A ratiometric fluorescent paper-based sensor for in situ visual detection of soil ammonium nitrogen

Abstract

The present invention relates to a ratio fluorescence paper-based sensor for in-situ visual detection of soil ammonium nitrogen, which is used for on-site real-time monitoring of soil ammonium nitrogen status. In the presence of potassium sulfite, 1,2-phthalaldehyde can be combined with NH4 + A derivatization reaction occurs at room temperature to generate a derivative with blue fluorescence, and a self-reference background fluorescent material (red CdTe quantum dots) is introduced to construct a ratiometric fluorescence paper-based sensor. Ratiometric fluorescence detects NH4 based on the change in fluorescence intensity ratio. + , can eliminate the interference of various environmental factors, and at the same time has a multi-level color change effect to make data interpretation more accurate and more conducive to naked eye visual detection. The present invention can monitor the real-time status of soil ammonium nitrogen as conveniently as the use of pH test paper, for NH4 + The detection provides a very effective and practical technical means, and has good application prospects in the field of rapid detection of ammonium nitrogen in soil.

Description

Technical Field

[0001] The invention relates to a ratiometric fluorescent paper-based sensor for in-situ visual detection of soil ammonium nitrogen, which is used for on-site real-time monitoring of the status of soil ammonium nitrogen. Background Art

[0002] Ammonium nitrogen (NH4 + ) is one of the main forms of effective nitrogen in the soil and is an important indicator for crop growth and development, nutritional management, and production environment monitoring. On the one hand, nitrogen is a large amount of nutrient elements necessary for crop growth and development. On the other hand, the application of large amounts of chemical nitrogen fertilizers in agricultural production has also exacerbated environmental problems such as eutrophication caused by nitrogen pollution. Therefore, it is an urgent task to develop a low-cost, universal, real-time sensing technology for soil ammonium nitrogen. At present, soil ammonium nitrogen (NH4 + The standard method for determining the content of ) is to extract fresh soil samples with potassium chloride solution, and the NH4 + Under alkaline conditions and the presence of hypochlorite ions, it reacts with phenol to generate a water-soluble dye indigo blue, which is then detected by spectrophotometry. In addition, the traditional chemical colorimetric method for ammonium nitrogen detection uses Nessler reagent (potassium mercury iodide) to react with different concentrations of NH4 + The effect changes from yellow to yellow-brown. This method is easily interfered by metal ions, sulfides and other factors, and mercury is harmful to human body and environment. + The detection has gradually developed the high performance liquid chromatography (HPLC) column derivatization method, which is detected by the HPLC fluorescence detector, with strong specificity and high sensitivity. In addition, other NH4 + Detection methods such as electrochemical method and bio-enzymatic method are also relatively common. The main problem with traditional detection methods is that the detection equipment is expensive and bulky, and is only suitable for laboratory testing, and cannot meet the needs of on-site and real-time detection; and the detection process is cumbersome, and its operation, calculation and interpretation require professionals, making it difficult to apply universally and at low cost. The biggest problem in actual soil ammonium nitrogen detection is the low natural concentration and the complexity of the sample. The pH and the types and contents of interfering substances are different in different environments, so the requirements for high sensitivity, selectivity and anti-interference ability of the detection method are getting higher and higher. With the development of national high-standard farmland construction and production area health assessment work, there is an urgent need for the development and commissioning of simplified and rapid detection technology and sensors for soil ammonium nitrogen. Summary of the Invention

[0003] In order to solve the above problems in the prior art, the present invention provides a method for rapid online detection of soil ammonium nitrogen (NH4 + ) ratio fluorescence paper-based sensor. In the presence of potassium sulfite, 1,2-benzenedicarboxaldehyde can react with NH4 +A derivatization reaction occurs at room temperature to generate a derivative with blue fluorescence. A ratiometric fluorescence sensor is constructed by introducing a self-reference background fluorescent material (CdTe quantum dots). Ratiometric fluorescence detects NH4 based on the change in fluorescence intensity ratio. + , which can eliminate interference from various environmental factors, and has a multi-stage color change effect to make data interpretation more accurate and more conducive to naked eye visualization detection, and the ratio fluorescence solution is evenly fixed on the non-fluorescent filter paper to obtain a paper-based sensor. + The extract was dropped onto the paper-based sensor to react (response time was less than 2 minutes). Under 365nm UV light, the fluorescence color of the paper-based sensor changed with NH4 + Concentration is directly related to the reference standard NH4 + The corresponding content can be read from the solution's color response. This eliminates the need for large instruments and enables rapid, low-cost, and universal detection of ammonium nitrogen in soil and water, regardless of time or location.

[0004] Specifically, the present invention provides a ratiometric fluorescent paper-based sensor for in-situ visual detection of soil ammonium nitrogen. The sensor is obtained by uniformly immobilizing a ratiometric fluorescent mixed solution on non-fluorescent filter paper. The ratiometric fluorescent mixed solution comprises a CdTe quantum dot solution (system 1), a 1,2-benzenedicarboxaldehyde solution, and a K2SO3 solution. The ratiometric fluorescent mixed solution can be prepared by separately preparing a 1,2-benzenedicarboxaldehyde solution and a K2SO3 solution, then mixing them to form system 2, which is then mixed with system 1. Among them, the concentration of the 1,2-benzenedicarboxaldehyde solution is 0.015-0.025M, preferably 0.016-0.020M, more preferably 0.018M, the concentration of the K2SO3 solution is 0.010-0.020M, preferably 0.012-0.018M, more preferably 0.015M, the 1,2-benzenedicarboxaldehyde solution and the K2SO3 solution are mixed in a volume ratio of 1.2-1.5:1, preferably 1.3-1.4:1 to form system 2; the volume ratio of system 2 to system 1 is 2.2-2.8:1, preferably 2.4-2.5:1. The 1,2-benzenedicarboxaldehyde solution and the K2SO3 solution are prepared and used immediately or sealed and stored in a dark place at 4°C. The ratiometric fluorescent paper-based sensor of the present invention can be used for the detection of ammonium nitrogen in soil. In a specific application, the soil sample extract is taken and dripped onto the ratiometric fluorescent paper-based sensor, and the fluorescence color change is observed under a 365nm ultraviolet lamp, and compared with the standard NH4 + The concentration of ammonium nitrogen in the soil extract can be read by the response color of the solution.

[0005] In practice, fluorescence spectrophotometer is commonly used to measure NH4 +The method requires the cooperation of instruments and equipment such as spectrophotometers and takes a long time to detect, which is not suitable for the requirements of on-site rapid detection. On the other hand, there is also a method that obtains ammonia gas by reacting ammonium ions with strong bases and then captures the ammonia gas for detection. The present invention can realize the on-site rapid and direct detection of NH4 + The test paper is used for qualitative and semi-quantitative detection, and the fluorescence color is significant, which can be quickly judged by naked eyes. Specifically, the present invention prepares a ratio fluorescence test paper by combining CdTe red fluorescence background with 1,2-benzenedialdehyde and K2SO3 system to detect NH4 in soil extract. + , no need to add strong alkali to generate ammonia, which is more conducive to the safe promotion and application of products. In actual use, only the NH4 + Drop the soil extract on the test paper and let it react for 2 minutes. Observe the change in the fluorescence color of the test paper under a 365nm ultraviolet lamp and compare it with the standard color card to read the soil ammonium nitrogen concentration. The invention has a short reaction time and can quickly detect soil ammonium nitrogen without the help of other instruments and equipment. The ratio fluorescence technology used to add a CdTe red background can better overcome the influence of the external environment on the experiment. The color change of the test paper is very obvious and can be easily read with the naked eye. In addition, the inventors found that K2SO3 has stronger fluorescence stability than Na2SO3 in the detection of actual soil samples. Under strong ultraviolet light, the relatively stable fluorescence color of the test paper will be more conducive to the accurate reading of the data. And by optimizing the reaction concentration of 1,2-benzenedicarboxaldehyde and K2SO3, the test paper has a higher detection sensitivity during detection.

[0006] In the present invention, although the self-reference background fluorescent material CdTe quantum dots has a reference preparation method, due to different process conditions during preparation, CdTe quantum dots with different quantum levels or properties may be obtained. The CdTe quantum dots (about 4nm) obtained by the specific process of the present invention have strong red fluorescence under 365nm ultraviolet light. In addition, the present invention obtains NH4 with a usage style similar to pH test paper by selecting a specific concentration ratio of 1,2-benzenedialdehyde and K2SO3 and selecting the ratio of system 1 and system 2. + Test strips (such as Figure 8 ), in order to quickly and real-timely grasp the NH4 + The situation provides great convenience and also provides a basis for the subsequent NH4 + It provides a technical basis for the development of monitoring sensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 Comparative photos of CdTe background fluorescent material (system 1) under natural light (a) and 365nm ultraviolet light (b); under natural light (a) there is basically no color, while under ultraviolet light (b) there is a strong red color.

[0008] Figure 2 Transmission electron microscopy (TEM) image of CdTe.

[0009] Figure 3 System 2 and NH4 + Comparative photos of the fluorescent derivative after the reaction (taking 250 μM as an example) under natural light (a) and 365 nm ultraviolet light (b); under natural light (a) the derivative is essentially colorless, while under ultraviolet light (b) it has a strong blue color.

[0010] Figure 4 Under 365nm ultraviolet light irradiation, the fluorescence stability of soil extracts was determined using different probe systems (K2SO3 and Na2SO3).

[0011] Figure 5 Effects of different concentrations of OPA (a) and K2SO3 (b) on NH4 + The effect of the fluorescence intensity of the derivatives.

[0012] Figure 6 Effect of different pH on the fluorescence intensity ratio of the ratio probe.

[0013] Figure 7 Test of probe selectivity and anti-interference ability.

[0014] Figure 8 The ratiometric fluorescence paper-based sensor was used to detect different concentrations of NH4 under 365nm UV light. + The fluorescence color changes (above), from left to right with NH4 + As the concentration increases, the color gradually changes from red, pink, purple, and blue; the fluorescence color development of the two soil sample extracts dropped on the paper-based sensor (under 365nm ultraviolet light) (bottom). DETAILED DESCRIPTION

[0015] The following embodiments are further descriptions of the present invention to illustrate the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.

[0016] Example 1

[0017] The specific steps of the present invention are as follows:

[0018] (1) System 1 (CdTe red background fluorescent material): Under nitrogen protection, 0.1g tellurium powder and 0.07g sodium borohydride were mixed in 4mL deionized water and reacted in an ice-water bath to prepare NaHTe solution. 0.11g CdCl2·2.5H2O and 75μL mercaptopropionic acid were dissolved in 50mL deionized water, the pH was adjusted to 8.5 with NaOH solution, and the freshly prepared NaHTe solution was added under nitrogen protection. After stirring at room temperature for 20 minutes, it was refluxed at 100℃ for 10h under nitrogen protection. The reaction product was stored in a refrigerator at 4℃ in the dark. Before use, the CdTe quantum dot stock solution was diluted with 11 times the same volume of water to form System 1. Photos of CdTe quantum dots under natural light and ultraviolet light are shown in Figure 1. Figure 1 As shown. The transmission electron microscope image (TEM) of CdTe is as follows Figure 2 As shown, its morphology is a granular structure with relatively uniform distribution and a particle size of about 4 nm.

[0019] (2) System 2: Mix 1,2-benzenedialdehyde (0.018M) and K2SO3 (0.015M) in a volume ratio of 1.35:1. These two solutions can be used immediately after preparation. If they are to be used later, they must be sealed and stored in a dark place at 4°C. This system can specifically identify NH4 + The intensity of the blue fluorescence of the generated fluorescent derivative is similar to that of NH4 + The concentration is directly related to 250μM NH4 + For example, the photos of fluorescent derivatives under natural light and ultraviolet light are as follows Figure 3 shown.

[0020] (3) Ratio fluorescence paper-based sensor: 68 μL of system 1 and 165 μL of system 2 were mixed evenly, and the ratio fluorescence mixed solution was evenly fixed on non-fluorescent filter paper to obtain a paper-based sensor.

[0021] (4)NH4 + Standard solution: Prepare a 5mM ammonium nitrogen standard stock solution with NH4Cl and store it at 4℃; dilute the ammonium nitrogen standard solution with different concentrations. + The fluorescence color changes as Figure 8 Shown (above).

[0022] (5) Soil extraction and case detection: Dissolve 74.55g of potassium chloride in deionized water and dilute to 1L. Weigh 40g of two soil samples respectively and take 200ml of potassium chloride solution to extract NH4 + , to be tested. Example: Take the soil sample extract extracted with potassium chloride and use the prepared ratio fluorescence paper-based sensor to detect the soil extract. The results are as follows Figure 8 (below) shows the control standard NH4+ The response color of the solution ( Figure 8 The ammonium nitrogen concentration of the sample extract can be read as approximately 25 and 100 μM. This allows for low-cost, universal, and rapid detection of soil ammonium nitrogen at any time and in any location, without the need for large instruments.

[0023] Figure 4 This figure shows the fluorescence stability of soil extracts measured using different probe systems (K2SO3 and Na2SO3) under 365nm UV light. The upper curve represents the K2SO3 system, while the lower curve represents the Na2SO3 system. Comparing the determination of soil ammonium nitrogen using 1,2-benzenedicarboxaldehyde (OPA) in the presence of K2SO3 and Na2SO3, it was found that K2SO3 exhibited greater fluorescence stability than Na2SO3 under 365nm UV light. Stronger stability enhances detection reliability and accuracy in probe solutions and ratiometric test strips. Figure 5 Effects of different concentrations of OPA (a) and K2SO3 (b) on NH4 + The effect of the fluorescence intensity of the derivatives (its emission peak is at 445nm). + Taking 445 nm as an example, we compared the changes in fluorescence intensity at the 445 nm emission peak of different concentrations of OPA (Figure a) and different concentrations of K2SO3 (Figure b). The results showed that when the OPA concentration was 18 mM and the K2SO3 concentration was 15 mM, the detection results were the best, which was the optimal reaction ratio. Figure 6 The figure shows the effect of different pH values ​​on the fluorescence intensity ratio during ratiometric probe sensing. As shown in the figure, the pH value was adjusted from 2 to 11. The results show that the fluorescence intensity ratio of the ratiometric probe remains stable within the pH range of 4-11. Since the pH of conventional soils ranges from 4-9, this solution is fully capable of detecting conventional soils. Figure 7 (a) Ratio fluorescence probe for NH4 + and fluorescence spectra of various interfering ions, (b) Study on the selectivity and anti-interference ability of the probe (I 445 NH4 + The fluorescence intensity of the derivative after the reaction at the emission peak of 445 nm, I 639 is the fluorescence intensity of the CdTe red background at the 639nm emission peak, I 445 / I 639 is the fluorescence intensity ratio of the two). As shown in Figure a, the probe is sensitive to NH4 + There is a strong response to Na + ,K + ,Mg 2+ ,Ca 2+ ,Fe 2+ ,Cu 2+,Cl-,NO2 - ,NO3-,PO4 3- ,CO3 2- ,SO4 2- The possible coexisting interfering ions have basically no fluorescence response. The selectivity comparison is shown in Figure b, which shows that other interfering ions have basically no effect on the application of the probe. In the probe that has already been added with interfering ions, 250μM NH4 + The fluorescence intensity is the same as that of only adding NH4 + This proves that the fluorescent probe has good anti-interference performance against other ions.

[0024] The invention discloses a method for rapid in-situ detection of ammonium nitrogen (NH4 + ) fluorescent paper-based sensor, the reaction fluorescence color effect is very obvious, and has the characteristics of specificity, high sensitivity and high stability. Compared with other ammonium root detection methods, it is simpler, milder and safer, and can detect NH4 at room temperature. + It has a very fast and stable response time of less than 2 minutes. By constructing a ratio fluorescence sensor, it overcomes the difficulty in distinguishing different concentrations of NH4 when there is no red CdTe fluorescence background. + After reacting with system 2, the fluorescence color of the derivative changes from light blue to strong blue, and the ratio fluorescence sensor can achieve a wider fluorescence color change, from red, pink, purple, blue to strong blue, which is conducive to the accurate reading of the concentration of the sample to be tested. The present invention evenly fixes the ratio fluorescence mixed solution on non-fluorescent filter paper to obtain a paper-based sensor, and the detection cost is less than 1 yuan per time. It can be directly identified and detected with the naked eye under ultraviolet light, without the need for complex analytical equipment. Only a portable ultraviolet lamp worth about 20 yuan is needed to complete the detection, which greatly reduces the detection cost and significantly improves the stability. By reacting with NH4 + The concentration of ammonium nitrogen to be tested can be read by comparing with the fluorescence colorimetric card calibrated with the standard solution. The operation and interpretation do not require professionals, and can meet the low-cost, universal, and in-situ online detection of ammonium nitrogen. The present invention can monitor the real-time status (qualitative and semi-quantitative) of soil ammonium nitrogen in a convenient manner similar to the use of pH test paper, which is NH4 + The detection provides a very effective and practical technical means, and has good application prospects in the field of rapid detection of ammonium nitrogen in soil and water.

[0025] It should be noted that the technical contents of the present invention described above are only for the purpose of explaining and illustrating the technical essence of the present invention to enable those skilled in the art to understand the technical essence of the present invention. Therefore, the technical contents described above are not intended to limit the substantial protection scope of the present invention. The substantial protection scope of the present invention shall be based on the claims. Those skilled in the art should be aware that any modifications, equivalent substitutions, and improvements based on the substantial spirit of the present invention shall fall within the substantial protection scope of the present invention.

Claims

1. A ratiometric fluorescent paper-based sensor for in-situ visual detection of soil ammonium nitrogen, comprising a ratiometric fluorescent mixed solution uniformly fixed on non-fluorescent filter paper; the ratiometric fluorescent mixed solution comprises system 1, a 1,2-benzenedicarboxaldehyde solution, and a K2SO3 solution, wherein system 1 is a CdTe quantum dot solution; the 1,2-benzenedicarboxaldehyde solution has a concentration of 0.015-0.025 M, the K2SO3 solution has a concentration of 0.010-0.020 M, and the 1,2-benzenedicarboxaldehyde solution and the K2SO3 solution are mixed in a volume ratio of 1.2-1.5:1 to form system 2; the volume ratio of system 2 to system 1 is 2.2-2.8:

1.

2. The ratiometric fluorescent paper-based sensor according to claim 1, wherein: The ratio fluorescent mixed solution is prepared by the following method: 1,2-benzenedicarboxaldehyde solution and K2SO3 solution are prepared separately, and then mixed to form system 2, and then system 2 is mixed with system 1.

3. The ratiometric fluorescent paper-based sensor according to claim 1, wherein: The concentration of the 1,2-benzenedicarboxaldehyde solution is 0.016-0.020 M, the concentration of the K2SO3 solution is 0.012-0.018 M, the volume ratio of the 1,2-benzenedicarboxaldehyde solution to the K2SO3 solution is 1.3-1.4:1; the volume ratio of system 2 to system 1 is 2.4-2.5:

1.

4. The ratiometric fluorescent paper-based sensor according to claim 1, wherein: The 1,2-benzenedicarboxaldehyde solution and K2SO3 solution are prepared and used immediately or sealed and stored in a dark place at 4°C.

5. Use of the ratiometric fluorescent paper-based sensor according to any one of claims 1 to 4 in detecting ammonium nitrogen in soil.

6. The use according to claim 5, characterized in that Take the soil sample extract and drop it onto the ratio fluorescence paper-based sensor. Observe the color under UV light and compare it with the standard NH4 + The response color of the solution.

7. The use according to claim 6, characterized in that The soil sample extract is a potassium chloride solution to extract NH4 + And get.

8. The use according to claim 6, characterized in that The ultraviolet lamp is a 365 nm portable ultraviolet lamp.

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