A ratio fluorescence analysis method for artemisinin based on Zn-MOF in situ color development

Abstract

The present invention belongs to the field of fluorescence analysis technology, and specifically relates to an artemisinin ratio fluorescence analysis method based on Zn-MOF in-situ color development. The present invention uses Zn-MOF as a fluorescence reference, heme as a catalyst, and adds an o-phenylenediamine solution to construct an artemisinin ratio fluorescence sensing platform. The ratio fluorescence analysis method provided by the present invention is low in cost, simple to operate, has good selectivity, and high sensitivity. It can be used for the quantitative detection of artemisinin in biological samples, and at the same time provides a fast, simple, and accurate technical means for monitoring the clinical efficacy of artemisinin drugs and drug development.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescence analysis, and specifically relates to an artemisinin ratio fluorescence analysis method based on Zn-MOF in-situ color development. Background Art

[0002] Artemisinin is a sesquiterpene lactone compound with an endoperoxide bridge. It is a new antimalarial drug isolated from Artemisia annua with the characteristics of high efficiency and low toxicity. Combination therapy based on artemisinin treatment has become the best method for treating drug-resistant Plasmodium falciparum worldwide. At the same time, artemisinin also has certain therapeutic effects on hepatitis B, human leukemia, anti-tumor, antibacterial, and insecticide. However, excessive active ingredients in artemisinin drugs can cause side effects such as dizziness, nausea, anorexia, and vomiting. Therefore, designing a simple, highly selective, and sensitive artemisinin detection method is of great significance for monitoring clinical drug efficacy and drug development.

[0003] At present, methods for detecting artemisinin include chromatography, mass spectrometry, enzyme-linked immunosorbent assay, electrochemical method, chemiluminescence method, etc. Although most of these technologies have high sensitivity, some of them require expensive instruments and are complicated to operate, and some have poor stability and narrow detection range. Liu et al. (Analytica Chimica Acta, 2007, 581: 298-302) disclosed a high-performance liquid chromatography-evaporative light scattering method for detecting the content of artemisinin in Artemisia annua. This method first atomizes the column effluent to form an aerosol, then evaporates the solvent in a heated drift tube, and then detects the remaining non-volatile solute particles in a light scattering unit, but this method requires large-scale precision instruments and is relatively expensive. Spectroscopy has attracted widespread attention due to its high sensitivity, simple operation, and short response time. Since artemisinin has no special spectral characteristics, it is difficult to detect it directly by colorimetry and fluorescence methods, and it is usually necessary to introduce a luminescent probe for indirect detection. Chinese patent (CN 105784620 A) discloses an analytical method for colorimetric detection of artemisinin. This method uses single-stranded DNA stabilized nanogold colloids as probes. Ferrous ions can catalyze the reaction of artemisinin to produce free radicals that damage single-stranded DNA, causing changes in the color and absorbance of the solution, thereby constructing a sensing platform for artemisinin. This method has high sensitivity and is easy to operate, but the detection limit is high, the detection range is narrow, and DNA is expensive. Chinese patent (CN 108844933 A) discloses an analytical method for fluorescence detection of artemisinin. This method uses graphene quantum dots as fluorescent probes. In the presence of oxygen, the peroxide bridge structure of artemisinin catalyzes the oxidation of p-aminophenylboronic acid to generate p-iminobenzoquinone, and quenches the fluorescence of graphene quantum dots to construct a sensing platform for artemisinin. While having high sensitivity, the operation steps are greatly simplified, but this method is based on a single signal output and is easily interfered by the operating instrument and the external environment, thereby affecting the detection accuracy.

[0004] In recent years, metal organic frameworks (MOFs) have become one of the most popular new nanomaterials. Due to their unique properties such as adjustable topology, diverse structures, and large specific surface area, MOFs have broad application prospects in catalysis, drug delivery, bioimaging, and sensing. Among them, Zn-MOF has more coordination numbers, higher structural stability, and better fluorescence properties than MOFs composed of other metal ions. Due to these unique properties, a series of fluorescence analysis methods based on luminescent Zn-MOF have been designed. The design of ratio fluorescence analysis using luminescent Zn-MOF and in situ color reaction is still in its infancy, and research on its application in artemisinin detection has not been reported. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems of complex operation, high cost, single signal detection, etc. in the current artemisinin detection process, and provide an artemisinin ratio fluorescence analysis method based on Zn-MOF in situ color development. The method utilizes the unique luminescence properties of Zn-MOF and the catalytic cleavage effect of hemoglobin on the peroxide bridge structure of artemisinin to construct a simple and sensitive ratio fluorescence sensing platform, thereby realizing the qualitative and quantitative detection of artemisinin.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0007] The present invention provides an artemisinin ratio fluorescence analysis method based on Zn-MOF in situ color development, comprising the following steps:

[0008] (1) Preparation of Zn-MOF: Weigh terephthalic acid and 2-aminoterephthalic acid and dissolve them in N,N-dimethylformamide and mix them evenly. Pour the zinc acetate dihydrate solution dissolved in N,N-dimethylformamide into the solution under magnetic stirring. After stirring at room temperature for a period of time, collect the solid product by centrifugation and wash it alternately with water and ethanol. Put the purified Zn-MOF in an oven to dry to obtain a dry solid powder, which is then dissolved in deionized water to prepare a Zn-MOF aqueous solution.

[0009] (2) Establishment of a standard curve: add a series of artemisinin standard solutions of different concentrations to a centrifuge tube containing a buffer solution, and then add the heme solution, the o-phenylenediamine solution and the Zn-MOF aqueous solution prepared in step (1) in sequence, mix well, and react at room temperature in the dark for a period of time; draw a standard curve;

[0010] (3) Determination of artemisinin in samples: Replace the artemisinin standard solution with the sample to be tested, add it by the standard addition method, shake it to mix it evenly, and follow step (2) to convert the measured F 570 / F 443 Substituting the standard curve obtained in step (2) into the standard curve, the concentration of artemisinin in the sample to be tested is obtained.

[0011] Furthermore, in step (1), the mass ratio of terephthalic acid to 2-aminoterephthalic acid is 2:1 to 10:1; the concentration of terephthalic acid in N,N-dimethylformamide is 1 to 10 mg / mL; the concentration of zinc acetate dihydrate solution is 0.01 to 1 g / mL; the mass ratio of 2-aminoterephthalic acid to zinc acetate dihydrate is 1:100 to 10:100; and the concentration of the Zn-MOF aqueous solution is 1 mg / mL.

[0012] Furthermore, in step (1), the stirring time is 0.5 to 3 h.

[0013] Furthermore, in step (2), the buffer solution is an acetic acid-sodium acetate buffer solution with a pH of 4.3 to 7.8; and the concentration of the artemisinin standard solution is 0 to 150 µM.

[0014] Furthermore, in step (2), the concentration of heme is 1 to 15 µM; the concentration of o-phenylenediamine is 1 to 8 mM.

[0015] Furthermore, in step (2), the volume ratio of the artemisinin standard solution to the heme solution is 2:1 to 2:15; and the volume ratio of the heme solution, o-phenylenediamine solution and Zn-MOF solution is 1:1:1 to 15:8:10.

[0016] Furthermore, in step (2), the light-proof reaction time is 5 to 80 minutes.

[0017] Furthermore, in step (2), the specific process of drawing the standard curve is: under 390 nm excitation, recording the fluorescence emission spectrum in the range of 410 to 700 nm, and calculating the fluorescence intensity ratio F 570 / F 443 , with the logarithm of artemisinin concentration as the horizontal axis, F 570 / F 443 Plot a standard curve for the ordinate.

[0018] Furthermore, the detection range of artemisinin is 0.15 to 150 µM.

[0019] In the detection process of the present invention, the processing method of the sample to be tested is as follows: taking the compound naphtholquinoline phosphate tablet as an example, one compound naphtholquinoline phosphate tablet is ground into powder using an agate mortar, accurately weighed, and dissolved with ethanol. The obtained solution is ultrasonically dispersed, and then a clear filtrate is collected as the processed sample to be tested.

[0020] This invention uses Zn-MOF as a fluorescence reference and heme as a catalyst for the first time, and designs a new ratio fluorescence analysis method for the detection of artemisinin. The 2-aminoterephthalic acid ligand makes Zn-MOF have fixed fluorescence at 443 nm. After adding heme and o-phenylenediamine solution, the system shows a weak fluorescence signal at 570 nm. When artemisinin is present, heme can catalyze the breakage of the peroxide bridge of artemisinin and release a large amount of reactive oxygen, effectively oxidizing o-phenylenediamine to generate 2,3-diaminophenazine with orange-yellow fluorescence. Therefore, the fluorescence signal at 570 nm is significantly enhanced, while the fluorescence signal of Zn-MOF remains basically unchanged. The ratio of the fluorescence intensities of the two is used as the output signal, thereby constructing a ratio fluorescence sensing platform for artemisinin. The linear detection range of the sensing platform for artemisinin is 0.15 µM~150 µM, and the detection limit is as low as 50 nM, realizing the trace detection of artemisinin.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) Compared with single-signal fluorescent probes, the present invention is the first to use ratiometric fluorescent probes to detect artemisinin, which has good selectivity, high accuracy, and good reproducibility;

[0023] (2) The ratiometric fluorescence analysis method for detecting artemisinin using a luminescent metal organic framework and an in situ color development reaction as a dual emission probe provided by the present invention is simple to prepare, low in cost, and highly sensitive, and can be used for the quantitative detection of artemisinin in biological samples, with broad application prospects;

[0024] (3) The present invention applies the luminescent metal organic framework to the field of fluorescent drug analysis, and has great application potential in artemisinin clinical drug efficacy monitoring and drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the ratio fluorescence analysis method for detecting artemisinin of the present invention.

[0026] Figure 2 This is an electron scanning microscope characterization image of the Zn-MOF prepared in the ratio fluorescence analysis method for detecting artemisinin of the present invention.

[0027] Figure 3 It is an infrared spectrum diagram of the Zn-MOF prepared in the ratio fluorescence analysis method for detecting artemisinin of the present invention.

[0028] Figure 4 It is the XRD spectrum of the Zn-MOF prepared in the ratio fluorescence analysis method for detecting artemisinin of the present invention.

[0029] Figure 5It is the fluorescence emission spectrum of the Zn-MOF prepared in the ratio fluorescence analysis method for detecting artemisinin of the present invention at different excitation wavelengths.

[0030] Figure 6 It is a concentration curve diagram of the ratio fluorescence analysis method for detecting artemisinin of the present invention.

[0031] Figure 7 It is a standard curve diagram of the ratio fluorescence analysis method for detecting artemisinin of the present invention.

[0032] Figure 8 This is a graph showing the specificity of the detection results of the ratio fluorescence analysis method for detecting artemisinin according to the present invention. DETAILED DESCRIPTION

[0033] The present invention is specifically described below. It should be understood that the specific examples described herein are only used to explain the present invention and are not intended to limit the scope of the present invention. In addition, after reading the present invention, those skilled in the art may make various changes and modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

[0034] Example 1

[0035] A ratio fluorescence analysis method for detecting artemisinin, specifically as follows Figure 1 As shown, the steps are as follows:

[0036] (1) Preparation of Zn-MOF: Weigh 270 mg of terephthalic acid and 30 mg of 2-aminoterephthalic acid and dissolve them in 40 mL of N,N-dimethylformamide and mix them evenly. Pour zinc acetate dihydrate (1 g) solution dissolved in 20 mL of N,N-dimethylformamide into the mixture under magnetic stirring. After stirring at room temperature for 2 h, centrifuge at 8000 rpm for 10 min to collect the solid product, and wash it alternately with water and ethanol for 3 times. Dry the purified Zn-MOF in an oven at 45 °C to obtain a dry solid powder, which is then dissolved in deionized water to prepare a 1 mg / mL Zn-MOF aqueous solution.

[0037] (2) Establishment of the standard curve: Add a series of 2.5 mM artemisinin standard solutions (final concentrations ranging from 0 to 150 µM) to a centrifuge tube containing 420 µL of acetic acid-sodium acetate buffer (pH = 5.3), then add 25 µL of heme solution (100 µM), 25 µL of o-phenylenediamine solution (0.1 M) and 20 µL of the Zn-MOF (1 mg / mL) aqueous solution prepared in step (1) in sequence, mix well and react at room temperature in the dark for 60 minutes; record the fluorescence emission spectrum in the range of 410 to 700 nm under 390 nm excitation, and calculate the fluorescence intensity ratio.F 570 / F 443 , with the logarithm of artemisinin concentration as the horizontal axis, F 570 / F 443 Plot a standard curve for the ordinate.

[0038] from Figure 2 The electron scanning microscopy characterization image shows that the morphology of the prepared Zn-MOF is a clear cubic structure with an average size of about 200 nm. The Zn-MOF was characterized by infrared and XRD (see Figure 3 , 4 ), the Zn-MOF surface contains NH, OCO, and CN bonds, and the XRD characteristic peaks are basically consistent with the simulated MOF-5.

[0039] Figure 5 The fluorescence emission spectra of Zn-MOF prepared in Example 1 at different excitation wavelengths are shown in the figure. As shown in the figure, when the excitation wavelength is in the range of 360 to 420 nm, the fluorescence intensity decreases with the increase of the excitation wavelength, which is consistent with the luminescent properties of its ligand 2-aminoterephthalic acid.

[0040] like Figures 6-7 , Figure 6 The figure shows the change in fluorescence intensity after adding different concentrations of artemisinin standard solution (final concentration ranges from 0 to 150 µM). As the concentration of artemisinin increases, the reactive oxygen released by artemisinin catalyzed by heme increases, thereby increasing the amount of 2,3-diaminophenazine generated by the oxidation of o-phenylenediamine, and the fluorescence intensity at 570 nm is greatly enhanced. At the same time, the fluorescence intensity of Zn-MOF at 443 nm remains almost unchanged. Figure 7 The fluorescence intensity ratio is shown F 570 / F 443 The logarithm of the concentration of artemisinin shows a good linear relationship in the range of 0.15 to 150 µM: F 570 / F 443 = 0.953 + 0.567 lg C 青蒿素 (R 2 = 0.998) with a detection limit of 50 nM.

[0041] (3) Determination of artemisinin in samples: Grind the sample to be tested into powder, weigh accurately, and dissolve it in ethanol. Disperse the obtained solution by ultrasonication, and then collect the clear filtrate as the processed sample to be tested. Replace the artemisinin standard solution with the sample to be tested, add it by the standard addition method, shake it to mix it evenly, and follow step (2) to add the measured F 570 / F 443 Substituting the standard curve obtained in step (2) into the standard curve, the concentration of artemisinin in the sample to be tested is obtained.

[0042] 1. Effect Embodiment

[0043] Detection of actual samples of compound naphtholquinoline phosphate tablets: One tablet of compound naphtholquinoline phosphate tablet was ground into powder using an agate mortar, accurately weighed to 0.2632 g, and dissolved in 5 mL of ethanol. The resulting solution was ultrasonically dispersed for 5 min, and then the clear filtrate was collected and a recovery experiment was performed by adding three different concentrations (10, 40 and 90 µM) of artemisinin solution according to the steps described in step (2). As can be seen from Table 1 (samples 1, 2, and 3), the recovery rate was in the range of 97.0-107.5%, and the relative standard deviation (n=3) was less than 3%, indicating that this method has good accuracy and precision in the determination of artemisinin.

[0044] Table 1 Recovery test results

[0045]

[0046] 2. Selective analysis and detection methods for artemisinin

[0047] To a centrifuge tube containing 420 µL of acetic acid-sodium acetate buffer (pH=5.3), 10 µL of 50 µM artemisinin, calcium ions, magnesium ions, potassium ions, zinc ions, cobalt ions, sodium ions, chloride ions, nitrate ions, sulfate ions, phosphate ions, starch, lactose, glucose, lysine, cysteine ​​and magnesium stearate were added, and then 25 µL of heme solution (100 µM), 25 µL of o-phenylenediamine solution (0.1 M) and 20 µL of Zn-MOF (1 mg / mL) solution were added in sequence. After mixing evenly, the mixture was reacted at room temperature in the dark for 60 minutes. Under 390 nm excitation, the fluorescence emission spectrum in the range of 410-700 nm was recorded, and the fluorescence intensity ratio was calculated. F 570 / F 443 .Depend on Figure 8 It can be seen that the fluorescence intensity ratio of the system after adding these substances F 570 / F443 There was no significant change, except after artemisinin was added F 570 / F 443 The detection method of the present invention has good selectivity and is not interfered by some common metal cations, anions and biological molecules.

[0048] Comparative Example 1

[0049] This comparative example uses high performance liquid chromatography-evaporative light scattering to detect artemisinin. The main working principle is to atomize the column effluent to form an aerosol, then evaporate the solvent in a heated drift tube, and then detect the remaining non-volatile solute particles in the light scattering unit. However, this method has a high detection limit, expensive instruments, and complex equipment.

[0050] Comparative Example 2

[0051] This comparative example uses an electrochemical method to detect artemisinin, selectively oxidizing p-aminophenylboronic acid with artemisinin to generate p-aminophenol that is easily detected by electrochemical means, and then detecting the generated p-aminophenol to detect the content of artemisinin. This method has good selectivity and a wide detection range, but a high detection limit and poor stability.

[0052] Comparative Example 3

[0053] The comparative example uses chemiluminescence to detect artemisinin, where artemisinin combines with heme and luminol in an alkaline solution to generate a chemiluminescence signal proportional to the concentration of artemisinin. However, this method has a narrow detection range and poor repeatability.

[0054] Comparative Example 4

[0055] This comparative example uses a fluorescence method to detect artemisinin, and its main feature is that graphene quantum dots are used as fluorescent probes. In the presence of oxygen, the peroxide bridge structure of artemisinin catalyzes the oxidation of p-aminophenylboronic acid to generate p-iminobenzoquinone, while quenching the fluorescence of graphene quantum dots. It has high sensitivity and is easy to operate. However, this method is based on a single signal output and is easily interfered by the operating instrument and the external environment, thereby affecting the detection accuracy.

[0056] Effect Example

[0057] The detection range and detection limit were determined by the method used in Example 1 and Comparative Examples 1-4. The specific results are shown in Table 2.

[0058] Table 2

[0059]

[0060] The results in Table 2 show that the present invention uses a luminescent metal organic framework and an in situ color development reaction to combine the ratio to detect artemisinin, which has the characteristics of high sensitivity, good selectivity, wide detection range, and high accuracy. Compared with Comparative Examples 1 and 2, the detection limit of the present invention is 1-3 orders of magnitude lower. Compared with Comparative Examples 3 and 4, the detection range of the present invention is wider and the results are more accurate. The present invention uses a luminescent metal organic framework and an in situ color development reaction to construct a dual-emission fluorescent probe, avoiding complex synthetic reactions, saving time and cost, and being low-toxic and pollution-free, with broader application prospects.

Claims

1. A method for ratio fluorescence analysis of artemisinin based on Zn-MOF in situ color development, characterized in that: The following steps are involved: (1) Preparation of Zn-MOF: Weigh terephthalic acid and 2-aminoterephthalic acid, dissolve in N,N-dimethylformamide and mix well, pour zinc acetate dihydrate solution dissolved in N,N-dimethylformamide into the mixture under magnetic stirring; stir at room temperature for 0.5-3h, collect the solid product by centrifugation, and wash it alternately with water and ethanol; dry the purified Zn-MOF in an oven to obtain a dry solid powder, which is then dissolved in deionized water to prepare a Zn-MOF aqueous solution; The mass ratio of terephthalic acid to 2-aminoterephthalic acid is 2:1 to 10:1; the concentration of terephthalic acid in N,N-dimethylformamide is 1 to 10 mg / mL; the concentration of zinc acetate dihydrate solution is 0.01 to 1 g / mL; the mass ratio of 2-aminoterephthalic acid to zinc acetate dihydrate is 1:100 to 10:100; the concentration of the Zn-MOF aqueous solution is 1 mg / mL; (2) Establishment of standard curve: add a series of artemisinin standard solutions of different concentrations to a centrifuge tube containing buffer, and then add heme solution, o-phenylenediamine solution and Zn-MOF aqueous solution prepared in step (1) in sequence, mix well and react at room temperature in the dark; draw the standard curve; The specific process of drawing the standard curve is as follows: under 390 nm excitation, record the fluorescence emission spectrum in the range of 410 to 700 nm, and calculate the fluorescence intensity ratio F 570 / F 443, with the logarithm of artemisinin concentration as the horizontal axis, F 570 / F 443 is the ordinate for drawing the standard curve; (3) Determination of artemisinin in samples: Replace the artemisinin standard solution with the sample to be tested, add it by the standard addition method, shake it to mix it evenly, and follow step (2) to convert the measured F 570 / F Substitute 443 into the standard curve obtained in step (2) to obtain the concentration of artemisinin in the sample to be tested.

2. The method for ratio fluorescence analysis of artemisinin according to claim 1, characterized in that: In step (2), the buffer solution is an acetic acid-sodium acetate buffer solution with a pH of 4.3 to 7.8; and the concentration of the artemisinin standard solution is 0 to 150 µM.

3. The method for ratio fluorescence analysis of artemisinin according to claim 1, characterized in that: In step (2), the concentration of heme is 1 to 15 µM; the concentration of o-phenylenediamine is 1 to 8 mM.

4. The method for ratio fluorescence analysis of artemisinin according to claim 1 or 3, characterized in that: In step (2), the volume ratio of the artemisinin standard solution to the heme solution is 2:1 to 2:15; the volume ratio of the heme solution, o-phenylenediamine solution and Zn-MOF solution is 1:1:1 to 15:8:

10.

5. The method for ratio fluorescence analysis of artemisinin according to claim 1, characterized in that: In step (2), the light-proof reaction time is 5 to 80 minutes.

6. The method for ratio fluorescence analysis of artemisinin according to claim 1, characterized in that: The detection range of the artemisinin is 0.15 to 150 µM.

Images