A method of monitoring the trend of lipid oxidation

Abstract

The application provides a method for monitoring lipid oxidation tendency, and belongs to the technical field of fluorescent probe detection. The method utilizes the fluorescence characteristics of a BODIPY molecular probe, and through selecting a molecular probe with a specific structure, directional reaction occurs between the BODIPY molecular probe and oxygen free radicals generated by lipid oxidation at a specific pH value. The conjugated system of the BODIPY molecular probe is destroyed, the half-chlorin structure is broken, and the fluorescence color changes from red to blue-green. According to the change of the fluorescence color before and after the reaction, the visualization monitoring of the lipid oxidation tendency and spatial distribution is realized through fluorescence imaging.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe detection technology, specifically relating to a method for monitoring lipid oxidation trends. Background Technology

[0002] With economic development, people have increasingly higher requirements for food quality. Meat, plant-based foods, and oils, as important food categories, generate a large number of oxygen free radicals during storage. Oxygen free radicals are highly reactive, leading to lipid oxidation and affecting the nutrition, flavor, and safety of the ingredients.

[0003] Current detection methods characterize the degree of lipid oxidation using malondialdehyde (MDA) values ​​(TBARs method) and point of effect (POV) values. MDA is one of the most important products generated during the oxidation of most unsaturated fatty acids. Its presence in small amounts in meat produces a putrid odor and it is considered a major marker of lipid oxidation. The TBARs method is the primary technique for quantifying MDA, measuring the colorimetric reaction of a complex formed by thiobarbituric acid (TBA) with MDA. However, the reaction with TBA is not unique to MDA; various aldehydes and other oxidation products also react with TBA. Therefore, this method detects all substances that react with TBA. Before performing the TBARs method, MDA must be completely extracted. Acid extraction is a commonly used method, which is relatively simple to operate, but has a significant drawback: the extract can extract colored components and dissolve proteins, resulting in a colored or turbid extract, leading to inaccurate detection values. The POV (Potential Oxidation Value) is a key indicator for measuring the initial degree of oxidation in oils and fats. It directly reflects the content of hydroperoxides in the oil and fats, measured by iodometric titration. The lower the value, the less oxidative damage the oil surface has undergone, and the fresher the quality. However, as oxidation progresses, hydroperoxides decompose, and the POV value decreases. Therefore, the POV value is not a reliable indicator for accurately detecting the oxidation trend of lipids. In practical applications, multiple indicators are usually used in combination rather than relying on a single indicator to more comprehensively and accurately determine the degree of oxidation.

[0004] More importantly, the aforementioned traditional detection methods are all destructive, requiring pretreatment such as homogenization and extraction of samples. These methods fail to preserve the original tissue morphology and make it difficult to obtain spatial distribution information on the degree of oxidation within the sample. This results in a complex detection process with poor timeliness, making it difficult to meet the needs for timely monitoring of lipid oxidation trends and in-depth understanding of spatial distribution differences in samples. Summary of the Invention

[0005] The purpose of this invention is to provide a method for monitoring lipid oxidation trends. The method provided by this invention is simple, rapid, and highly sensitive, meeting the need for visual monitoring of lipid oxidation trends. It obtains spatial distribution information of oxidation while preserving the original appearance of the sample tissue, overcoming the shortcomings of traditional detection methods that are highly destructive and unable to obtain spatial distribution information.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for monitoring lipid oxidation trends, comprising: The sample to be tested is mixed with the probe solution and then incubated to obtain an incubated sample; the pH value of the probe solution is 4-9; the molecular probe in the probe solution is a boron fluoride dipyrrole molecular probe with the following structural formula: ; After the incubated sample was embedded, it was frozen and sliced ​​sequentially to obtain the sample to be tested; Fluorescence imaging was performed on the sample to be tested; By comparing the imaging colors of different test samples, if green light is enhanced and red light is weakened, the oxidation degree of the corresponding test sample is more severe.

[0007] Preferably, the concentration of the molecular probe in the probe solution is 1~10 μmol / L.

[0008] Preferably, the mass ratio of the sample to the probe solution is 1 g:(2~5) mL.

[0009] Preferably, the solvent of the probe solution is a mixture of dimethyl sulfoxide and phosphate buffer solution.

[0010] Preferably, the volume ratio of the dimethyl sulfoxide to the phosphate buffer solution is 1:(1~99).

[0011] Preferably, the incubation is performed by standing in a dark environment for 3 to 30 minutes.

[0012] Preferably, the excitation wavelength for the fluorescence imaging is 480~590nm.

[0013] Preferably, the freezing process involves immersing the embedded sample in liquid nitrogen to solidify the embedding agent.

[0014] Preferably, the thickness of the sample to be tested is 0.5~1μm.

[0015] Preferably, the source of the sample to be tested includes meat, lipid-containing plant products, or oils.

[0016] This invention provides a method for monitoring lipid oxidation trends, comprising: The sample to be tested is mixed with the probe solution and then incubated to obtain an incubated sample; the pH value of the probe solution is 4-9; the molecular probe in the probe solution is a boron fluoride dipyrrole molecular probe with the following structural formula: ; After the incubated sample was embedded, it was frozen and sliced ​​sequentially to obtain the sample to be tested; Fluorescence imaging was performed on the sample to be tested; By comparing the fluorescence imaging colors of different test samples, increased green light and decreased red light indicate a more severe degree of oxidation in the corresponding test sample. This invention utilizes the fluorescence properties of boron dipyrrole (BODIPY) molecular probes. By selecting molecular probes with specific structures, a directed reaction occurs with oxygen free radicals generated by lipid oxidation at a specific pH value. The conjugated system of the BODIPY molecular probe is disrupted, the hemicyanine structure breaks, and the fluorescence color changes from red to blue-green. Based on the change in fluorescence color before and after the reaction, fluorescence imaging enables visual monitoring of the lipid oxidation trend and spatial distribution. The results of the examples show that the BODIPY molecular probe has a good response to the concentration of oxygen free radicals generated by lipid oxidation and a low detection limit. Attached Figure Description

[0017] Figure 1 This is a diagram illustrating the reaction mechanism between the BODIPY molecular probe and ROO in this invention. Figure 2 The BODIPY molecular probe and ONOO in this invention - Reaction mechanism diagram; Figure 3 These are fluorescence images of meat samples stored for different times in Example 1 of the present invention; Figure 4 This is a fluorescence image processing diagram from Test Example 1 of the present invention; Figure 5 The correlation curve between relative fluorescence intensity and POV and TBARS values ​​obtained in Test Example 1 of this invention is shown. Figure 6 The UV absorption spectrum of the BODIPY molecular probe; Figure 7 The fluorescence spectrum of the BODIPY molecular probe; Figure 8 The curves showing the rate of change of the BODIPY molecular probe response value at different pH values; Figure 9 Different ONOO in this invention - Fluorescence spectrum at 490 nm excitation concentration; Figure 10 ONOO in this invention - Concentration and I 509 / I 588 The standard curve; Figure 11 The following are fluorescence spectra of different ROO concentrations excited at 490 nm in this invention; Figure 12 The concentration of ROO· and I in this invention509 / I 588 The standard curve; Figure 13 The following are fluorescence spectra of different ROO concentrations excited at 544 nm in this invention; Figure 14 The concentration of ROO· and I in this invention 588 The standard curve. Detailed Implementation

[0018] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0019] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or those with conventional purity in the field of fluorescent probe detection.

[0020] This invention provides a method for monitoring lipid oxidation trends, comprising: The sample to be tested is mixed with the probe solution and then incubated to obtain an incubated sample; the pH value of the probe solution is 4-9; the molecular probe in the probe solution is a boron fluoride dipyrrole molecular probe with the following structural formula: ; After the incubated sample was embedded, it was frozen and sliced ​​sequentially to obtain the sample to be tested; Fluorescence imaging was performed on the sample to be tested; By comparing the fluorescence imaging colors of different test samples, if green light is enhanced and red light is weakened, the oxidation degree of the corresponding test sample is more severe.

[0021] In this invention, the sample to be tested is mixed with a probe solution and then incubated to obtain an incubated sample.

[0022] In this invention, the source of the sample to be tested preferably includes meat, lipid-containing plant products, or oils, and more preferably meat. Since meat is a solid substance with diverse lipid types and complex oxidation behavior, the detection of its oxidation degree is more difficult. Using meat as the sample to be tested helps to demonstrate the speed of the method of this invention.

[0023] In this invention, the molecular probe in the probe solution is a boron fluoride dipyrrole molecular probe with the following structural formula: .

[0024] The free radicals generated by lipid oxidation are mainly peroxy radicals (ROO•) and peroxynitrites (ONOO). - In this invention, the reaction mechanism of the BODIPY molecular probe with oxygen free radicals is as follows: Figure 1 , Figure 2As shown: The reactants are the BODIPY fluorescent probe, as well as peroxy radicals (ROO•) and peroxynitrite (ONOO•). - Two target analytes. When the probe reacts with ROO•, the conjugated double bond of ROO• reacts with the probe to form an intermediate. After subsequent reactions, the conjugated double bond is oxidatively cleaved, and the final products are a BODIPY aldehyde derivative and N-ethylbenzothiazol-2-one. The probe reacts with ONOO•... - During the reaction, intermediates are formed through addition and elimination, and after oxygenation and decomposition, the same two end products as described above are generated.

[0025] The BODIPY molecular probe exhibits red fluorescence at 588 nm, in conjunction with ONOO - After the ROO· reaction, the conjugated system is destroyed, the hemicyanine structure breaks, and it emits blue-green fluorescence at 509 nm.

[0026] In this invention, the concentration of the molecular probe in the probe solution is preferably 1~10 μmol / L, more preferably 3~6 μmol / L; as one embodiment of this invention, the concentration of the molecular probe in the probe solution can be 2 μmol / L, 4 μmol / L, 5 μmol / L, 7 μmol / L, or 8 μmol / L. A concentration of the molecular probe in the probe solution within the above range is beneficial for fluorescence intensity testing.

[0027] In this invention, the solvent of the probe solution is preferably a mixture of dimethyl sulfoxide (DMSO) and phosphate buffered saline (PBS); the volume ratio of DMSO to PBS is preferably 1:(1~99), more preferably 1:(50~99); as one embodiment of this invention, the volume ratio of DMSO to PBS can be 1:5, 1:10, 1:20, 1:40, 1:60, 1:80, 1:90, 1:95, or 1:99. DMSO can effectively dissolve the BODIPY molecular probe, and PBS can adjust the concentration of the molecular probe and the pH value of the solution; a volume ratio of DMSO to PBS within the above range is beneficial to improving detection accuracy.

[0028] In this invention, the pH value of the probe solution is 4-9, preferably 5-8; as one embodiment of this invention, the pH value of the probe solution can be 4, 5, 6, 7, 7.5, 8, or 9. When the pH value of the probe solution is within the above range, the BODIPY molecular probe can undergo a directed reaction with oxygen free radicals, and the fluorescence imaging will show different colors.

[0029] In this invention, the preferred mass-to-volume ratio of the sample to the probe solution is 1 g:(2~5) mL, more preferably 1 g:3 mL. A mass-to-volume ratio within this range facilitates the reaction between oxygen free radicals in the sample and molecular probes in the probe solution, further improving detection accuracy. As one embodiment of this invention, when the sample is a solid, it can be a cube with a side length of 1 cm.

[0030] In this invention, the incubation is preferably carried out in a light-protected environment for 3-30 minutes, more preferably 5-20 minutes. Standing in a light-protected environment facilitates the reaction between oxygen free radicals in the sample and the molecular probes in the probe solution, reducing the decomposition of oxygen free radicals. The standing time within the above range ensures a complete reaction, further improving detection accuracy.

[0031] After obtaining the incubation sample, the present invention embeds the incubation sample and then freezes and slices it sequentially to obtain the sample to be tested.

[0032] As one embodiment of the present invention, the embedding can be performed by covering the sample to be tested with OCT embedding agent; when it is not possible to slice immediately, the incubated sample can be fixed in 4% paraformaldehyde.

[0033] In this invention, the freezing process is preferably performed by immersing the embedded sample in liquid nitrogen to solidify the embedding agent; using liquid nitrogen for freezing can achieve rapid freezing and further improve the accuracy of detection.

[0034] In this invention, the thickness of the sample to be tested is preferably 0.5~1μm. A thickness within this range is beneficial for fluorescence imaging and improves detection accuracy.

[0035] The present invention does not have any particular limitation on the method of slicing, as long as the sample to be tested can meet the testing requirements.

[0036] After obtaining the sample to be tested, the present invention performs fluorescence imaging on the sample to be tested.

[0037] In this invention, the excitation wavelength for fluorescence imaging is preferably 480-590 nm, more preferably 500-560 nm; as one embodiment of this invention, the excitation wavelength for fluorescence imaging can be 488 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 545 nm, or 561 nm. The characteristic peak of the BODIPY molecular probe is in the range of 480-590 nm, and an excitation wavelength within this range can excite the BODIPY molecular probe to produce fluorescence.

[0038] As one embodiment of the present invention, the fluorescence imaging instrument can be a laser confocal microscope with an excitation wavelength of 488 nm or 561 nm.

[0039] After performing fluorescence imaging on the test sample, the present invention compares the fluorescence imaging colors of different test samples. Enhanced green light indicates a more severe degree of oxidation of the corresponding test sample.

[0040] This invention utilizes the fluorescence properties of BODIPY molecular probes. By selecting molecular probes with specific structures, a directed reaction occurs with oxygen free radicals generated by lipid oxidation at a specific pH value. The conjugated system of the BODIPY molecular probe is destroyed, the hemicyanine structure breaks, and the fluorescence color changes from red to blue-green. Based on the change in fluorescence color before and after the reaction, the trend of lipid oxidation can be monitored.

[0041] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0042] Example 1 A method for monitoring lipid oxidation trends, comprising the following steps: Multiple cube-shaped meat samples with sides of 1 cm were refrigerated at 4℃ for 0–96 h, with one sample taken every 24 h. After sampling, the meat sample was immersed in 3 mL of 5 μmol / L BODIPY molecular probe solution (solvent: 1:99 DMSO and pH 7.5 PBS) and incubated in the dark for 5 min. After rinsing with PBS, the sample was immediately embedded with OCT embedding medium, covering the meat sample. The sample was then placed in liquid nitrogen to solidify the embedding medium, and then sectioned. The sections were imaged under a laser confocal microscope (excitation wavelengths of 488 nm and 561 nm). The results are shown below. Figure 3 As shown.

[0043] from Figure 3 It can be seen that by directly observing red and green light to judge the changes in lipid oxidation trends in meat samples, the gradual decrease in red light and the gradual increase in green light indicate that the level of oxygen free radical generation in the samples is gradually increasing, and correspondingly, the degree of oxidation is deepening. Figure 3 The study also revealed the spatial distribution of lipid oxidation within the meat-like tissue.

[0044] Test Example 1 Based on the fluorescence image acquired at an excitation wavelength of 561 nm in Example 1, a binary mask was generated using a thresholding method (>20) to segment the target region, as follows: Figure 4As shown; the average fluorescence intensity within the mask was calculated and corrected for background subtraction, and finally normalized using the control group as a benchmark to obtain the relative fluorescence intensity I. 588nm The values ​​are recorded in Table 1.

[0045] Table 1. Record of relative fluorescence intensity values ​​calculated based on fluorescence image mask.

[0046] Correlation analysis was performed between fluorescence intensity and two lipid oxidation indicators, POV and TBARS values, respectively. Figure 5 As shown. From Figure 5 It can be seen that I 588nm The values ​​showed a strong negative correlation with both POV and TBARS values, with correlations exceeding -0.9. Higher POV and TBARS values ​​indicated greater lipid oxidation. 588nm The decrease in the value corresponds to the increase in POV and TBARS values, indicating that the degree of lipid oxidation in meat samples is increased during storage. 588nm The value characterization results are consistent with the POV and TBARS value characterization results.

[0047] Test Example 2 The BODIPY molecular probe was dissolved in DMSO to prepare a stock solution (1 mmol / L). This stock solution was then diluted with PBS (pH 7.5) to prepare a 5 μmol / L probe solution (DMSO:PBS = 1:99). The solution was scanned using a UV-Vis spectrophotometer (scanning wavelength 300–800 nm) to obtain the UV absorption spectrum, as shown below. Figure 6 As shown.

[0048] from Figure 6 The characteristic peak of the BODIPY molecular probe can be seen at 545 nm. Further excitation at 544 nm using a fluorescence spectrophotometer yielded the fluorescence spectrum of the BODIPY molecular probe, as shown below. Figure 7 As shown. From Figure 7 It can be seen that the maximum emission wavelength of the BODIPY molecular probe is 588nm.

[0049] Test Example 3 The BODIPY molecular probe was dissolved in DMSO to prepare a stock solution (1 mmol / L). This stock solution was then mixed with disodium hydrogen citrate buffer solutions at pH values ​​of 4, 5, 6, 7, 8, 9, and 10 to prepare 5 μmol / L probe solutions. The solution was scanned every 1 minute using a UV-Vis spectrophotometer (scanning wavelength 300–800 nm) for 10 minutes. The probe response value at 0 minutes was recorded as A0 at different pH values, and the response value at different times was recorded as A. The rate of change of the probe response value at different pH values ​​was calculated using the formula (A-A0) / A0 (%). The results are shown below. Figure 8 As shown. From Figure 8 It can be seen that the BODIPY molecular probe has stable UV absorption in the pH range of 4 to 9, which covers the pH range of meat.

[0050] Test Example 4 Different concentrations of ONOO were added to 3 mL of 5 μmol / L probe solution (1:99 DMSO and pH 7.5 PBS). - The solution yields ONOO - Standard solutions with concentrations of 0 μmol / L, 2 μmol / L, 4 μmol / L, 6 μmol / L, 8 μmol / L, 10 μmol / L, 12 μmol / L, 14 μmol / L, 16 μmol / L, 18 μmol / L, and 20 μmol / L were incubated in a dark room for 5 min. The fluorescence intensity was immediately measured using a fluorescence spectrophotometer at an excitation wavelength of 490 nm. Figure 9 As shown, Figure 9 The curves of different colors represent the fluorescence curves of standard solutions of different concentrations; I is calculated based on the fluorescence intensity. 509 / I 588 The ratio values ​​were obtained and the data are recorded in Table 2. A standard curve was then plotted based on the data, as shown below. Figure 10 As shown; Table 2 Different ONOO - Table of fluorescence intensity records at 490nm excitation concentration

[0051] from Figure 10 It can be seen that the probe reacts with different concentrations of ONOO - The standard curve equation obtained after the solution reaction is y = 1.10026x - 1.96601 (R²). 2 =0.98943), and the detection limit calculated from 3δ / k is 0.056 μmol / L. The BODIPY molecular probe is effective against ONOO. - The solution shows a good response to changes in concentration and has a low detection limit.

[0052] Test Example 5 Different concentrations of ROO· solution were added to 3 mL of 5 μmol / L probe solution (1:99 DMSO and pH 7.5 PBS) to obtain standard solutions with ROO· concentrations of 0 μmol / L, 50 μmol / L, 100 μmol / L, 150 μmol / L, 200 μmol / L, 250 μmol / L, 300 μmol / L, 350 μmol / L, 400 μmol / L, 450 μmol / L, 500 μmol / L, 550 μmol / L, 600 μmol / L, 650 μmol / L, 700 μmol / L, 750 μmol / L, 800 μmol / L, 850 μmol / L, and 900 μmol / L. The solutions were incubated in the dark for 5 min, and the fluorescence intensity was immediately measured using a fluorescence spectrophotometer at an excitation wavelength of 490 nm. Figure 11 As shown, Figure 11 The curves of different colors represent the fluorescence curves of standard solutions of different concentrations; I is calculated based on the fluorescence intensity. 509 / I 588 The ratio values ​​were obtained and the data are recorded in Table 3. A standard curve was then plotted based on the data, as shown below. Figure 12 As shown.

[0053] Table 3. Fluorescence intensity records at 490 nm for different ROO concentrations.

[0054] from Figure 12 The standard curve equation obtained after the probe reacts with ROO· solutions of different concentrations is y = 0.00368x + 0.0438 (R·). 2 =0.9968), and the detection limit is calculated to be 167.89 μmol / L based on 3δ / k. The BODIPY molecular probe has a good response to the concentration change of ROO· solution.

[0055] Test Example 6 Different concentrations of ROO· solution were added to 3 mL of 5 μmol / L probe solution (solvents were DMSO at a volume ratio of 1:99 and PBS at pH 7.5) to obtain standard solutions with ROO· concentrations of 0 μmol / L, 50 μmol / L, 100 μmol / L, 150 μmol / L, 200 μmol / L, 250 μmol / L, 300 μmol / L, 350 μmol / L, 400 μmol / L, 450 μmol / L, 500 μmol / L, 550 μmol / L, 600 μmol / L, 650 μmol / L, 700 μmol / L, 750 μmol / L, 800 μmol / L, 850 μmol / L, and 900 μmol / L. The solutions were incubated in the dark for 5 min, and the fluorescence intensity was immediately measured using a fluorescence spectrophotometer at an excitation wavelength of 544 nm. Figure 13 As shown, Figure 13 The curves of different colors represent the fluorescence curves of standard solutions of different concentrations. The data obtained are recorded in Table 4. Standard curves were plotted based on the data, as shown below. Figure 14 As shown; Table 4. Fluorescence intensity records at 544 nm for different ROO concentrations.

[0056] from Figure 14 The standard curve equation obtained after the probe reacts with ROO· solutions of different concentrations is y = 56295 - 52.67x (R 2 =0.9998), and the detection limit is calculated to be 12.6 μmol / L based on 3δ / k. The probe has a good response to the concentration change of ROO· solution and the detection limit is low.

[0057] As can be seen from the above embodiments, the present invention uses the BODIPY molecular probe to effectively monitor the lipid oxidation trend of meat samples.

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for monitoring lipid oxidation trends, comprising: The sample to be tested is mixed with the probe solution and then incubated to obtain the incubated sample. The pH value of the probe solution is 4-9; The molecular probe in the probe solution is a boron fluoride dipyrrole molecular probe, with the following structural formula: ； After the incubated sample was embedded, it was frozen and sliced ​​sequentially to obtain the sample to be tested; Fluorescence imaging was performed on the sample to be tested; By comparing the fluorescence imaging colors of different test samples, if green light is enhanced and red light is weakened, the oxidation degree of the corresponding test sample is more severe.

2. The method according to claim 1, characterized in that, The concentration of the molecular probe in the probe solution is 1~10 μmol / L.

3. The method according to claim 2, characterized in that, The mass ratio of the sample to the probe solution is 1 g:(2~5) mL.

4. The method according to claim 1, characterized in that, The solvent for the probe solution is a mixture of dimethyl sulfoxide and phosphate buffer solution.

5. The method according to claim 4, characterized in that, The volume ratio of the dimethyl sulfoxide to the phosphate buffer solution is 1:(1~99).

6. The method according to claim 1, characterized in that, The incubation process involves placing the food in a dark environment for 3 to 30 minutes.

7. The method according to claim 1, characterized in that, The excitation wavelength for the fluorescence imaging is 480~590nm.

8. The method according to claim 1, characterized in that, The freezing process involves immersing the embedded sample in liquid nitrogen to solidify the embedding agent.

9. The method according to claim 1, characterized in that, The thickness of the sample to be tested is 0.5~1μm.

10. The method according to claim 1, characterized in that, The sources of the samples to be tested include meat, lipid-containing plant products, or oils.

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