Up-conversion fluorescent molecular probe for detecting content of hydrogen sulfide in pickled and smoked food

Abstract

The invention relates to a near-infrared up-conversion fluorescent molecular probe for detecting the content of hydrogen sulfide in food. The probe molecule has a structure as shown in a formula (1), can be used as a fluorescent probe to detect hydrogen sulfide in food through an up-conversion luminescence mechanism, and can be used for detecting the content of hydrogen sulfide in pickled and smoked food.

Description

Technical Field

[0001] This invention belongs to the field of spectral analysis, specifically relating to an upconversion fluorescent molecular probe for detecting hydrogen sulfide content in pickled and smoked foods. Background Technology

[0002] Hydrogen sulfide (H2S) is a gas with a smell similar to rotten eggs. It is a nerve agent; inhalation concentrations greater than 300 mg / m³ are dangerous. 3 Hydrogen sulfide can cause irritation to the respiratory tract and eyes. Inhaling high concentrations of hydrogen sulfide for a short period can lead to sudden death. Studies have found that its primary target organs are the central nervous system and respiratory system, and it can also cause damage to multiple organs, including the heart. The brain and mucous membranes are the most sensitive tissues to hydrogen sulfide; therefore, respiratory and eye damage are common. High concentrations of hydrogen sulfide can directly stimulate chemoreceptors in the carotid sinus and aortic region, causing reflex respiratory depression. This is often seen in long-term, high-volume consumption of foods containing hydrogen sulfide.

[0003] Hydrogen sulfide can be produced during the production and fermentation of various foods in the food industry, especially as an essential preservative additive in some pickled and smoked foods. Long-term consumption of food containing residual hydrogen sulfide can cause many diseases. Therefore, developing hydrogen sulfide detection technologies for the food industry is essential for food safety control.

[0004] In recent years, fluorescent probe technology based on molecular recognition has shown a rapid development trend in the field of hydrogen sulfide detection. Fluorescent probes have advantages such as high sensitivity, good selectivity, rapid response, and good visualization, especially their non-invasiveness and real-time imaging capabilities, making them widely used in studies of hydrogen sulfide distribution and signaling pathways in organisms. On the other hand, fluorescent probes for hydrogen sulfide detection in food are also gradually gaining attention. However, due to the complex background interference in food, the application of these fluorescent probes often requires complex pretreatment processes, thus limiting their application scope and probe performance. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a fluorescent probe for detecting hydrogen sulfide and to apply it to the detection of hydrogen sulfide content in food samples.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] Design of a fluorescent probe for detecting hydrogen sulfide.

[0008] A fluorescent probe with the chemical formula shown in formula (1) was designed using pentamethylcyanine dye as the fluorescent backbone. This probe exhibits near-infrared fluorescence at 700 nm, which can avoid background fluorescence in most visible light regions.

[0009] Furthermore, by selecting long-wavelength excitation, the upconversion luminescence signal of the probe can be obtained, further avoiding interference from fluorescence signals in complex backgrounds.

[0010] Using benzoindole salt as the recognition unit, the nucleophilic reaction of hydrogen sulfide on the carbon atom adjacent to the nitrogen atom of the indole salt is utilized to achieve a change in the probe signal, thereby realizing the detection of hydrogen sulfide concentration.

[0011] Sensing properties of a near-infrared upconversion fluorescent molecular probe for hydrogen sulfide.

[0012] The steps include: dissolving the fluorescent probe in DMSO to prepare a stock solution, and diluting it with deionized water to prepare an aqueous solution containing 1% DMSO for testing to study various sensing properties.

[0013] 1) Add hydrogen sulfide samples of different concentrations and conduct UV-Vis and fluorescence spectroscopy studies on the prepared test solutions. Obtain the corresponding curves of absorbance and fluorescence emission intensity versus hydrogen sulfide concentration, and calculate the detection limit.

[0014] 2) Add different anion test solutions and conduct selective studies using fluorescence spectroscopy.

[0015] 3) Add hydrogen sulfide test solution to the solution in 2) and study the anti-interference performance by fluorescence spectroscopy.

[0016] A near-infrared upconversion fluorescent molecular probe. Used to detect hydrogen sulfide content in food.

[0017] Furthermore, the fluorescent probe provided by this invention is used to detect the hydrogen sulfide content in smoked meat samples. The detection method includes:

[0018] 1) Prepare smoked meat test samples using freeze-drying method, and then prepare test solutions for the smoked meat samples.

[0019] 2) Add the above actual sample solution to the test solution containing the probe, perform fluorescence spectroscopy test, and obtain the hydrogen sulfide content of the actual sample.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] The fluorescent probe of this invention is simple to synthesize, exhibits good selectivity and anti-interference performance for hydrogen sulfide, high sensitivity, and good linearity. Utilizing the upconversion property of molecules, background signal interference from actual samples can be removed, while simultaneously improving the detection sensitivity of the spectral signal for hydrogen sulfide, thus enabling effective detection of trace amounts of hydrogen sulfide in pickled products. Attached Figure Description

[0022] Figure 1 This is the hydrogen NMR spectrum of the probe described in this invention.

[0023] Figure 2 The UV-Vis absorption spectra of the probe of this invention in different concentrations of hydrogen sulfide and the relationship between the absorbance ratio and the hydrogen sulfide concentration (detection wavelength 675 nm) are shown.

[0024] Figure 3 The fluorescence spectra of the probe of the present invention in different concentrations of hydrogen sulfide and the relationship between the fluorescence ratio and the concentration of hydrogen sulfide (detection wavelength 700 nm) are shown.

[0025] Figure 4 The fluorescence signal changes of the probe of the present invention in the presence of different anions and the response properties to hydrogen sulfide based thereon (detection wavelength 700 nm).

[0026] Figure 5 The upconversion fluorescence spectra of the probe of the present invention in different concentrations of hydrogen sulfide and the relationship between the fluorescence ratio and the hydrogen sulfide concentration (detection wavelength 700 nm) are shown.

[0027] Figure 6 This table shows the hydrogen sulfide content, recovery rate, and standard deviation calculated from the fluorescence detection signals of different smoked meat samples based on the probe of this invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1: Probe Synthesis Method

[0030]

[0031] The intermediate ethylbenzoindole iodide (1 g, 2.68 mmol) and malondialdehyde diphenylamidine hydrochloride (0.35 mg, 1.34 mmol) were added to a 50 mL two-necked flask, along with sodium acetate (868 mg, 10.5 mmol), acetic anhydride (18 mL), and acetic acid (2 mL). The mixture was heated to 140 °C under nitrogen protection, and the color change of the solution was observed and detected by TLC during the reaction. After stopping the reaction, the mixture was cooled to room temperature, neutralized to pH 7 with sodium bicarbonate, and extracted three times with DCM. The organic phases were combined, dried over anhydrous magnesium sulfate, and the solvent was evaporated to dryness using a rotary evaporator. The solution was purified by column chromatography with an eluent of DCM / MeOH at a volume ratio of 30:1 to obtain a metallic green solid, which is the probe molecule proposed in this invention, with a synthesis yield of 55%. 1HNMR (400MHz, Methanol-d4) δppm: 1.48 (t, J=7.28Hz, 5H), 2.01-2.10 (m, 9H), 4.31 (q, J=7.11Hz, 3H), 6.31-6.44 (m, 2H), 6.63-6.74(m,1H),7.52(t,J=7.53Hz,2H),7.60-7.70(m,3H),8.00-8.08(m,3H),8.25-8.31(m,2H),8.33-8.44(m,2H), such as Figure 1 As shown.

[0032] Example 2: This invention further provides a performance testing scheme for the detection of hydrogen sulfide using a fluorescent molecular probe. The steps are as follows:

[0033] 1) Add the prepared fluorescent probe (31.9 mg) to a 5 mL volumetric flask, then add DMSO to dissolve and dilute to volume to prepare a 1 mM stock solution. Add sodium sulfide heptahydrate (12.0 mg) to a 5 mL volumetric flask, then add deionized water to dissolve and dilute to volume to prepare a 10 mM stock solution.

[0034] 2) Take 20 μL of probe stock solution and add it to a four-sided quartz cuvette containing 1980 μL of deionized water to obtain a deionized water test solution containing 1% DMSO, in which the probe concentration is 10 μM. Collect the absorption and fluorescence spectra of the sample.

[0035] 3) Add 20 μL of probe stock solution to a four-sided quartz cuvette, and add different volumes of deionized water and sodium sulfide stock solution to obtain a series of test solutions with sodium sulfide concentrations of 1.25, 2.5, 3.75, 5, 6.25, 7.5, 8.875, 10, 15, 20, 25, 30, and 35 μM, maintaining a total test solution volume of 2 mL (containing 10 μM probe). Collect the absorption and fluorescence spectra, obtaining the absorbance at 675 nm and the fluorescence intensity at 700 nm. By comparing these values ​​with those of the probe solution without hydrogen sulfide in step 2), obtain a graph showing the relationship between the concentration of hydrogen sulfide in the solution and absorbance and fluorescence intensity. See [link to graph]. Figure 2 and 3 .

[0036] 3) Take 20 μL of probe stock solution and add it to a tetrahedral quartz cuvette containing 1960 μL of deionized water. Then add 20 μL of sodium salt stock solution of various ions (Cl). - ,Br - ClO4 - H2PO4 - HCO3 - HPO4 2- IO4- NO2 - SO3 2- SO4 2- The test solutions contained 10 μM probe and 100 μM other ions (all at a concentration of 10 mM). Fluorescence spectra of the samples were collected, and the fluorescence intensity at 700 nm was recorded. The changes in probe fluorescence signal in the presence of different ions were calculated.

[0037] 4) Take 20 μL of probe stock solution and add it to a tetrahedral quartz cuvette containing 1940 μL of deionized water. Then add 20 μL of sodium salt stock solution of various ions (Cl). - ,Br - ClO4 - H2PO4 - HCO3 - HPO4 2- IO4 - NO2 - SO3 2- SO4 2- The sample was prepared by adding 20 μL of sodium sulfide stock solution to a sample containing 10 μM probe, 100 μM other ions, and 10 μM sodium sulfide. Fluorescence spectra of the samples were collected, and the fluorescence intensity at 700 nm was recorded. The changes in the fluorescence signal of the probe to sodium sulfide in the presence of different ions were calculated. A bar chart was plotted for steps 3) and 4), see [link to bar chart]. Figure 4 .

[0038] Example 3: This invention further provides a detection scheme for detecting hydrogen sulfide content in solution using upconversion fluorescence technology. The steps are as follows:

[0039] 1) Add the prepared fluorescent probe (31.9 mg) to a 5 mL volumetric flask, then add DMSO to dissolve and dilute to volume to prepare a 1 mM stock solution. Add sodium sulfide heptahydrate (12.0 mg) to a 50 mL volumetric flask, then add deionized water to dissolve and dilute to volume to prepare a 1 mM stock solution.

[0040] 2) Take 20 μL of probe stock solution and add it to a four-sided quartz cuvette containing 1980 μL of deionized water to obtain a deionized water test solution containing 1% DMSO, in which the probe concentration is 10 μM. Collect the absorption and fluorescence spectra of the sample.

[0041] 3) Add 20 μL of probe stock solution to a four-sided quartz cuvette, and add different volumes of deionized water and sodium sulfide stock solution to obtain a series of test solutions with sodium sulfide concentrations of 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, and 3.5 μM, maintaining a total test solution volume of 2 mL (containing 10 μM probe). Using a 785 nm laser, collect fluorescence spectra in the 650–725 nm range, obtaining the fluorescence intensity of the sample at 700 nm. Compare this intensity with the values ​​from the probe solution without hydrogen sulfide in step 2) to obtain a graph showing the relationship between the concentration of hydrogen sulfide in the solution and the upconversion fluorescence intensity. See [link to graph]. Figure 5 .

[0042] Implementation Example 4:

[0043] This invention further provides a detection scheme for hydrogen sulfide content in food using a near-infrared upconversion fluorescent molecular probe. The steps are as follows:

[0044] 1) Add the prepared fluorescent probe (31.9 mg) to a 5 mL volumetric flask, then add DMSO to dissolve and dilute to volume to prepare a 1 mM stock solution, and store it in a refrigerator for later use.

[0045] 2) Take the smoked meat sample to be tested, freeze-dry, pulverize and sieve. Weigh 500 mg of the sieved powder, add 10 mL of deionized water, and shake at 37 degrees Celsius for 30 minutes. Filter through a 0.22 μm aqueous phase filter membrane to obtain a clear solution, and then dilute to 50 mL with deionized water. Store the obtained stock solution in a refrigerator for later use.

[0046] 3) Take 20 μL of the probe stock solution prepared in step 1), add it to 1960 μL of deionized water, and then add 20 μL of the stock solution of different samples prepared in step 2). This yields a test solution containing a probe concentration of 10 μM. Collect the fluorescence spectrum, obtain the fluorescence intensity at 700 nm, and compare it with the working curve to obtain the hydrogen sulfide content of the sample.

[0047] 4) Perform three tests on different samples, and calculate the hydrogen sulfide content, recovery rate, and relative deviation of each sample. Results are shown below. Figure 6 .

[0048] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A near-infrared upconversion fluorescent molecule for hydrogen sulfide detection, characterized in that, It has the structural formula shown in equation (Ⅰ):

2. The near-infrared fluorescent molecule for hydrogen sulfide detection as described in claim 1 can be used for colorimetric detection of hydrogen sulfide concentration.

3. The near-infrared fluorescent molecule for hydrogen sulfide detection as described in claim 1 can be used for the fluorescence method to detect hydrogen sulfide concentration.

4. The near-infrared fluorescent molecule for hydrogen sulfide detection described in claim 1 can be used to detect the concentration of hydrogen sulfide in pickled and smoked food samples by upconversion fluorescence method.

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