Eu-polyaspartate fluorescent sensor, preparation method thereof and application thereof in detection of tetracycline and copper ion

Abstract

A europium chelated polyaspartic acid biopolymer for high sensitivity and selective Tc and Cu 2+ ratio fluorescence detection is disclosed. EDTA functionalized PASP is used which binds Eu 3+ ions with high affinity. Fluorescein dye is also attached to the PASP backbone allowing it to act as an internal reference signal. Tc and Cu 2+ ratio fluorescence detection is achieved in the micromolar concentration range. The detection limit for Tc is 10 nM and for Cu 2+ is 333 nM.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological chemical industry, and particularly relates to a preparation method of Eu-polyaspartic acid fluorescent probe and application thereof. BACKGROUND

[0002] Tetracycline (TC) is a broad-spectrum antibiotic with low price, high antibacterial activity, good oral absorption, low toxicity, and is widely used in animal husbandry, aquaculture and individual therapy. The maximum residue limit of TC in milk is 225 nm as regulated by the European Union. Unfortunately, the misuse of TC as a veterinary drug for preventing bacterial infection or as a feed additive for promoting growth in animal farming has led to drug residues in food (such as milk, meat, eggs, fish and honey), which poses a serious threat to human health, including hepatotoxicity, gastrointestinal disorders, allergic reactions and bacterial resistance to antibiotics. However, methods such as high performance liquid chromatography (HPLC), capillary electrophoresis (CE), liquid chromatography-mass spectrometry (LC-MS), chemiluminescence, photoelectrochemistry, etc. have limitations due to complex sample pretreatment steps and expensive instruments. Therefore, it is crucial to develop and establish a simple, economical, rapid and sensitive method for the determination of TC in food and environment.

[0003] Copper ions are a kind of trace elements widely existing in nature and play an important role in biological processes. Copper is the third largest metal ion in the human body next to iron. It helps the formation of red blood cells, as well as maintaining nerve cells and the immune system. The intake of copper also reduces the chances of cardiovascular disease and osteoporosis. In addition, the presence of copper is essential for the normal development and normal functioning of the brain, as well as serving as a co-factor for many enzymes, but the concentration of Cu 2+ must be maintained within a certain range in the organism. Excessive use of copper-containing reagents in agriculture or discharge of copper-containing industrial waste into the environment can seriously pollute soil and water resources, and also easily accumulate in the human body through the food chain, affecting human health. Excessive copper content can cause gastrointestinal disorders, kidney or liver damage, and disrupt cell metabolism. It can also inhibit the activity of various enzymes, triggering a series of mental illnesses such as Alzheimer's disease, Wilson's disease, and Munchausen syndrome. Various analytical techniques have been developed for the detection of copper ions, such as atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS), electroanalytical methods, ultraviolet-visible spectrophotometry (UV-VIS), and fluorescence methods. However, some of these methods require expensive instruments, complex sample pretreatment, high cost or long time-consuming. Therefore, it is of great significance to develop a simple, rapid and low-cost analytical method to achieve sensitive and selective detection of copper ions. SUMMARY

[0004] The technical problem solved by the present application is to provide a new europium chelated polyaspartic acid biopolymer for high-sensitivity and selective Tc and Cu 2+ fluorescence detection.

[0005] The present application first provides a preparation method of Eu-polyaspartic acid fluorescence sensor, comprising the following steps:

[0006] (1) Dissolve polyaspartic acid PASP in bicarbonate buffer, then add DMTMM and diethylene triamine DETA, adjust the pH value to 8-9; stir the mixture at room temperature for reaction, purify in a dialysis bag, and the dialysis liquid is bicarbonate buffer, to prepare PASP-DETA;

[0007] The mass ratio of PASP, DMTMM and DETA is 1:(5-7):(8-9);

[0008] The pH is preferably 8.5;

[0009] The stirring reaction time is 8-16h, preferably 12h;

[0010] (2) Dissolve EDTA disodium salt in bicarbonate buffer, add DMTMM, adjust the pH value to 8-9, add PASP-DETA prepared in step (1), and stir at room temperature for reaction, then purify the obtained mixture in a dialysis bag, and slowly convert the bicarbonate buffer into deionized water, to prepare PASP-EDTA solution;

[0011] The mass ratio of EDTA-2Na, DMTMM and PASP-DETA is (10-20):1:(1-10);

[0012] Stir for 8-16h, preferably 12h;

[0013] (3) Dissolve fluorescein sodium in water, add 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole DEC and N-hydroxysuccinimide NHS for reaction activation, add the activated fluorescein sodium to the PASP-EDTA solution prepared in step (2), and stir at low speed for reaction, then purify in a dialysis bag, and the dialysis liquid is deionized water, to prepare PASP-FL-EDTA solution;

[0014] The mass ratio of fluorescein sodium, EDC, NHS and PASP-EDTA is (5-10):(1-3):(1-2):(1-10);

[0015] The reaction activation time is 30min;

[0016] The low-speed stirring reaction time is 6-24h, preferably 12h;

[0017] The purification time is 12 h.

[0018] (4) adding Eu 3+ solution into the PASP-FL-EDTA solution prepared above, stirring at low speed, purifying with a dialysis bag, using deionized water as the dialysis liquid, freeze-drying the dialysis liquid, and obtaining the fluorescent sensor.

[0019] Eu 3+ The concentration of the Eu 3+ solution is 0.1-0.3 g / mL, preferably 0.18 g / mL;

[0020] Eu 3+ The mass ratio of the Eu 2+ solution to the PASP-FL-EDTA is (1.5-4):1, preferably 1:2;

[0021] The reaction time is 1-6 h, preferably 3 h;

[0022] The purification time is 12 h.

[0023] The Eu-polyaspartic acid fluorescent sensor prepared by the above method is used for the detection of tetracycline and copper ions. The test is carried out under the condition that the pH is 9.

[0024] The application discloses a europium chelated polyaspartic acid biopolymer, which is used for high-sensitivity and selective ratio fluorescence detection of Tc and Cu 2+ EDTA functionalized PASP is used, which can be strongly coordinated with Eu 3+ ions. Fluorescein dye is also attached to the PASP main chain, allowing it to act as an internal reference signal. When TC is continuously added, the fluorescence intensity of the fluorescein dye at 519 nm remains unchanged due to the antenna effect, while the emission intensity of Eu 3+ ions at 617 nm linearly increases. When Cu 2+ is added, the concentration of the cation detected at 617 nm is linearly related to the fluorescence intensity reduced by the static quenching of Eu 3+ . Therefore, ratio fluorescence detection of Tc and Cu 2+ is realized in the micromolar concentration range. The detection limit of TC is 10 nM, and the detection limit of Cu 2+ is 333 nM. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 (a) the fluorescence spectra of the PFE solution in the presence of different concentrations of TC; (b) the linear relationship between the fluorescence intensity ratio (I617 / I519) and the concentration of TC; and (c) the chromaticity diagram of CIE in the presence of different concentrations of TC.

[0026] Figure 2 (a) the fluorescence spectra of the PFE solution in the presence of different concentrations of Cu 2+Fluorescence spectra of PFE-TC solution with different concentrations. (b) The ratio of fluorescence intensity (I519 / I617) versus Cu 2+ concentration. (c) CIE chromaticity diagram at different concentrations of Cu 2+ concentration. (c) CIE chromaticity diagram at different concentrations of Cu DETAILED DESCRIPTION

[0027] The preferred embodiments of the present application will be described hereinafter with reference to the accompanying drawings, in which, for purposes of

[0028] Example 1

[0029] A preparation method of a Eu-polyaspartate fluorescent sensor, the specific steps are as follows:

[0030] (1) First, dissolve 0.1 g of PASP in 10 mL of 10 mM bicarbonate buffer in a round-bottom flask using a magnetic stirrer, then add 0.6 g of DMTMM and 0.87 g of DET, and adjust the pH value to 8.5. The mixture is stirred at room temperature for 12 hours, and the obtained mixture is purified in a dialysis bag (MW 1000 Da) for 12 hours, with bicarbonate buffer as the dialysate. The purified PASP-DET solution is prepared, with a solution volume of 15 mL and a PASP-DET mass of 0.18 g in the solution, which is stored at 4°C for standby.

[0031] (2) Dissolve 1.35 g of EDTA disodium salt in 10 mL of bicarbonate buffer, add 0.11 g of DMTMM, adjust the pH value to 8.5, and react for 10 min to activate the carboxyl group of EDTA. Then add the prepared PASP-DET solution, and stir at room temperature for 12 h. The obtained mixture is purified in a dialysis bag for 12 hours, with bicarbonate buffer slowly converted into deionized water, to prepare a PASP-EDTA solution, with a PASP-EDTA mass of 0.3 g in the solution, a solution volume of 30 mL, and storage at 4°C for standby.

[0032] (3) Dissolve 0.75 g of fluorescein sodium in 10 mL of deionized water, and add 0.19 g of EDC and 0.12 g of NHS to activate the carboxyl group for 30 min. Add the activated fluorescein sodium to the PASP-EDTA solution prepared in step (2). Stir at low speed for 12 hours. Purify with a dialysis bag (MW 1000 DA) for 12 hours, with deionized water as the dialysate. Prepare a PASP-FL-EDTA solution, with a PASP-FL-EDTA mass of about 0.45 g in the solution, a solution volume of 50 mL, and storage at 4°C for standby.

[0033] (4) Dissolve 0.9 g of europium chloride hexahydrate in 5 mL of deionized water. 3+ The solution was slowly added dropwise to the PASP-FL-EDTA solution prepared above, with low-speed stirring for 3 hours. The solution was purified using a dialysis bag (MW 1000DA). The solution was purified using a dialysis bag (MW 1000DA) for 12 hours, with deionized water as the dialysate. The dialysate was freeze-dried to obtain the fluorescent sensor.

[0034] Example 2

[0035] The fluorescent sensor prepared in Example 1 was used to detect tetracycline. The sensor was dispersed in a 0.05M Tris-HCl buffer solution (pH 9.0) to a final concentration of 1 mg / mL. A series of TC concentration gradients were prepared using a 0.05M Tris-HCl buffer solution (pH 9.0). 500 μL of each TC solution at different concentrations was added to 10 μL of the PFE sensor. The total volume of the mixed solution remained constant. The solution was incubated at room temperature for 5 minutes, and then the fluorescence spectrum was recorded.

[0036] like Figure 1 As shown in the figure, (a) fluorescence spectra of PFE solution in the presence of different concentrations of TC; (b) linear relationship between fluorescence intensity ratio (I617 / I519) and TC concentration; (c) CIE chromaticity diagram in the presence of different concentrations of TC. The fluorescence spectrum of PFE is mainly dominated by the emission of fluorescein, and the emission center of fluorescein is located at 519nm. After adding TC solution of different concentrations (0-20μM), the fluorescence intensity of PFE remains stable, while Eu 3+ The luminescence intensity at 592, 617, 652 and 696 nm is significantly enhanced, which is due to the transfer of Tc to Eu 3+ The nanoprobe exhibits efficient energy transfer, known as the "antenna effect." Within the 0-20 μM range, the fluorescence intensity ratio of I617 / I519 exhibits a good linear relationship with Tc concentration (I617 / I519 = 0.9137Tc - 0.16471), with an R² of 0.996. Based on these linear data, the nanoprobe's limit of detection (LOD, 3σ / K, where σ is the standard deviation of the blank solution and K is the slope of the calibration curve) for TC is 10 nM, significantly lower than the minimum concentration (225 nM) specified by the European Union and the U.S. Food and Drug Administration (FDA) (676 nM).

[0037] Example 3

[0038] The fluorescence sensor prepared in Example 1 was used to analyze the effect of tetracycline and Cu 2+Detection. The sensor was dispersed in Tris-HCl buffer solution (0.05 M, pH 9.0) with a final concentration of 1 mg / ml. Tc was 20 μM, and Cu was prepared in a series of gradient concentrations in Tris-HCl buffer solution (0.05 M, pH 9.0) 2+ . 500 μL of 20 μM TC solution was taken, 10 μL of PFE sensor was added, and 500 μL of Cu with different concentrations was added after 5 min of reaction 2+ . The fluorescence spectrum was recorded.

[0039] As shown in Figure 2 : (a) fluorescence spectra of PFE-TC solution with different Cu 2+ contents. (b) linear relationship between fluorescence intensity ratio (I519 / I617) and Cu 2+ concentration. (c) chromaticity diagram of CIE in the presence of different concentrations of Cu 2+ . The fluorescence intensity at 617 nm was gradually quenched with the increase of Cu 2+ concentration (1-100 μm). In the range of 1-100 μm, the fluorescence ratio of I519 / I617 had a good linear relationship with Cu 2+ concentration, and the regression equation was I519 / I617 = 0.0674 + 0.00562 (R2 = 0.994), and the detection limit was 333 nM, which was much lower than the maximum limit of Cu 2+ concentration in drinking water (20 μM) specified by the United States Environmental Protection Agency (EPA). The sensitivity of this method can meet the requirements of environmental monitoring.

[0040] Principles, steps, etc. not explicitly described in the present application can be obtained by those skilled in the art through conventional technical means, and therefore will not be described. Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. A method for preparing a Eu-polyaspartic acid fluorescence sensor, characterized in that: The following steps are involved: (1) Polyaspartic acid (PASP) is dissolved in bicarbonate buffer, and then DMTMM and diethylenetriamine (DETA) are added to adjust the pH to 8-9; the mixture is stirred at room temperature and purified in a dialysis bag, with the dialysate being bicarbonate buffer, to prepare a PASP-DETA solution; (2) dissolving EDTA disodium salt in bicarbonate buffer, adding DMTMM, adjusting the pH to 8-9, adding PASP-DETA prepared in step (1), stirring and reacting at room temperature, purifying the obtained mixture in a dialysis bag, and slowly converting the bicarbonate buffer into deionized water to prepare a PASP-EDTA solution; (3) dissolving sodium fluorescein in water, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide DEC and N-hydroxysuccinimide NHS for reaction activation, adding the activated sodium fluorescein to the PASP-EDTA solution prepared in step (2), stirring at a low speed for reaction, and purifying with a dialysis bag, wherein the dialyzate is deionized water, to prepare a PASP-FL-EDTA solution; (4) Containing Eu 3+ The solution was slowly added dropwise to the PASP-FL-EDTA solution prepared above, stirred at a low speed for reaction, purified using a dialysis bag, the dialysate was deionized water, and the dialysate was freeze-dried to obtain a fluorescent sensor.

2. The method for preparing a Eu-polyaspartic acid fluorescence sensor according to claim 1, characterized in that: In step (1): The mass ratio of PASP, DMTMM, and DETA is: 1:(5-7):(8-9); pH 8.5; The stirring reaction time is 8-16h.

3. The method for preparing a Eu-polyaspartic acid fluorescence sensor according to claim 1, characterized in that: In step (2): The mass ratio of EDTA-2Na, DMTMM, and PASP-DETA is (10-20):1:(1-10); The reaction was stirred for 8-16 hours.

4. The method for preparing a Eu-polyaspartic acid fluorescence sensor according to claim 1, characterized in that: In step (3): The mass ratio of fluorescein sodium, EDC, NHS and PASP-EDTA is (5-10):(1-3):(1-2):(1-10); The reaction activation time was 30 min; The reaction time with low-speed stirring is 6-24h; The purification time was 12 h.

5. The method for preparing a Eu-polyaspartic acid fluorescence sensor according to claim 1, characterized in that: In step (4): Containing Eu 3+ The concentration of the solution is 0.1-0.3g / mL; Eu 3+ The mass ratio of PASP-FL-EDTA is (1.5-4):1; The reaction time under low-speed stirring is 1-6h; The purification time was 12 h.

6. The method for preparing a Eu-polyaspartic acid fluorescence sensor according to claim 5, characterized in that: In step (4): Containing Eu 3+ The concentration of the solution is 0.18 g / mL; Eu 3+ The mass ratio with PASP-FL-EDTA is 1:2; The reaction time was 3 h with low-speed stirring. 7 . A Eu-polyaspartic acid fluorescence sensor prepared according to the preparation method according to any one of claims 1 to 6 .

8. Use of the Eu-polyaspartic acid fluorescence sensor according to claim 7 for detecting tetracycline and copper ions.

9. The use according to claim 8, characterized in that The assay was performed at pH 9.

10. The use according to claim 8 or 9, characterized in that The detection limit of tetracycline is 10 nM, and Cu 2+ The detection limit was 333 nM.

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