PH fluorescent probe and application thereof in food microorganism detection

Abstract

The invention provides a pH fluorescent probe and application thereof in food microbiological detection, the chemical structure of the probe is (E)-6 '-(diethylamino)-4'-(2, 4-dimethoxybenzylidene)-2-(2-morpholinoethyl)-1 ', 2', 3 ', 4'-tetrahydrospiro [isoquinoline-1, 9 '-anthracene]-3-ketone, and the structural formula of the probe is shown in the description. The preparation method comprises the following steps: (1) carrying out acid catalytic condensation on cyclohexanone and 4-diethylaminoketonic acid; (2) carrying out aldehyde ketone condensation with 3, 4-dimethoxybenzaldehyde; and (3) finally, carrying out an amidation reaction with 4-(2-aminoethyl) morpholine under the mediation of BOP. The pH fluorescent probe has a good linear relationship between the fluorescence intensity and the pH value within the pH range of 5-8, and completely covers the whole food spoilage process (pH 5.8-7.5); after 4 times of acid-base circulation, the fluorescence recovery rate is gt; therefore, the composite material has excellent reversibility and stability; and the response steady-state value within 1 minute can reach 90% or above. The probe provided by the invention realizes rapid detection, provides an efficient tool for real-time and sensitive monitoring of food freshness, and can be extensively applied to microbial spoilage detection of foods such as aquatic products and dairy products.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing, specifically relating to a pH fluorescent probe and its application in food microbial testing. Background Technology

[0002] Food safety is a major fundamental issue concerning people's livelihood, directly related to consumers' health and safety. Consuming unsafe food not only causes acute, subacute, or chronic health hazards, resulting in enormous personal suffering and a heavy social medical burden, but also affects social harmony, stability, and healthy economic development. Among the many evaluation indicators for food safety, food freshness is one of the core qualities. During storage, food is affected by microorganisms (such as bacteria and mold) and endogenous enzymes; on the other hand, a series of complex biochemical reactions occur within the food (such as protein degradation and fat oxidation), leading to spoilage, flavor deterioration, destruction of nutritional components, and even the production of toxic substances that threaten human health. Therefore, accurate and timely detection of food freshness is a crucial link in ensuring food safety.

[0003] Among numerous indicators of food spoilage, pH changes are an extremely important and universally applicable signal. Taking meat as an example, after slaughter, glycogen in the animal's body ferments to produce lactic acid, while adenosine triphosphate (ATP) decomposes to produce acidic substances such as phosphoric acid, causing an initial drop in pH. As storage time increases, microorganisms multiply rapidly and decompose proteins and other components, producing alkaline volatile substances such as ammonia and amines, which causes a significant increase in pH. Therefore, the trajectory of pH changes can sensitively and intuitively reflect the spoilage process of meat. However, traditional pH detection methods have significant limitations in the food industry: electrode methods require frequent calibration, have long response times, and are prone to drift due to membrane fouling in oily matrices (such as meat). The test strip method has low sensitivity (resolution ≥ 0.5 pH units) and cannot meet the detection requirements of weak pH changes in the early stage of spoilage (such as the initial spoilage of pork only decreases by 0.05~0.1 pH units). Although existing fluorescent probes can partially replace traditional methods, they still have the following key defects: (1) Narrow linear range: Most probes only cover pH 6.0~7.5 and cannot detect the entire process of pork spoilage (pH 5.8 to 7.5); (2) Poor reversibility: The fluorescence recovery rate after acid-base cycling is often less than 80%; (3) Poor kinetic performance: Triple base stacked probes require 35 min to reach 90% signal saturation.

[0004] Therefore, there is an urgent need to develop a pH fluorescent probe that can cover the typical pH range of the entire food spoilage process, has good reversibility, and can respond quickly, so as to effectively ensure food safety, reduce food waste, and recover huge economic losses. This has significant scientific significance and practical application value. Summary of the Invention

[0005] To address the aforementioned technical problems, the first objective of this invention is to provide a pH fluorescent probe.

[0006] A second objective of this invention is to provide a method for preparing the pH fluorescent probe.

[0007] A third objective of this invention is to provide the application of the pH fluorescent probe in the detection of microorganisms in food.

[0008] Technical solution: A pH fluorescent probe with the chemical structure (E)-6'-(diethylamino)-4'-(2,4-dimethoxybenzyl)-2-(2-morpholinoethyl)-1',2',3',4'-tetrahydrospiro[isoquinoline-1,9'-anthraphen]-3-one, and the chemical structural formula is as follows:

[0009]

[0010] Preferably, the pH fluorescent probe is a pH fluorescent probe based on the isoquinoline-spiroanthrone structure.

[0011] Furthermore, the isoquinoline-spiroanthrone structure is a tetrahydrospiro[isoquinoline-1,9'-anthrene]-3-one structure.

[0012] The preparation method of the above-mentioned pH fluorescent probe includes the following steps:

[0013] S1. Cyclohexanone and 4-diethylaminoketo acid were condensed in concentrated sulfuric acid. After the reaction was completed, the mixture was cooled to room temperature and transferred to ice water with stirring. 70% HClO4 was added to produce a red precipitate. After filtration, washing, drying, and separation by chromatography, compound 1 was obtained.

[0014] S2. Compound 1 and 3,4-dimethoxybenzaldehyde were condensed in acetic acid. After the reaction was completed, the mixture was cooled to room temperature and then rotary evaporated to obtain compound 2.

[0015] S3. Compound 2 and BOP were added to dichloromethane, and 4-(2-aminoethyl)morpholine was added dropwise. The mixture was stirred at room temperature, and after extraction with dichloromethane, washing with water and NaCl solution, drying with anhydrous Na2SO4, and rotary evaporation, the pH fluorescent probe FCE was obtained.

[0016] Preferably, the molar ratio of cyclohexanone to 4-diethylaminoketo acid in step S1 is 1.5~2.5:1.

[0017] Preferably, in step S1, the mass-to-volume ratio of concentrated sulfuric acid to 4-diethylaminoketo acid is 1:5~10.

[0018] Preferably, the temperature of the condensation reaction in step S1 is 90~110℃, and the time of the condensation reaction is 2~6 h.

[0019] Preferably, the volume ratio of 70% HClO4 to 4-diethylaminoketo acid in step S1 is 2.5~3:1.

[0020] Preferably, the molar ratio of compound 1 and 3,4-dimethoxybenzaldehyde in step S2 is 1:1.1~1.5.

[0021] Preferably, the temperature of the condensation reaction in step S2 is 110~120℃, and the time of the condensation reaction is 15~18h.

[0022] Preferably, the molar ratio of compound 2, BOP and 4-(2-aminoethyl)morpholine in step S3 is 1:1.5~2:8~15.

[0023] Preferably, the reaction time in step S3 is 12-15 h.

[0024] The above-mentioned pH fluorescent probe FCE is used in the detection of microorganisms in food.

[0025] Beneficial effects:

[0026] 1. The linear response range (pH 5.0~8.0) of the pH fluorescent probe FCE provided by this invention not only fully covers the entire process of meat spoilage (pH 5.8~7.5), but also encompasses the typical range of environmental pH changes caused by various microbial metabolic activities. This enables accurate and continuous monitoring of the meat spoilage process and provides an effective detection window for environmental pH changes caused by various microbial metabolic activities. During food spoilage and fermentation, microbial metabolism is often accompanied by significant pH changes. For example, lactic acid bacteria fermentation can significantly lower the environmental pH, while many spoilage bacteria produce ammonia and amines when decomposing proteins, thus causing a pH increase. The linear response range of the FCE probe of this invention precisely covers the typical pH changes caused by these key biochemical processes. Therefore, it is not only suitable for meat freshness assessment, but can also be widely applied to fermentation process monitoring, environmental microbial activity research, and other biological, chemical, and food detection scenarios involving dynamic pH changes, demonstrating good versatility and practical value.

[0027] 2. The pH fluorescent probe FCE provided by this invention maintains a fluorescence signal recovery rate of over 95% after four cycles of strong acid and strong alkali (pH 1.0 ⇌ 10.0), demonstrating excellent reversibility and stability. This characteristic is crucial for scenarios requiring long-term, continuous monitoring of dynamic changes in microbial activity, such as composting processes, bioreactors, or soil microbial activity monitoring, where the pH of these environments may fluctuate frequently due to microbial community succession.

[0028] 3. The pH fluorescent probe FCE provided by this invention achieves a steady-state value of over 90% within 1 minute, demonstrating excellent rapid response capability. This enables the probe to capture rapid pH changes caused by microbial metabolic activities in near real-time, providing timely feedback for process control.

[0029] 4. The pH fluorescence probe (FCE) provided by this invention enables dynamic and visual monitoring of the food spoilage process. By tracking changes in fluorescence intensity in real time, the FCE probe can clearly reflect the dynamic pH trajectory dominated by microbial metabolic activity during storage, achieving continuous and non-destructive monitoring of the spoilage process. For example, many pathogenic microorganisms (such as molds and bacteria) that cause fruit and vegetable spoilage alter the pH value of the microenvironment during infection; the FCE probe can be integrated into smart packaging films or labels. In wastewater treatment and eutrophication monitoring, microbial respiration, nitrification / denitrification, and other processes are accompanied by pH changes. The FCE probe can be used for online monitoring of pH profiles at different stages within a reactor or to assess the intensity of microbial metabolic activity in natural water bodies.

[0030] 5. The pH fluorescent probe FCE provided by this invention can achieve accurate early warning and degree of spoilage initiation. Under storage conditions of 4℃, the fluorescence signal first decreases and then increases, and the inflection point of this change can serve as a key early warning signal for the early stage of spoilage; while the signal entering a high plateau period indicates that spoilage is irreversible, thus enabling accurate differentiation of different stages of spoilage.

[0031] 6. The pH fluorescent probe FCE provided by this invention can reveal the microscopic mechanism of the putrefaction process. By analyzing the fluorescence trajectory, the succession pattern of the microbial community can be deduced: the initial fluorescence inhibition corresponds to acid production by lactic acid bacteria, and the later fluorescence surge corresponds to alkali production by protein-decomposing bacteria, thus intuitively revealing the intrinsic microbial activity mechanism of putrefaction from a chemical perspective. Attached Figure Description

[0033] Figure 1 The synthesis route diagrams for the pH fluorescent probe FCE in Examples 1-4 are shown.

[0034] Figure 2 The UV-Vis absorption spectrum of the pH fluorescent probe FCE in Example 1 is shown.

[0035] Figure 3 The pH response fluorescence spectrum of the pH fluorescent probe FCE in Example 1;

[0036] Figure 4 Example 1: The fluorescence intensity-pH linear relationship of the pH fluorescent probe FCE;

[0037] Figure 5 The fluorescence titration spectrum of the pH fluorescent probe FCE in Example 1 is shown.

[0038] Figure 6 The results of the reversibility test of the pH fluorescent probe FCE in Example 1 are shown.

[0039] Figure 7 The results are the response time test results of the pH fluorescent probe FCE in Example 1;

[0040] Figure 8 The following are line graphs (A) and bar graphs (B) showing the pH changes of pork over time under fluorescence spectrophotometer and pH meter conditions in Example 1.

[0041] Figure 9 This is a graph showing the color changes of pork over ten days at -20℃ (above) and 4℃ (below). Detailed Implementation

[0043] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments:

[0044] Example 1

[0045] This embodiment describes a method for preparing a pH fluorescent probe (FCE), including the following steps:

[0046] S1. 1.32 mL of cyclohexanone (12.74 mmol) was added dropwise to 14 mL of concentrated sulfuric acid (0 °C). Under vigorous stirring, 2.0 g of 4-diethylaminoketo acid (6.4 mmol) was added all at once. The mixture was then heated to 90 °C and reacted for 2 h. After the reaction was completed, the reactants were cooled to room temperature and poured into 30 g of ice water while stirring. Immediately afterward, 1.4 mL of 70% HClO4 was added, and a large amount of red precipitate was produced. After filtration, the solid was washed with 40 mL of ice water, dried, and separated by chromatography (DCM:MeOH=30:1, v / v) to obtain 3.68 g of red solid, which is compound 1.

[0047] S2. 144.48 mg of compound 1 (0.42 mmol) and 83.089 mg of 3,4-dimethoxybenzaldehyde (0.5 mmol) were dissolved in 35 mL of acetic acid and heated to 110 °C. The reaction was continued until the reactants were completely consumed. After the reaction was completed, the mixture was cooled to room temperature and rotary evaporated to obtain 0.201 g of black solid, which was compound 2.

[0048] S3. Add 500 mg of compound 2 and 620.75 mg of BOP to 22.5 mL of dichloromethane, then add 1275 mg of 4-(2-aminoethyl)morpholine dropwise. Stir the reaction at room temperature for about 12 h. Detect the reaction by TLC. If the reaction is not complete, add another 1275 mg of 4-(2-aminoethyl)morpholine. After the reaction is complete, add excess dichloromethane for extraction. Wash 2-3 times with water and 2-3 times with saturated NaCl solution. Add 5 g of anhydrous Na2SO4 to dry, and remove the solvent by rotary evaporation. The final product is (E)-6'-(diethylamino)-4'-(2,4-dimethoxybenzyl)-2-(2-morpholinoethyl)-1',2',3',4'-tetrahydrospiro[isoquinoline-1,9'-anthraphen]-3-one.

[0049] Example 2

[0050] This embodiment describes a method for preparing a pH fluorescent probe (FCE), including the following steps:

[0051] S1. 1.20 mL of cyclohexanone (approximately 11.6 mmol) was added dropwise to 12 mL of concentrated sulfuric acid (0 °C). Under vigorous stirring, 1.8 g of 4-diethylaminoketo acid (approximately 5.8 mmol) was added all at once. The mixture was then heated to 90 °C and reacted for 2 h. After the reaction was completed, the mixture was cooled to room temperature and poured into 30 g of ice water with stirring. Immediately afterward, 1.3 mL of 70% HClO4 was added, and a large amount of red precipitate was produced. After filtration, the solid was washed with 40 mL of ice water, dried, and separated by chromatography (DCM:MeOH = 30:1, v / v) to obtain approximately 3.2 g of red solid, which is compound 1.

[0052] S2. Dissolve 130 mg of compound 1 (approximately 0.38 mmol) and 75 mg of 3,4-dimethoxybenzaldehyde (approximately 0.45 mmol) in 30 mL of acetic acid, heat to 110 °C, and react until the reactants are completely consumed. After cooling, rotary evaporate to obtain approximately 0.18 g of black solid, which is compound 2.

[0053] S3. Add 450 mg of compound 2 and 560 mg of BOP to 20 mL of dichloromethane, then add 1150 mg of 4-(2-aminoethyl)morpholine dropwise. Stir the reaction at room temperature for about 12 h, monitor the reaction progress by TLC, and add 1150 mg of 4-(2-aminoethyl)morpholine if necessary. After the reaction is complete, extract with excess dichloromethane, wash with water 2-3 times, wash with saturated NaCl solution 2-3 times, dry with 6 g of anhydrous Na2SO4, and remove the solvent by rotary evaporation to obtain the final product (E)-6'-(diethylamino)-4'-(2,4-dimethoxybenzyl)-2-(2-morpholinoethyl)-1',2',3',4'-tetrahydrospiro[isoquinoline-1,9'-anthraphen]-3-one.

[0054] Example 3

[0055] This embodiment describes a method for preparing a pH fluorescent probe (FCE), including the following steps:

[0056] S1. 0.66 mL of cyclohexanone (approximately 6.4 mmol) was added dropwise to 14 mL of concentrated sulfuric acid (0 °C). Under vigorous stirring, 2.0 g of 4-diethylaminoketo acid (6.4 mmol, in equimolar ratio with cyclohexanone) was added all at once. The mixture was then heated to 90 °C and reacted for 2 h. After the reaction was completed, the mixture was cooled to room temperature and poured into 30 g of ice water with stirring. Immediately afterward, 1.4 mL of 70% HClO4 was added, producing a red precipitate. The precipitate was filtered, washed with 40 mL of ice water, dried, and separated by column chromatography to obtain approximately 2.5 g of red solid, which was compound 1.

[0057] S2. 144.48 mg of compound 1 (0.42 mmol) and 66.47 mg of 3,4-dimethoxybenzaldehyde (0.4 mmol, aldehyde content insufficient) were dissolved in 40 mL of acetic acid and heated to 110 °C. TLC monitoring showed that the reaction was slow and byproducts were formed. After the reaction was completed, the mixture was cooled and rotary evaporated to obtain approximately 0.15 g of black solid, which was compound 2.

[0058] S3. 500 mg of compound 2 and 500 mg of BOP (the amount of condensing agent was insufficient) were added to 22.5 mL of dichloromethane, followed by the dropwise addition of 1275 mg of 4-(2-aminoethyl)morpholine. The mixture was stirred at room temperature for about 12 h. TLC analysis showed that a significant amount of the starting material remained. After the reaction was completed, the mixture was extracted, washed, dried, and rotary evaporated to obtain approximately 0.422 g of a purplish-black solid. The final product was (E)-6'-(diethylamino)-4'-(2,4-dimethoxybenzyl)-2-(2-morpholinoethyl)-1',2',3',4'-tetrahydrospiro[isoquinoline-1,9'-anthraphen]-3-one.

[0059] Example 4

[0060] This embodiment describes a method for preparing a pH fluorescent probe (FCE), including the following steps:

[0061] S1. 2.64 mL of cyclohexanone (approximately 25.5 mmol) was added dropwise to 10 mL of concentrated sulfuric acid (0°C). Under vigorous stirring, 2.0 g of 4-diethylaminoketo acid (6.4 mmol) was added all at once. The mixture was then heated to 90°C and reacted for 2 h. After the reaction was complete, the mixture was cooled and processed in the same manner as in Example 1. Post-treatment yielded approximately 3.0 g of a red solid, but the product contained a significant amount of unreacted material, increasing the difficulty of chromatographic separation.

[0062] S2. 144.48 mg of compound 1 (0.42 mmol) and 83.089 mg of 3,4-dimethoxybenzaldehyde (0.5 mmol) were dissolved in 50 mL of acetic acid and heated to 110 °C. The reaction time was extended, and after the reaction was completed, approximately 0.17 g of black solid was obtained by rotary evaporation.

[0063] S3. 500 mg of compound 2 and 620.75 mg of BOP were added to 22.5 mL of dichloromethane, followed by the dropwise addition of 800 mg of 4-(2-aminoethyl)morpholine. The mixture was stirred at room temperature for about 12 h. TLC analysis showed that the reaction was incomplete. After post-treatment, about 0.48 g of the final product was obtained as a dark red oily crude product. Further purification was required to obtain 0.341 g of the final product (E)-6'-(diethylamino)-4'-(2,4-dimethoxybenzyl)-2-(2-morpholinoethyl)-1',2',3',4'-tetrahydrospiro[isoquinoline-1,9'-anthracite]-3-one.

[0064] Table 1. Production of FCE probes

[0065]

[0066] like Figure 1 The diagram shows the synthesis route of the pH fluorescent probe prepared in Example 1, which consists of three steps: (A) Synthesis of compound 1, (B) Synthesis of compound 2, and (C) BOP-mediated coupling to obtain the final product pH fluorescent probe FCE.

[0067] like Figure 2 As shown, the pH-dependent optical properties of the pH fluorescent probe FCE were investigated in phosphate buffer at room temperature. Its UV-Vis absorption spectrum showed that the absorption peak intensity at 463 nm continuously increased with increasing pH (pH 1.0~13.0), providing a physical basis for the high sensitivity response of the pH fluorescent probe FCE (selecting 463 nm excitation).

[0068] like Figure 3 As shown, the fluorescence spectrum under 463 nm excitation confirms that the fluorescence change of the pH fluorescent probe FCE originates from the intramolecular charge transfer (ICT) effect regulated by the protonation state of isoquinoline nitrogen atoms, and is synergistically affected by the stereoelectronic effect of the spironanthrone rigid framework. Specifically, under acidic conditions (pH < 5.0), isoquinoline nitrogen protonation (-NH...) + =) Suppressing the ICT effect results in weak fluorescence at 585 nm (quantum yield Φ < 0.1). Gradual deprotonation in neutral to weakly alkaline environments (pH 5.0~8.0) enhances ICT and increases fluorescence intensity.

[0069] like Figure 4 As shown, the fluorescence intensity of the pH fluorescent probe FCE increases linearly at 585 nm. In an alkaline environment (pH>8.0), complete deprotonation saturates the ICT, and the fluorescence intensity stabilizes (fluctuation <3%).

[0070] like Figure 5 As shown, the linear response range covers the physiologically critical pH range (R0). 2 >0.99), and within the pH range of 5.0 to 8.0, the fluorescence intensity showed a good linear relationship with the pH value.

[0071] like Figure 6 As shown, the pH fluorescent probe FCE passed the test of 4 acid-base cycles (pH 1.0 ⇌ 10.0), and the probe fluorescence recovery rate was >95%, proving that its protonation / deprotonation process is highly reversible.

[0072] like Figure 7 As shown, the environmental adaptability of the pH fluorescent probe FCE stems from its structural advantages. The fluorescence intensity difference at 585nm between pH 1.0 and 10.0 is <5%, which is attributed to: the hydrophobic cavity of spiroanthrone shielding ion interference and the steric hindrance effect of morpholine ethyl protecting the isoquinoline active site. It can respond quickly, with a steady-state fluorescence value of over 90% within 1 min.

[0073] Example 5 This embodiment describes a method for detecting microorganisms using the pH fluorescent probe FCE during meat spoilage, including the following steps:

[0074] S1. Take the pH fluorescent probe FCE prepared in Example 1 and prepare a 1 mM probe stock solution with DMSO for later use;

[0075] S2. Fresh pork samples were minced and evenly divided into three groups, and stored at 4℃ and -20℃ respectively. 10.00 g (accurate to 0.01 g) of samples were taken at regular intervals every day, 100 mL of distilled water was added, and after high-speed homogenization, the samples were allowed to stand and filtered to obtain the filtrate to be tested.

[0076] S3. Take 2.975 mL of the filtrate to be tested and add 25 μL of probe stock solution (final concentration 10 μM). Immediately use a fluorescence spectrophotometer to measure the fluorescence intensity at 585 nm at an excitation wavelength of 463 nm. Convert the fluorescence value to pH value using the pre-established fluorescence intensity-pH standard curve.

[0077] To further illustrate the technical effects of the present invention, a comparative example is also provided, as follows:

[0078] Comparative Example 1

[0079] The difference between this comparative example and Example 5 is that this comparative example uses a pH meter to detect the pH value of the filtrate to be tested, and each sample is measured in parallel 3 times and the average value is taken. The remaining steps are the same as in Example 5.

[0080] like Figure 8As shown, a systematic comparison of the line graph (A) and bar graph (B) clearly reveals the comprehensive performance advantages of the pH fluorescence probe FCE (Example 5) of this invention compared to the traditional pH meter method (Comparative Example 1). The 4℃ storage group showed a clear fluorescence intensity inflection point on day 3, with the corresponding pH value dropping from an initial 5.8 to 5.6 and then rising back to above 6.0, indicating the onset of spoilage. The 20℃ storage group showed a significantly accelerated spoilage process, with a fluorescence inflection point appearing at the end of day 1, and the pH value exceeding 6.5 on day 2. In contrast, the -20℃ control group maintained stable fluorescence intensity and pH value throughout the experiment. In line graph (A), the pH change trends measured by the two methods are generally consistent, verifying the reliability of the probe detection. The fluorescence method curve shows an earlier and smoother "inflection point" from decreasing to increasing in the early stages of spoilage (approximately day 2-3), a crucial turning point captured approximately one day earlier than the pH meter method. This clearly demonstrates the outstanding value of this invention in high-sensitivity monitoring, achieving early warning of the spoilage process dominated by microbial metabolism. The bar chart (B) further confirms from a statistical analysis perspective that, throughout the storage period, the pH detection values ​​at each time point using the fluorescence method not only showed no significant deviation from the traditional method, but also exhibited smaller data dispersion (error bars), indicating that this method has superior repeatability and stability. Compared with the traditional pH meter method, the pH fluorescence probe method provided by this invention demonstrates significant advantages in monitoring meat spoilage: high sensitivity: it can detect minute initial changes that are difficult for a pH meter to distinguish, and can clearly detect a slight decrease in pH from 5.8 to 5.6 (reflected by changes in fluorescence intensity); timely warning: it can capture the "inflection point" signal marking the start of spoilage earlier. In Example 5, a clear fluorescence inflection point appeared on the 3rd day, providing an early warning. In Comparative Example 1, the pH value did not stabilize above 6.2 until the 4th day, at which point the meat had already clearly spoiled, resulting in a delayed warning. The above description demonstrates that the FCE probe prepared by this invention can reflect the dynamic changes in pH during the pork spoilage process in real time through changes in fluorescence signals. Its detection results are consistent with the traditional pH meter method, and it has higher sensitivity, enabling accurate early warning of the spoilage initiation point and effective discrimination of the degree of spoilage. Therefore, this invention not only provides a new detection tool, but also surpasses traditional methods in detection performance, and has important application value in the field of real-time monitoring of food safety.

[0081] like Figure 9 As shown, the color changes of pork over ten days at -20℃ (above) and 4℃ (below) showed that at 4℃, the color of pork changed from bright red to brownish-green over storage time, and the deterioration stage strictly corresponded to the dynamic trajectory of pH first decreasing and then increasing; while the color and pH of the control group at -20℃ remained stable, which proved that the above changes were indeed dominated by microbial activity.

[0082] Comparative Example 2

[0083] This comparative example is a quinalidine derivative fluorescent probe QH used for detecting strong acid pH environments. The preparation method includes the following steps:

[0084] S1. Mix 6-hydroxyquinalidine, chloroacetylhexylamine and cesium carbonate in N,N-dimethylformamide (DMF) solvent and stir at 85~95℃ for 1~3 hours;

[0085] After the reaction was completed, the mixture was cooled and filtered to remove the residue. The filtrate was concentrated by rotary evaporation and then purified by column chromatography using a mixed solvent of ethyl acetate and n-hexane to obtain the target fluorescent probe QH.

[0086] The quinalidine derivative fluorescent probe QH has a simple structure, is specific to a narrow strong acid range, and has a direct synthetic route with mild conditions. It exhibits activity against H+ within a narrow pH range of 2.4–4.4. + The probe exhibits high sensitivity and specificity in fluorescence response to concentration changes and has been applied to fluorescence imaging of Saccharomyces cerevisiae cells. However, the response range of this probe is strictly limited to strongly acidic environments (pH < 4.5), and it cannot cover physiological or common pathological microenvironments (such as pH 5.0–7.4). At the same time, it relies on a "switch" mode of single fluorescence intensity change, which is easily affected by factors such as probe concentration and environmental interference, and has inherent limitations in accurate and quantitative imaging in complex biological systems.

[0087] Comparative Example 3

[0088] This comparative example is a red fluorescent silicon nanoparticle probe for wide-range pH detection, and its preparation method includes the following steps:

[0089] S1. Using 3-aminopropyltrimethoxysilane (APTMS) as the silicon source and surface modification precursor, 2-nitro-4-aminodiphenylamine (NAP) as the red fluorescent chromophore and pH-responsive unit, and dithiothreitol (DTT) as the reducing agent, the three components were added to an ethanol solvent in a specific ratio (e.g., APTMS: 2.0~4.0 mL, NAP: 0.38~0.76 g, DTT: 0.11~0.22 g) and mixed uniformly by ultrasonication.

[0090] S2. The reaction solution was then placed in a microwave reactor and reacted at 150-152°C for 10-12 minutes. After the reaction was completed, the crude product solution was placed in a dialysis bag with a molecular weight cutoff of 3000-10000 and purified by dialysis in deionized water for 3-6 days to completely remove unreacted small molecule impurities, and finally water-soluble red fluorescent silicon nanoparticle probes (SiNPs) were obtained.

[0091] Red fluorescent silicon nanoparticle probes (SiNPs) are suitable for wide-range pH detection. The synthesis method is simple and efficient, exhibiting excellent linear fluorescence response over an extremely wide pH range of 3.0–9.0. Their fluorescence intensity significantly increases with decreasing pH and has been successfully applied to pH fluorescence imaging in living cells such as human cervical cancer (HeLa) cells, demonstrating their potential in the biomedical field. However, as a nanomaterial system, this probe has the following inherent limitations: First, while its ultra-wide operating range (covering strong acids to weak bases) is universal, it also results in insufficient sensitivity and discrimination for subtle changes near physiological pH (e.g., pH 6.8–7.4) or specific pathological microenvironments (e.g., early lysosomes or tumor extracellular matrix at pH 5.0–6.5), making precise local pH quantification difficult. Secondly, its response mechanism remains a single fluorescence intensity (turn-on) enhancement mode dependent on environmental acidity regulation. Signal output is easily interfered with by complex factors such as local nanoparticle aggregation concentration, differences in cellular uptake efficiency, and non-specific adsorption, posing a challenge to the reliability of absolute quantitative imaging in living organisms or deep tissues. Furthermore, although red light emission reduces biological background interference, its nanoscale size (~2.13 nm) and surface properties may affect its transmembrane transport mechanism, subcellular organelle targeting precision, and long-term in vivo metabolic behavior, introducing uncertainties in the pursuit of organelle-specific detection and clinical translation. In addition, batch-to-batch variations in the synthesis of nanomaterials are more difficult to control precisely than those of small organic molecules, potentially affecting the consistency of their performance.

[0092] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A pH fluorescent probe, characterized in that: Its chemical structure is (E)-6'-(diethylamino)-4'-(2,4-dimethoxybenzyl)-2-(2-morpholinoethyl)-1',2',3',4'-tetrahydrospiro[isoquinoline-1,9'-anthraphen]-3-one, and its chemical structural formula is:

2. The pH fluorescent probe according to claim 1, characterized in that: The pH fluorescent probe is a pH fluorescent probe based on the isoquinoline-spiroanthrone structure, which is a tetrahydrospiro[isoquinoline-1,9'-anthrene]-3-one structure.

3. A method for preparing a pH fluorescent probe according to claim 1 or 2, characterized in that, Includes the following steps: S1. Cyclohexanone and 4-diethylaminoketo acid were condensed in concentrated sulfuric acid. After the reaction was completed, the mixture was cooled to room temperature and transferred to ice water with stirring. 70% HClO4 was added to produce a red precipitate. After filtration, washing, drying, and separation by chromatography, compound 1 was obtained. S2. Compound 1 and 3,4-dimethoxybenzaldehyde were condensed in acetic acid. After the reaction was completed, the mixture was cooled to room temperature and then rotary evaporated to obtain compound 2. S3. Compound 2 and BOP were added to dichloromethane, and 4-(2-aminoethyl)morpholine was added dropwise. The mixture was stirred at room temperature, and the pH fluorescent probe was obtained by dichloromethane extraction, washing with water and NaCl solution, drying with anhydrous Na2SO4, and rotary evaporation.

4. The preparation method according to claim 3, characterized in that: In step S1, the molar ratio of cyclohexanone to 4-diethylaminoketo acid is 1.5~2.5:1, the mass-to-volume ratio of concentrated sulfuric acid to 4-diethylaminoketo acid is 1:5~10, the condensation reaction temperature is 90~110℃, the condensation reaction time is 2~6 h, and the volume ratio of 70% HClO4 to 4-diethylaminoketo acid is 2.5~3:

1.

5. The preparation method according to claim 3, characterized in that: In step S2, the molar ratio of compound 1 and 3,4-dimethoxybenzaldehyde is 1:1.1~1.5, the condensation reaction temperature is 110~120℃, and the condensation reaction time is 15~18h.

6. The preparation method according to claim 3, characterized in that: In step S3, the molar ratio of compound 2, BOP and 4-(2-aminoethyl)morpholine is 1:1.5~2:8~15, and the reaction time is 12~15 h.

7. The application of a pH fluorescent probe according to claim 1 or 2 in the detection of microorganisms in food.

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