a BPCL ultra-weak luminescence analyzer equipped with a photomultiplier tube;

[0079] (3) The final standard curve range was 0.1 μg / mL-10 μg / mL, and the detection limit was determined to be 0.002 μg / mL, resulting in the regression equation I = 1208.59 [SP] + 362.74.

[0080] 2. Testing process

[0081] (1) 0.9 g of cigarette smoke was burned in a natural environment and the smoke was pumped into a closed round-bottom flask containing 50 ml of methanol for 3 minutes to simulate the smoking process. After the smoke was completely dissolved, the cigarette smoke sample was obtained;

[0082] (2) The cigarette smoke sample was mixed with the BPNs from Example 1 and placed in a luminescent dish in the reaction system. A 1:1 PBS buffer solution was added, followed by the introduction of 0.01 mol / L potassium permanganate. The chemiluminescent signal generated by the reaction was recorded using an ultra-weak luminescence analyzer. The resulting CL signal intensity was substituted into the regression equation I = 1208.59 [SP] + 362.74, yielding a calculated scopoletin content of 2.55 μg / mL.

[0083] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments to better understand how the present invention achieves its objectives and solves related technical problems:

[0084] 1. Reagents

[0085] Bulk black phosphorus (BP) was purchased from Chengdu Kelong Chemical Co., Ltd., China; scopoletin (SP) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., China; and potassium permanganate was purchased from Chongqing Chuandong Chemical Co., Ltd., China.

[0086] 2 Instruments

[0087] Chemiluminescence signals were detected using an ultraweak chemiluminescence instrument (BPCL-2-TGG, Guangzhou Weiguang Technology Co., Ltd., China). TEM images of BPNs were taken using a Tecnai G2 F20 S-Twin (Thermo Fisher Scientific, USA) with an accelerating voltage set to 200 kV. Fourier transform infrared (FT-IR) spectra were measured using a VERTEX70 infrared spectrometer (Brooks, Germany). Electron paramagnetic resonance (EPR) spectra were measured on a Bruker E-500 instrument. Zeta potential values were determined using a JS94 H2 instrument.

[0088] Preparation method of 3BPNs chemical photoluminescence probe

[0089] 15 mg of bulk black phosphorus was placed in a 100 mL round-bottom flask and 30 mL of distilled water was added. The flask was then sealed and ultrasonically exfoliated for 8 hours in a 40 kHz ultrasonic cleaner (water bath temperature 16-25°C). The resulting mixture was centrifuged at 12,000 rpm for 10 minutes, and 90% of the suspension was collected to obtain a stable BPNs chemiluminescent probe.

[0090] TEM image characterization of 4 pairs of BPNs chemical light-emitting probes

[0091] The TEM image experiment of BPNs chemical light emitting probe is shown in the attached figure. Figure 1 As shown, it is a nanosheet structure, indicating that the nanosheet material (BPNs chemical photoluminescence probe) was successfully synthesized.

[0092] Reactive oxygen species scavenging and chemiluminescent detection of 5BPNs chemiluminescent probe loaded with scopoletin

[0093] 5.1 Loading method of scopoletin on BPNs chemical photoluminescence probe

[0094] Firstly, Fourier transform infrared spectroscopy (FT-IR) was used to characterize the loading mode of scopoletin (SP) on BPNs chemical photoluminescence probe ( Figure 2 A). At 3446cm -1 and 1597cm -1 The absorption bands at 2718cm belong to -OH and P=O bonds, indicating that there is a small amount of oxidation on the surface of BPNSs chemical light emitting probe. -1 and 2829cm -1 The -CH3 vibration of scopoletin is at 1135 cm -1 and 1706cm -1 The stretching vibrations of COC and C=O are at 773cm -1 and 1354cm -1 Bending vibrations of OH and stretching vibrations of POH were observed; the absorption peaks of -OH and P=O bonds were also observed to be enhanced and broadened. FT-IR experiments confirmed the existence of hydrogen bonding interactions between BPNs and scopolamine. Then, the zeta potential of the BPNs chemical photoluminescence probe before and after drug loading was characterized. Figure 2 As shown in Figure B, the potentials of BPNs and scopoletin are -25.99 mV and -50.60 mV, respectively. After loading scopoletin, the potential of BPNs / scopoletin becomes -30.83 mV. This is because scopoletin is loaded onto the BPNs chemical photoluminescent probe through hydrogen bonding, thereby shielding part of the negative charge of scopoletin.

[0095] Chemiluminescence properties of 5.2BPNs loaded with scopoletin

[0096] The present invention utilizes a traditional chemiluminescence device and adopts a peristaltic pump static injection method for detection, including an injection system, a reaction system and a detection system. The main function of the injection system is to introduce 200 μL of potassium permanganate solution into the reaction system through a peristaltic pump. 600 μL of BPNs chemiluminescent probe and 200 μL of scopolamine solution are mixed and placed in a luminescent dish of the reaction system, and react with the solution introduced by the peristaltic pump to form a reaction system. The luminescence signal generated by the reaction is monitored by a BPCL ultra-weak luminescence analyzer equipped with a photomultiplier tube. The working voltage of the photomultiplier tube is set to -1000 V, and the data integration time of the BPCL ultra-weak luminescence analyzer is 0.1 s. The chemiluminescence curve of the mixture of potassium permanganate and BPNs / scopolamine is shown in the attached figure. Figure 3As shown in Figure A, the reaction of potassium permanganate with the BPNs / scopoletin mixture resulted in a strong chemiluminescent signal. In addition, the chemiluminescent response of BPNs / potassium permanganate and other organic compounds with similar scopoletin structures was also measured. Except for scopoletin, BPNs / potassium permanganate showed relatively weak chemiluminescent signals for other compounds, proving that the BPNs chemiluminescent probe has good selectivity for scopoletin ( Figure 3 B).

[0097] 5.3 Active oxygen scavenging properties of BPNs-loaded scopoletin composites

[0098] First, free radical scavenging experiments demonstrated that the singlet oxygen generated in the BPNs / scopolamine-potassium permanganate system ( 1 O2), hydroxyl radicals (·OH) and superoxide anions ( · O2-) plays an important role in chemiluminescence ( Figure 4 A). Then, electron paramagnetic resonance (EPR) experiments were used to study the scavenging effect of BPNs / scopolamine on reactive oxygen species in the chemiluminescence system. Figure 4 As shown in B, scopoletin can eliminate the 1 O2, after loading scopoletin onto the surface of BPNs chemical light-emitting probe, its 1 In addition, the mixed solution of scopoletin and potassium permanganate can produce ·OH, and when scopoletin is loaded onto the surface of BPNs chemical light-emitting probe, it can effectively remove the ·OH generated by the mixed solution ( Figure 4 C). The chemical probe nitro blue tetrazolium chloride (NBT) was used to study · The conversion of O2-, such as Figure 4 As shown in D, BPNs loaded with scopolamine can effectively clear · O2-, which may promote the mutual conversion between reactive oxygen species. EPR and chemical probe experiments have shown that BPNs chemical light-emitting probes loaded with scopolamine have efficient reactive oxygen species scavenging ability.

[0099] 5.4 Detection of Scopolamine in Cigarettes and Cigarette Smoke

[0100] Finally, the applicability of the CL method was verified by measuring the scopolamine content in cigarettes and smoke. Cigarettes were purchased from supermarkets and scopolamine was extracted from the cigarettes using ultrasound-assisted technology; the smoke from the burning cigarettes was dissolved in an appropriate amount of methanol solvent. The CL sensor constructed as follows ( Figure 5 ) The actual sample solution obtained was subjected to scopoletin detection and recovery experiments.

[0101] 5.4.1 Construction of Chemiluminescence Platform:

[0102] (1) SP standard solutions with concentrations of 0.1, 0.3, 0.5, 1, 3, 5, 8, and 10 μg / mL were prepared and then mixed with 600 μL of BPNs solution (prepared in Example 1) to obtain 8 composite materials of BPNs chemical light-emitting probe loaded with scopoletin at different concentrations.

[0103] (2) The composite materials of 8 different concentrations of BPNs chemiluminescent probes loaded with scopolamine obtained in (1) were placed in a luminescent dish of the reaction system, and then 0.01 mol / L potassium permanganate was introduced externally to react with them, and the chemiluminescent signal was monitored by a BPCL ultra-weak luminescence analyzer equipped with a photomultiplier tube.

[0104] (3) The final standard curve range was 0.1 μg / mL-10 μg / mL, and the detection limit was 0.002 μg / mL.

[0105] 5.4.2 Specific testing process:

[0106] (1) First, a cigarette extract (Example 12) or a cigarette smoke sample (Example 13) was mixed with the BPNs chemiluminescent probe (prepared in Example 1) and placed in a luminescent dish of the reaction system. A PBS buffer solution of 1:1 was added, and then 0.01 mol / L potassium permanganate was introduced externally. The chemiluminescent signal generated by the reaction was simultaneously recorded using an ultra-weak luminescence analyzer. The obtained CL signal intensity was substituted into the regression equation I = 1208.59 [SP] + 362.74, and the scopoletin detected in the cigarette extract and cigarette smoke was calculated to be 0.62 μg / mL and 2.55 μg / mL, respectively.

[0107] (2) Then, SP solutions of known concentrations (0.1, 0.5, and 1.0 μg / mL) were added to cigarette extracts or cigarette smoke samples using the standard addition method. The same experimental steps as in (1) were used to detect the CL emissions of BPNs mixed with samples of varying spiked concentrations and then reacted with 0.01 mol / L potassium permanganate. The concentrations were calculated based on the standard curve. The recoveries and relative standard deviations (RSDs) of the CL method in actual samples are shown in Table 1. The recoveries of scopoletin ranged from 92.6% to 101.6%.

[0108] Table 1: Recovery and determination results of SP in cigarettes and cigarette smoke samples

[0109]

[0110]

[0111] 6 Conclusion

[0112] This invention develops a new chemiluminescence technology to study the reactive oxygen species scavenging mechanism of the drug scopolamine. Black phosphorus nanosheets (BPNs chemiluminescent probes) with oxygen-containing functional groups on their surfaces were designed. These nanosheets are loaded with scopolamine through hydrogen bonding interactions and then react with potassium permanganate to effectively amplify the chemiluminescent signal. Experiments also demonstrated that scopolamine loaded with BPNs chemiluminescent probes can effectively scavenge singlet oxygen, hydroxyl radicals, and superoxide anions. Furthermore, a highly selective chemiluminescent platform was constructed for the detection of scopolamine in cigarettes and cigarette smoke, with a recovery rate between 92.6% and 101.6%, demonstrating the reliability of this method. This invention marks the first use of chemiluminescence to study the reactive oxygen species scavenging mechanism of scopolamine, providing a new perspective for understanding the pharmacological and physiological activities of scopolamine.

Claims

1. A BPNs chemiluminescent probe, characterized by: The BPNs chemiluminescent probe is synthesized using bulk black phosphorus as raw material through water bath ultrasonic exfoliation. The specific preparation steps are as follows: 15-25 mg of bulk black phosphorus was placed in a round-bottom flask, 30-50 mL of distilled water was added, and the flask was sealed. The flask was then placed in an ultrasonic cleaner with an operating frequency of 30-50 kHz for ultrasonic stripping for 7-9 hours. The water bath temperature in the ultrasonic cleaner was 10-30°C to obtain a black mixture. The black mixture was collected and centrifuged at a speed of 10,000-15,000 rpm for 5-15 minutes. 85-95% of the suspension was collected to obtain BPNs rich in oxygen functional groups, i.e., BPNs chemical photoluminescence probes.

2. The BPNs chemiluminescent probe according to claim 1, wherein: The BPNs chemical photoluminescence probe is synthesized from bulk black phosphorus by water bath ultrasonic exfoliation. The specific preparation steps are as follows: 15-20 mg of bulk black phosphorus was placed in a round-bottom flask, 30-40 mL of distilled water was added, and the flask was sealed. The flask was then placed in an ultrasonic cleaner with an operating frequency of 35-45 kHz for ultrasonic stripping for 7.5-8.5 hours. The water bath temperature in the ultrasonic cleaner was 16-25°C to obtain a black mixture. The black mixture was collected and centrifuged at 12000 rpm for 8-12 minutes. 90% of the suspension was collected to obtain a BPNs suspension rich in oxygen functional groups.

3. The BPNs chemiluminescent probe according to claim 2, wherein: The BPNs chemical photoluminescence probe is synthesized from bulk black phosphorus by water bath ultrasonic exfoliation. The specific preparation steps are as follows: 15 mg of bulk black phosphorus was placed in a 100 mL round-bottom flask, 30 mL of distilled water was added, and the flask was sealed. The flask was then placed in an ultrasonic cleaner operating at a frequency of 40 kHz for ultrasonic stripping for 8 h. The water bath temperature in the ultrasonic cleaner was 16-25°C to obtain a black mixture. The black mixture was collected and centrifuged at 12,000 rpm for 10 min. 90% of the suspension was collected to obtain a BPNs suspension rich in oxygen functional groups.

4. A composite material of BPNs chemiluminescent probe loaded with scopoletin according to claim 1, characterized in that: The composite material is a BPNs chemiluminescent probe loaded on scopoletin through hydrogen bonding to obtain a BPNs-loaded scopoletin composite material. The composite material has a high-efficiency active oxygen scavenging ability and can react with potassium permanganate to produce a strong chemiluminescent signal. The specific preparation method of the composite material is to place 500-700 μL of BPNs chemical light emitting probe in a quartz reaction cell, and add 150-250 μL of 5-15 μg / mL scopoletin solution to obtain the composite material.

5. The composite material according to claim 4, wherein: The specific preparation method of the composite material is to place 600 μL of BPNs suspension in a quartz reaction cell, and add 200 μL of 10 μg / mL scopoletin solution to obtain a BPNs-loaded scopoletin composite material.

6. A chemiluminescence detection method, characterized in that: The chemiluminescence detection method comprises placing 800 μL of the composite material of the BPNs chemiluminescent probe loaded with scopoletin in claim 4 or 5 in a chemiluminescent dish, adopting a peristaltic pump static injection method, externally introducing 200 μL of potassium permanganate to generate a chemiluminescent signal, using a chemiluminescent signal detection instrument to collect the light signal, and quantitatively analyzing the concentration of scopoletin based on the signal intensity.

7. The chemiluminescent detection method according to claim 6, wherein: The chemiluminescence signal detection instrument sets the operating voltage of the photomultiplier tube to -1000 V, and the data integration time of the BPCL ultra-weak luminescence analyzer to 0.1 s.

8. A method for detecting scopoletin in cigarettes or cigarette smoke using the BPNs chemiluminescent probe according to any one of claims 1 to 3.

9. The detection method according to claim 8, wherein: The method for detecting scopoletin in the cigarette is carried out according to the following steps: (1) Scopoletin standard solutions with concentrations of 0.1, 0.3, 0.5, 1, 3, 5, 8, and 10 μg / mL were prepared, and then mixed with 600 μL of BPNs chemiluminescent probe to obtain 8 BPNs-loaded scopoletin composites with different concentrations; (2) The eight BPNs-loaded scopoletin composite materials with different concentrations were placed in the luminescent dishes of the reaction system, and then 0.01 mol / L potassium permanganate was introduced externally to react with them. At the same time, a BPCL ultra-weak luminescence analyzer equipped with a photomultiplier tube was used to monitor the chemiluminescence signal, construct a chemiluminescence platform, draw a standard curve, and confirm the regression equation; (3) 5 g of cigarette smoke was added to 10 mL of n-hexane and ultrasonicated at 18-25°C for 15 min at a frequency of 40 kHz and a power of 500 W. The extract was then centrifuged at 4500 rpm for 4 min to remove triglycerides. This step was repeated twice to obtain a crude cigarette extract. (4) adding 50 mL of methanol to the crude cigarette extract, ultrasonicating the solution at 18-25°C for 30 min at a frequency of 40 kHz and a power of 500 W, then filtering the solution through a Buchner funnel and finally filtering the solution through a 0.22 μm filter membrane to obtain the cigarette extract; (5) The cigarette extract was mixed with the BPNs chemiluminescent probe and placed in a luminescent dish of the reaction system. A PBS=11 buffer solution was added, and then 0.01 mol / L potassium permanganate was introduced. At the same time, an ultra-weak luminescence analyzer was used to record the chemiluminescent signal generated by the reaction. The obtained chemiluminescent signal intensity was substituted into the regression equation to calculate the scopoletin content in the cigarette extract.

10. The detection method according to claim 8, wherein: The method for detecting scopoletin in cigarette smoke is carried out according to the following steps: (1) Scopoletin standard solutions with concentrations of 0.1, 0.3, 0.5, 1, 3, 5, 8, and 10 μg / mL were prepared, and then mixed with 600 μL of BPNs chemiluminescent probe to obtain 8 BPNs-loaded scopoletin composites with different concentrations; (2) The eight BPNs-loaded scopoletin composite materials with different concentrations were placed in the luminescent dishes of the reaction system, and then 0.01 mol / L potassium permanganate was introduced externally to react with them. At the same time, a BPCL ultra-weak luminescence analyzer equipped with a photomultiplier tube was used to monitor the chemiluminescence signal, construct a chemiluminescence platform, draw a standard curve, and confirm the regression equation; (3) 0.9 g of cigarette smoke was burned in a natural environment, and smoke was pumped into a closed round-bottom flask containing 50 mL of methanol for 3 minutes to simulate the smoking process; after the smoke was completely dissolved, a cigarette smoke sample was obtained; (4) A cigarette smoke sample was mixed with the BPNs chemiluminescent probe and placed in a luminescent dish of the reaction system. A PBS=11 buffer solution was added, and then 0.01 mol / L potassium permanganate was introduced. At the same time, an ultra-weak luminescence analyzer was used to record the chemiluminescent signal generated by the reaction. The obtained chemiluminescent signal intensity was substituted into the regression equation to calculate the scopoletin content in the cigarette smoke.

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