A near-infrared fluorescent probe for detecting viscosity of liquid food and a preparation method and application thereof

Abstract

The application discloses a near-infrared fluorescent probe for detecting viscosity of liquid food, and a preparation method and application thereof. The fluorescent probe is 2-(2-6-(diethylamino)-1,2-dihydrocyclopenta[b]chromen-3-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indol-3-ium salt. The fluorescent probe contains two kinds of rotor elements, i.e. a vinyl bond and a single bond, which is beneficial to sensitive response of the probe to viscosity change. The probe has a long emission wavelength in a high-viscosity solvent, which can reach 787 nm, and has strong fluorescence penetration, low background interference and high signal-to-noise ratio. In addition, the probe has good selectivity for the high-viscosity solvent and high sensitivity, and therefore, the probe can be applied to detection of viscosity of liquid food.

Description

Technical Field

[0001] This invention belongs to the field of viscosity analysis and detection of liquid food, specifically relating to a near-infrared fluorescent probe for detecting viscosity, its preparation method, and its application. Background Technology

[0002] Liquid food safety is a major public health issue worldwide. Liquid spoilage typically occurs during long-term transportation and storage, often leading to significant economic losses and even serious public health problems. Various foodborne illnesses can be caused by liquid spoilage, making liquid safety inspections crucial. Commercial liquid foods are typically composed of various nutritional additives, such as cations, anions, amino acids, glucose, and vitamins. These basic components play a vital role in maintaining the internal and external balance of liquid foods. Simultaneously, the growth of bacteria, molds, and yeasts during liquid food spoilage depends on these nutrients, resulting in significant changes to the liquid's microenvironment. Viscosity, as an important microenvironmental physical parameter, plays a key role in the spoilage process because the detection of viscosity increases is directly related to the assessment of the degree of spoilage.

[0003] Traditional viscosity detection methods, such as viscometer measurements, typically require complex pretreatment processes, time-consuming detection procedures, and involve significant sample consumption and damage. Furthermore, these methods are highly dependent on operator skills, the ability to process experimental results, and the equipment used, severely impacting the accuracy of the results. In recent years, fluorescent probes have attracted considerable attention due to their low cost, ease of operation, high sensitivity, non-destructive testing, and real-time imaging capabilities, and are widely used in biochemical analysis and fluorescence tracing. In addition, near-infrared fluorescent probes with long-wavelength emission offer advantages such as low levels of autofluorescence interference and high imaging signal-to-noise ratio, making them an effective means of monitoring certain physical or chemical indicators in the environment.

[0004] Several fluorescent probes for viscosity detection have been developed. For example, patent CN 114957041 A, "A Fluorescent Probe for Detecting the Viscosity of Industrial Lubricating Oil and Its Preparation Method and Application," designs a DA-structured fluorescent probe for viscosity detection formed by the Nevon condensation of salicylaldehyde and 2-(5,6-dichloro-3-oxoinden-1-ylidene)malonitrile. The ethyl ester group on the aromatic ring of this probe provides it with appropriate lipophilicity, flexibility, and steric hindrance, resulting in good dispersibility of the probe molecule in lubricating oil, making it suitable for detecting the viscosity of industrial lubricating oil. However, the fluorescence intensity of this probe in a 99% glycerol / water system is less than 10 times higher than that in a 0% glycerol / water system, reflecting its poor sensitivity to viscosity response and making it difficult to apply to the detection of minute viscosity changes in liquid microenvironments. Furthermore, the short emission wavelength of this probe, located in the visible light region, limits its application to some extent. Patent CN 115215864 A, "A Fluorescent Probe for Detecting Beverage Viscosity and Its Preparation Method and Application," develops a viscosity-responsive fluorescent probe using bromobenzimidazole and indolonium as double electron-withdrawing groups and methoxyphenyl as an electron-donating group. As the proportion of the high-viscosity solvent glycerol increases, the probe's fluorescence at 570 nm intensifies, thus enabling viscosity detection. Although this probe achieves a 40-fold fluorescence enhancement and can sensitively respond to changes in viscosity, its absorption and emission wavelengths are relatively short, not reaching the near-infrared region. Fluorescent probes with near-infrared emission wavelengths can minimize interference from the autofluorescence of substances such as proteins and cellulose in food, thereby improving detection accuracy. Patent CN 114790388 A, "Application of Tetrahydronaphthone as a Near-Infrared Fluorescent Probe in Lipid Droplet Imaging," prepares a molecule with a tetrahydronaphthone derivative group as an electron donor and a conjugated electron-withdrawing p-nitrophenylacetonitrile (DA) structure for fluorescent imaging of lipid droplets in cells or tissues. Although the fluorescence probe emits in the near-infrared region, its fluorescence enhancement is only about 12 times, making it difficult to sensitively respond to changes in the viscosity of the liquid microenvironment. Therefore, preparing a fluorescence probe that simultaneously possesses near-infrared emission wavelength and significantly enhanced fluorescence intensity at high viscosity is of great significance for the easy, accurate, and sensitive detection of viscosity changes in liquid foods. Summary of the Invention

[0005] To address the shortcomings and deficiencies of the prior art, the present invention aims to provide a near-infrared fluorescent probe for detecting the viscosity of liquid food, its preparation method, and its application. Specifically, it provides a near-infrared fluorescent probe capable of detecting the viscosity of liquid food, along with a preparation method for the probe and its application in detecting viscosity changes during the spoilage and deterioration of liquid food. The fluorescent probe of the present invention exhibits long-wavelength near-infrared emission in high-viscosity solvents, mitigating the fluorescence interference problem inherent in short-wavelength probes. Furthermore, the probe contains two rotor units, which facilitates a sensitive response to viscosity changes in liquid food.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A near-infrared fluorescent probe for detecting the viscosity of liquid foods, the probe having the molecular formula C 33 H 35 N2O + The specific structure is as follows:

[0008]

[0009] This invention provides a method for preparing a near-infrared fluorescent probe for detecting the viscosity of liquid food, comprising the following steps:

[0010] Compounds 6-(diethylamino)-1,2-dihydrocyclopentano[b]chromene-3-carboxaldehyde, 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-onium salt, and sodium acetate were dissolved in acetic anhydride. The reaction system was then purged with an inert gas to maintain an inert atmosphere, and the reaction was carried out under heating with stirring. After the reaction was completed, the fluorescent probe was obtained by separation and purification.

[0011] Preferably, the molar ratio of the compound 6-(diethylamino)-1,2-dihydrocyclopentane-3-carboxaldehyde to 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-onium salt is 1:(1-1.2).

[0012] Preferably, the molar ratio of the compound 6-(diethylamino)-1,2-dihydrocyclopentano[b]chromene-3-carboxaldehyde to sodium acetate is 1:(2-2.5).

[0013] Preferably, the amount of acetic anhydride added per mmol of compound 6-(diethylamino)-1,2-dihydrocyclopentano[b]chromene-3-carboxaldehyde is (8-10) mL.

[0014] Preferably, the inert atmosphere is achieved by evacuating the system and purging it with nitrogen, repeating this process at least three times, wherein the inert gas is nitrogen.

[0015] Preferably, the temperature of the stirring reaction under heating is (80-90)℃.

[0016] Preferably, the stirring reaction time under heating is (0.5-1) h.

[0017] Preferably, the purification method is silica gel chromatography.

[0018] Preferably, the eluent used in the silica gel chromatography is dichloromethane / methanol.

[0019] The present invention also provides the application of the near-infrared fluorescent probe for detecting the viscosity of liquid food in detecting viscosity changes during the spoilage and deterioration of liquid food.

[0020] Compared with existing technologies, the outstanding advantages of the fluorescent probe provided by this invention include:

[0021] (1) The fluorescent probe of this invention has an ADD-type structure, thus possessing a longer emission wavelength. Upon response to a high-viscosity solvent (glycerol), the intramolecular motion is restricted, and the intramolecular electron push-pull effect is significantly enhanced, resulting in near-infrared fluorescence emission. Compared to some other short-wavelength probes, this probe can effectively reduce the autofluorescence interference that may be generated by substances such as proteins and cellulose in liquid foods. Furthermore, since the emission is in the near-infrared region, the detection depth is greatly increased, enabling effective detection of changes in the viscosity of liquid foods.

[0022] (2) The fluorescent probe of the present invention contains multiple freely rotating rotor units, which can sensitively detect changes in viscosity. As the ambient viscosity increases, the free rotation of vinyl bonds and single bonds is restricted, and the fluorescence intensity increases accordingly. Changes in viscosity can be characterized by changes in fluorescence intensity.

[0023] (3) The fluorescent probe of the present invention has strong anti-interference ability and does not respond to some ions, amino acids and food additives that may be present in liquid food. It also has good selectivity for high viscosity solvents (glycerol). Therefore, when this probe is applied to the viscosity detection of liquid food, it is simple to operate, fast, accurate and reliable.

[0024] (4) The fluorescent probe of the present invention has good chemical stability. Its molecular structure is simple, and the vinyl bond and the single bond on the diethylamino bond can act as an intramolecular rotor to achieve viscosity responsiveness. No other more complex rotor structure is required. Therefore, the preparation process of this probe is simple, the reaction conditions are mild, and the manufacturing cost is low, making it suitable for large-scale production and use. Attached Figure Description

[0025] Figure 1 This is a synthetic route diagram of the fluorescent probe HCY-V of the present invention.

[0026] Figure 2 This is the 1H NMR spectrum of the fluorescent probe HCY-V in Example 1.

[0027] Figure 3 This is a high-resolution mass spectrum of the fluorescent probe HCY-V in Example 1.

[0028] Figure 4 The fluorescence spectra of the fluorescent probe HCY-V in Application Example 1 in response to viscosity changes in glycerol / water mixed solutions of different volume ratios are shown.

[0029] Figure 5 This is a linear fit graph of the fluorescence intensity of the fluorescent probe HCY-V at 787 nm and its viscosity as a logarithmic function in Application Example 1.

[0030] Figure 6 This is a selective test diagram of the fluorescent probe HCY-V in Application Example 2.

[0031] Figure 7 This is the fluorescence emission spectrum of raspberry juice solutions stored at room temperature for different days using the fluorescent probe HCY-V in Example 3.

[0032] Figure 8 This is a viscosity graph of raspberry juice solutions stored at room temperature for different numbers of days in Application Example 3. Detailed Implementation

[0033] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0034] The synthesis route of the near-infrared fluorescent probe for detecting the viscosity of liquid food of the present invention is as follows: Figure 1 As shown.

[0035] Example 1

[0036] 67.29 mg (0.25 mmol) of 6-(diethylamino)-1,2-dihydrocyclopentane[b]chromene-3-carboxaldehyde, 87.76 mg (0.25 mmol) of 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-onium salt and 41 mg (0.5 mmol) of sodium acetate were dissolved in 2 mL of acetic anhydride to obtain a mixed solution. Nitrogen gas was introduced to maintain the reaction system under a nitrogen atmosphere, and the mixture was stirred and heated to 85 °C for 0.5 h. After the reaction was completed, the mixed solution was cooled to room temperature and evaporated by rotary evaporation. The resulting solid was purified by silica gel column chromatography (using dichloromethane / methanol as the eluent, V / V = 30:1) to obtain 121.28 mg of fluorescent probe (yield: 80.56%).

[0037] Characterized by proton nuclear magnetic resonance spectroscopy:1 H NMR (400MHz, CDCl3) δ8.21(d,J=14.2Hz,1H),8.17(d,J=8.6Hz,1H),7.92-7.97(m,2H ),7.62(t,J=7.0Hz,1H),7.50(d,J=2.9Hz,1H),7.47(d,J=8.2Hz,1H),7.33-7.36(m,2 The hydrogen NMR spectrum of the probe is as follows: 6.73 (d, J = 2.6 Hz, 1H), 6.71 (s, 1H), 6.05 (d, J = 14.2 Hz, 1H), 4.39–4.44 (m, 2H), 3.5–3.55 (m, 4H), 3.03 (s, 4H), 2.05 (s, 6H), 1.52 (t, J = 7.3 Hz, 3H), 1.28 (t, J = 7.2 Hz, 6H). Figure 2 As shown

[0038] Furthermore, it was further verified by high-resolution mass spectrometry: HR-MS (ESI, m / z): theoretically calculated molecular mass-to-charge ratio C 34 H 37 N2O + [M] + The mass-to-charge ratio (MMR) of the probe is 489.2906, and the actual molecular mass-to-charge ratio is 489.2922. The high-resolution mass spectrum of this probe is shown below. Figure 3 As shown.

[0039] Example 2

[0040] 134.58 mg (0.5 mmol) of 6-(diethylamino)-1,2-dihydrocyclopentane[b]chromene-3-carboxaldehyde, 210.62 mg (0.6 mmol) of 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-onium salt and 90.2 mg (1.1 mmol) of sodium acetate were dissolved in 4.5 mL of acetic anhydride to obtain a mixed solution. Nitrogen gas was introduced to maintain the reaction system under a nitrogen atmosphere, and the mixture was stirred and heated to 90 °C for 45 min. After the reaction was completed, the mixed solution was cooled to room temperature and evaporated by rotary evaporation. The resulting solid was purified by silica gel column chromatography (using dichloromethane / methanol as the eluent, V / V = 30:1) to obtain 231.06 mg of fluorescent probe (yield: 76.74%).

[0041] The fluorescent probe obtained in this embodiment has the same characterization results as in Example 1.

[0042] Example 3

[0043] 269.16 mg (1 mmol) of 6-(diethylamino)-1,2-dihydrocyclopentane[b]chromene-3-carboxaldehyde, 386.14 mg (1.1 mmol) of 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-onium salt and 205 mg (2.5 mmol) of sodium acetate were dissolved in 10 mL of acetic anhydride to obtain a mixed solution. Nitrogen gas was introduced to keep the reaction system under a nitrogen atmosphere, and the mixture was stirred and heated to 80 °C for 1 h. After the reaction was completed, the mixed solution was cooled to room temperature and evaporated by rotary evaporation. The resulting solid was purified by silica gel column chromatography (using dichloromethane / methanol as the eluent, V / V = 30:1) to obtain 423.11 mg of fluorescent probe (yield: 70.26%).

[0044] The fluorescent probe obtained in this embodiment has the same characterization results as in Example 1.

[0045] Application Example 1

[0046] Viscosity response test of fluorescent probe HCY-V:

[0047] The fluorescent probe HCY-V prepared in Example 1 was dissolved in DMSO to prepare a test stock solution with a probe concentration of 1 mM. 30 μL of the probe stock solution was dissolved in mixed solutions of glycerol / water with different volume ratios for viscosity response testing. Maintaining a total test volume of 3.0 mL, and using a wavelength of 720 nm as the excitation wavelength, the viscosity of the system was changed by varying the volume fraction of glycerol. Viscosity response fluorescence spectra were measured in mixed solutions with 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99% glycerol volume percentages. At room temperature, the viscosities of these eleven test systems were 1 cP, 2.3 cP, 3.7 cP, 5.4 cP, 14.45 cP, 30.5 cP, 60.1 cP, 109 cP, 219 cP, 488 cP, and 1412 cP, respectively. The obtained fluorescence spectra are shown below. Figure 4 As shown, a plot of the logarithm of fluorescence intensity at 787 nm versus the logarithm of viscosity is presented. Figure 4 As shown. From Figure 4 As can be seen, the fluorescence intensity of the solution gradually increases with the increase of the volume fraction of glycerol in the solution (i.e., the viscosity gradually increases). When the volume fraction of glycerol reaches 99%, the fluorescence intensity reaches its maximum value, which is about 34 times higher than that of the 0% glycerol solution system. This indicates that the fluorescence intensity of the fluorescent probe HCY-V changes significantly with viscosity. Meanwhile, from... Figure 5As can be seen, this fluorescent probe is quite sensitive to viscosity changes. The logarithmic relationship between its fluorescence intensity and the logarithmic relationship between the solution viscosity and the fluorescence intensity shows a good linear relationship; that is, as the viscosity increases, its fluorescence intensity gradually increases. The sensitivity is calculated to be 0.29134 using the Forster-Hoffmann equation, and the logarithmic function between the fluorescence intensity and viscosity at 787 nm is logI. 787 =2.23857 + 0.29134logη, the logarithm of the fluorescence intensity at 787 nm shows a good linear relationship with the logarithm of the viscosity in the range of 0.57-3.15, indicating that the fluorescent probe HCY-V has good sensitivity to viscosity. The above test results show that the fluorescent probe HCY-V of this invention has a good response to viscosity and can characterize viscosity changes through changes in fluorescence intensity, making it suitable for detecting the viscosity of liquid foods.

[0048] Application Example 2

[0049] Selectivity testing of the fluorescent probe HCY-V:

[0050] Some of the substances being detected include: ions (K+) + Na + Ca + Mg 2+ Fe 2+ Fe 3+ Zn 2+ NO3 3- PO4 2- SO4 2- The following substances were dissolved in deionized water to obtain corresponding solutions (viscosity values ​​less than 2 cP): amino acids (GSH, Cys), food additives (glucose, fructose, vitamin C, disodium hydrogen phosphate, and dehydrated sodium citrate). 30 μl of the probe stock solution from Application Example 1 was added to 2.97 ml of glycerol to prepare a glycerol (containing 1% DMSO) test sample (viscosity value 1412 cP). Similarly, 30 μL of the probe stock solution from Application Example 1 was added to the solutions of the above-mentioned analytes (containing 1% DMSO), while an aqueous solution containing only the fluorescent probe (containing 1% DMSO, viscosity value 1 cP) was used as a control group. During testing, the concentration of the fluorescent probe HCY-V was maintained at 10 μM in all test samples, the concentration of each analyte was 100 μM, the volume percentage of methanol in each test sample was 1%, and the total volume of each group of test samples was maintained at 3 mL. The fluorescence intensity of each group of test samples at 787 nm was measured, and a bar graph of the test results was plotted. The results are shown below. Figure 6 As shown. From Figure 6As can be seen, the fluorescence intensity of the test groups with the addition of various other substances did not change significantly. Only in the viscous glycerol solution (containing 1% DMSO) was a significant increase in fluorescence intensity observed. The experimental results indicate that the fluorescent probe HCY-V has a good selective response to viscosity; only changes in viscosity can cause changes in fluorescence intensity. Furthermore, this fluorescent probe has high chemical stability and exhibits inertness to a variety of chemical substances, making it suitable for use in the complex microenvironment of liquid foods. The aforementioned components will not interfere with the fluorescence signal.

[0051] Application Example 3

[0052] Application of the fluorescent probe HCY-V in detecting viscosity changes during the spoilage of liquid food:

[0053] To track the spoilage process of liquid food, raspberry juice was stored at room temperature for 8 days. Fluorescence spectra were measured on days 0, 2, 4, 6, and 8. A raspberry juice test sample (containing 1% DMSO) was prepared by adding 30 μL of the probe stock solution from Application Example 1 to 2.97 mL of raspberry juice. F0 and F2 were measured. n These are the fluorescence intensity values ​​of raspberry juice on day 0 and day n, where 0 < n < 9. The results are as follows: Figure 7 As shown. From Figure 7 It can be seen that the fluorescence intensity increases with the degree of spoilage of the raspberry juice, increasing by 21.22% on day 8 compared to day 0. Meanwhile, the viscosity values ​​of the raspberry juice, η0 and η, were measured using a viscometer on days 0, 2, 4, 6, and 8. n These are the viscosity values ​​of raspberry juice on day 0 and day n, where 0 < n < 9. The results are as follows: Figure 8 .Depend on Figure 8 It can be seen that the viscosity of raspberry juice increases with the number of storage days, with a 15.63% increase on day 8 compared to day 0, indicating that the greater the degree of spoilage, the higher the viscosity. These results demonstrate that the fluorescent probe HCY-V can accurately assess the viscosity level during the spoilage process of liquid food using fluorescence technology, thereby distinguishing between fresh and spoiled liquids.

[0054] The above examples are preferred embodiments of the present invention, but the embodiments of the invention are not limited to the above examples. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An application of a near-infrared fluorescent probe in detecting viscosity changes during the spoilage of liquid food, characterized in that... The molecular formula of the probe is C 34 H 37 N2O + It has the following structural formula: The probe has a maximum emission wavelength of 787 nm in 99% glycerol and can detect viscosity changes during the spoilage of liquid food.

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