Fluorescent sensor for detecting hepatocellular carcinoma biomarker GCA and method and application thereof

Abstract

The present invention discloses a fluorescent sensor for detecting hepatocellular carcinoma biomarker GCA, and a method and application thereof. The fluorescent sensor is a metal organic gel material Eu-MOG. Weigh 0.0541-0.2162 mg 1,4-NDC and 0.1115-0.4461 mg Eu(NO3)3·6H2O, respectively dissolve them in DMF, add 150-300 μL triethylamine to the DMF solution of 1,4-NDC until all are dissolved, mix the DMF solution of 1,4-NDC and the DMF solution of Eu(NO3)3·6H2O, shake evenly and let stand for a period of time to obtain the metal organic gel Eu-MOG; first, the analyte can be detected in serum and the detection process is simple and the response time is fast. Secondly, compared with other methods reported in the literature, it does not require expensive equipment and has low cost. Finally, under the interference of other metal ions and amino acids, the detection effect is still not affected.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry, and in particular to a fluorescent sensor for detecting GCA, a biomarker of hepatocellular carcinoma, and a method and application thereof; a novel Eu-MOG material with stable fluorescent properties is specifically designed, which uses 1,4-NDC=1,4-naphthalenedicarboxylic acid as a ligand and Eu(NO3)3·6H2O as a metal node, and can be used for detecting a biomarker (GCA) of hepatocellular carcinoma (HCC) in serum with high selectivity, high sensitivity and high stability. Background Art

[0002] Glycocholic acid (GCA) plays an important role as a conjugated bile acid in reflecting hepatobiliary diseases. Many researchers have found that GCA is a promising biomarker for the diagnosis of liver diseases, especially hepatocellular carcinoma (HCC). For healthy people, the concentration of GCA in the blood is less than 3 μg / mL. In addition, there is a correlation between the level of glycocholic acid and the severity of liver disease. Therefore, routine determination of glycocholic acid is of great significance for the early diagnosis, monitoring and detection of HCC.

[0003] At present, some methods have been established for the determination of GCA in serum and urine, such as high performance liquid chromatography (HPLC); the limit of detection (LOD) is reported as 5.6 μg / mL, ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS); LOD = 0.5 ng / mL, liquid chromatography-electrospray tandem mass spectrometry (LC-MS-MS); LOD = 10 ng / mL, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS); LOD = 4.2 μg / mL. Despite the advantages of high sensitivity and selectivity, instrumental methods require extensive sample preparation and cleanup procedures, which become laborious and time-consuming, requiring advanced infrastructure to support complex instruments, as well as specific expertise and well-trained operators. In order to simplify and reduce the cost of the detection process, it is very necessary to develop a material with a simple preparation process, high response to GCA and low detection limit as soon as possible. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a fluorescent sensor for detecting hepatocellular carcinoma biomarker GCA and a method and application thereof in view of the deficiencies in the prior art.

[0005] The technical solution of the present invention is as follows:

[0006] A fluorescent sensor for detecting hepatocellular carcinoma biomarker GCA, wherein the fluorescent sensor is a metal organic gel material Eu-MOG.

[0007] The preparation method of the fluorescent sensor comprises the following steps: weighing 0.0541-0.2162 mg 1,4-NDC and 0.1115-0.4461 mg Eu(NO3)3·6H2O in the following proportions, dissolving them in DMF respectively, dropping 150-300 μL triethylamine into the DMF solution of 1,4-NDC until all the triethylamine is dissolved, mixing the DMF solution of 1,4-NDC and the DMF solution of Eu(NO3)3·6H2O, shaking them evenly and letting them stand for a period of time to obtain the metal organic gel Eu-MOG.

[0008] The fluorescent sensor material is prepared by the preparation method.

[0009] The fluorescent sensor material is used in detecting the hepatocellular carcinoma biomarker GCA.

[0010] The fluorescent sensor material is used in preparing a reagent for detecting a hepatocellular carcinoma biomarker, GCA.

[0011] The concentration of GCA is 10 -2 moL / L.

[0012] Testing for GCA:

[0013] In order to detect the specific recognition of Eu-MOG to GCA, 25 μL of Eu-MOG solution was added to serum, DL-methionine, L-phenylalanine, L-glutamic acid, L-glutamine, L-methionine, L-tyrosine, L-leucine, L-proline, L-tryptophan, L-serine, L-threonine, L-asparagine, L-valine, L-isoleucine, L-histidine, glycine, calcium chloride, potassium chloride, magnesium chloride, sodium chloride, zinc chloride, urea, glucose, lithocholic acid, deoxycholic acid, cholic acid, and glycocholic acid solutions, respectively. When Eu-MOG solution was added to the glycocholic acid solution, it was found that under an excitation wavelength of 330 nm, the fluorescence intensity at 420 nm was significantly enhanced, the fluorescence intensity at 618 nm was reduced, and the solution changed from red to blue, which indicates the ability of this material to specifically recognize GCA.

[0014] In order to explore the anti-interference properties of Eu-MOG to GCA, the fluorescence response of Eu-MOG material to GCA was tested when the above 27 solutions coexisted with GCA. It was found that the presence of other interferences did not affect the detection effect of the material on GCA.

[0015] To evaluate the sensitivity of Eu-MOG, a quantitative fluorescence titration experiment was performed. As the concentration of GCA increased, the fluorescence intensity of the solution at 420nm increased significantly, and the fluorescence intensity at 618nm gradually decreased. When the concentration of GCA increased from 0μL to 10μL, the fluorescence intensity of the solution at 420nm gradually increased. This shows that Eu-MOG can be highly sensitive to GCA as a fluorescent probe. Based on the standard deviation (σ) of three repeated measurements of the fluorescence intensity of the blank solution, the detection limit (3σ / Ksv) was calculated to be 110.9ppm.

[0016] In order to explore the time response of the sensor to the detected substance GCA, a fluorescence experiment of the response time was carried out. It was found that when the response time was 2 minutes, the solution had a significant color change from red to blue.

[0017] The present invention has the following advantages:

[0018] First, the analytes can be detected in serum with a simple detection process and a fast response time.

[0019] Secondly, compared with other methods reported in the literature, it does not require expensive equipment and is low in cost.

[0020] Finally, the detection effect is not affected by the interference of other metal ions and amino acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 :Synthesis of Eu-MOG sensor at room temperature.

[0022] Figure 2 : Fluorescence spectrum of Eu-MOG solution.

[0023] Figure 3 : Fluorescence sensing diagram of Eu-MOG solution to different detection objects.

[0024] Figure 4 : Fluorescence images when other metal ions, amino acids and GCA exist simultaneously.

[0025] Figure 5 : Fluorescence titration experiment diagram of Eu-MOG solution and GCA.

[0026] Figure 6 : Time response diagram of Eu-MOG solution to the detector GCA. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific examples. The present invention uses DL-methionine, L-phenylalanine, L-glutamic acid, L-glutamine, L-methionine, L-tyrosine, L-leucine, L-proline, L-tryptophan, L-serine, L-threonine, L-asparagine, L-valine, L-isoleucine, L-histidine, and glycine as amino acids, calcium chloride, potassium chloride, magnesium chloride, sodium chloride, and zinc chloride as metal ions, urea, and glucose, and uses lithocholic acid, deoxycholic acid, cholic acid, and glycocholic acid as bile acids to selectively detect glycocholic acid using the synthesized Eu-MOG solution.

[0028] Example 1

[0029] First, prepare the ligand solution: weigh 1,4-NDC (0.0541 mg) and dissolve it in DMF (1 ml), sonicate for 2 minutes, then drop triethylamine (150 μL) into the DMF solution of 1,4-NDC, and continue sonicating for 2 minutes.

[0030] Then prepare the metal solution: weigh Eu(NO3)3·6H2O (0.1115 mg) and dissolve it in DMF (1 ml), and dissolve it by ultrasonic for 2 minutes.

[0031] Finally, the ligand solution and the metal solution were mixed, shaken evenly and allowed to stand for two hours to obtain an invertible metal organic gel. Figure 1 This is a physical picture of Eu-MOG. Figure 2 This is the fluorescence spectrum of Eu-MOG solution.

[0032] Example 2

[0033] First, prepare the ligand solution: weigh 1,4-NDC (0.2162 mg) and dissolve it in DMF (2 ml), sonicate for 2 minutes, then drop triethylamine (300 μL) into the DMF solution of 1,4-NDC, and continue sonicating for 2 minutes.

[0034] Then prepare the metal solution: weigh Eu(NO3)3·6H2O (0.4461 mg) and dissolve it in DMF (2 ml), and dissolve it by ultrasonic for 2 minutes.

[0035] Finally, the ligand solution and the metal solution were mixed, shaken evenly and allowed to stand for two hours to obtain an invertible metal organic gel.

[0036] Example 3: Detection of GCA using Eu-MOG as a fluorescence sensor

[0037] Preparation of Eu-MOG solution: 100 mg Eu-MOG was dissolved in 5 mL serum.

[0038] 25 μL of Eu-MOG solution was added to 750 μL of 0.01 mol / L serum, DL-methionine, L-phenylalanine, L-glutamic acid, L-glutamine, L-methionine, L-tyrosine, L-leucine, L-proline, L-tryptophan, L-serine, L-threonine, L-asparagine, L-valine, L-isoleucine, L-histidine, glycine, calcium chloride, potassium chloride, magnesium chloride, sodium chloride, zinc chloride, urea, glucose, lithocholic acid, deoxycholic acid, cholic acid, and glycocholic acid solutions, respectively. Figure 3 It is shown that GCA can significantly enhance the fluorescence emission peak of Eu-MOG solution at 420nm.

[0039] To 25 μL Eu-MOG solution, 2 mL, 0.01 mol / L of DL-methionine, L-phenylalanine, L-glutamic acid, L-glutamine, L-methionine, L-tyrosine, L-leucine, L-proline, L-tryptophan, L-serine, L-threonine, L-asparagine, L-valine, L-isoleucine, L-histidine, glycine, calcium chloride, potassium chloride, magnesium chloride, sodium chloride, zinc chloride, urea, glucose, lithocholic acid, deoxycholic acid, and cholic acid were added, and then 1 mL, 2*10 -3 mol / L of GCA, so that other substances also exist when GCA exists. Figure 4 After adding the mixed substances, the fluorescence intensity of all solutions at 420nm increased significantly. The presence of surface interferents did not affect the detection effect of Eu-MOG on GCA.

[0040] In the Eu-MOG solution, when the concentration of GCA increased from 0 μL to 10 μL, the fluorescence intensity of the solution at 420 nm gradually increased. Figure 5 As the concentration of GCA increases, the fluorescence intensity of the Eu-MOG solution gradually increases, indicating that Eu-MOG can be highly sensitive to GCA as a fluorescent probe. Figure 5 Based on the data in , the detection limit (3σ / Ksv) was calculated to be 110.9 ppm based on the standard deviation (σ) of three repeated measurements of the fluorescence intensity of the blank solution.

[0041] The response time was tested when 100 μL of Eu-MOG solution was added to 3 ml of 0.01 mol / L GCA solution. Figure 6 It is reflected that when the response time is 2 minutes, the solution has obviously changed its color from red to blue.

[0042] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of the appended claims of the present invention.

Claims

1. A method for preparing a fluorescence sensor, characterized in that: The fluorescent sensor is a metal organic gel material Eu-MOG, and its preparation method includes the following steps: according to the following ratio, 0.0541 mg 1,4-NDC and 0.1115 mg Eu (NO3) 3 · 6H2O are weighed and dissolved in DMF respectively, 150 microliters of triethylamine is added dropwise to the DMF solution of 1,4-NDC until all the triethylamine is dissolved, the DMF solution of 1,4-NDC and the DMF solution of Eu (NO3) 3 · 6H2O are mixed, the two solutions are shaken evenly and then left to stand for a period of time to obtain the metal organic gel Eu-MOG.

2. A method for preparing a fluorescent sensor, characterized in that: The fluorescent sensor is a metal organic gel material Eu-MOG, and its preparation method includes the following steps: according to the following ratio, 0.2162 mg 1,4-NDC and 0.4461 mg Eu (NO3) 3 · 6H2O are weighed and dissolved in DMF respectively, 300 microliters of triethylamine are added dropwise to the DMF solution of 1,4-NDC until all the triethylamine is dissolved, the DMF solution of 1,4-NDC and the DMF solution of Eu (NO3) 3 · 6H2O are mixed, the two solutions are shaken evenly and then left to stand for a period of time to obtain the metal organic gel Eu-MOG.

3. A fluorescent sensor material prepared according to the preparation method of claim 1 or 2.

4. Use of the fluorescent sensor material according to claim 3 in the preparation of a reagent for detecting the hepatocellular carcinoma biomarker GCA.

Images