Novel water-soluble chitosan fluorescent probe and application thereof in ClO <-> and NO2 <-> ion detection

Abstract

The invention discloses a novel water-soluble chitosan fluorescent probe and application of the novel water-soluble chitosan fluorescent probe in ClO <-> and NO2 <-> ion detection. The structural formula of the water-soluble chitosan fluorescent probe is as follows: # imgabs0 #. The water-soluble chitosan fluorescent probe disclosed by the invention can realize qualitative and quantitative detection of ClO <-> and / or NO2 <-> ions through fluorescence spectrometry, and has good anti-interference performance, high selectivity and sensitivity in the presence of other analytes; meanwhile, the probe can be applied to test paper detection and shows huge potential in the aspect of detecting ClO <-> and NO2 <-> ions.

Description

Technical Field

[0001] The present invention belongs to the technical field of ion detection, and specifically relates to a novel water-soluble chitosan fluorescent probe and its - and NO 2 - 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 toxicant 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, pickled foods and drinking water. Because nitrite can interact with amines in the digestive tract, highly carcinogenic N-nitrosamine compounds are produced when people consume foods containing nitrite. Therefore, the development of a simple and effective nitrite detection technology is crucial to public health safety.

[0003] Hypochlorous acid is one of the most important reactive oxygen species (ROS) in the life system. It has a dual role in the life process. On the one hand, it can kill viruses and bacteria to ensure human health. On the other hand, excessive hypochlorous acid can oxidize biological molecules, leading to tissue damage, inflammation and the occurrence of various diseases. Therefore, studying the generation, regulation and action mechanism of hypochlorous acid, as well as developing highly selective and sensitive detection methods, is of great significance for further understanding its role in organisms and the occurrence mechanism of diseases.

[0004] The traditional detection method has a complicated pre-treatment process, the instruments and equipment used are expensive, and the detection takes a long time, which cannot meet the needs of rapid detection. Spectroscopy has the characteristics of rapid response, simple operation and low cost, so the use of fluorescent or colorimetric probes to qualitatively and quantitatively detect ions has become a hot topic of research. Although the organic small molecule fluorescent probe detection method is easy to operate, highly sensitive, easy to observe, selective, and fast to detect, it cannot detect ions directly in the aqueous phase because organic compounds have low solubility in water. Organic reagents need to be mixed with water for detection. Therefore, the pre-treatment process for detecting ions will be more cumbersome, and because organic reagents need to be added when detecting ions, it will also cause secondary pollution to the environment.

[0005] In order to avoid the above defects, 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, and have 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, and has been widely used in various fields due to its good biological properties. Therefore, it is necessary to provide a novel fluorescent probe for detecting ClO - and NO 2 - Ionic water-soluble chitosan fluorescent probes and preparation methods and applications thereof 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 NO 2 - Application in ion detection. The technical problem to be solved is to synthesize molecules that can detect ClO - and NO 2 - 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 ratio of the nuclear magnetic H spectrum area of ​​the probe synthesized at the optimal feed ratio. Under the same detection effect, the larger the ratio of x / y, the smaller 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% acetic acid aqueous solution, and TTG (4.45 mg, 0.147 mmol) dissolved in DMSO was added, and the mixture was heated to reflux at 65 °C for 48 h. After cooling, the reaction solution was dialyzed in DMSO solution for 72 h to remove unreacted TTG, and then dialyzed in distilled water for 3 days (MWCO: 3500 Da), 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 discloses a water-soluble chitosan fluorescent probe for use in preparing a detection reagent capable of qualitatively or quantitatively detecting ClO - and / or NO 2 - .

[0016] Furthermore, fluorescence spectroscopy was performed in an aqueous medium to detect ClO by changes in fluorescence intensity. - and / or NO 2 - 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 the ion is detected, the detection limit is 0.59 μM; 2 - 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 NO 2 - 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 NO 2 - The fluorescence intensity is slightly reduced. Therefore, the pH of the detection system can be adjusted to achieve ClO - and NO 2 - 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 NO 2 - Qualitative detection.

[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 NO 2 - 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 NO 2- Rapid identification and quantitative detection of ions, and for ClO - and / or NO 2 - 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 NO 2 - Ions show great potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of the water-soluble chitosan fluorescent probe of the present invention and its precursor aldehyde TTG.

[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 are added to a 1% acetic acid aqueous solution (λex=338nm).

[0026] Figure 4 The water-soluble chitosan fluorescent probe of the present invention is used to detect ClO under different pH conditions. - and NO 2 - 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 fluorescence emission spectrum (λex = 338nm); Figure b shows the fluorescence intensity of the fluorescent probe at the fluorescence emission wavelength of 550nm and ClO - Linear relationship between ion concentrations.

[0028] Figure 6 Figure a shows the water-soluble chitosan fluorescent probe of the present invention in a 1% acetic acid aqueous solution with 0-35 μM ion concentration of NO 2 - ion after fluorescence emission spectrum (λex = 338nm); Figure b shows the fluorescence intensity of the fluorescent probe at the fluorescence emission wavelength of 550nm and NO 2 - Linear relationship between ion concentrations.

[0029] Figure 7Figure a is a bar graph 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 bar graph 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 in 1% acetic acid aqueous solution with different analytes added (λex=338nm); Representative fluorescent probe compounds were added to 1% acetic acid aqueous solution with different analytes and ClO - Fluorescence intensity of ions (λex=338nm). Representative fluorescent probe compounds were added to 1% acetic acid aqueous solution with different analytes and NO 2 - Fluorescence intensity of ions (λex=338nm).

[0030] Figure 8 The pictures are of the test paper made of the water-soluble chitosan fluorescent probe of the present invention under a 365nm ultraviolet lamp when different ions are added.

[0031] Fig. 9 The test paper made of the water-soluble chitosan fluorescent probe of the present invention is first added with NaOH and then with ClO - and NO 2 - The picture was taken under 365nm UV light.

[0032] Fig.10 The test strip made of the water-soluble chitosan fluorescent probe of the present invention is added with different concentrations of ClO - Ion image under 365nm UV light.

[0033] Fig.11 The test strip made of the water-soluble chitosan fluorescent probe of the present invention is added with different concentrations of NO 2 - Ion image under 365nm UV light. DETAILED DESCRIPTION

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

[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 aqueous 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 hours to remove unreacted TTG, and then dialyzed in distilled water for 3 days (MWCO: 3500 Da), and the dialyzate was replaced every 12 hours. Finally, the product was freeze-dried to obtain 220 mg of the target product TTGN with a yield of 73%.

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

[0038] Figure 2 The 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 on the left and right are relatively sharp and are at 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: Water-soluble chitosan fluorescent probe for ClO - and NO 2 - 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 a 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 - , NO 2 - , NO 3 - , HS - , S 2 O 3 2- , SO 4 2- , H2 O 2 , Hcy, Cys, GSH, SCN - , Cl - ,Br - , I - , CO 3 2- , HCO 3 - , SO 3 2- , HSO 3 - , 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 it well and measure the fluorescence spectrum of the solution. Figure 3 As shown, it can be seen that the probe can specifically recognize ClO - and NO 2 - , and almost no response to other ions. The results show that the fluorescent probe is sensitive to ClO - and NO 2 - Highly selective.

[0041] Example 3: Water-soluble chitosan fluorescent probe for ClO under different pH conditions - and NO 2 - Identification of ions

[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 - , NO 2 - ) aqueous solution, shake it well and measure the fluorescence spectrum of the solution. Figure 4As shown in the figure, it can be seen that in the 1% acetic acid aqueous solution of the probe, when the system pH is ≤ 2, ClO - and NO 2 - 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 NO 2 - The fluorescence intensity is slightly reduced. Therefore, the pH of the probe solution can be adjusted to achieve ClO - and NO 2 - Distinguish between the two ions.

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

[0044] 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. Drop the probe solution into each test tube and take ClO - (0-22.5 μM) was dripped 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 probe (0.03 g / L) and ClO - (0-22.5 μM) has a good linear relationship R 2 = 0.995, which indicates that the fluorescent probe can quantitatively detect ClO in the fluorescence spectrum. - According to LOD=3σ / k, it is calculated that the fluorescence probe in the fluorescence spectrum is ClO - The detection limit is 0.59 μM. Where σ is the standard deviation of the blank measurement, 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 NO 2 - Concentration correlation

[0046] Weigh the water-soluble chitosan fluorescent probe of the present invention and dissolve it in 1% acetic acid aqueous solution to prepare a 0.03 g / L probe solution. Drop the probe solution into each test tube and take NO 2 - (0-35 μM) was dripped 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. 2- (0-35μM) has a good linear relationship R 2 = 0.993, which indicates that the fluorescent probe can quantitatively detect NO in the fluorescence spectrum. 2 - According to LOD=3σ / k, it is calculated that the fluorescence probe in the fluorescence spectrum is 2 - The detection limit is 0.92 μM. Where σ is the standard deviation of the blank measurement value, k is the fluorescence intensity of NO 2 - The slope of the concentration graph.

[0047] Example 6: Water-soluble chitosan fluorescent probe for ClO - and NO 2 - 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 - , NO 2 - , NO 3 - , HS - , S 2 O 3 2- , SO 4 2- , H 2 O 2 , Hcy, Cys, GSH, SCN - , Cl - ,Br - , I - , CO 3 2- , HCO 3 - , SO 3 2- , HSO 3 - , CH3COO - , Fe 3+ , Ca 2+ , Cu 2+ , Al 3+ ,Mg 2+ , Ag + , K + , Na + , Ba2+ ,Cr 3+ , Hg 2+ , Zn 2+ , Fe 2 + , Pb 2+ ) solution, immediately add the same volume and concentration of ClO - or NO 2 - , 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 indicates that the fluorescent probe is sensitive to ClO - and NO 2 - 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 make it evenly adsorb the probe, and dried naturally to obtain a sample for detecting ClO. - and NO 2 - Fluorescent probe test paper. A 0.01 M aqueous solution containing different ions was added and observed under a 365 nm UV lamp. - and NO 2 - 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 NO 2 - The solution was observed under 365 nm UV light and NO 2 - The test paper fluorescence of the solution remained unchanged. - The test paper fluorescence of the solution is significantly quenched ( Fig. 9 ), indicating that the probe can be prepared into a test strip for ClO - and NO 2 - Qualitative detection and differentiation.

[0052] Ten copies of the prepared fluorescent probe test paper were added with different concentrations of ClO - and NO 2 -The solution was irradiated with a 365nm portable ultraviolet lamp to observe the fluorescence intensity. The fluorescent probe test paper can well distinguish different concentrations of ClO - and NO 2 - Ionic solution. The lowest detectable ClO - The ion concentration is 5mM, and the results are shown in Fig.10 The lowest NO detected 2 - The ion concentration is 7.5mM, and the results are shown in Fig.11 .

Claims

1. A water-soluble chitosan fluorescent probe, characterized in that Its structural formula is as follows: ； 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: After chitosan was dissolved in acetic acid aqueous solution, TTG dissolved in DMSO was added, and the mixture was heated to reflux for reaction. After the reaction was completed, the mixture was cooled, and the reaction solution was dialyzed in DMSO solution for 72 hours to remove unreacted TTG. The reaction solution was then dialyzed in 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, characterized in that: The concentration of the acetic acid aqueous solution is 1%.

4. The 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 is used to prepare a test strip, which is observed under a 365nm ultraviolet lamp, and the ClO is detected by the change of the intensity and color of the fluorescence. - and / or NO2 - Qualitative detection.

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