阅读说明：本技术 基于银-金属有机骨架材料作为荧光探针应用于叶酸检测的方法 (Method for applying silver-metal organic framework material as fluorescent probe to folic acid detection ) 是由 李妍 杨斌 李欣書 王璐 丁斌 于 2019-10-08 设计创作，主要内容包括：本发明涉及一种基于银-金属有机骨架材料作为荧光探针应用于叶酸检测的方法,该材料具有很好的水稳定性和卓越的荧光性能,能够高选择性、高灵敏性地检测叶酸,并且成功地应用于实际样品的检测。与传统的检测方法相比,这种基于MOFs的荧光探针具有灵敏度高、响应时间短、可操作性强等优点。本发明中该方法检测叶酸的传感机制主要归因于内滤效应,因为叶酸的紫外吸收光谱和银-金属有机骨架材料的荧光发射光谱两者之间存在光谱重叠,叶酸可以猝灭其荧光。本发明公开了利用银-金属有机骨架材料作为荧光探针应用于叶酸检测的详细分析工作,且具有选择性好和灵敏度高的特点。(The invention relates to a method for applying a silver-metal organic framework material as a fluorescent probe to folic acid detection, wherein the material has good water stability and excellent fluorescence property, can detect folic acid with high selectivity and high sensitivity, and is successfully applied to the detection of actual samples. Compared with the traditional detection method, the fluorescent probe based on the MOFs has the advantages of high sensitivity, short response time, strong operability and the like. The sensing mechanism for detecting folic acid by the method is mainly attributed to the internal filtering effect, and folic acid can quench the fluorescence of folic acid because the ultraviolet absorption spectrum of folic acid and the fluorescence emission spectrum of the silver-metal organic framework material have spectrum overlap. The invention discloses a detailed analysis work of folic acid detection by using a silver-metal organic framework material as a fluorescent probe, and has the characteristics of good selectivity and high sensitivity.)

1. the method for applying the silver-metal organic framework material as the fluorescent probe to folic acid detection is characterized by comprising the following steps of:

1) preparation of standard stock solution:

（a）100 mg L^-1standard solution of silver-metal organic framework material: weighing 0.0030 g of silver-metal organic framework material, dispersing the silver-metal organic framework material in 30 mL of deionized water, ultrasonically dispersing for 5 min, and storing in the shade for later use;

(b) standard 8 mM folic acid solution: weighing 0.1059 g folic acid (Mr: 441.4) and dissolving in 30 mL deionized water, shaking to dissolve completely, and storing in the shade for use;

(c) Tris-HCl standard buffer solution (pH 7.0, 100 mM): weighing 0.7780 g of trihydroxyaminomethane-hydrochloric acid (Tris-HCl, Mr = 157.6) and dissolving in 50 mL of deionized water, shaking to completely dissolve, and adjusting to pH7.0 by using a pH meter;

2) folate detection

(a) 400 muL of silver-metal organic framework material suspension (100 mg L) was sequentially added to the centrifuge tube, respectively^-1) 400 muL of Tris-HCl standard buffer solution (pH 7.0, 100 mM), diluting to 4 mL with deionized water, and standing for 10 min;

then, measuring and recording the fluorescence emission spectrum of the system solution and the fluorescence emission intensity at the emission wavelength of 350 nm by using a fluorescence spectrophotometer;

(b) 400 muL of silver-metal organic framework material suspension (100 mg L) was sequentially added to the centrifuge tube, respectively^-1) 400 muL of Tris-HCl standard buffer solution (pH 7.0, 100 mM), 200 muL of a series of folic acid standard solutions with different concentrations (2-400 muM), finally respectively diluting to 4 mL with deionized water, uniformly mixing, and standing for reaction for 10 min;

then, measuring and recording the fluorescence emission spectrum of the system solution and the fluorescence emission intensity at the emission wavelength of 350 nm by using a fluorescence spectrophotometer;

(c) according to the change value (I-I) between two recorded fluorescence emission intensities₀) And its corresponding folate concentration (C: mu M) can be fitted to obtain a linear equation, and the leaves in the unknown solution or the actual sample can be calculated through the change value of the fluorescence emission intensity in the system solution and the linear equationThe content of acid;

wherein the silver-metal organic framework material described above refers to: { [ Ag ]₂(abtz)₂(ClO₄)] . (ClO₄) }; abtz means: 1- (4-aminobenzyl) -1, 2, 4-benzotriazole organic ligand.

2. The method for detecting folic acid according to claim 1, characterized in that the fitted linear equation is I-I₀Linear range of 0.1-20 mu M, lowest detection limit of 68.1 nM, R of 15.64C-0.32²The value was 0.992.

3. The method for detecting folic acid according to claim 1, which is characterized in that folic acid can be sensitively detected by using a silver-metal organic framework material and can be applied to the analysis and detection of folic acid content in human serum and plasma actual samples.

4. The method for detecting folic acid of claim 1, which has good selectivity and anti-interference capability.

Technical Field

The invention belongs to the field of preparation of metal organic framework materials and fluorescence detection, and particularly relates to a method for detecting folic acid with high sensitivity by taking a silver-metal organic framework material with high water stability as a fluorescence probe.

Background

Metal Organic Frameworks (MOFs) are a new class of porous materials with high crystallinity, also known as Porous Coordination Polymers (PCPs), and have potential application prospects, so that they have become an important research direction in material chemistry at present. The essence of the MOFs material is that a matrix framework with expansibility is assembled by metal ions (ion clusters) and organic ligands through strong coordination bonds. At present, MOFs have the advantages of flexible binding sites, large specific surface area, large pore volume, adjustable pore diameter and the like, and play an important role in the field of multifunctional materials. In recent years, due to its open channel structure, controllable aperture and abundant flexible sites, the luminescent MOFs material has attracted great interest and attention in the field of analytical sensing applications.

Folic acid is a water-soluble compound of vitamin B group, and plays an important role in the normal operation of various physiological functions of human body. Folic acid can promote the production and maintenance of new cells during their division and growth. In addition, it facilitates the synthesis of thymine and purine (essential components of DNA and RNA), prevents the alteration of DNA, and thus prevents cancer. It is also a key component of the hematopoietic system and is believed to be a coenzyme that controls heme synthesis and production. Thus, abnormal levels of folate in biological fluids can affect normal physiological processes, causing several related diseases. For example, folate deficiency can lead to megaloblastic anemia, leukopenia, cardiovascular disease, fetal neural tube defects, and some psychiatric disorders. At present, the traditional methods for detecting folic acid include high performance liquid chromatography, colorimetric method, electrochemical method, ultraviolet absorption spectrometry, capillary electrophoresis and the like. These methods still have some disadvantages and limitations, such as time-consuming detection, complicated modification process, low sensitivity and expensive instrument. Therefore, the development of a reliable and effective analysis method applied to quantitative detection of folic acid has important significance for diagnosis and prevention of related physiological diseases.

As a functional porous coordination polymer, the metal organic framework material has excellent optical properties and good chemical stability, and has been applied to sensing applications of a series of analytes or conditions such as metal ions, biological small molecules, nitro explosives, biomarkers, pH, and temperature. According to the invention, the silver-metal organic framework material is successfully prepared by a hydrothermal method and is used as a fluorescent probe, and the work of applying the silver-metal organic framework material to the fluorescence analysis and detection of folic acid is not reported at present.

The invention relates to a method for applying a silver-metal organic framework material to folic acid analysis and detection, and folic acid can effectively quench fluorescence of the silver-metal organic framework material due to spectral overlap (internal filtering effect) between an ultraviolet absorption spectrum of folic acid and a fluorescence emission spectrum of the silver-metal organic framework material. According to the change value (I-I) of the fluorescence emission intensity₀) The folic acid concentration corresponding thereto (C: mu M) can be fitted to obtain a good linear relation, so that the aim of rapid and sensitive detection can be fulfilled.

Wherein the silver-metal organic framework material described above refers to: { [ Ag ]₂(abtz)₂(ClO₄)] . (ClO₄) }; abtz means: 1- (4-aminobenzyl) -1, 2, 4-benzotriazole organic ligand.

Disclosure of Invention

The invention relates to a method for detecting folic acid content in biological fluid based on a silver-metal organic framework material with high water stability as a fluorescent probe, and the folic acid can effectively quench fluorescence emission of the silver-metal organic framework material due to spectral overlap (internal filtering effect) between an ultraviolet absorption spectrum of the folic acid and a fluorescence emission spectrum of the silver-metal organic framework material, and can realize analysis and detection of folic acid selectivity and sensitivity according to a change value of fluorescence intensity of a system solution.

In order to achieve the aim, the invention discloses a method for selectively detecting folic acid by taking a silver-metal organic framework material with high water stability as a fluorescent probe, which is characterized by comprising the following steps of:

1) preparation of Standard stock solutions

（a）100 mg L^-1Standard solution of silver-metal organic framework material: weighing silver-metal with mass of 0.0030 gDispersing the organic framework material in 30 mL of deionized water, ultrasonically dispersing for 5 min, and storing in the shade for later use;

(b) standard 8 mM folic acid solution: weighing 0.1059 g folic acid (Mr: 441.4) and dissolving in 30 mL deionized water, shaking to dissolve completely, and storing in the shade for use;

(c) Tris-HCl standard buffer solution (pH 7.0, 100 mM): weighing 0.7780 g of trihydroxyaminomethane-hydrochloric acid (Tris-HCl, Mr = 157.6) in 50 mL of deionized water, shaking the solution to completely dissolve the trihydroxyaminomethane-hydrochloric acid, and adjusting the pH value to 7.0 by using a pH meter;

2) folate detection

(a) 400 muL of silver-metal organic framework material suspension (100 mg L) was sequentially added to the centrifuge tube, respectively^-1) 400 muL of Tris-HCl standard buffer solution (pH 7.0, 100 mM) is added with deionized water to be constant volume to 4 mL, and the mixture is kept stand for 10 min. Then, measuring and recording the fluorescence emission spectrum of the system solution and the fluorescence emission intensity at the emission wavelength of 350 nm by using a fluorescence spectrophotometer;

(b) 400 muL of silver-metal organic framework material suspension (100 mg L) was sequentially added to the centrifuge tube, respectively^-1) 400 mu Tris-HClL standard buffer solution (pH 7.0, 100 mM), 200 mu L folic acid standard solution with different concentrations (2-400 mu M), finally respectively diluting to 4 mL with deionized water, uniformly mixing, and standing for reaction for 10 min. Then, measuring and recording the fluorescence emission spectrum of the system solution and the fluorescence emission intensity at the emission wavelength of 350 nm by using a fluorescence spectrophotometer;

(c) according to the change value (I-I) between two recorded fluorescence emission intensities₀) And its corresponding folate concentration (C: mu M) can be fitted to obtain a linear equation, and the content of folic acid in the unknown solution or the actual sample can be calculated through the change value of the fluorescence emission intensity in the system solution and the linear equation;

the silver-metal organic framework material in the invention refers to: { [ Ag ]₂(abtz)₂(ClO₄)] . (ClO₄) }; abtz means: 1- (4-aminobenzyl) -1, 2, 4-benzotriazole organic ligands。

The method for detecting folic acid is characterized in that a fitted linear equation is I-I₀Linear range of 0.1-20 mu M, lowest detection limit of 68.1 nM, R of 15.64C-0.32²The value was 0.992.

The invention utilizes silver-metal organic framework material to detect folic acid with high sensitivity, and can be applied to analysis detection and labeled recovery of folic acid content in human serum and plasma actual samples.

The invention further discloses a test with high sensitivity for detecting the folic acid by the method, and the result shows that the folic acid detected by the method has higher selectivity and anti-interference capability, the method has lower detection limit, and the method is successfully applied to the analysis and detection of human serum and plasma actual samples, and obtains satisfactory standard-adding recovery rate.

The invention is described in more detail below:

the invention discloses a method for selectively detecting folic acid by taking a silver-metal organic framework material with high water stability as a fluorescent probe, which is characterized by comprising the following steps of:

(1) preparation of Standard stock solutions

100 mg L^-1Standard solution of silver-metal organic framework material: weighing 0.0030 g of silver-metal organic framework material, dispersing the silver-metal organic framework material in 30 mL of deionized water, ultrasonically dispersing for 5 min, and storing in the shade for later use;

standard 8 mM folic acid solution: weighing 0.1059 g folic acid (Mr: 441.4) and dissolving in 30 mL deionized water, shaking to dissolve completely, diluting step by step to obtain a series of folic acid standard solutions with different concentrations (2-400 μ M), and storing in the dark for use;

Tris-HCl standard buffer solution (pH 7.0, 100 mM): 0.7780 g of trihydroxyaminomethane-hydrochloric acid (Tris-HCl, Mr = 157.6) was weighed out and dissolved completely in 50 mL of deionized water, followed by adjustment to pH7.0 with a pH meter.

(2) Sequentially and respectively adding 400 mu L of silver-metal organic framework material suspension into the centrifuge tube100 mg L^-1) And adding 400 mu L of Tris-HCl standard buffer solution (pH 7.0 and 100 mM), diluting to 4 mL with deionized water, uniformly mixing, and standing for 10 min. The fluorescence emission spectrum of the system solution and the fluorescence emission intensity at an emission wavelength of 350 nm were then measured and recorded with a spectrofluorometer.

(3) 400 muL of silver-metal organic framework material suspension (100 mg L) was sequentially added to the centrifuge tube, respectively^-1) 400 muL of Tris-HCl standard buffer solution (pH 7.0, 100 mM), 200 muL of a series of folic acid standard solutions with different concentrations (2-400 muM), finally, deionized water is respectively used for constant volume to 4 mL, and the solutions are mixed uniformly and are kept still for reaction for 10 min. The fluorescence emission spectrum of the system solution and the fluorescence emission intensity at an emission wavelength of 350 nm were measured and recorded using a spectrofluorometer.

(4) The change in fluorescence emission intensity (I-I) was recorded by two times₀) And its corresponding folate concentration (C: mu M) can be fitted to obtain a linear equation, wherein the fitted linear equation is I-I₀Linear range of 0.10-20 mu M, lowest detection limit of 68.1 nM, R of 15.64C-0.32²The value was 0.992. Therefore, the change value (I-I) can be determined according to the fluorescence intensity in the system solution₀) And the fitted linear equation can calculate the folic acid content in the unknown solution or actual sample (C: μ M).

(5) In order to verify the sensitivity of the method for detecting folic acid by using the silver-metal organic framework material as the fluorescent probe, the selectivity and the anti-interference capability are tested. The result shows that the method has good selectivity and strong anti-interference capability, and can detect the folic acid with high sensitivity.

(6) The method is also applied to the analysis and detection of the folic acid content in the actual human serum and plasma samples, and the satisfactory standard-adding recovery rate is obtained, which indicates that the method has higher practicability.

The silver-metal organic framework material in the invention refers to: { [ Ag ]₂(abtz)₂(ClO₄)] . (ClO₄) }; abtz means: 1- (4-aminobenzyl) -1, 2, 4-benzotriazole organic ligand.

The method for detecting folic acid by adopting the silver-metal organic framework material as the fluorescent probe has the positive effects that:

(1) the silver-metal organic framework material with high water stability is used as the fluorescent probe, so that the complex modification/functionalization process can be avoided, the silver-metal organic framework material can be directly used as the probe to detect the folic acid in the biological fluid, and the operation is quick and simple.

(2) The fluorescence analysis method for detecting folic acid based on the silver-metal organic framework material disclosed by the invention has the advantages of wide detection linear range (0.10-20 mu M), low detection limit (68.1 nM), good selectivity, high sensitivity, strong anti-interference capability and the like.

(3) The analysis method disclosed by the invention can be well applied to the analysis and detection of human serum and plasma actual samples, and has higher practicability.

The analytical method disclosed in the present invention is also compared with different methods for detecting folic acid, and the advantages are clearly shown in the following table 1:

drawings

FIG. 1 is a powder diffraction XRD pattern for a silver-metal organic framework material used in the present invention;

FIG. 2 is an XRD pattern of a silver-metal organic framework material under acidic and basic conditions;

FIG. 3 is a graph of fluorescence excitation spectrum (curve 1) and a fluorescence emission spectrum (curve 2) using a silver-metal organic framework material in the present invention;

FIG. 4 is a graph of the ultraviolet absorption spectrum (curve 1) of folic acid and the fluorescence emission spectrum (curve 2) of a silver-metal organic framework material according to the present invention;

FIG. 5 is a graph showing the fluorescence intensity variation curve (curve 1) of the silver-metal organic framework material at different concentrations and the fluorescence quenching efficiency curve (curve 2) of the silver-metal organic framework material at different concentrations in the present invention;

FIG. 6 is a graph showing the effect of different pH values on fluorescence of a silver-metal organic framework material (curve 1) and the effect of different pH values on fluorescence quenching of a mixed system of a silver-metal organic framework material and folic acid (curve 2);

FIG. 7 is a graph of the quenching effect of folic acid on the fluorescence of a silver-metal organic framework material over time;

FIG. 8 is a graph of fluorescence quenching spectra of different concentrations of folic acid on silver-metal organic framework materials;

FIG. 9 is a graph of a linear fit of a silver-metal organic framework material used in the present invention to detect folic acid;

FIG. 10 is a bar graph of the selective recognition of the application of silver-metal organic framework material in folic acid detection in the present invention.

FIG. 11 is a bar graph of the anti-interference ability of the silver-metal organic framework material applied to folic acid detection in the present invention.

Detailed Description

The invention is described in detail by the following figures and specific embodiments. The technical means used in the present invention are all known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

AgClO reagent used in the following examples₄Diethyl ether and methanol were purchased from the institute for optochemical and polishing industries, Tianjin. 1- (4-aminobenzyl) -1, 2, 4-benzotriazole organic ligands were purchased from Sigma Aldrich trade, Inc. Tris-HCl (Tris-HCl) was purchased from Biotechnology Inc., Beijing ancient China. Folic acid was purchased from Oncoka technologies, Inc. of Beijing. Citric acid, dopamine, glucose, fructose and urea were all purchased from Guang Compound technology development, Inc., Tianjin. The desired amino acid (glutamic acid, phenylalanine, methionine, lysine, histidine, serine)) Are purchased from Guangdong chemical research institute, Tianjin and are BR biochemical reagents with the purity of 99 percent.