Preparation method and application of proportional fluorescent probe for detecting anthrax biomarker

Abstract

The invention belongs to the field of fluorescence analytical chemistry, and discloses a preparation method and application of a proportional fluorescent probe for detecting an anthrax biomarker. The synthesis process comprises the following steps: 1) preparing CDs; 2) preparing a CDs (at) Eu < 3 + > proportional fluorescent probe solution; and (3) adding a to-be-detected sample solution into the proportional fluorescent probe solution, and detecting the DPA concentration under a fixed excitation wavelength. In the detection process, after the bacillus anthracis spore marker is added, the emission peak at 615 nm is enhanced. The probe disclosed by the invention can be used for detecting the bacillus anthracis spore marker in a fluorescence mode, is more convenient in practical application, and has higher accuracy as the result of the detection mode is verifiable.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence analytical chemistry, and more particularly to a ratiometric fluorescence probe based on europium-modified carbon dots for detecting the anthrax biomarker dipicolinic acid (DPA). Background Art

[0002] Anthrax, as an infectious disease, can be caused by rotting animal carcasses, contaminated feed, contaminated water, and infected plants. In addition, anthrax spores can enter the human body through the intestines or respiratory tract directly, leading to breathing difficulties, diarrhea, vomiting, and skin rashes. If not diagnosed in time, it may be life-threatening. Therefore, detecting the main metabolite DPA of anthrax spores is crucial for ensuring ecological and public health safety as well as disease diagnosis. Currently, the conventional methods for DPA determination are relatively mature, mainly including surface-enhanced Raman scattering, liquid chromatography-tandem mass spectrometry (LC-MS / MS), and hydrophilic interaction chromatography. Although these methods can accurately determine the content of DPA, their high costs, complex operations, difficult maintenance, environmental limitations, and other disadvantages greatly limit their practical applications. Therefore, there is an urgent need to develop a rapid on-site DPA detection method with high portability, simplicity, and sensitivity. The emergence of fluorescent nanomaterials has changed people's views on pollutant detection. To achieve this concept, great efforts have been made in material design. Currently, most of the reported methods only provide a single change in fluorescence output, and there is background fluorescence in the operating environment, which brings significant interference and results in poor detection accuracy.

[0003] To solve this problem, researchers have reported the pioneering basis for detecting DPA using a ratiometric fluorescence sensing mechanism. Since then, a large number of ratiometric fluorescence sensors for DPA detection have been developed, using fluorescent materials as the internal signal and combining lanthanide elements, such as nanosheets, silicon quantum dots, metal-organic frameworks, covalent organic frameworks, etc. However, low quantum yields, low water solubility, and material toxicity pose significant challenges to environmental DPA detection. Therefore, developing high-performance fluorescent nanomaterials is the key and urgent requirement for constructing environmentally friendly and simple DPA sensors. CDs are a new type of carbon-based nanomaterial with many excellent properties. It is generally believed that its low toxicity and high water solubility enable it to have wide applications in biological imaging and environmental detection. However, the complexity of the experimental process leads to a wide variety of synthetic materials, reducing the yield of the target product and ultimately affecting the fluorescence quantum yield of the probe.

[0004] Therefore, developing a ratiometric fluorescence probe for detecting DPA is an urgent problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a preparation method and application of a ratiometric fluorescence probe for detecting anthrax biomarkers, specifically a ratiometric fluorescence probe based on europium-modified carbon dots for detecting DPA (2,6-dipicolinic acid).

[0006] The present invention strictly controls the reaction time and temperature, and prepares water-soluble, good fluorescence performance, and environmentally friendly functionalized carbon quantum dots (CDs) by a dry-heat method; uses rare earth element europium to functionalize the surface of CDs to construct a ratiometric fluorescence probe based on the change of dual-channel fluorescence. After adding DPA, the blue-green fluorescence of the probe is quenched, and due to the antenna effect, the dual-channel fluorescence response causes the solution to turn red. In addition, a paper sensor for quantitative detection of DPA was fabricated using a smartphone equipped with a color block recognition system, which can rapidly detect DPA on-site in the range of 10 - 120 μM.

[0007] One of the purposes of the present invention is to provide a preparation method of a ratiometric fluorescence probe for detecting anthrax biomarkers, including the following steps:

[0008] S1. Preparation of CDs:

[0009] S11. Stir 3,9-perylene diacid, boric acid, and urea evenly to obtain a mixture solution;

[0010] S12. Place the mixture solution in a drying oven for reaction, and cool to room temperature after the reaction is completed;

[0011] S13. Dissolve the reaction mixture in water, centrifuge, separate the solid and liquid, and obtain the supernatant;

[0012] S14. Transfer the supernatant to a dialysis bag with a molecular weight of 1000 Da, dialyze for 12 - 36 h, and freeze-dry to obtain an orange-red solid powder of CDs;

[0013] S2. Preparation of CDs@Eu 3+ ratiometric fluorescence probe;

[0014] S21. Dissolve europium chloride hexahydrate in the CDs solution, stir at room temperature for 5 - 12 h, mix evenly to obtain a mixed solution;

[0015] S22. Dialyze the mixed solution in pure water using a dialysis bag with a molecular weight of 1000 Da for 24 - 48 h to obtain the CDs@Eu 3+ ratiometric fluorescence probe, and store the obtained probe solution at 4 °C.

[0016] Preferably, in step S11, the mass ratio of 3,9-perylene diacid, boric acid, and urea is 0.2 - 0.3:4.0 - 5.0:9.0 - 10.0.

[0017] The beneficial effects of adopting the above mass ratio are as follows: promoting the full contact of materials and improving the reaction yield.

[0018] Preferably, in step S12, the temperature of the reaction is 150 - 180 °C and the time is 5 - 8 h.

[0019] The beneficial effects of adopting the above conditions are as follows: improving the substrate utilization rate, accelerating the product generation, avoiding the deterioration, decomposition or generation of excessive impurities of the product due to improper reaction time and temperature, thereby ensuring that the material produces stronger fluorescence.

[0020] Preferably, in step S13, the dosage relationship between the reaction mixture and water is 1 - 5 g: 20 - 40 mL; the centrifugation is at 10000 rpm for 10 - 30 min.

[0021] Preferably, in step S14, the conditions for freeze-drying are that the material temperature is -4 - 0 °C and the freeze-drying time is 3 - 5 days.

[0022] Preferably, in step S21, the concentration of the CDs solution is 0.01 - 0.05 g / L, and the concentration of europium chloride hexahydrate in the CDs solution is 5 - 15 g / L.

[0023] More preferably, the concentration of the CDs solution is 0.01 g / L, and the concentration of europium chloride hexahydrate in the CDs solution is 5 - 10 g / L.

[0024] The beneficial effects of adopting the above dosage relationship are as follows: promoting the full reaction of europium chloride hexahydrate and CDs, and improving the sensitivity of the probe solution for detecting DPA.

[0025] Preferably, in step S22, the concentration of the CDs@Eu 3+ ratiometric fluorescence probe solution is 0.01 - 0.1 g / L.

[0026] The second object of the present invention is to provide an application of a fluorescence-mode ratiometric fluorescence probe in detecting anthrax spore markers, and the specific steps are as follows:

[0027] Adjust the pH value of the CDs@Eu 3+ ratiometric fluorescence probe to 7 - 8, add the sample to be tested, incubate at room temperature for 2 min, and measure the DPA concentration at a fixed excitation wavelength.

[0028] Furthermore, the anthrax spore marker is DPA.

[0029] Preferably, the concentration of DPA in the sample to be tested is 0 - 12 mM, the measured DPA concentration is 0 - 120 μM; the fixed excitation wavelength is 251 nm, and a fluorescence emission spectrum with a scanning range of 450 - 650 nm is obtained.

[0030] Preferably, the dosage relationship between the sample to be measured and the CDs@Eu 3+ ratio fluorescence probe solution is 1 - 12 mM: 0.01 - 0.05 g / L.

[0031] Preferably, it further includes using the emission wavelength F 615 / F 487 ratio as the ratio fluorescence value.

[0032] Furthermore, the working curve during measurement is the standard curve of the fluorescence intensity ratio and the DPA concentration.

[0033] Furthermore, when the CDs@Eu 3+ ratio fluorescence probe solution detects DPA, the fluorescence intensity value of CDs decreases, and the Eu 3 + fluorescence in combination with DPA gradually increases.

[0034] Preferably, when detecting DPA, the material is irradiated with 251 nm excitation light, and the fluorescence of the solution changes from blue - green to red.

[0035] More preferably, a paper sensor for the quantitative detection of DPA is fabricated using a smartphone equipped with a color block recognition system, which can rapidly detect DPA on - site within the range of 10 - 120 μM.

[0036] The technical solution of the present invention is based on the inner filter effect between Eu 3+ and DPA, which causes the red fluorescence of the fluorescence probe to turn on, while the blue - green fluorescence of CDs is quenched. The CDs@Eu 3+ probe is reacted with different concentrations of DPA under neutral pH conditions. When DPA reacts with CDs@Eu 3+ , its red fluorescence originating from CDs@Eu 3+ increases with the increase in the concentration of the analyte, while the blue - green fluorescence of the reference material CDs decreases. By the linear relationship presented by the ratio data of the fluorescence intensity at 615 nm to the fluorescence intensity at 487 nm and the concentration of DPA, a ratio fluorescence analysis method for detecting DPA in the sample is established.

[0037] From the above - mentioned technical solutions, it can be seen that compared with the prior art, the beneficial effects achieved by the present invention are as follows: The present invention is a ratio fluorescence probe for anthrax spore markers based on Eu 3+ functionalized CDs. During the detection process, after adding the anthrax bacillus spore marker, the emission peak at 615 nm is enhanced. The probe can detect the anthrax bacillus spore marker through the fluorescence mode, which is more convenient in practical applications, and the results of the detection mode can be verified, with higher accuracy. Brief Description of the Drawings

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on the provided drawings.

[0039] Figure 1 It is CDs coordinated with Eu 3+ and CDs@Eu 3+ Schematic diagram for detecting DPA;

[0040] Figure 2 Rear view and top view photos of CDs after reaction;

[0041] Figure 3 TEM image of CDs;

[0042] Figure 4 It is CDs@Eu 3+ Selectivity and anti-interference ratio fluorescence diagram, where the selected ions are all metal chlorides and sodium salts;

[0043] Figure 5 It is CDs@Eu 3+ Selectivity and anti-interference ratio fluorescence diagram, and the interfering substances are all common antibiotics and DPA structure analogs;

[0044] Figure 6 It is CDs@Eu 3+ Fluorescence intensity diagram of CDs@Eu in solutions with different pH values;

[0045] Figure 7 Fluorescence intensity value diagram after adding different concentrations of DPA to CDs;

[0046] Figure 8 It is the relationship diagram between DPA concentration and I 615 / I 487 ratio. Detailed implementation manners

[0047] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0048] Embodiment 1

[0049] Preparation of a ratio fluorescence probe for detecting anthrax biomarkers

[0050] Preparation of CDs:

[0051] Add 240 mg of 3,9-perylene acid, 4.8 g of boric acid, and 9.6 g of urea into a 50 mL beaker, mix thoroughly, and incubate in an oven at 180 °C for 6 hours. Figure 2 As shown, the product after the reaction is puff-shaped. The obtained expanded material was dissolved in 20 mL of water and centrifuged at 10,000 rpm for 20 minutes to separate the large particles from the liquid. The supernatant was transferred to a dialysis bag with a molecular weight of 1,000 Daltons and dialyzed for 36 hours to remove water-soluble molecules. Finally, the dialysate was frozen into ice cubes, placed in a freeze dryer and the cold well temperature facility was set to -74°C, the material temperature was -4 to 0°C, and the material was frozen for 3 days to obtain orange-red CDs. Figure 3 ,The material was characterized using transmission electron microscopy, and it was not difficult to find that the CDs particles were uniform and had a size of 5nm.

[0052] CDs@Eu 3+ The synthesis method is as follows:

[0053] 0.7329 g of europium chloride hexahydrate was added to 60 mL of water containing 0.0011 g of CDs and stirred evenly at room temperature for 12 hours. The reaction mixture was dialyzed for 48 hours using a dialysis bag with a molecular weight cutoff of 1000 Daltons to remove uncomplexed Eu 3 + The obtained CDs@Eu3+ probe solution was stored at 4°C for later use.

[0054] Example 2

[0055] Preparation of CDs:

[0056] 200 mg of 3,9-perylene acid, 4 g of boric acid and 9 g of urea were added to a 50 mL beaker, mixed thoroughly and incubated in an oven at 150 °C for 5 hours. The obtained expanded material was dissolved in 20 mL of water and centrifuged at 10,000 rpm for 10 minutes to separate the large particles from the liquid. The supernatant was transferred to a dialysis bag with a molecular weight of 1,000 Daltons and dialyzed for 36 hours to remove water-soluble molecules. Finally, the dialysate was frozen into ice cubes, placed in a freeze dryer and the cold well temperature facility was set to -74 °C, the material temperature was -4 to 0 °C, and the frozen material was freeze-dried for 3 days to obtain orange-red CDs.

[0057] CDs@Eu 3+ The synthesis method is as follows:

[0058] Add 0.3000 g of europium(III) chloride hexahydrate to 60 mL of water containing 0.0011 g of CDs, and stir evenly at room temperature for 12 hours. Dialyze the reaction mixture for 48 hours using a dialysis bag with a molecular weight cut-off of 1000 Da to remove uncomplexed Eu 3 + . The resulting CDs@Eu3+ probe solution is stored at 4 °C for future use.

[0059] Example 3

[0060] Preparation of CDs:

[0061] Add 300 mg of 3,9-perylenedicarboxylic acid, 5 g of boric acid, and 10 g of urea to a 50 mL beaker, mix well, and incubate in an oven at 180 °C for 6 hours. Dissolve the obtained expanded material in 40 mL of water, centrifuge at 10000 rpm for 40 minutes to separate large particles from the liquid. Transfer the supernatant to a dialysis bag with a molecular weight of 1000 Da and dialyze for 36 hours to remove water-soluble molecules. Finally, freeze the dialysate into ice cubes, place them in a freeze dryer, set the cold well temperature facility to -74 °C, the material temperature to -4 to 0 °C, and freeze-dry the frozen material for 5 days to obtain orange-red CDs.

[0062] CDs@Eu 3+ The synthesis method is as follows:

[0063] Add 0.9000 g of europium(III) chloride hexahydrate to 60 mL of water containing 0.0011 g of CDs, and stir evenly at room temperature for 12 hours. Dialyze the reaction mixture for 48 hours using a dialysis bag with a molecular weight cut-off of 1000 Da to remove uncomplexed Eu 3 + . The resulting CDs@Eu3+ probe solution is stored at 4 °C for future use.

[0064] Experiment 1: Selectivity and anti-interference test

[0065] Selectivity and anti-interference are key indicators for evaluating the performance of CDs@Eu 3+ in detecting DPA. To investigate the selectivity of CDs@Eu 3+ in different elemental environments, common metal ions (Zn 2+ , Ni 2+ , Na + , Mn 2+ , K + , Cu 2+ , Cr 3+ , Ca 2+ , Ba 2 + and Al 3+ ) and anions (S -, NO2 - , HS - , HPO4 2- , HCO3 - , CO3 2- , CH3COO - and Br - ), The performance of the probe for detecting DPA was evaluated with antibiotics and DPA structural analogs.

[0066] CDs@Eu 3+ Interfering substances at the same concentration were added to the solution. As Figure 4 shown, the I 615 / I 487 values of most ions fluctuated by no more than 0.1. In contrast, Figure 5 the ratio of antibiotics to structural analogs fluctuated around 0.2.

[0067] Interfering substances at the same concentration were added to the CDs@Eu 3+ solution containing DPA. The I 615 / I 487 values of most ions fluctuated by no more than 0.1. In contrast, the ratio of antibiotics and structural analogs exceeded 1 because their structural complexity significantly affected the fluorescence intensity of the probe.

[0068] Example 5

[0069] CDs@Eu 3+ The route for detecting DPA is as follows:

[0070] To detect DPA, 990 μL of the CDs@Eu 3+ stock solution (0.07 mg / mL) was mixed with 10 μL of DPA solutions at different concentrations (0 - 12 mM) to prepare 1 mL of reaction solution with a DPA concentration range of 0 - 120 μM.

[0071] To achieve the best effect for CDs and Eu 3+ , the pH of the CDs@Eu 3+ solution was adjusted to neutral, as Figure 6 shown. After incubation in a test tube at room temperature for 2 minutes, measurements were taken at room temperature (20 °C). The fluorescence spectrum was recorded at an excitation wavelength of 251 nm.

[0072] The sensitivity of the CDs@Eu 3+ probe to DPA was evaluated by tracking the fluorescence emission intensity when different concentrations of DPA were added to the probe solution.

[0073] As Figure 7 shown, as the concentration of DPA increased, Eu 3+The emission at 615 nm gradually increases, while the fluorescence emission of CDs at 487 nm is inhibited by DPA and shows a downward trend. Therefore, the fluorescence color of the probe solution changes from turquoise to red.

[0074] Figure 8 Shows the relationship between different concentrations of DPA and the fluorescence ratio I 615 / I 487 . The criterion of three - times the blank signal deviation was adopted. The fluorescence ratio of high - concentration DPA obtained by fitting shows a non - linear correlation. The DPA concentration and the ratio can be expressed as Y = 1.071e 0.031X -0.659, with R 2 being 0.99393. After reducing the DPA concentration, the two show an obvious linear correlation, with R 2 being 0.99462.

[0075] The detection limit of this probe is as low as 7.566 nM. In summary, the CDs@Eu 3+ solution has high visual selectivity for DPA detection, providing a potential solution for on - site qualitative identification of DPA.

[0076] Experiment 2: Practicality and feasibility test

[0077] Using a smartphone equipped with a color - block recognition system, 0.07 mg / mL CDs@Eu 3+ solution was dropped onto commercial filter paper and dried at room temperature (20 - 25 °C) to fabricate a paper - based sensor for quantitative detection of DPA, which can rapidly detect DPA on - site in the range of 10 - 120 μM.

[0078] The practicality and feasibility of the paper - based sensor for DPA detection were further verified through actual samples such as tap water, lake water, and milk.

[0079] Before detection, all samples were centrifuged at 8000 rpm for 20 minutes to remove larger impurities, and then filtered through a 0.22 - μm membrane to remove larger particles. Subsequently, the samples were spiked to two DPA concentrations (30 and 60 μM).

[0080] For each sample, including the blank, fluorescence was measured three times and the mean value and standard deviation were given.

[0081] Table 1 Content, recovery rate (n = 3), and standard deviation of HIAA in actual samples:

[0082]

[0083] It is worth noting that no significant fluorescence enhancement was observed in the samples without DPA peak formation. As shown in Table 1, the recovery rates of the DPA-spiked samples were close to 100% (ranging from 91% to 100.17%).

[0084] The foregoing description of the disclosed embodiments enables those skilled in the art to practice or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A preparation method of a ratio fluorescence probe for detecting anthrax biomarkers, characterized in that, It includes the following steps: S1. Prepare CDs: S11. Stir 3,9-perylene diacid, boric acid and urea evenly to obtain a mixture solution; S12. React the mixture solution at a certain temperature, and cool it to room temperature after the reaction is completed; S13. Dissolve the reaction mixture in water, centrifuge to obtain a supernatant; S14. Dialyze and freeze-dry the supernatant to obtain a solid powder of CDs; S2. Preparation of CDs@Eu 3+ Ratio fluorescence probe; S21. Dissolve europium chloride hexahydrate in the CDs solution, stir evenly to obtain a mixed solution; S22. Dialyze the mixed solution in pure water to obtain the CDs@Eu 3+ ratiometric fluorescence probe.

2. The preparation method of a ratiometric fluorescence probe for detecting anthrax biomarkers according to claim 1, wherein In step S11, the mass ratio of the 3,9-perylene diacid, boric acid and urea is 0.2 - 0.3:4.0 - 5.0:9.0 - 10.

0.

3. The preparation method of a ratio fluorescence probe for detecting anthrax biomarkers according to claim 1, characterized in that, In step S12, the temperature of the reaction is 150 - 180 °C and the time is 5 - 8 h.

4. The preparation method of a ratiometric fluorescence probe for detecting anthrax biomarkers according to claim 1, characterized in that, In step S13, the centrifugation is at 10000 rpm for 10 - 30 min.

5. The preparation method of a ratio fluorescence probe for detecting anthrax biomarkers according to claim 1, characterized in that, In step S14, the conditions for freeze-drying are that the material temperature is -4 - 0 °C and the freeze-drying time is 3 - 5 d.

6. The preparation method of a ratiometric fluorescence probe for detecting anthrax biomarkers according to claim 1, characterized in that, In step S21, the concentration of the CDs solution is 0.01 - 0.05 g / L; The concentration of the europium chloride hexahydrate in the CDs solution is 5 - 15 g / L.

7. The preparation method of a ratiometric fluorescence probe for detecting anthrax biomarkers according to claim 1, characterized in that, In steps S14 and S22, the dialysis is both carried out using a dialysis bag with a molecular weight cut-off of 1000 Da; among them, the dialysis time in step S14 is 12 - 36 h, and the dialysis time in step S22 is 24 - 48 h.

8. Use of the proportional fluorescence probe for detecting anthrax biomarkers according to any one of claims 1-7, characterized in that, Specifically: Adjust the pH value of the CDs@Eu 3+ ratiometric fluorescent probe to 7 - 8, add the sample to be measured, incubate at room temperature for 2 min, and measure the DPA concentration at a fixed excitation wavelength.

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