WO2021035654A1

WO2021035654A1 - Method for preparing double-doped metal-organic framework complex ratiometric fluorescent baicalin probe

Info

Publication number: WO2021035654A1
Application number: PCT/CN2019/103465
Authority: WO
Inventors: 桂日军; 姜晓文; 金辉; 孙玉娇
Original assignee: 青岛大学
Priority date: 2019-08-27
Filing date: 2019-08-30
Publication date: 2021-03-04
Also published as: CN110849861A; CN110849861B; CN110376176A

Abstract

A method for preparing a ratiometric fluorescent baicalin probe based on a silver nanocluster AgNCs/black phosphorus quantum dot BPQDs double-doped metal organic framework MOF complex. Blue fluorescent BPQDs are reacted with a MOF precursor to prepare a BPQDs/MOF complex, red fluorescent AgNCs are added and the reaction is continued, BPQDs are encapsulated in MOF, and the AgNCs are adsorbed in MOF pores. Baicalin is added into an aqueous dispersion of an AgNCs/BPQDs/MOF complex containing catalase, and the baicalin generates H₂O₂ under the action of catalase. H₂O₂ causes fluorescence quenching of AgNCs, but has little effect on the fluorescence of BPQDs encapsulated in MOF. A linear relationship between a fluorescent emission peak intensity ratio I_BPQDs/I_AgNCs and the concentration of baicalin is fitted to construct a ratiometric fluorescent probe for efficient detection of baicalin.

Description

Method for preparing double-doped metal-organic framework complex ratio fluorescent bacein probe

Technical field:

The invention belongs to the technical field of preparation of metal organic framework composite materials and ratio fluorescent probes, and specifically relates to a method for preparing ratio fluorescent bacitin probes based on silver nanoclusters/black phosphorous quantum dots double-doped metal organic framework composites , The probe prepared by it can be used for the highly sensitive and selective quantitative detection of bacitrin.

Background technique:

Baicalin is a flavonoid compound extracted from the dried rhizome of the plant Scutellaria baicalensis Georgi. Baicin has significant biological activity, and has the effects of antibacterial, anti-inflammatory, cholesterol-lowering, anti-thrombosis, relieving asthma, purging fire and detoxification, hemostasis, and antispasmodic. Bacitin is a specific inhibitor of mammalian liver salivase, which has the effect of regulating certain diseases, and also has a strong anti-cancer response physiological effect. Baicin also has certain side effects on the human body, which are mainly reflected in the bitter cold injures the stomach, and those with spleen and stomach deficiency and cold are not suitable for consumption. Baicin is very low in toxicity and will not have obvious adverse reactions to the human body at general doses. Certain patients may have stomach discomfort, diarrhea and other reactions. People with allergies may have vesicular drug eruption. When bazine glycoside injection preparations are used in large doses, the human body will experience low-grade fever, muscle aches, and decreased white blood cells. Illegal addition of an excessive amount of bacitrin in drugs can cause damage to the human body, which is very necessary for accurate detection.

The analytical methods for detecting bacitrin mainly include electrochemical methods, chromatography and so on. After consulting the literature, Sheng et al. have prepared a cobalt nanoparticle-doped aminated graphene-modified electrode for the electrochemical detection of bacitin (Kai Sheng, Lu Wang, Huichao Li, Lina Zou, Baoxian Ye. Green synthesized Co nanoparticles doped amino-graphene modified electrode and its application towards determination of baicalin. Talanta, 2017, 164, 249-256); Wang et al. used liquid chromatography-mass spectrometry/mass spectrometry to determine baicalin in rat plasma ( Ying Wang, Yifan Zhang, Juan Xiao, Ranchi Xu, Qiangli Wang, Xinhong Wang.Simultaneous determination of baicalin, baicalein, wogonoside, wogonin, scutellarin, berberine, coptisine, ginsenoside Rb1, coptisine, ginsenoside Rb1 and ex- gins of government MS/MS and its application to a pharmacokinetic study. Biomedical Chromatography, 2018, 32, e4083). Up to now, no domestic and foreign patent reports related to the quantitative detection of bacitrin have been retrieved.

The current analytical method for detecting bazine glycosides is mainly chromatography, but this method generally suffers from problems such as long time-consuming, complicated operation, harsh conditions, and high cost. In contrast, chemical and biosensor detection methods such as electrochemical sensors have excellent performance such as easy operation, high sensitivity, and good selectivity. At present, the detection of baclidin is mainly an electrochemical sensor method, in which the detection of baclidin relies on a single electrochemical signal output. Generally, the intensity of a single signal is susceptible to interference from factors such as background, reagents, systems, and environmental conditions, causing fluctuations in the measurement results. However, the dual signal ratio processing to obtain the ratio value of the signal strength can have a self-calibration function, which effectively eliminates the interference of self and background signals, thereby improving the accuracy and reliability of the detection results.

Based on this, the present invention reports a ratio fluorescent probe based on silver nanoclusters AgNCs/black phosphorous quantum dots BPQDs double-doped metal-organic framework MOF complex AgNCs/BPQDs/MOF for detecting bacitin. In the aqueous dispersion system of the complex containing catalase, bacitrin is added, and bacitrin generates hydrogen peroxide under the catalysis of catalase, which causes the fluorescence of AgNCs in the complex to be quenched. The fluorescence of BPQDs has little effect. Therefore, using the fluorescence of BPQDs as the reference signal and the fluorescence of AgNCs as the response signal, the linear relationship between the _{fluorescence emission peak intensity ratio I BPQDs} /I _{AgNCs and bacitin concentration is constructed to construct} Ratio Fluorescent Bacein Probe. So far, there have not been reports on the metal-organic framework composites doped with silver nanoclusters/black phosphorous quantum dots, and the domestic and foreign literature and patents using ratiometric fluorescent probes to detect bacitin.

Summary of the invention:

The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art, and design a simple method, low cost, high sensitivity and high selectivity based on silver nanocluster/black phosphor quantum dot double doped metal organic framework composite Preparation method of ratio fluorescent bacitin probe.

In order to achieve the above objective, the present invention relates to a preparation process of a ratio fluorescent bacitin probe based on a silver nanocluster/black phosphorous quantum dot double-doped metal-organic framework composite including the following steps:

1. The preparation method of double-doped metal-organic framework complex ratio fluorescent bacitin probe, characterized in that the method specifically includes the following steps:

(1) Preparation of black phosphorous quantum dots BPQDs: Weigh 10 mg of black phosphorous crystals and add them to 30 mL of nitromethylpyrrolidone, sonicate for 30 minutes to form a dispersion, transfer the dispersion to a micro high pressure reactor, and heat it to At 140°C, the reaction was continuously stirred for 12 hours. The reaction mixture was centrifuged at 3500 rpm for 15 min to remove larger size products, and then centrifuged at 13000 rpm for 15 min to obtain a precipitate. The precipitate was washed three times with ethanol and distilled water, and dried in vacuum to obtain BPQDs, which were stored in the dark and nitrogen for later use. The average size of the BPQDs was 1 to 5 nm.

(2) Preparation of silver nanocluster AgNCs: Weigh 40 mg of lipoic acid powder into 20 mL of distilled water, stir well, add ^{0.1 mL of freshly prepared sodium borohydride with a concentration of 2 mol L -1} , and stir quickly for 30 minutes to form a homogeneous mixture. Under rapid stirring, 0.4 mL of silver nitrate with a concentration of 0.05 mol L ^-1 was added to the mixed solution, and then 0.3 mL of sodium borohydride with a concentration of 2 mol L ^{-1 was} added dropwise, and the reaction was maintained for 90 min with rapid stirring to obtain the product AgNCs. The dispersion should be stored in the dark and at 4°C for later use. The average size of AgNCs is 10-20nm.

(3) Preparation of silver nanoclusters/black phosphorus quantum dots/metal organic framework AgNCs/BPQDs/MOF composite: Weigh 2mg BPQDs into 10mL 2-methylimidazole ethanol solution, stir for 10min to form a mixed solution, add 10mL six The hydrated zinc nitrate aqueous solution was stirred for 30 min to obtain a brown precipitate, which was washed three times with ethanol and distilled water, and centrifuged at 3500 rpm for 15 min to prepare an aqueous dispersion of the BPQDs/MOF complex. Add 5mL AgNCs dispersion to this dispersion dropwise, stir and react for 2h, the product solution is centrifuged to obtain a precipitate, the precipitate is washed and dried to prepare AgNCs/BPQDs/MOF composite, which is protected from light and nitrogen Store at 4°C for later use. The mass concentrations of 2-methylimidazole, zinc nitrate hexahydrate and AgNCs are respectively 1～5g L ^-1 , 1～10 g L ^-1 and 1～10 mg L ^-1 .

(4) Add catalase to the aqueous dispersion of AgNCs/BPQDs/MOF complex, stir evenly, adjust the pH to 7.4 with phosphate buffer, then add bacitrin and stir to form a homogeneous mixture. After the light is stable for 5 minutes, measure the fluorescence emission spectra of the homogeneous mixture at different concentrations of _baclidin , and fit the linear relationship between the fluorescence emission peak intensity ratio I BPQDs /I _AgNCs and the concentration of baclidin to construct a ratio fluorescent probe , Used for the quantitative detection of bacitrin. Among them, the concentrations of AgNCs/BPQDs/MOF complex, catalase and ^{bacitin are 1-10 mg mL -1} , 5-10 mU L ^-1 and 0.01-100 μg mL ^-1 , respectively. The linear detection range of bacitin concentration is It is 0.01～100μg mL ^-1 , and the detection limit is 1～10ng mL ^-1 .

The effect of the present invention is: a ratio fluorescent probe based on a metal organic framework AgNCs/BPQDs/MOF complex doped with silver nanoclusters/black phosphorus quantum dots is reported, which is used for quantitative detection of bacitin. Firstly, blue fluorescent BPQDs and MOF precursors were reacted to prepare BPQDs/MOF complexes, and then red fluorescent AgNCs were added to continue the reaction, and BPQDs and AgNCs were gradually embedded in the MOF structure. BPQDs are encapsulated in MOF, while AgNCs are adsorbed in MOF pores. Baicin is added to the aqueous dispersion of AgNCs/BPQDs/MOF compound with catalase added. Baicin is catalyzed by catalase to generate hydrogen peroxide. The oxidation of hydrogen peroxide leads to the fluorescence of AgNCs. Quenched, BPQDs are encapsulated in MOF, which has little effect on their fluorescence. Taking the fluorescence of BPQDs as the reference and the fluorescence of AgNCs as the response signal, the ratio of the fluorescence emission peak intensity I _BPQDs /I AgNCs and the linear relationship between the concentration of _{baclidin were fitted to construct a ratio fluorescent baclidin probe.} Compared with the prior art, the method of the present invention is easy to operate, has strong anti-interference ability of ratio fluorescent signal, high sensitivity and good selectivity, and can be used as a novel ratio fluorescent probe for the highly sensitive and selective detection of bacein. .

Description of the drawings:

Figure 1 is a schematic diagram of the preparation of a ratio fluorescent probe based on a silver nanocluster/black phosphorous quantum dot double-doped metal-organic framework composite and the principle diagram of bacitin detection;

Figure 2 (a) is the measurement of the fluorescence emission spectra of the ratio fluorescent probe system under different bacitin concentrations;

_{Figure 2(b) shows the} ratio of fluorescence emission peak intensity I BPQDs /I _{AgNCs corresponding to different bacitin concentrations} , i.e. I ₅₃₀ /I ₆₃₀ , fitting the linear relationship between different ratio values and bacitin concentrations.

detailed description:

The present invention will be described in detail below with reference to the drawings and specific embodiments.

Example 1:

This embodiment relates to a method for preparing a ratio fluorescent bacein probe based on a silver nanocluster/black phosphorous quantum dot double-doped metal-organic framework complex. The preparation process and the principle diagram of the ratio fluorescent detection of bacein are shown in Figure 1 As shown, the specific process steps are as follows:

Preparation of BPQDs: Weigh 10 mg of black phosphorus crystals and add 30 mL of nitrogen methyl pyrrolidone, sonicate for 30 minutes to form a dispersion, transfer the dispersion to a micro high pressure reactor, heat to 140°C under nitrogen protection, and continue stirring for 12 hours . The reaction mixture was centrifuged at 3500 rpm for 15 minutes to remove larger-sized products, and then centrifuged at 13000 rpm for 15 minutes to obtain a precipitate. The precipitate was washed three times with ethanol and distilled water, and dried in vacuum to obtain BPQDs, which were stored in the dark and nitrogen for use. The average size of BPQDs was 2 nm.

Preparation of AgNCs: Weigh 40 mg of lipoic acid powder into 20 mL of distilled water, stir well, add ^{0.1 mL of freshly prepared sodium borohydride with a concentration of 2 mol L -1} , and stir quickly for 30 minutes to form a homogeneous mixture. Add 0.4 mL of silver nitrate with a concentration of 0.05 mol L ^{-1 to} the mixed solution, and then add 0.3 mL of sodium borohydride with a concentration of 2 mol L ^-1 dropwise, keep the rapid stirring reaction for 90 minutes, and prepare the product AgNCs dispersion. When stored at 4℃, the average size of AgNCs is 10 nm.

Preparation of AgNCs/BPQDs/MOF complex: Weigh 2 mg of BPQDs and add 10 mL ^{of 2-methylimidazole ethanol solution with a concentration of 1 g L -1} , stir for 10 min to form a mixed solution, and add 10 mL of 2 g L ^-1 of hexahydrate nitric acid The zinc aqueous solution was stirred for 30 min to prepare a brown precipitate, which was washed three times with ethanol and distilled water, and centrifuged at 3500 rpm for 15 min to prepare an aqueous dispersion of the BPQDs/MOF complex. Add 5 mL of aqueous AgNCs dispersion with a concentration of 2 mg L ^-1 dropwise to this dispersion, stir and react for 2 hours, the product solution is centrifuged to obtain a precipitate, and the precipitate is washed and dried to prepare an AgNCs/BPQDs/MOF complex. Store it in the dark, under nitrogen protection and at 4°C for later use.

Add catalase to the AgNCs/BPQDs/MOF complex aqueous dispersion, stir well, adjust the pH to 7.4 with phosphate water buffer, then add bacitrin, stir evenly to form a homogeneous mixture, which is stable in a dark place After 5 minutes, measure the fluorescence emission spectra of the homogeneous mixture at different concentrations of _baclidin (as shown in Figure 2(a)), and fit the linearity between the ratio of fluorescence emission peak intensity I BPQDs /I _{AgNCs and baclidin concentration} The relationship (as shown in Figure 2(b)), a ratio fluorescent probe was constructed for the quantitative detection of bacein. Among them, the concentrations of AgNCs/BPQDs/MOF complex, catalase and ^{bacitin are 1 mg mL -1} , 5 mU L ^-1 and 0.1-50 μg mL ^-1 , respectively. The linear detection range of bacitin concentration is 0.1-50 μg mL ^-1 , the detection limit is 4ng mL ^-1 .

Example 2: This example relates to the preparation process of the ratio fluorescent bacein probe based on the silver nanocluster/black phosphorus quantum dot double-doped metal-organic framework composite and the schematic diagram of the principle of ratio fluorescent detection of bacein, BPQDs and The process steps for preparing AgNCs are the same as in Example 1, wherein the average size of BPQDs is 3 nm, and the average size of AgNCs is 12 nm. Other specific process steps are as follows:

Preparation of AgNCs/BPQDs/MOF complex: Weigh 2 mg of BPQDs and add 10 mL ^{of 2-methylimidazole ethanol solution with a concentration of 2 g L -1} , stir for 10 min to form a mixed solution, and add 10 mL of 3 g L ^-1 of hexahydrate nitric acid The zinc aqueous solution was stirred for 30 min to prepare a brown precipitate, which was washed three times with ethanol and distilled water, and centrifuged at 3500 rpm for 15 min to prepare an aqueous dispersion of the BPQDs/MOF complex. Add 5 mL of aqueous AgNCs dispersion with a concentration of 3 mg L ^-1 dropwise to this dispersion, stir and react for 2 hours, the product solution is centrifuged to obtain a precipitate, and the precipitate is washed and dried to prepare AgNCs/BPQDs/MOF complex. Store it in the dark, under nitrogen protection and at 4°C for later use.

Add catalase to the AgNCs/BPQDs/MOF complex aqueous dispersion, stir well, adjust the pH to 7.4 with phosphate water buffer, then add bacitrin, stir evenly to form a homogeneous mixture, which is stable in a dark place After 5 minutes, measure the fluorescence emission spectra of the homogeneous mixture at different concentrations of _baclidin (as shown in Figure 2(a)), and fit the linearity between the ratio of fluorescence emission peak intensity I BPQDs /I _{AgNCs and baclidin concentration} The relationship (as shown in Figure 2(b)), a ratio fluorescent probe was constructed for the quantitative detection of bacein. Among them, the concentrations of AgNCs/BPQDs/MOF complex, catalase and ^{bacitin are 2 mg mL -1} , 7 mU L ^-1 and 0.1-100 μg mL ^-1 , respectively. The linear detection range of bacitin concentration is 0.1-100 μg mL ^-1 , the detection limit is 5ng mL ^-1 .

Example 3: This example relates to the preparation process of the ratio fluorescent bacitin probe based on the silver nanocluster/black phosphorus quantum dot double-doped metal-organic framework complex and the schematic diagram of the principle of ratio fluorescent detection of bacein, BPQDs and The process steps for preparing AgNCs are the same as in Example 1, wherein the average size of BPQDs is 5 nm, and the average size of AgNCs is 15 nm. Other specific process steps are as follows:

Preparation of AgNCs/BPQDs/MOF complex: Weigh 2 mg of BPQDs and add 10 mL ^{of 2-methylimidazole ethanol solution with a concentration of 4 g L -1} , stir for 10 min to form a mixed solution, and add 10 mL of 5 g L ^-1 of hexahydrate nitric acid The zinc aqueous solution was stirred for 30 min to prepare a brown precipitate, which was washed three times with ethanol and distilled water, and centrifuged at 3500 rpm for 15 min to prepare an aqueous dispersion of the BPQDs/MOF complex. ^{Add 5mL of aqueous AgNCs dispersion with a concentration of 8mgL -1} dropwise to this dispersion, stir and react for 2h. The product solution is centrifuged to obtain a precipitate. The precipitate is washed and dried to prepare AgNCs/BPQDs/MOF complex. It should be stored under protection from light, nitrogen and 4°C for later use.

Add catalase to the AgNCs/BPQDs/MOF complex aqueous dispersion, stir well, adjust the pH to 7.4 with phosphate water buffer, then add bacitrin, stir evenly to form a homogeneous mixture, which is stable in a dark place After 5 minutes, measure the fluorescence emission spectra of the homogeneous mixture at different concentrations of _baclidin (as shown in Figure 2(a)), and fit the linearity between the ratio of fluorescence emission peak intensity I BPQDs /I _{AgNCs and baclidin concentration} The relationship (as shown in Figure 2(b)), a ratio fluorescent probe was constructed for the quantitative detection of bacein. Among them, the concentrations of AgNCs/BPQDs/MOF complex, catalase and ^{bacitin are 5 mg mL -1} , 10 mU L ^-1 and 0.01-50 μg mL ^-1 , respectively. The linear detection range of bacitin concentration is 0.01-50 μg mL ^-1 , the detection limit is 2ng mL ^-1 .

Claims

A method for preparing a ratio fluorescent bacitin probe based on a silver nanocluster/black phosphorous quantum dot double-doped metal-organic framework composite, which is characterized in that the method specifically includes the following steps:

(1) Preparation of black phosphorous quantum dots BPQDs: Weigh 10 mg of black phosphorous crystals and add them to 30 mL of nitromethylpyrrolidone, sonicate for 30 minutes to form a dispersion, transfer the dispersion to a micro high pressure reactor, and heat it to The reaction mixture was continuously stirred at 140°C for 12 hours. The reaction mixture was centrifuged at 3500 rpm for 15 minutes to remove larger-size products, and then centrifuged at 13000 rpm for 15 minutes to obtain a precipitate. The precipitate was washed three times with ethanol and distilled water, and then dried under vacuum. Obtain BPQDs and store them in the dark and nitrogen for later use. The average size of BPQDs is 1～5nm;

(2) Preparation of silver nanocluster AgNCs: Weigh 40 mg of lipoic acid powder into 20 mL of distilled water, stir well, add 0.1 mL of freshly prepared sodium borohydride with a concentration of 2 mol L -1 , and stir quickly for 30 minutes to form a homogeneous mixture. Under rapid stirring, 0.4 mL of silver nitrate with a concentration of 0.05 mol L -1 was added to the mixed solution, and then 0.3 mL of sodium borohydride with a concentration of 2 mol L -1 was added dropwise, and the reaction was maintained for 90 min with rapid stirring to obtain the product AgNCs. Dispersion solution, stored in the dark and 4℃ for later use, the average size of AgNCs is 10-20nm;

(3) Preparation of silver nanoclusters/black phosphorus quantum dots/metal organic framework AgNCs/BPQDs/MOF composite: Weigh 2mg BPQDs into 10mL 2-methylimidazole ethanol solution, stir for 10min to form a mixed solution, add 10mL six Aqueous zinc nitrate solution was hydrated and stirred for 30 minutes to obtain a brown precipitate, washed three times with ethanol and distilled water, centrifuged at 3500 rpm for 15 minutes to prepare an aqueous dispersion of BPQDs/MOF complex, and 5mL AgNCs dispersion was added dropwise to this dispersion After stirring for 2h, the product solution was centrifuged to obtain a precipitate. The precipitate was washed and dried to prepare AgNCs/BPQDs/MOF complex, which was stored in the dark, protected by nitrogen and at 4℃ for later use, among which 2-methyl The mass concentrations of imidazole, zinc nitrate hexahydrate and AgNCs are respectively 1～5g L -1 , 1～10g L -1 and 1～10mg L -1 ;

(4) Add catalase to the aqueous dispersion of AgNCs/BPQDs/MOF complex, stir evenly, adjust the pH to 7.4 with phosphate buffer, then add bacitrin and stir to form a homogeneous mixture. After the light is stable for 5 minutes, measure the fluorescence emission spectra of the homogeneous mixture at different concentrations of baclidin , and fit the linear relationship between the fluorescence emission peak intensity ratio I BPQDs /I AgNCs and the concentration of baclidin to construct a ratio fluorescent probe , Used for the quantitative detection of baclidin, in which the concentrations of AgNCs/BPQDs/MOF complex, catalase and baclidin are 1-10 mg mL -1 , 5-10 mU L -1 and 0.01-100 μg mL -respectively 1. The linear detection range of bacitin concentration is 0.01-100μg mL -1 , and the detection limit is 1-10ng mL -1 .