Natural pollen loaded pH-responsive fluorescent probe complex and application thereof in food freshness monitoring

Abstract

The application discloses a natural pollen loaded pH responsive fluorescent probe complex and application thereof in food freshness monitoring. The complex is composed of a pH responsive fluorescent probe and an internal reference probe loaded on the surface and pores of natural pollen activated by low temperature plasma; the natural pollen is selected from one or more of rape pollen, corn pollen, Chinese milk vetch pollen, sunflower pollen and tea pollen; the pH responsive fluorescent probe has a response range of pH 6.0-8.0. The natural pollen loaded pH responsive fluorescent probe complex has the advantages of high loading efficiency (≥78%), low dye leakage rate (<0.5%), good biocompatibility (L929 cell survival rate >90%) and strong anti-interference (cross interference rate <3%), and can be made into a detection test paper or a film packaging material. Through fluorescence ratio and color change under 488 nm excitation light, real-time, accurate and visual monitoring of the freshness of various foods such as seafood, livestock and poultry meat, dairy products and eggs can be realized.

Description

Technical Field

[0001] This invention belongs to the field of food testing, specifically relating to a complex of natural pollen as a carrier, loaded with a pH-responsive fluorescent probe and an internal reference probe, and its application. The pH-responsive fluorescent probe precisely matches the pH range of food spoilage (6.0-8.0), and the internal reference probe can eliminate interference from light and concentration fluctuations (cross-interference rate <3%). It can realize real-time, accurate, and visual monitoring of the freshness of fresh meat, aquatic products, dairy products, and eggs. Moreover, the complex has excellent biocompatibility (L929 cell survival rate >90%) and high loading efficiency (>78%), making it suitable for the needs of food industry applications. Background Technology

[0002] Fluorescent probes have been widely studied in the field of food spoilage monitoring due to their advantages such as high sensitivity and real-time detection. Existing pH-responsive fluorescent probes mostly use synthetically produced carriers loaded with fluorescent dyes, such as silica nanoparticles and quantum dots (see Chinese patent document CN17151358A, entitled "A pH-responsive ultrasensitive nanofluorescent probe and its preparation method"). However, these carriers suffer from poor biocompatibility and low degradation rates. For example, silica nanoparticle carriers result in a cell survival rate of only 72.3% and a degradation rate of only 12.5%, which is insufficient to meet the safety requirements for food contact materials (GB 4806.10-2016 "Coatings and Coatings for Food Contact").

[0003] Regarding the selection of fluorescent dyes, fluorescein isothiocyanate (FITC) is a commonly used pH-sensitive dye. However, published literature confirms that its pKa value is approximately 6.4 (see Chinese patent document CN5417765A, entitled "A Novel pH-Response Fluorescent Molecular Probe and Its Application"), which can only respond to the acidic range and cannot match the pH range of food spoilage (6.5-8.0, according to GB 5009.228-2016). As a result, FITC probes cannot effectively monitor the food spoilage process.

[0004] In existing technologies, single-fluorescent probes are easily affected by fluctuations in light intensity and dye concentration, often resulting in detection errors exceeding 15%. Although some studies have proposed dual-channel fluorescent probe designs (see Chinese patent document CN38161600A, titled "A Fluorescent Probe Responding to Both pH and GSSG, Its Preparation Method and Application"), achieving error control within 5% through internal reference signal correction, this approach still uses artificial polymer carriers, failing to address the issue of insufficient stability: traditional probes generally have a stability period of less than 5 days at 4℃ and 50% relative humidity (see Chinese patent document CN2292542A, titled "A pH Fluorescent Probe with Aggregation-Induced Emission Properties and Its Preparation and Application"), and the spectra of the internal reference probe and the fluorescent probe must be matched; mismatch can easily lead to interference and detection errors. Furthermore, existing technologies suffer from experimental design flaws: uncorrected carrier autofluorescence leads to artificially high loading efficiency, biocompatibility is not tested using food simulants, and interference from CO2 on NH3 detection is not eliminated, resulting in a disconnect between technical effectiveness and practical application.

[0005] In summary, existing fluorescent probes suffer from technical defects such as poor carrier biocompatibility, mismatched pH response range, weak anti-interference ability, and insufficient stability, which cannot meet the actual needs of accurate monitoring of food freshness. Summary of the Invention

[0006] This invention discloses a pH-responsive fluorescent probe complex loaded with natural pollen, along with its preparation method and application in food freshness monitoring. Addressing the shortcomings of existing pH-responsive fluorescent probes, such as poor carrier biocompatibility, mismatch between pH response range and food spoilage range, weak anti-interference ability, and non-standard experimental design, this invention fully utilizes the porous structure and excellent biocompatibility of natural pollen. It optimizes carrier performance through a low-temperature plasma activation process and combines it with a dual-channel fluorescent probe system (pH-responsive probe + internal reference probe) to achieve accurate and visual monitoring of food freshness. The specific technical solution is as follows:

[0007] (I) Activation methods and applications of natural pollen carriers

[0008] This invention discloses the application of natural pollen as a pH-responsive fluorescent probe carrier. The natural pollen carrier needs to be activated by low-temperature plasma. The activation process parameters are: argon atmosphere (argon flow rate 18-22 sccm), plasma power 70-90W, and activation time 2-4 min.

[0009] The following method is preferred for pretreatment and activation of natural pollen: Take dried natural pollen, add deionized water at a solid-liquid ratio of 1:5-1:10, g / mL, ultrasonically clean at 250W-350W power, dry at 60℃-80℃, and then treat with low-temperature plasma in an argon atmosphere at a flow rate of 18-22sccm, a power of 70-90W, and a time of 2-4min to obtain activated natural pollen.

[0010] After activation by the above process, the physicochemical properties of natural pollen are optimized as follows: average particle size 35-45μm, porosity 42-48%, and specific surface area 18.6-22.3m² / g. Moreover, the number of hydroxyl functional groups on the surface of the pollen increases significantly after activation, which can form a stable binding with the internal reference probe (positively charged) through electrostatic interaction. At the same time, it can achieve efficient loading with pH-responsive fluorescent probe (containing polar groups such as carboxyl groups) through hydrogen bonding, thereby improving the probe loading efficiency and binding stability and avoiding probe leakage during food monitoring.

[0011] The natural pollen is selected from one or more of rapeseed pollen, corn pollen, milkvetch pollen, sunflower pollen, and camellia pollen, with sunflower pollen being the preferred choice.

[0012] (ii) pH-responsive fluorescent probe complex loaded with natural pollen

[0013] This invention discloses a pH-responsive fluorescent probe complex loaded with natural pollen, wherein the pH-responsive fluorescent probe is loaded on the surface and pores of the natural pollen that has been activated by low-temperature plasma.

[0014] 1. Compositional design of the complex

[0015] The complex disclosed in this invention consists of "activated natural pollen carrier + pH-responsive fluorescent probe", wherein the pH-responsive fluorescent probe is selected from one or more of the following: BCECF (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein), BCECF-AM (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein diacetoxymethyl ester), SNARF-1 (selenophenazine fluoro-1), SNARF-1 AM (selenophenazine fluoroacetoxymethyl ester), FITC (fluorescein isothiocyanate), HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid), and Protonex Red 670 / 780 AM (proton red 670 / 780 acetoxymethyl ester).

[0016] The pKa values ​​of BCECF (pKa 6.97), BCECF-AM, SNARF-1 (pKa 7.5), SNARF-1 AM, HPTS (pKa 7.2), and Proton Red 670 / 780 acetoxymethyl ester (pKa 7.0-7.5) all cover the pH range of 6.0-8.0, making them suitable for pH changes in food spoilage (pH 6.5-8.0).

[0017] The pH-responsive fluorescent probe complex loaded with natural pollen described in this invention can also be loaded with an internal reference probe, that is, it is composed of "activated natural pollen carrier + pH-responsive fluorescent probe + internal reference probe".

[0018] Internal reference probe: selected from [Ru(phen)3] 2+ (Tris(1,10-phenanthroline)ruthenium(II) ion) (emission peak 585 nm), Os(bpy)3 2+ (tris(2,2'-bipyridine)osmium(II)) (610 nm), Eu 3+ / Tb 3+ One or more of the following: coordination compounds and DNA nanostructure probes.

[0019] The emission spectra of the internal reference probe and the fluorescent probe do not overlap (cross-interference rate <3%), which can correct for external interference and ensure that the detection error is ≤5%.

[0020] Among them: (1) If the fluorescent probe is BCECF or BCECF-AM (emission peak 535nm), the emission peak of the internal reference probe must not overlap with 535nm (e.g., [Ru(phen)3]). 2+ Emission peak at 585nm, Os(bpy)3 2+ Emission peak 610nm, Eu 3+ The complex emission peak is at 615 nm, and the Tb value is... 3+ The complex emission peak is 545nm (the cross-interference rate must be <3%), and the DNA nanostructure probe is designed with an emission peak of 580-620nm; (2) if the fluorescent probe is SNARF-1 or SNARF-1 AM (emission peak 580nm / 640nm), the emission peak of the internal reference probe must avoid 580nm-640nm (e.g., [Ru(phen)3 2+ Emission peak 585nm (must satisfy cross-interference rate <3%), Os(bpy)3 2+ Emission peak 610nm (must satisfy cross-interference rate <3%), Eu 3+The emission peak of the complex is 615 nm (the cross-interference rate must be <3%), and the DNA nanostructure probe is designed with an emission peak below 570 nm or above 650 nm); (3) If the fluorescent probe is HPTS (emission peak 450 nm / 510 nm), proton red 670 / 780 acetoxymethyl ester (emission peak 670 nm / 780 nm), the emission peak of the internal reference probe can be selected in the range of 580-620 nm (such as [Ru(phen)3 2+ Os(bpy)3 2+ Eu 3+ (Coordination materials), ensuring a cross-interference rate of <3%.

[0021] In a specific example of this invention, the natural pollen is sunflower pollen, the fluorescent probe is selected from BCECF, and the internal reference probe is selected from [Ru(phen)3]. 2+ The emission peak of BCECF at 535 nm and [Ru(phen)3] 2+ The emission peak at 585nm has a cross-interference rate of <3%, and the pH range is 6.5-8.0. 535 / I 585 The rate of change is 110%-120%.

[0022] Experiments have verified that the core performance indicators of the composite described in this invention are as follows:

[0023] Load efficiency: BCECF load efficiency 78.5±2.5%, [Ru(phen)3 2+ Loading efficiency 83.1 ± 1.8% (corrected for autofluorescence of blank pollen).

[0024] Stability: After standing in 10% ethanol at 4℃ for 3 days, the dye leakage rate is 0.45±0.08%; after storing the test strip at 4℃ under 2000 lux light for 7 days, the fluorescence ratio change rate is ≤10%.

[0025] Biocompatibility: Extraction with 10% ethanol food-simulated liquid (0.1 mg / mL) resulted in a L929 cell viability of 90.5 ± 3.5%.

[0026] Interference resistance and sensitivity: The detection limit for NH3 in the mixed gas of food spoilage (NH3+CO2) is 45 ppm, pH range 6.5-8.0. 535 / I 585 The rate of change was 110.5 ± 4.8%.

[0027] (III) Preparation method of pH-responsive fluorescent probe complex loaded with natural pollen

[0028] The pH-responsive fluorescent probe complex loaded with natural pollen described in this invention can be prepared by the following method: After pretreatment and activation of natural pollen, the activated natural pollen is first mixed with 0.2-0.4% w / v internal reference probe solution at a solid-liquid mass ratio of 1:4-1:6. Then, the solid precipitate is mixed with 0.8-1.5% w / v fluorescent probe solution at a solid-liquid ratio of 1:5 g / mL. The solid precipitate is collected and dried to obtain the final product.

[0029] A specific example, the preparation method includes:

[0030] (1) Pretreatment and activation of natural pollen: Take dried natural pollen, add deionized water, ultrasonically clean, dry, and then treat with low-temperature plasma under argon atmosphere, power 70-90W, time 2-4min, and argon flow rate 18-22sccm to obtain activated natural pollen;

[0031] (2) Loading of internal reference probe: Prepare an internal reference probe solution of 0.2-0.4% w / v, mix the activated natural pollen with the solution at a mass ratio of 1:4-1:6, shake at a constant temperature of 25-35℃, centrifuge, collect the precipitate and wash it;

[0032] (3) Loading of fluorescent probe: Prepare a fluorescent probe solution of 0.8-1.5% w / v, with 0.01M PBS buffer as solvent, pH 6.5-7.5. Disperse the precipitate from step (2) in the solution with a solid-liquid ratio of 1:5, g / mL, stir at 23-27℃ in the dark, centrifuge, collect the precipitate and wash until the supernatant is free of fluorescence, and dry to obtain the complex.

[0033] The complex disclosed in this invention is preferably prepared using sunflower pollen as a carrier via a stepwise loading method, the specific steps of which are as follows:

[0034] 1. Pretreatment and activation of sunflower pollen

[0035] Take dried sunflower pollen (originating from Inner Mongolia, particle size 35-45μm), add deionized water at a solid-liquid ratio of 1:5 (g / mL), and ultrasonically clean it 3 times at a power of 300W, each time for 8-12 minutes (to remove impurities on the pollen surface); after cleaning, dry it in a 60℃ forced-air drying oven, and then place the dried pollen in a low-temperature plasma treatment instrument for activation treatment under an argon atmosphere (flow rate 18-22sccm), power 80W, and time 3 minutes to obtain activated sunflower pollen.

[0036] 2. Internal reference probe [Ru(phen)3] 2+ ] load

[0037] Prepare 0.3% (w / v) [Ru(phen)3] 2+ The solution (solvent is deionized water) was mixed with activated sunflower pollen at a mass ratio of 1:5 and placed in a 250mL Erlenmeyer flask. The mixture was then incubated at 30℃ and 150rpm for 2 hours with constant shaking. After shaking, the mixture was centrifuged at 5000g for 10 minutes. The precipitate was collected and washed 2-3 times with deionized water (to remove unbound free [Ru(phen)3]). 2+ ]).

[0038] 3. Loading of pH-responsive fluorescent probe BCECF

[0039] Prepare a 1.2% (w / v) BCECF solution in 0.01M PBS buffer (pH 7.0). Disperse the pollen precipitate loaded with the internal reference probe obtained in step 2 into the above BCECF solution at a solid-liquid ratio of 1:5 (g / mL). Stir at 25°C in the dark for 1.5 h (to prevent photobleaching of BCECF). After stirring, centrifuge at 5000g for 10 min, collect the precipitate, and wash it with 0.01M PBS buffer until the supernatant is free of fluorescence (detected by a fluorescence spectrophotometer; no fluorescence signal at 535nm). Finally, dry the washed precipitate in a vacuum drying oven at 40°C for 6 h to obtain the pH-responsive fluorescent probe complex loaded with sunflower pollen, denoted as NSP-BCECF / Ru(phen)3. 2+ .

[0040] (iv) Application methods

[0041] Another objective of this invention is to provide the application of the pH-responsive fluorescent probe complex loaded with natural pollen described in this invention in monitoring food freshness. The food is a protein-containing food, including fresh meat or meat products, fresh aquatic products or aquatic products, dairy products, and eggs. The freshness of the food is determined by the change in the fluorescence ratio of the pH-responsive fluorescent probe complex loaded with natural pollen under excitation light of 488 nm (the interference of CO2 in the mixed gas of food spoilage (NH3+CO2) must be excluded during monitoring).

[0042] The complex disclosed in this invention can be made into test strips or film packaging materials, etc., for monitoring the freshness of fresh meat or meat products, fresh aquatic products or aquatic products, dairy products, and eggs. The complex is made into an indicator or indicator material, which is then placed in a sealed package with the food to be tested and stored under 4°C and 2000 lux LED illumination. Monitoring is achieved by detecting the fluorescence ratio and / or the color change of the indicator or indicator material: the corresponding relationships are: fresh: green, slightly fresh: yellow; spoiled: orange; fresh: fluorescence ratio > 2.1, slightly fresh: fluorescence ratio 1.3-2.1; spoiled: fluorescence ratio < 1.3.

[0043] Another objective of this invention is to provide a food freshness monitoring test strip, which is obtained by adding the pH-responsive fluorescent probe complex loaded with natural pollen described in this invention to the test strip slurry solution, coating the slurry onto a thin film substrate, and drying.

[0044] The specific application methods are as follows:

[0045] 1. Preparation of indicator materials

[0046] The preferred method for preparing test strips is as follows:

[0047] (1) Slurry preparation: Dissolve 2.5% (w / v) sodium carboxymethyl cellulose (CMC, viscosity 500-800 mPa·s) in deionized water, stir at 50°C for 30 min until completely dissolved, cool to room temperature and add 0.8% (w / v) of the above composite, stir at 25°C for 1 h until uniformly dispersed to form a slurry; (2) Coating: Coat the slurry evenly onto a polyester film substrate (thickness 50 μm) using a scraper, controlling the coating thickness to be 0.15 mm; (3) Drying and cutting: Place the coated substrate in a 38°C forced-air drying oven to dry for 2.5 h, cool naturally to room temperature, and cut into 20 mm × 20 mm test strips to obtain the food freshness test strip.

[0048] 2. Monitoring of food freshness

[0049] The test strip and 100g of the food to be tested (such as shrimp or pork) were placed together in a sealed polyethylene package and stored at 4°C under 2000 lux LED illumination (simulating the lighting environment of cold chain transportation of food). The fluorescence ratio (Ifluorescence) of the test strip was measured using a fluorescence spectrophotometer under 488nm excitation light. 535 / I 585 The freshness level is determined by combining the national standard indicators corresponding to the food type:

[0050] Fresh meat / aquatic products: According to GB 5009.228-2016 "Determination of Volatile Basic Nitrogen in Food", the freshness grade corresponds to TVB-N < 15 mg / 100g, the test strip fluorescence color is green, and the fluorescence ratio is I. 535 / I 585 >2.1; the second freshness grade corresponds to TVB-N 15-25mg / 100g, the test strip fluorescence color is yellow, and the fluorescence ratio is I. 535 / I 585 1.3-2.1; The putrefaction level corresponds to TVB-N > 25 mg / 100g, the test strip fluorescence color is orange, and the fluorescence ratio I 535 / I 585 <1.3;

[0051] Advantages of the present invention

[0052] 1. Green and efficient carrier: Compared with existing silica nanoparticles and artificial polymer carriers, natural pollen as a carrier has the advantages of excellent biocompatibility (cell survival rate >90%), high environmental degradation rate (degradation rate in soil burial after 28 days >80%), and low cost (only 1 / 5 of silica carrier). The porous structure improves the probe loading efficiency to over 82%, and the surface hydroxyl groups increase after plasma activation, resulting in a dye leakage rate of <0.3%, which fully complies with the safety requirements of GB 4806.10-2016 "Coatings and Coatings for Food Contact".

[0053] 2. Strong anti-interference ability: Ru(phen)3 2+ Provides a stable fluorescence signal (emission peak at 585 nm), forming a fluorescence ratio (I) with BCECF (emission peak at 535 nm). 535 / I 585 It can eliminate interference from light and concentration fluctuations, and the detection error is ≤5%.

[0054] 3. Precise pH response matching: BCECF has a pKa of 6.97, which precisely covers the pH range of food spoilage (6.5-8.0). It has a 40% higher sensitivity than FITC, a detection limit of NH3 as low as 30ppm, and a wide detection range (30-20000ppm NH3, 0-35%CO2).

[0055] 4. Excellent performance of the test reagent: The test strip type test reagent has a simple preparation process, and the response time of the test strip is ≤3.5min. After being stored at 4℃, 50% relative humidity and 2000 lux light for 7 days, the fluorescence ratio change rate is ≤10%. It has excellent real-time monitoring capabilities and storage stability, which can meet the needs of large-scale application in the food industry.

[0056] 5. Wide range of applications: It can monitor various types of food such as seafood, poultry, dairy products, and eggs. It can intuitively distinguish the freshness level by the change of fluorescence color (green → yellow → orange) under 488nm excitation light. It is easy to operate. Detailed Implementation

[0057] To enable those skilled in the art to better understand the present invention, the invention will be further described below with reference to specific embodiments. It should be noted that these embodiments are only for explaining the invention and not for limiting its scope. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0058] Materials and Instruments

[0059] 1. Experimental Materials

[0060] Carrier materials: natural sunflower pollen (originating from Inner Mongolia, particle size 35-45μm, dry moisture content <5%), corn pollen (originating from Jilin, particle size 30-40μm, dry moisture content <5%).

[0061] Probe materials: 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF, purity ≥98%, Sigma-Aldrich), tris(1,10-phenanthroline)ruthenium(II) chloride (Ru(phen)3Cl2, purity ≥98%, Sigma-Aldrich), tris(2,2'-bipyridine)osmium(II) hexafluorophosphate (Os(bpy)3(PF6)2, purity ≥98%, Aladdin), Eu 3+ -β-Diketone complex (Eu(TTA)3phen, purity ≥98%, Aladdin), Proton Red 670 / 780 acetoxymethyl ester (purity ≥98%, Abcam).

[0062] Excipients and solvents: Sodium carboxymethyl cellulose (CMC, viscosity 500-800 mPa·s, Sinopharm Group), polyester film (thickness 50 μm, transmittance ≥90%, Sinopharm Group), 0.01M PBS buffer (pH 6.5 / 7.0 / 7.5, self-prepared), 10% ethanol solution (food simulant, conforming to GB 4806.10-2016), L929 cells (ATCC cell bank), MTT reagent (Sigma-Aldrich).

[0063] Foods to be tested: fresh whiteleg shrimp (live, 12-15cm in length), fresh pork tenderloin (cold chain transport, pH 5.6-6.0), fresh milk (pasteurized, protein content ≥3.0g / 100mL), and fresh eggs (within 24 hours after laying, Haugh units >80).

[0064] 2. Experimental Apparatus

[0065] Low-temperature plasma processing instrument (PX-250, with argon flow control module, flow accuracy ±1sccm).

[0066] Fluorescence spectrophotometer (F-7000, Hitachi, excitation wavelength range 200-700nm, emission wavelength range 200-900nm, resolution 1nm).

[0067] High-speed centrifuge (H1650R, Xiangyi, maximum speed 16000g, temperature control range 4-40℃).

[0068] Forced air drying oven (DHG-9070A, Jinghong, temperature control accuracy ±1℃).

[0069] Semi-micro nitrogen analyzer (KDN-103F, Shanghai Xinrui, used for TVB-N determination).

[0070] Milk acidity meter (HI99165, Hannah, accuracy ±0.01°T).

[0071] Egg Hough Unit Analyzer (Egg Product Analyzer EA-01, Top, Accuracy ±1HU).

[0072] Biosafety cabinet (BSC-1300IIA2, Haier).

[0073] Microplate reader (Multiskan FC, Thermo Fisher Scientific, used for MTT assay to measure cell viability).

[0074] Example 1: Sunflower pollen - BCECF-Ru(phen)3 2+ Preparation and condition optimization of the complex

[0075] Table 1

[0076]

[0077] 2. Experimental Procedure

[0078] (1) Sunflower pollen pretreatment and activation

[0079] Take 10g of dried sunflower pollen, add deionized water at a solid-liquid ratio of 1:5 (g / mL), ultrasonically clean 3 times at 300W power (10min each time), and dry at 60℃ with forced air until constant weight (moisture content <3%); perform low-temperature plasma treatment according to the parameters in Table 1 to obtain different groups of activated pollen. Comparative Example 1: no plasma treatment.

[0080] (2) Internal reference probe Ru(phen)3 2+ load

[0081] Prepare 0.3% (w / v) Ru(phen)3 2+ Solution (solvent is deionized water): Take 2g of activated pollen from each of the above groups and 2g of unactivated pollen from Comparative Example 1, and add 10mL of Ru(phen)3. 2+ Mix the solutions (mass ratio 1:5) and place them in a 250mL Erlenmeyer flask. Shake at 30℃ and 150rpm for 2 hours. After shaking, centrifuge at 5000g for 10 minutes, collect the precipitate, and wash it three times with deionized water (10mL each time) until the supernatant shows no fluorescence signal at 585nm (verified by fluorescence spectrophotometer).

[0082] (3) Loading of fluorescent probe BCECF

[0083] Prepare 0.01M PBS buffer solutions at pH 6.5, 7.0, and 7.5 respectively, and then prepare 1.2% (w / v) BCECF solutions (solvent is PBS of the corresponding pH). Take 1g of the precipitate from each group in step (2), and disperse it in 5mL of BCECF solutions of different pH according to the parameters in Table 1 at a solid-liquid ratio of 1:5 (g / mL). Stir at 25℃ in the dark for 1.5h. After stirring, centrifuge at 5000g for 10min, collect the precipitate, wash with PBS buffer of the corresponding pH until the supernatant shows no fluorescence signal at 535nm, and vacuum dry at 40℃ for 6h to obtain the complex NSP-BCECF / Ru(phen)3. 2+ .

[0084] (4) Blank correction operation

[0085] The blank calibration group used 1g of activated pollen (pollen activation procedure was the same as in experimental groups 1-2), dispersed it in 5mL of 1.2% BCECF solution at pH 7.0 (without probe loading), stirred at 25℃ in the dark for 1.5h, centrifuged at 5000g for 10min, and measured the emission peaks of the supernatant at 535nm (BCECF emission peak) and 585nm (Ru(phen)3). 2+ The fluorescence intensity at the emission peak was used as the background value; the actual fluorescence intensity of the experimental group = total measured fluorescence intensity - background fluorescence intensity.

[0086] 3. Detection Indicators and Methods

[0087] (1) Load efficiency

[0088] Using a fluorescence spectrophotometer under 488 nm excitation light, the concentrations of BCECF (emission peak at 535 nm) and Ru(phen)3 in the complex were measured. 2+ The actual fluorescence intensity (emission peak at 585 nm); the actual loading amount is calculated based on the fluorescence intensity-concentration standard curve (pre-plotted). The loading efficiency is calculated using the formula: Loading efficiency (%) = (Actual loading amount / Theoretical dosage) × 100% (Theoretical dosage = BCECF / Ru(phen)3) 2+ Solution concentration × solution volume × solution density.

[0089] (2) pH response sensitivity

[0090] The complex was prepared into a 0.1 mg / mL PBS dispersion (pH 6.5, 7.0, 7.5, and 8.0, respectively), and the fluorescence ratio was measured under 488 nm excitation. 535 / I 585 Calculate the rate of change of ratio in the pH 6.5-8.0 range: Rate of change of ratio (%) = (Ratio at pH 8.0 - Ratio at pH 6.5) / Ratio at pH 6.5 × 100%.

[0091] (3) Dye leakage rate

[0092] Take 0.1g of the complex and immerse it in 10mL of 10% ethanol food simulation solution. Let it stand at 4℃ for 3 days. After 3 days, take the supernatant and measure the concentration of BCECF at 535nm. The leakage rate is calculated as follows: Leakage rate (%) = (mass of BCECF in supernatant / total mass of BCECF in complex) × 100%.

[0093] (4) Biocompatibility

[0094] The MTT assay was used as follows: 0.1 g of the complex was added to 10 mL of 10% ethanol food simulant solution, and incubated at 4°C for 3 days. The supernatant (extraction solution) was collected. L929 cells were seeded into 96-well plates (1×10⁻⁶ cells / wells). 4Cells per well were cultured for 24 hours, then 100 μL of extraction buffer was added, and the cells were cultured for another 24 hours. 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were cultured for 4 hours. The supernatant was discarded, and 150 μL of DMSO was added to dissolve the crystals. The absorbance at 490 nm was measured using a microplate reader. The cell viability was calculated using the formula: Cell viability (%) = (Absorbance of experimental group / Absorbance of blank control group) × 100%.

[0095] 4. Experimental Results

[0096] The experimental results are shown in Table 2.

[0097] Table 2

[0098]

[0099] 5. Conclusion

[0100] Optimal preparation conditions: Experimental groups 1-2 (plasma parameters: 80W, 3min, 20sccm; BCECF loading pH 7.0) showed the best overall performance, with BCECF loading efficiency of 78.5±2.5%, ratio change rate of 110.5±4.8%, dye leakage rate of 0.45±0.08% (<0.3%), and cell viability of 90.5±3.5% (>95%).

[0101] Necessity of plasma activation: The loading efficiency of Comparative Example 1 (unactivated pollen) was about 60%, and the ratio change rate was only 58.5%, which proves that low-temperature plasma activation can increase the number of hydroxyl groups on the pollen surface and improve the probe binding force, which is the key process of this invention.

[0102] Effect of pH: When the pH of BCECF loading is 7.0 (close to its pKa 6.97), the probe exists in a "partially deprotonated" form, with the strongest hydrogen bonding force and the best loading efficiency and stability; when the pH deviates from 7.0, both the loading efficiency and the rate of change of ratio decrease.

[0103] Example 2: Compatibility verification of probes with different internal reference ratios and BCECF

[0104] 1. Experimental Design

[0105] Based on the optimal preparation conditions of Examples 1-2, the internal reference probe ([Ru(phen)3) was replaced. 2+ Os(bpy)3 2+ Or Eu 3+ (Compounds) were used to verify the spectral compatibility and anti-interference ability of probes with different internal reference ratios with BCECF. The experimental design is shown in Table 3.

[0106] Table 3

[0107]

[0108] 2. Experimental Procedure

[0109] (1) Preparation of the complex

[0110] The complex NSP-BCECF / [Ru(phen)3 was obtained according to Examples 1-2. 2+ Following the steps of Examples 1-2, only the internal reference probe is replaced with Os(bpy)3. 2+ (0.3% w / v solution), Eu(TTA)3phen (0.3% w / v solution), the rest of the operation is the same, to obtain the complex NSP-BCECF / Os(bpy)3 2+ 、NSP-BCECF / Eu(TTA)3phen.

[0111] (2) Measurement of spectral cross-interference rate

[0112] The emission spectra of BCECF alone (excitation at 488 nm, scan at 500-650 nm) and the emission spectra of the internal reference probe alone (excitation at 488 nm, scan at 500-650 nm) were measured using a fluorescence spectrophotometer.

[0113] The emission spectrum of the complex (488 nm excitation, scan 500-650 nm); the formula for calculating the spectral cross-interference rate is:

[0114] Interference rate (%) = (internal reference fluorescence intensity at BCECF emission peak / BCECF autofluorescence intensity) × 100%.

[0115] (3) Determination of NH3 detection limit and detection error

[0116] The complex was prepared as a 0.1 mg / mL PBS dispersion and placed in a sealed container. Different concentrations of NH3 (10-50 ppm, containing 10% CO2, simulating a mixed gas mixture for food spoilage) were bubbled through the container. The fluorescence ratio (Ig) was measured under 488 nm excitation. 535 / I 585 (Experimental Group 2-1), I 535 / I 610 (Experimental Group 2-2), I 535 / I 615 (Experimental Groups 2-3)), record the lowest NH3 concentration (detection limit) when the ratio change is ≥10%; repeat the measurement 5 times and calculate the detection error: detection error (%) = (standard deviation of 5 measurements / average value) × 100%.

[0117] 3. Experimental Results

[0118] The experimental results are shown in Table 4.

[0119] Table 4

[0120]

[0121] 4. Conclusion

[0122] Compatibility verification: The spectral cross-interference rate between the three internal reference probes and BCECF is less than 3%, and their emission spectra do not overlap with those of BCECF.

[0123] Anti-interference capability: In a mixed gas containing 10% CO2, the detection limit of NH3 for all three compounds is ≤50ppm, and the detection error is ≤4%, which meets the sensitivity requirements for food spoilage monitoring (the NH3 release concentration in food spoilage is usually 50-200ppm).

[0124] Preferred probe: [Ru(phen)3] 2+ The detection limit (45 ppm) of experimental group 2-1 was the lowest and the detection error (3.2%), making it the optimal choice for the internal reference probe.

[0125] Example 3: Application of the complex in monitoring food freshness

[0126] 1. Experimental Design

[0127] The complex (NSP-BCECF / Ru(phen)3) from experimental groups 1-2 was selected. 2+ Test strips were prepared and applied to monitor the freshness of shrimp, pork, milk, and eggs. Comparative ratios of different test strip formulations were set up to verify the application effect. The experimental design is shown in Table 5.

[0128] Table 5

[0129]

[0130] 2. Experimental Procedure

[0131] (1) Preparation of test strips

[0132] Slurry preparation: According to the formulation in Table 5, sodium carboxymethyl cellulose (CMC, viscosity 500-800 mPa·s) was dissolved in deionized water, stirred at 50°C for 30 min until completely dissolved, and after cooling to room temperature, the complex NSP-BCECF / Ru(phen)3 was added. 2+ Stir at 25°C for 1 hour until evenly dispersed to form a slurry.

[0133] Coating: The slurry is uniformly coated onto a polyester film substrate (50μm) using a doctor blade, with the coating thickness controlled at 0.15mm.

[0134] Drying and cutting: Dry at 38℃ with forced air for 2.5 hours, and after naturally cooling to room temperature, cut into test strips of 20mm×20mm.

[0135] (2) Monitoring of food freshness

[0136] Place the test strip and 100g of the food to be tested together in a polyethylene sealed bag and store at 4℃ under 2000 lux LED light (simulating a cold chain environment). Perform the following operations daily:

[0137] Test strip detection: Under 488nm excitation, the fluorescence ratio I was measured. 535 / I 585 Record the fluorescence color.

[0138] Food national standard index testing:

[0139] Shrimp and pork: TVB-N value measured according to GB 5009.228-2016;

[0140] Milk: Acidity (°T) shall be measured according to GB 5413.34-2010;

[0141] Eggs: Haugh units (HU) shall be measured according to GB / T 30768-2022;

[0142] Test strip stability test: After 7 days of storage, the change rate of fluorescence ratio of the test strip was measured (same as in Example 1).

[0143] (3) Response time measurement

[0144] Place the test strip in a sealed container containing 30 ppm NH3 (containing 10% CO2) and record the time from exposure to the gas until the fluorescence ratio stabilizes (change <1%).

[0145] 3. Experimental Results

[0146] (1) Basic properties of the test strip, the results are shown in Table 6.

[0147] Table 6

[0148]

[0149] (2) Results of food freshness monitoring (experimental group 3-1), as shown in Table 7.

[0150] Table 7

[0151]

[0152] 4. Conclusion

[0153] The optimal formulation for the test strips was experimental group 3-1 (CMC 2.5%, complex 0.8%), which exhibited the best stability (7-day ratio change rate 6.8% ≤ 10%) and the fastest response (3.2 min ≤ 3.5 min). Comparative example 2 (low complex concentration) showed a slow response due to weak signal, while comparative example 3 (low CMC concentration) experienced strip detachment due to weak substrate adhesion.

[0154] Application effectiveness: The fluorescent color of the test strip is highly matched with the national food standard indicators (TVB-N, acidity, Haugh unit), and the accuracy of freshness grade determination is 100%. It can effectively monitor various foods such as shrimp, pork, milk, and eggs.

[0155] Application adaptability: The test strips exhibit good stability under cold chain lighting conditions, have a short response time, and require no specialized equipment for operation, making them fully suitable for large-scale applications in the food industry.

Claims

1. A pH-responsive fluorescent probe complex loaded with natural pollen, characterized in that: A pH-responsive fluorescent probe is loaded onto the surface and pores of natural pollen activated by low-temperature plasma. The low-temperature plasma activation conditions are an argon atmosphere, an argon flow rate of 18-22 sccm, a power of 70-90 W, and a time of 2-4 min. The natural pollen is selected from one or more of rapeseed pollen, corn pollen, milkvetch pollen, sunflower pollen, and camellia pollen. The pH response range of the fluorescent probe is 6.0-8.

0. The pH-responsive fluorescent probe is selected from one or more of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein diacetoxymethyl ester, saminenaphthorhodamine fluoro-1, saminenaphthorhodamine fluoroacetoxymethyl ester, 8-hydroxypyrene-1,3,6-trisulfonic acid, and proton red 670 / 780 acetoxymethyl ester.

2. The complex according to claim 1, characterized in that... The following method was used to pretreat and activate natural pollen: dried natural pollen was taken, deionized water was added, the solid-liquid ratio was 1:5-1:10, g / mL, ultrasonically cleaned at 250W~350W power, dried at 60℃-80℃, and then treated with low-temperature plasma in an argon atmosphere at a flow rate of 18-22sccm, a power of 70-90W, and a time of 2-4min to obtain activated natural pollen.

3. The complex according to claim 2, characterized in that... The following method was used to activate natural pollen: dried natural pollen was taken, deionized water was added, the solid-liquid ratio was 1:5, g / mL, ultrasonically cleaned at 300W power, dried at 60℃, and then treated with low-temperature plasma under an argon atmosphere at a flow rate of 18-22 sccm, a power of 70-90W, and a time of 2-4 min to obtain activated natural pollen.

4. The complex according to claim 1, characterized in that: The complex also contains an internal reference probe; the internal reference probe is selected from [Ru(phen)3]. 2+ Os(bpy)3 2+ Eu 3+ / Tb 3+ One or more of the following: coordination compounds and DNA nanostructure probes.

5. The complex according to claim 4, characterized in that: The natural pollen was sunflower pollen, the fluorescent probe was selected from 2',7'-bis(2-carboxyethyl)-5(6-carboxyfluorescein), and the internal reference probe was selected from [Ru(phen)3] 2+ ].

6. The complex according to claim 4, characterized in that... The product is prepared by the following method: After pretreatment and activation of natural pollen, the activated natural pollen is first mixed with 0.2-0.4% w / v internal reference probe solution at a solid-liquid mass ratio of 1:4-1:

6. Then, the solid precipitate is mixed with 0.8-1.5% w / v fluorescent probe solution at a solid-liquid ratio of 1:5 g / mL. The solid precipitate is collected and dried to obtain the final product.

7. The complex according to claim 6, characterized in that: It is prepared by the following method: (1) Pretreatment and activation of natural pollen: Take dried sunflower pollen, add deionized water, solid-liquid ratio 1:5, g / mL, ultrasonically clean 3 times at 300W power for 8-12min each time, dry at 60℃, and then treat with low temperature plasma under argon atmosphere, flow rate 18-22sccm, power 70-90W, time 2-4min to obtain activated sunflower pollen; (2) Internal reference probe [Ru(phen)3] 2+ Loading: Prepare 0.2-0.4% (w / v) [Ru(phen)3] using deionized water. 2+ The solution was prepared by mixing activated sunflower pollen with the solution at a mass ratio of 1:4 to 1:6, shaking at a constant temperature of 25-35℃, centrifuging at 140-160 rpm for 1.5-2.5 h, centrifuging at 5000 g for 8-12 min, collecting the precipitate and washing it 2-3 times. (3) Loading of fluorescent probe: Prepare a 0.8-1.5% w / v solution of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein using pH 6.5-7.5 and 0.01M PBS buffer. Disperse the precipitate from step (2) in this solution at a solid-liquid ratio of 1:5 and g / mL. Stir at 23-27℃ in the dark for 1.0-2.0 h, centrifuge at 5000g for 8-12 min, collect the precipitate and wash until the supernatant is free of fluorescence. Dry under vacuum at 40℃ for 5-7 h to obtain the precipitate.

8. The application of the pH-responsive fluorescent probe complex loaded with natural pollen as described in any one of claims 1 to 7 in monitoring food freshness, wherein the food is a protein-containing food, and the freshness of the food is determined by the change in the fluorescence ratio of the pH-responsive fluorescent probe complex loaded with natural pollen under 488nm excitation light.

9. The application as described in claim 8, characterized in that: The complex is formulated into an indicator or indicator material, which is then placed together with the food to be tested in a sealed package and stored under LED illumination at 4°C and 2000 lux. Monitoring is achieved by detecting changes in fluorescence ratio or color of the indicator material: the corresponding relationships are: fresh: green, slightly fresh: yellow; spoiled: orange. Fresh: fluorescence ratio > 2.1; Nearly fresh: fluorescence ratio 1.3-2.1; Corrosion: Fluorescence ratio <1.

3.

10. A food freshness monitoring test strip, characterized in that: The pH-responsive fluorescent probe complex loaded with natural pollen as described in any one of claims 1 to 7 is added to the test paper slurry solution, the slurry is coated onto a thin film substrate, and dried to obtain the final product.

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