A novel water-soluble chitosan fluorescent probe and its application in detection of ClO - and NO2 - ions

Abstract

The application discloses a novel water-soluble chitosan fluorescent probe and application of the probe in ClO ‑ and NO2 ‑ ion detection. The water-soluble chitosan fluorescent probe has the structural formula as shown in the following. The water-soluble chitosan fluorescent probe can realize qualitative and quantitative detection of ClO ‑ and / or NO2 ‑ ions by a fluorescence spectrum method, and has good anti-interference, high selectivity and sensitivity in the presence of other analytes; meanwhile, the probe can be applied to test paper detection, and has great potential in the detection of ClO ‑ and NO2 ‑ ions.

Description

Technical Field

[0001] The present invention belongs to the field of ion detection technology, and specifically relates to a novel water-soluble chitosan fluorescent probe and its - and NO2 - Applications in ion detection. Background Art

[0002] Nitrite is one of the most common nitrogen-containing compounds widely present in the human environment and plays an important role in the production of organic synthesis, agricultural chemicals, pharmaceuticals and many other industrial products. However, nitrite is also a food poison and water pollutant that is harmful to humans and aquatic life. Excessive nitrite in water can cause fish and other aquatic life to die from lack of oxygen. Nitrite in food is generally found in meat products, preserved foods and drinking water. Because nitrite can interact with amines in the digestive tract, when people consume foods containing nitrite, highly carcinogenic N-nitrosamine compounds are produced. Therefore, the development of a simple and effective nitrite detection technology is crucial to public health safety.

[0003] Hypochlorous acid (HOCl) is a crucial reactive oxygen species (ROS) in living systems, playing a dual role in life. On the one hand, it kills viruses and bacteria, ensuring human health. On the other hand, excessive HOCl can oxidize biomolecules, leading to tissue damage, inflammation, and various diseases. Therefore, studying the generation, regulation, and mechanisms of action of HOCl, as well as developing highly selective and sensitive detection methods, is crucial for further understanding its role in living organisms and the mechanisms of disease.

[0004] Traditional detection methods require complex pretreatment processes, expensive instrumentation, and lengthy detection times, failing to meet the demands of rapid detection. Spectroscopic methods, on the other hand, offer rapid response, ease of operation, and low cost. Consequently, the use of fluorescent or colorimetric probes for the qualitative and quantitative detection of ions has become a research hotspot. While organic small molecule fluorescent probe detection methods offer ease of operation, high sensitivity, ease of observation, good selectivity, and rapid detection, they cannot be directly detected in the aqueous phase due to the low solubility of organic compounds in water. Detection requires mixing an organic reagent with water. Consequently, the pretreatment process for ion detection is cumbersome, and the addition of organic reagents can also cause secondary environmental pollution.

[0005] To avoid the above-mentioned drawbacks, we are eager to develop new fluorescent probes that can detect ions in pure water. Chitosan contains a large number of free amino and hydroxyl groups, which are easy to modify and functionalize. It also has excellent properties such as biodegradability and biocompatibility. Chitosan has been widely used in the field of functional materials due to its low cost and environmental performance. It is also widely used in various fields due to its good biological properties. Therefore, it is necessary to provide a new fluorescent probe for detecting ClO. - and NO2 - Ionic water-soluble chitosan fluorescent probes and their preparation methods and applications are issues that need to be urgently addressed by those skilled in the art. Summary of the Invention

[0006] The present invention aims to provide a novel water-soluble chitosan fluorescent probe and its - and NO2 - Application in ion detection. The technical problem to be solved is to synthesize molecules that can detect ClO - and NO2 - A new water-soluble chitosan fluorescent probe for ions.

[0007] The novel water-soluble chitosan fluorescent probe of the present invention has the following structural formula:

[0008] ;

[0009] Where x=23, y=117.

[0010] The values ​​of x and y are calculated based on the H NMR spectrum area ratio of the probe synthesized at the optimal feed ratio. Under the same detection effect, the larger the x / y ratio, the lower the concentration of the probe used.

[0011] The preparation method of the novel water-soluble chitosan fluorescent probe of the present invention comprises the following steps:

[0012] 300 mg of chitosan was dissolved in 15 mL of 1% aqueous acetic acid, and TTG (4.45 mg, 0.147 mmol) dissolved in DMSO was added. The reaction was heated under reflux at 65°C for 48 hours. After cooling, the reaction solution was dialyzed against DMSO for 72 hours to remove unreacted TTG. The solution was then dialyzed against distilled water (MWCO: 3500 Da) for 3 days, with the dialyzate replaced every 12 hours. Finally, the product was freeze-dried to obtain the target product, TTGN.

[0013] The synthetic route is as follows:

[0014] .

[0015] The invention provides a water-soluble chitosan fluorescent probe for use in preparing a detection reagent capable of qualitatively or quantitatively detecting ClO- and / or NO2 - .

[0016] Furthermore, fluorescence spectroscopy was performed in an aqueous medium to detect ClO by changes in fluorescence intensity. - and / or NO2 - Qualitative or quantitative detection of ions, with strong anti-interference ability for a variety of analytes.

[0017] The aqueous medium is a 1% acetic acid aqueous solution.

[0018] The novel water-soluble chitosan fluorescent probe is used as a detection reagent for ClO - When ion detection is used, the detection limit is 0.59 μM; and for NO2 - When ion detection was used, the detection limit was 0.92 μM.

[0019] In the detection system, when the system pH is ≤2, add ClO - and NO2 - After that, the fluorescence was quenched and the fluorescence intensity decreased significantly. When the pH of the system was adjusted to 4-12, only when ClO was added - Fluorescence quenching, adding NO2 - The fluorescence intensity is slightly reduced. Therefore, the pH of the detection system can be adjusted to achieve the effect of ClO - and NO2 - Distinguish between the two ions.

[0020] The novel water-soluble chitosan fluorescent probe of the present invention can be used to prepare test strips for use, and can be observed under a 365nm ultraviolet lamp to detect ClO by changes in the intensity and color of the fluorescence. - and / or NO2 - Qualitative detection of .

[0021] The beneficial effects of the present invention are embodied in:

[0022] The water-soluble chitosan fluorescent probe of the present invention can realize the detection of ClO by fluorescence spectroscopy. - and / or NO2 - The water-soluble chitosan fluorescent probe of the present invention can be used to detect ClO in a medium similar to pure water (1% acetic acid aqueous solution). - and / or NO2 - Rapid identification and quantitative detection of ions, and ClO - and / or NO2 - Ion recognition has high selectivity and good anti-interference ability, and the obvious color change phenomenon can be recognized by naked eyes. At the same time, the present invention can be applied to test paper detection, in the detection of trace ClO - and / or NO2 -Ions show great potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The nuclear magnetic resonance hydrogen spectra of the water-soluble chitosan fluorescent probe of the present invention and its precursor aldehyde TTG are shown.

[0024] Figure 2 The infrared spectra of the water-soluble chitosan fluorescent probe and chitosan of the present invention are shown in Figure 1. A is chitosan; b is the water-soluble chitosan fluorescent probe of the present invention.

[0025] Figure 3 The fluorescence emission spectra of the water-soluble chitosan fluorescent probe of the present invention when different analytes were added to a 1% acetic acid aqueous solution (λex=338 nm).

[0026] Figure 4 The water-soluble chitosan fluorescent probe of the present invention is used to detect ClO under different pH conditions. - and NO2 - Fluorescence intensity comparison of ion recognition (λex=338nm).

[0027] Figure 5 Figure a shows the water-soluble chitosan fluorescent probe of the present invention in a 1% acetic acid aqueous solution with 0-22.5 μM ClO ion concentration added. - ion after the fluorescence emission spectrum (λex = 338nm); Figure b shows the fluorescence intensity of the fluorescent probe at a fluorescence emission wavelength of 550nm and the ClO - Linear relationship with ion concentration.

[0028] Figure 6 Figure a shows the water-soluble chitosan fluorescent probe of the present invention in a 1% acetic acid aqueous solution with 0-35μM NO2 ion concentration added. - ion after the fluorescence emission spectrum (λex = 338nm); Figure b shows the fluorescence intensity of the fluorescent probe at the fluorescence emission wavelength of 550nm and NO2 - Linear relationship with ion concentration.

[0029] Figure 7 Figure a is a histogram showing the anti-interference of the water-soluble chitosan fluorescent probe of the present invention when different metal ions are added to a 1% acetic acid aqueous solution; Figure b is a histogram showing the anti-interference of the fluorescent probe when different non-metal ions are added to a 1% acetic acid aqueous solution; represents the fluorescence intensity of the fluorescent probe when different analytes were added to 1% acetic acid aqueous solution (λex = 338 nm); Representative fluorescent probe compounds were added to 1% acetic acid aqueous solution with different analytes and ClO - Fluorescence intensity of ions (λex=338nm). Fluorescent probe compound in 1% acetic acid solution with different analytes and NO2 - Fluorescence intensity of ions (λex= 338 nm).

[0030] Figure 8 Pictures of test paper made of the water-soluble chitosan fluorescent probe of the present application under 365 nm ultraviolet light when different ions were added.

[0031] Figure 9 Pictures of test paper made of the water-soluble chitosan fluorescent probe of the present application under 365 nm ultraviolet light when NaOH was added first and then ClO - and NO2 - .

[0032] Figure 10 Pictures of test paper made of the water-soluble chitosan fluorescent probe of the present application under 365 nm ultraviolet light when different concentrations of ClO - ions were added.

[0033] Figure 11 Pictures of test paper made of the water-soluble chitosan fluorescent probe of the present application under 365 nm ultraviolet light when different concentrations of NO2 - ions were added. DETAILED DESCRIPTION

[0034] The present application can be further illustrated by the following examples, but is not limited to the examples only.

[0035] Example 1: Preparation and characterization of water-soluble chitosan fluorescent probe

[0036] After 300 mg of chitosan was dissolved in 15 mL of 1% acetic acid solution, TTG (0.045 g, 0.147 mmol) was added, and the reaction was heated to reflux at 65°C for 48 h. After cooling, the reaction solution was dialyzed in DMSO solution for 72 h, and the unreacted TTG was removed. Then, the product was dialyzed in distilled water for 3 days (MWCO: 3500 Da), and the dialysis solution was replaced every 12 h. Finally, the product was freeze-dried to obtain the target product 220 mg, and the yield of the target product TTGN was 73%.

[0037] Figure 1 NMR hydrogen spectrum of the water-soluble chitosan fluorescent probe of the present application and its precursor aldehyde. There is no peak of aldehyde H around 10 ppm in the NMR hydrogen spectrum of TTGN, indicating that the unreacted TTG has been completely removed by dialysis. The peak at 8-9 ppm in the NMR hydrogen spectrum of the probe represents the H on the benzene ring, proving the successful synthesis of the product.

[0038] Figure 2The infrared spectra of the water-soluble chitosan fluorescent probe TTGN and raw chitosan of the present invention are shown in Figure 1. The characteristic absorption peak of chitosan is 3430 cm -1 (stretching vibrations of NH and OH), 2930 cm -1 and 2870 cm -1 (Stretching vibration of CH). Compared with the starting material, the product has a 3430 cm -1 The peaks around 620 cm -1 The CH bending vibration peaks of the benzene ring appeared on the left and right, proving the successful synthesis of the new water-soluble chitosan fluorescent probe.

[0039] Example 2: Fluorescent probe of water-soluble chitosan for ClO - and NO2 - Specific response

[0040] Accurately weigh a certain amount of the new water-soluble chitosan fluorescent probe and prepare a probe standard solution with a concentration of 0.03 g / L using 1% acetic acid aqueous solution. Take 2 mL of the probe solution in a quartz cuvette, and then add 20 μL of a 1.0×10 -2 mol / L of various analytes (Blank, ClO - , NO2 - , NO3 - , HS - , S2O3 2- , SO4 2- ,H2O2,Hcy,Cys,GSH,SCN - , Cl - , Br - , I - , CO3 2- ,HCO3 - , SO3 2- , HSO3 - , CH3COO - , Fe 3+ , Ca 2+ , Cu 2+ , Al 3+ , Mg 2+ , Ag + , K + , Na + , Ba 2+ , Cr 3 + , Hg 2+ , Zn 2+ , Fe 2+ , Pb 2+ ) aqueous solution, shake well and measure the fluorescence spectrum of the solution. Figure 3As shown, it can be seen that the probe can specifically recognize ClO - and NO2 - , and has almost no response to other ions. The results show that the fluorescent probe is sensitive to ClO - and NO2 - Highly selective.

[0041] Example 3: Fluorescent probe of water-soluble chitosan for ClO under different pH conditions - and NO2 - Ion identification

[0042] The water-soluble chitosan fluorescent probe of the present invention was weighed and dissolved in 1% acetic acid aqueous solution to prepare a 0.3 g / L probe solution. 200 μL of the probe solution and 1.8 mL of buffer solutions of different pH values ​​and 20 μL of a 1.0×10 -2 mol / L of various analytes (Blank, ClO - , NO2 - ) aqueous solution, shake well and measure the fluorescence spectrum of the solution. Figure 4 As shown in the figure, it can be seen that in the 1% acetic acid aqueous solution of the probe, when the pH of the system is ≤ 2, ClO - and NO2 - After that, the fluorescence was quenched and the fluorescence intensity decreased significantly. When the pH of the probe aqueous solution was adjusted to 4-12, only when ClO was added - Fluorescence quenching, adding NO2 - The fluorescence intensity is slightly reduced. Therefore, the pH of the probe solution can be adjusted to achieve the effect of ClO - and NO2 - Distinguish between the two ions.

[0043] Example 4: Fluorescence intensity of water-soluble chitosan fluorescent probe and ClO - Correlation of concentration

[0044] Weigh the water-soluble chitosan fluorescent probe of the present invention and dissolve it in 1% acetic acid aqueous solution to prepare 0.03g / L probe solution. Add the probe solution dropwise to each test tube and take ClO - (0-22.5 μM) was dropped into the test tube above. The fluorescence spectra of these test solutions were measured at 550 nm (e.g. Figure 5 As shown in Figure 2, the fluorescence intensity of the probe gradually weakened with the increase of solution concentration. The fluorescence intensity of the probe (0.03 g / L) and ClO - (0-22.5 μM) has a good linear relationship R 2 = 0.995, which shows that the fluorescent probe can quantitatively detect ClO in the fluorescence spectrum. -According to LOD=3σ / k, the fluorescence probe is calculated to be ClO in the fluorescence spectrum. - The detection limit is 0.59 μM. Where σ is the standard deviation of blank measurements, k is the fluorescence intensity relative to ClO - The slope of the concentration graph.

[0045] Example 5: Fluorescence intensity of the new water-soluble chitosan fluorescent probe and NO2 - Correlation of concentration

[0046] Weigh the water-soluble chitosan fluorescent probe of the present invention and dissolve it in 1% acetic acid aqueous solution to prepare 0.03 g / L probe solution. - (0-35 μM) was dropped into the test tube above. The fluorescence spectra of these test solutions were measured at 550 nm (e.g. Figure 6 As shown in Figure 2, the fluorescence intensity of the probe gradually weakened with the increase of solution concentration. The fluorescence probe (0.03 g / L) and NO2 - (0-35μM) has a good linear relationship R 2 = 0.993, which shows that the fluorescent probe can quantitatively detect NO2 in the fluorescence spectrum. - According to LOD=3σ / k, the fluorescence probe is calculated to be NO2 in the fluorescence spectrum. - The detection limit is 0.92 μM. Where σ is the standard deviation of the blank measurement value, k is the fluorescence intensity of NO2 - The slope of the concentration graph.

[0047] Example 6: Fluorescent probe of water-soluble chitosan for ClO - and NO2 - Anti-interference of ion recognition

[0048] The water-soluble chitosan fluorescent probe of the present invention was weighed and dissolved in 1% acetic acid aqueous solution to prepare a 0.03 g / L probe solution. 2 mL of the probe solution and 20 μL of a 1.0×10 -2 mol / L of various analytes (Blank, ClO - , NO2 - , NO3 - , HS - , S2O3 2- , SO4 2- , H2O2, Hcy, Cys, GSH, SCN - , Cl - , Br - , I - , CO3 2- ,HCO3 - , SO32- , HSO3 - , CH3COO - , Fe 3+ , Ca 2+ , Cu 2+ , Al 3+ , Mg 2+ , Ag + , K + , Na + , Ba 2+ , Cr 3+ , Hg 2+ , Zn 2+ , Fe 2 + , Pb 2+ ) solution, immediately add the same volume and concentration of ClO - or NO2 - , shake well, and measure its fluorescence emission spectrum at an excitation wavelength of λ = 338nm. The results show that the interferences have almost no effect on the fluorescence intensity of the fluorescent probe (such as Figure 7 ), which shows that the fluorescent probe is sensitive to ClO - and NO2 - Ion recognition has good anti-interference ability.

[0049] Example 7: Preparation and detection of water-soluble chitosan fluorescent probe test paper

[0050] The water-soluble chitosan fluorescent probe of the present invention was weighed and dissolved in 1% acetic acid aqueous solution to prepare a 2 g / L probe solution. The cut blank filter paper was soaked in the 2 g / L probe solution to allow it to evenly adsorb the probe and dried naturally to obtain a sample for detecting ClO. - and NO2 - Fluorescent probe test paper. Add 0.01 M aqueous solution containing different ions and observe under 365 nm UV light. Only when ClO is added - and NO2 - The test paper of the solution will show obvious fluorescence quenching ( Figure 8 ).

[0051] First drop a drop of NaOH solution on the prepared fluorescent probe test paper, then add ClO - and NO2 - The solution was observed under 365nm UV light and NO2 was added - The test paper fluorescence of the solution remained unchanged, and ClO - The fluorescence of the test paper of the solution is obviously quenched ( Figure 9 ), indicating that the probe can be prepared into a test paper for ClO - and NO2 -Qualitative detection and differentiation.

[0052] Ten fluorescent probe test papers were added with different concentrations of ClO - and NO2 - The solution was irradiated with a 365nm portable UV lamp to observe the fluorescence intensity. This fluorescent probe paper can well distinguish different concentrations of ClO - and NO2 - Ionic solution. The lowest detectable ClO - The ion concentration is 5mM, and the results are shown in Figure 10 The lowest NO2 detected - The ion concentration is 7.5mM, and the results are shown in Figure 11 .

Claims

1. A water-soluble chitosan fluorescent probe, characterized in that Its structural formula is shown below: ； Where x=23, y=117.

2. The method for preparing the water-soluble chitosan fluorescent probe according to claim 1, characterized in that The steps include: Chitosan was dissolved in acetic acid aqueous solution, and TTG dissolved in DMSO was added, and the mixture was heated to reflux for reaction. After the reaction, the mixture was cooled and dialyzed against DMSO for 72 hours to remove unreacted TTG. The mixture was then dialyzed against distilled water for 3 days, and the product was freeze-dried to obtain the target product, TTGN. The synthetic route is as follows: 。 3. The preparation method according to claim 2, wherein: The concentration of the acetic acid aqueous solution is 1%.

4. Use of the water-soluble chitosan fluorescent probe according to claim 1 in preparing a detection reagent, characterized in that: The detection reagent qualitatively or quantitatively detects ClO - and / or NO2 - .

5. The use according to claim 4, characterized in that: Fluorescence spectrometry was performed in aqueous media to detect ClO by changes in fluorescence intensity. - and / or NO2 - Qualitative or quantitative detection of ions.

6. The use according to claim 5, characterized in that: The aqueous medium is a 1% acetic acid aqueous solution.

7. The use according to claim 4, characterized in that: The detection reagent is used for ClO - When ion detection is performed, the detection limit is 0.59 μM; the detection reagent is used for NO2 - When ion detection was used, the detection limit was 0.92 μM.

8. The use according to claim 4, characterized in that: The water-soluble chitosan fluorescent probe was used to prepare a test paper, which was observed under a 365nm ultraviolet lamp, and the ClO was detected by the changes in the intensity and color of the fluorescence. - and / or NO2 - Qualitative detection of .

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