Ratio fluorescent probe and ratiometric fluorescent test paper for field detection of farmland nitrogen as well as preparation method and application of ratiometric fluorescent probe and ratiometric fluorescent test paper

Abstract

The invention relates to the technical field of detection of nitrogen in soil, and particularly discloses a ratiometric fluorescent probe and ratiometric fluorescent test paper for on-site detection of farmland nitrogen as well as a preparation method and application of the ratiometric fluorescent probe and the ratiometric fluorescent test paper. The ratiometric fluorescent probe for field detection of farmland nitrogen comprises a system 1 and a system 2, the system 1 is a CdTe quantum dot solution, and the system 2 is a mixed solution of a 3, 4-thiophenedicarboxaldehyde (TDA) solution and a Na2SO3 solution. The ratiometric fluorescent test paper is obtained by uniformly fixing the ratiometric fluorescent probe solution on non-fluorescent filter paper in a soaking manner, and has very good specific recognition capability on NH4 < + >, and the multi-gear color changing effect is beneficial to naked eye visual detection. The fluorescent test paper can be used similar to pH test paper, so that on-site detection is more convenient and portable, the detection cost is lower, and a novel very effective and practical technical means is provided for NH4 < + > detection.

Description

Technical Field

[0001] This invention relates to the field of nitrogen detection technology in soil, and particularly to a ratiometric fluorescent probe, ratiometric fluorescent test paper, preparation method and application for on-site detection of nitrogen in farmland. Background Technology

[0002] Ammonium nitrogen (NH4) + Ammonium nitrogen (AM) is a commonly used form of nitrogen fertilizer in agricultural production, playing a vital role in plant growth and the ecological nitrogen cycle. Insufficient nitrogen in farmland soil affects soil fertility, while excessive use leads to excessive nitrogen accumulation, which is easily lost into water bodies through rainwater runoff or irrigation, causing eutrophication and affecting the survival and reproduction of aquatic organisms. Therefore, timely and accurate understanding of ammonium nitrogen content in farmland is crucial for analyzing soil fertility and reducing agricultural non-point source pollution.

[0003] Currently, the amount of ammonium nitrogen (NH4) in the soil + The standard method for determining the content of NH4+ is the indophenol blue method, the principle of which is... + In a strongly alkaline medium, NH4 reacts with hypochlorite and phenol to form water-soluble indophenol blue, which is then detected spectrophotometrically. However, this method has a long reaction time and requires specialized experimental equipment, making it suitable only for laboratory applications. In recent years, electrochemical methods have been used to detect NH4. + It has gradually become a research hotspot in this field, utilizing potential difference to explore the concentration of NH4 in solution. + The potential relationship between ions and the reference electrode of the sensing probe. In addition, other NH4... + Detection methods such as ion chromatography and Nessler method are also common. However, traditional detection methods rely on large analytical instruments and specialized technicians, and the reagents used may have a certain degree of toxicity, or be limited by low detection sensitivity, making it difficult to achieve universal application. In addition, practical applications also face problems such as the complex composition of soil containing various impurity ions and interfering substances. Therefore, high demands are placed on the high sensitivity, selectivity, and anti-interference ability of the detection methods. Thus, the development of a new method for detecting ammonium nitrogen in soil that is rapid, convenient, sensitive, reliable, and economical has significant scientific importance and great application value. Summary of the Invention

[0004] Based on this, the purpose of this invention is to provide a ratiometric fluorescent probe, ratiometric fluorescent test strip, preparation method, and application for on-site detection of nitrogen in farmland, specifically for NH4. + The detection provides a very effective and practical technical means, making on-site detection of ammonium nitrogen more convenient and portable, and cheaper. It has broad application prospects in the on-site visual detection of soil ammonium nitrogen.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a ratiometric fluorescent probe for on-site detection of nitrogen in farmland, comprising system 1 and system 2. System 1 is a CdTe quantum dot solution, and system 2 is a mixture of 3,4-thiophene dicarboxaldehyde (TDA) solution and Na₂SO₃ solution. The experimental mechanism of this invention is that, in the presence of sodium sulfite, 3,4-thiophene dicarboxaldehyde can react with NH₄⁻. + A derivatization reaction occurs under heating conditions to generate a derivative with strong yellow fluorescence. Simultaneously, a ratiometric fluorescent probe is constructed by introducing a stable red background fluorescent material (CdTe quantum dots).

[0006] As a further improvement to the above-described scheme of the present invention, the volume ratio of system 1 to system 2 is 0.8-1.6:1, preferably 1:1.

[0007] As a further improvement to the above-described scheme of the present invention, in system 2, the volume ratio of 3,4-thiophene dicarboxaldehyde solution to Na2SO3 solution is 0.25-5:1, preferably 3:1. In system 2, the concentration of 3,4-thiophene dicarboxaldehyde solution is 0.02-0.07M, preferably 0.04-0.07M, more preferably 0.055M; the concentration of Na2SO3 solution is 0.01-0.1M, preferably 0.06-0.1M, more preferably 0.08M.

[0008] The present invention also provides a ratio fluorescence test strip, which is obtained by immersing non-fluorescent filter paper in the ratio fluorescence probe for on-site detection of nitrogen in farmland as described above, and then drying it.

[0009] The present invention also provides an application of the ratio fluorescent test strip as described above in the field detection of nitrogen in farmland.

[0010] As a further improvement to the above-mentioned solution of the present invention, the application includes the following steps: taking soil sample extract and dropping it onto the ratiometric fluorescent test paper, heating it, and observing the fluorescence color under ultraviolet light, comparing it with the standard NH4. + The concentration of ammonium nitrogen in a soil sample can be obtained by observing the fluorescence response color of the solution.

[0011] As a further improvement to the above-mentioned solution of the present invention, the heating is performed at 20-70°C for 15 minutes, preferably at 55°C for 15 minutes.

[0012] As a further improvement to the above-described solution of the present invention, the ultraviolet lamp is a 365nm portable ultraviolet lamp.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The test strip containing the ratiometric fluorescent probe for on-site detection of nitrogen in farmland according to this invention can directly detect NH4 in the soil. +The test strip utilizes ratio fluorescence technology, constructed by adding CdTe to create a red fluorescent background, to achieve multiple levels of fluorescence color-changing effects. This results in more accurate data interpretation and facilitates naked-eye visual detection. In practical use, simply place the strip containing NH4... + Soil extract is dropped onto test paper, which is then placed on a self-heating heat pack to react. The fluorescence color change of the test paper is observed under a 365 nm ultraviolet lamp, and compared with a standard color chart to read the soil ammonium nitrogen concentration. This invention is highly sensitive, pollution-free, and low-cost. It enables on-site detection of ammonium nitrogen in farmland soil without the need for large-scale instruments, providing a new technical means for on-site detection of ammonium nitrogen in farmland, and also laying the foundation for subsequent NH4... + The development of monitoring sensors provided key technological support. Attached Figure Description

[0014] Figure 1 This is a fluorescence color photograph of CdTe quantum dots under ultraviolet light irradiation in Example 1 of the present invention; Figure 2 This is a TEM image of the CdTe quantum dots in Example 1 of the present invention; Figure 3 System 2 of Example 1 of this invention is mixed with NH4 at different concentrations. + Fluorescence image of the fluorescent derivative after reaction under 365nm ultraviolet light; Figure 4 The ratiometric fluorescent test strip prepared in Example 1 of this invention reacts with standard NH4 at different concentrations under ultraviolet light irradiation. + The change in the fluorescent color of the reaction; Figure 5 The concentrations of TDA solution and Na2SO3 solution affect the concentration of NH4+. + The effect of derivative fluorescence intensity; Figure 5 (a) Adding NH4 to TDA solutions of different concentrations + After fluorescence intensity, Figure 5 (b) Adding NH4 to Na2SO3 solutions of different concentrations + The fluorescence intensity after; Figure 6 NH4 was added to probe solutions containing different volume ratios of TDA solution and Na2SO3 solution. + The fluorescence intensity after; Figure 7 NH4 was added to system 2 prepared in Example 1 of this invention. + The fluorescence intensity at different reaction temperatures; Figure 8 The fluorescent probe solution prepared in Example 1 of this invention is effective against NH4+. + Results of the analysis of the selectivity and anti-interference ability of the detection; Figure 8(a) Ratio-fluorescent probe for NH4 + Fluorescence spectra of various interfering ions, Figure 8 (b) Study on the selectivity and anti-interference capability of the probe (I) 546 NH4 + The fluorescence intensity of the derivative at the 546 nm emission peak after the reaction, I 654 I represents the fluorescence intensity of CdTe against a red background at the emission peak at 654 nm. 546 / I 654 (This refers to the ratio of their fluorescence intensities). Figure 9 The ratiometric fluorescent test paper prepared in Example 1 of this invention is used to test the levels of NH4 in two soil samples. + Photographs of the detected fluorescence colors. Detailed Implementation

[0015] To facilitate understanding of the present invention, a more comprehensive description will be given below with reference to specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0017] Example 1 This embodiment proposes a ratiometric fluorescent probe for detecting ammonium nitrogen, which includes system 1 and system 2. System 1 is a CdTe quantum dot solution, and system 2 contains 3,4-thiophene dicarboxaldehyde solution (TDA solution) and Na2SO3 solution.

[0018] The preparation method of System 1 in this embodiment is as follows: 0.0365g of cadmium chloride hemipentahydrate was added to 35mL of deionized water, and 0.1g of trisodium citrate was added under stirring; 0.01g of potassium tellurite was dissolved in 5mL of deionized water and added to the above solution. 0.078mL of mercaptoacetic acid and 0.05g of sodium borohydride were added under thorough stirring. The mixture was magnetically stirred at room temperature until homogeneous. The mixed solution was placed in a high-pressure reactor lined with polytetrafluoroethylene and heated at 180°C for 80min to obtain the CdTe quantum dot stock solution. Before use, the CdTe quantum dot stock solution was diluted 11 times with an equal volume of deionized water and labeled as System 1. Figure 1 The image shows the fluorescence color of the CdTe quantum dots in this embodiment under ultraviolet light irradiation; Figure 2The image shows a transmission electron microscope (TEM) image of CdTe quantum dots in this embodiment. The results show that the material has a granular morphology, is relatively uniformly distributed, and has an average particle size of about 4.1 nm.

[0019] The preparation method of system 2 in this embodiment is as follows: 3,4-thiophene dicarboxaldehyde solution with a concentration of 0.055M and Na2SO3 solution with a concentration of 0.08M are mixed at a volume ratio of 3:1 to obtain system 2.

[0020] The method for preparing the ratiometric fluorescent probe for detecting ammonium nitrogen in this embodiment is as follows: System 1 and System 2 are mixed at a volume ratio of 1:1.

[0021] This embodiment also provides a ratio fluorescence test paper, which is prepared by mixing 100 μL of system 1 and 100 μL of system 2 evenly, and then uniformly fixing the ratio fluorescence mixed solution on non-fluorescent filter paper by immersion, thereby obtaining the ratio fluorescence test paper.

[0022] Examples 2-7 The difference between Examples 2-7 and Example 1 is that the concentrations of the 3,4-thiophene dicarboxaldehyde solution in system 2 of Examples 2-7 are 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, and 0.07M, respectively.

[0023] Examples 8-12 The difference between Examples 8-12 and Example 1 is that the concentrations of Na2SO3 solution in system 2 of Examples 8-12 are 0.01M, 0.02M, 0.04M, 0.06M, and 0.1M, respectively.

[0024] Examples 13-18 The difference between Examples 13-18 and Example 1 is that in System 2 of Examples 13-18, the volume ratio of 3,4-thiophene dicarboxaldehyde solution to Na2SO3 solution is 0.25:1, 0.5:1, 1:1, 2:1, 4:1, and 5:1, respectively.

[0025] Application Example 1 A 10 mM ammonium nitrogen standard stock solution was prepared using NH4Cl and stored at 4°C. Ammonium nitrogen standard solutions of different concentrations were obtained by diluting the solutions with the 10 mM stock solution. 100 μL of each of the different concentrations of ammonium nitrogen standard solutions were added to 200 μL of system 2 from Example 1, and the fluorescence color was observed under a 365 nm UV lamp, yielding the results shown below. Figure 3 The different concentrations of NH4 shown + A photograph of the generated fluorescent derivative under ultraviolet light. It can be seen that system 2 of the present invention can recognize NH4. + The intensity of the yellow fluorescence of the generated fluorescent derivative is related to that of NH4. +The concentration is directly related to NH4 + As the concentration gradually increases, the fluorescence gradually transitions from colorless to a bright yellow.

[0026] Application Example 2 7 μL of ammonium nitrogen standard solutions of different concentrations were dropped onto the ratiometric fluorescent test paper prepared in Example 1. After heating at 55 °C for 15 min, the fluorescence color change was observed under a 365 nm UV lamp. The results are as follows. Figure 4 As shown. From Figure 4 It can be seen that, along with NH4 + As the concentration increases, the fluorescence color gradually changes from purplish-red, orange, to yellow.

[0027] Application Example 3 1 mL of 250 μM ammonium nitrogen standard solution was added to 2 mL of the probe solutions prepared in Examples 1-12. After heating at 55 °C for 15 min, the samples were detected using an Agilent fluorescence spectrometer (emission wavelength 546 nm) to obtain the following results. Figure 5 The different concentrations of TDA solutions shown Figure 5 a) Na2SO3 solutions of different concentrations ( Figure 5 b) Fluorescence intensity change at the 546 nm emission peak. Figure 5 It can be seen that the fluorescence intensity of the detection result is the highest when the TDA solution concentration is 0.055M and the Na2SO3 solution concentration is 0.08M, which are the optimal reaction conditions.

[0028] Application Example 4 1 mL of 250 μM ammonium nitrogen standard solution was added to the probe solutions prepared in Examples 1 and 13-18, respectively. After heating at 55 °C for 15 min, the samples were detected using an Agilent fluorescence spectrometer (emission wavelength 546 nm). The results were as follows: Figure 6 The fluorescence intensity changes at the 546 nm emission peak are shown for different volume ratios. Figure 6 It can be seen that when the TDA concentration and Na2SO3 concentration are optimal, the fluorescence intensity reaches its maximum when the volume ratio of TDA solution to Na2SO3 solution is 3:1, which is the best reaction ratio.

[0029] Application Example 5 Take 7 x 2 mL of the fluorescent probe solution prepared in Example 1 and add 1 mL of 250 μM ammonium nitrogen standard solution to each. After heating at different temperatures for 15 min, perform spectral detection using an Agilent fluorescence spectrometer (emission wavelength 546 nm) to obtain the following results. Figure 7 The fluorescence intensity change is shown. From Figure 7It can be seen that, under the optimal reaction ratio, when different heating temperatures are applied and the fluorescence intensity at the 546nm emission peak is compared, the detection result is best when the heating temperature is 55℃, which is the optimal reaction temperature.

[0030] Application Example 6 NH4 + Analysis of selectivity and anti-interference capability of the detection: Take 1 mL of pure aqueous solution, 1 mL of 250 μM ammonium nitrogen standard solution, and 1 mL of 250 μM interfering aqueous solution. The interfering aqueous solution contains Na. + K + Mg 2+ Ca 2+ Fe 2+ Zn 2+ Cl - NO2 - NO3 - PO4 3- CO3 2- SO4 2- Verify the ratiometric fluorescent probe of this application for NH4 + Selective response.

[0031] Simultaneously, an anti-interference test was set up. Twelve groups of 1 mL 250 μM ammonium nitrogen standard solutions were added to the selectively prepared interfering aqueous solution. Spectroscopic measurements were performed using an Agilent fluorescence spectrometer, and the fluorescence intensity ratio (Ig) was used as the spectral data. 546 / I 654 , where: I 546 NH4 + The fluorescence intensity of the derivative at the 546 nm emission peak after the reaction, I 654 I represents the fluorescence intensity of CdTe against a red background at the emission peak at 654 nm. 546 / I 654 Using the ratio of fluorescence intensity between the two as an indicator, the presence or absence of interfering substances is compared and analyzed. The ratio of fluorescent probes to NH4+ is used in this study. + The impact of the response. The experiment was repeated three times, and the average value was taken. Origin 2021 software was used for data processing and error analysis.

[0032] like Figure 8 As shown in (a), the probe of the present invention is used for NH4 + It has a strong response, and to Na + K + Mg 2+ Ca 2+ Fe 2+ Zn 2+ Cl - NO2 -NO3 - PO4 3- CO3 2- SO4 2- Common interfering ions generally show no fluorescence response. Their selectivity and anti-interference capabilities are comparable to those of... Figure 8 As shown in (b), this probe system is only effective against NH4. + It exhibits a fluorescent response; 250 μM NH4+ is then added to the probe containing the interfering ions. + The fluorescence intensity recovered to the level achieved with only the addition of NH4. + The fluorescence intensity at that time indicates that the fluorescent probe of the present invention is sensitive to NH4. + It exhibits excellent selectivity and strong resistance to interference from other ions.

[0033] Application Example 7 Detection of ammonium nitrogen in actual soil samples: Dissolve 74.55g of potassium chloride in deionized water and bring the volume to 1L to prepare a 1M potassium chloride solution. Weigh 5g of each of the two farmland soil samples, add 25mL of the 1M potassium chloride solution to each, and shake to extract NH4 from the soil. + Two types of soil sample extracts were obtained. These two soil sample extracts were then tested using the ratiometric fluorescent test paper prepared in Example 1. Heating conditions were provided by a self-heating heat pack. The test results are as follows: Figure 9 As shown below, compare with the standard solution colorimetric card ( Figure 9 By observing the sample extract (see above), the concentration of ammonium nitrogen in the extract can be read as approximately 50 µM and 200 µM. This demonstrates that low-cost and universally applicable on-site detection of soil ammonium nitrogen can be achieved without the need for large, specialized, and precise instruments.

[0034] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0035] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A ratiometric fluorescent probe for on-site detection of nitrogen in farmland, characterized in that, It includes System 1 and System 2, wherein System 1 is a CdTe quantum dot solution and System 2 is a mixed solution of 3,4-thiophene dicarboxaldehyde solution and Na2SO3 solution.

2. The ratiometric fluorescent probe for on-site detection of nitrogen in farmland according to claim 1, characterized in that, The volume ratio of system 1 to system 2 is 0.8-1.6:

1.

3. The ratiometric fluorescent probe for on-site detection of nitrogen in farmland according to claim 1, characterized in that, In system 2, the volume ratio of 3,4-thiophene dicarboxaldehyde solution to Na2SO3 solution is 0.25-5:1; in system 2, the concentration of 3,4-thiophene dicarboxaldehyde solution is 0.02-0.07M, and the concentration of Na2SO3 solution is 0.01-0.1M.

4. A method for preparing a ratiometric fluorescent probe for on-site detection of nitrogen in farmland as described in any one of claims 1-3, characterized in that, It includes the following steps: mixing 3,4-thiophene dicarboxaldehyde solution and Na2SO3 solution to form system 2, and then mixing system 1 with system 2.

5. A ratiometric fluorescent test strip, characterized in that, It is obtained by immersing non-fluorescent filter paper in a ratio fluorescent probe for on-site detection of nitrogen in farmland as described in any one of claims 1-3, and then drying it.

6. The application of the ratio fluorescent test strip as described in claim 5 in the field detection of nitrogen in farmland.

7. The application according to claim 6, characterized in that, The application The procedure includes the following steps: taking soil sample extract and dropping it onto the ratiometric fluorescent test paper, heating it, and observing the fluorescence color under a UV lamp, comparing it with the standard NH4. + The concentration of ammonium nitrogen in a soil sample can be obtained by observing the fluorescence response color of the solution.

8. The application according to claim 7, characterized in that, The heating is performed at 20-70℃ for 15 minutes.

9. The application according to claim 7, characterized in that, The ultraviolet lamp is a 365nm portable ultraviolet lamp.

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