4-hexylresorcinol detection using a ratiometric fluorescent probe and a preparation method thereof

Abstract

The application discloses a ratio type fluorescent probe for 4-hexyl resorcinol detection and a preparation method thereof, relates to the field of food safety detection technology, and comprises up-conversion nanoparticles and fast blue B salt; the up-conversion nanoparticles are NaYF4:Yb,Er@NaYF4 up-conversion nanoparticles with a core-shell structure. The application introduces the fast blue B salt as a 4HR specific recognition unit, combines the up-conversion luminescence characteristics of the up-conversion nanoparticles, and constructs a visual detection system; near-infrared (980 nm) is used to excite the up-conversion nanoparticles, so that the interference of background fluorescence is effectively eliminated; and based on the rapid color reaction between the fast blue B salt and 4HR and the luminescence response of the up-conversion nanoparticles, the detection time is greatly shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and in particular to a ratiometric fluorescent probe for detecting 4-hexylresorcinol and a preparation method thereof. Background Art

[0002] 4-Hexylresorcinol (4HR) is a commonly used antioxidant, preservative, and enzymatic browning inhibitor in the food industry. It is widely used in the preservation of aquatic products, fruits, and vegetables, and in baked goods processing. However, problems such as improper use and excessive residues of 4HR have gradually emerged. Studies have shown that excessive intake of 4HR can interfere with the human endocrine system, disrupt hormone balance, and disrupt cellular metabolic homeostasis. Long-term exposure has been shown to pose a potential carcinogenic risk. Therefore, establishing an accurate and reliable 4HR detection method is essential for ensuring food safety and protecting public health.

[0003] Currently, the detection of 4HR mainly relies on chromatographic techniques (such as high-performance liquid chromatography and gas chromatography-mass spectrometry) and optical sensing methods. Among them, fluorescence and colorimetry have attracted widespread attention due to their unique advantages. Fluorescence detection methods are widely used due to their ultra-high sensitivity, while colorimetry is highly favored in real-time detection scenarios due to its simplicity of operation and visual visualization. However, traditional 4HR fluorescence detection methods rely on short-wavelength excitation sources and are extremely susceptible to strong background fluorescence interference from endogenous substances in biological tissues, food, or environmental samples. This significantly reduces the signal-to-noise ratio and severely limits its practical application potential. Therefore, developing a highly sensitive probe that can circumvent background interference has become a key approach to overcoming existing bottlenecks. Summary of the Invention

[0004] In order to solve the problems of large background fluorescence interference and long detection time in the existing 4HR detection method, the present invention provides a ratiometric fluorescent probe for 4-hexylresorcinol detection and a preparation method thereof, so as to realize rapid, sensitive and visual on-site detection of 4HR.

[0005] The technical problem to be solved by the present invention is achieved by adopting the following technical solutions:

[0006] The first object of the present invention is to provide a ratiometric fluorescent probe comprising upconversion nanoparticles and Fast Blue B salt.

[0007] Furthermore, the upconversion nanoparticles are NaYF4:Yb,Er@NaYF4 upconversion nanoparticles with a core-shell structure.

[0008] Furthermore, the ratiometric fluorescent probe further comprises a phosphate buffer. Preferably, the pH value of the phosphate buffer is 11.5. The phosphate buffer provides a stable alkaline environment for the color development reaction between 4HR and Fast Blue B salt.

[0009] A second object of the present invention is to provide a method for preparing the ratiometric fluorescent probe, comprising the following steps:

[0010] (1) NaYF4:Yb,Er upconversion nanoparticles were prepared by solvothermal reaction of yttrium source, ytterbium source, erbium source, sodium source, fluorine source and ligand;

[0011] (2) NaYF4:Yb,Er upconversion nanoparticles, yttrium source, sodium source, fluorine source and ligand were reacted by solvothermal reaction to prepare NaYF4:Yb,Er@NaYF4 upconversion nanoparticles with core-shell structure;

[0012] (3) Surface modification of NaYF4:Yb,Er@NaYF4 upconversion nanoparticles to obtain hydrophilic upconversion nanoparticles;

[0013] (4) The hydrophilic upconversion nanoparticles, Fast Blue B salt, and phosphate buffer are mixed uniformly to obtain a ratiometric fluorescent probe.

[0014] Furthermore, the yttrium source includes but is not limited to at least one of yttrium chloride, yttrium sulfate, yttrium nitrate, yttrium acetate and hydrates thereof.

[0015] Furthermore, the ytterbium source includes but is not limited to at least one of ytterbium chloride, ytterbium sulfate, ytterbium nitrate, ytterbium acetate and hydrates thereof.

[0016] Furthermore, the erbium source includes but is not limited to at least one of erbium chloride, erbium sulfate, erbium nitrate, erbium acetate and hydrates thereof.

[0017] Furthermore, the sodium source is sodium hydroxide (NaOH), which provides both a sodium source and an alkaline environment.

[0018] Furthermore, the fluoride source includes but is not limited to at least one of ammonium fluoride, sodium fluoride, and potassium fluoride.

[0019] Furthermore, the ligand includes but is not limited to at least one of oleic acid and oleylamine. The ligand can also serve as a solvent.

[0020] Furthermore, the solvent includes but is not limited to at least one of 1-octadecene and cyclohexane.

[0021] Furthermore, the solvothermal reaction is carried out at a temperature of 100-300° C. and for a time of 30-60 min.

[0022] Furthermore, the solvent thermal reaction is carried out in an inert atmosphere (such as argon) to prevent oxidation and reduce impurity generation.

[0023] Furthermore, the surface modification includes acid treatment to remove ligands and surface modification with hydrophilic molecules. Preferably, the acid treatment uses dilute hydrochloric acid with a pH of 0.8 to 1.2; the hydrophilic molecules include but are not limited to at least one of polyacrylic acid (PAA) and polyethylene glycol (PEG).

[0024] The third objective of the present invention is to provide an application of the ratiometric fluorescent probe in 4HR detection, which can be specifically applied to the detection of 4HR in food. Its applicability in complex biological matrices is verified by the standard addition method, enabling rapid quantitative detection of 4HR in food, and providing a new detection tool for food safety monitoring and prevention of preservative abuse.

[0025] Furthermore, the 4HR detection is performed by fluorescence detection at an excitation wavelength of 980 nm and an emission wavelength of 500-600 nm.

[0026] In the present invention, the upconversion nanoparticles can emit green (540 nm) upconversion luminescence (UCL) under near-infrared light excitation. Under alkaline conditions, Fast Blue B salt reacts rapidly with 4HR to generate a red product, which quenches the green UCL through the inner filter effect to form a detection signal, thereby realizing 4HR quantitative analysis with reduced background interference and rapid response.

[0027] The beneficial effects of the present invention are:

[0028] 1. The present invention constructs a visual detection system by introducing Fast Blue B salt as a 4HR-specific recognition unit and combining the upconversion luminescence characteristics of upconversion nanoparticles.

[0029] 2. The present invention uses near-infrared (980 nm) excitation upconversion nanoparticles to effectively eliminate the interference of background fluorescence, avoid false positive misjudgment, and improve the detection accuracy of the probe in complex matrices.

[0030] 3. The present invention is based on the rapid color development reaction of Fast Blue B salt and 4HR, combined with the luminescent response of upconversion nanoparticles, which shortens the detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the principle of using the probe of the present invention for 4HR detection;

[0032] Figure 2 The luminescence mechanism diagram (A), UV-visible absorption spectrum and UCL spectrum (B) of the UCNPs described in the present invention;

[0033] Figure 3 The effect of different concentrations of 4HR on the UCL spectrum of the probe (A); I 540 Linear fitting curve with 4HR concentration (B);

[0034] Figure 4 is the I of the probe in the presence of interfering substances 540 and UCL image (A) and UCL spectrum (B); 1-blank; 2-4HR; 3-Na + ,4-Cd 2+ , 5-K + , 6-Ca 2+ , 7-Fe 3+ ,8-Mg 2+ ,9-Cl - ,10-SO4 2- , 11-glucose, 12-glycine. DETAILED DESCRIPTION

[0035] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below with reference to specific embodiments and illustrations. Example

[0036] The preparation methods of upconversion nanoparticles and hydrophilic upconversion nanoparticle solutions refer to Example 1 of patent CN119269488B "Reversible background-free hydrogel nanosensor and intelligent sensing platform for acetone detection".

[0037] Preparation of the probe: 100 μL of Fast Blue B saline solution (2.5 mM) was ultrasonically mixed with 1600 μL of phosphate buffer (pH = 11.5) and 200 μL of hydrophilic upconversion nanoparticle solution (10 mg / mL) to obtain the probe solution.

[0038] Example 2

[0039] The probe solution was prepared by ultrasonically mixing 100 μL of Fast Blue B saline solutions of varying concentrations (0, 0.5, 1, 1.5, 2, 2.5, 3, and 3.5 mM) with 1600 μL of phosphate buffer (pH 11.5) and 200 μL of a 10 mg / mL hydrophilic upconversion nanoparticle solution. Then, 100 μL of a 50 μM 4HR aqueous solution was added. Under 980 nm laser excitation, UCL spectra were recorded from 500 to 600 nm using a Fluorolog-3 fluorescence spectrometer, and absorbance spectra were recorded from 400 to 700 nm using a Shimadzu UV-2550 spectrophotometer. The results showed that as the concentration of Fast Blue B salt increased from 0 mM to 2.5 mM, the UCL intensity at 540 nm continued to decrease and the absorbance at 485 nm continued to increase; when the concentration of Fast Blue B salt continued to increase to 3 mM and 3.5 mM, the absorbance no longer changed. Therefore, 2.5 mM was selected as the optimal concentration of Fast Blue B salt.

[0040] Example 3

[0041] Probe solutions were prepared by ultrasonically mixing 100 μL of Fast Blue B saline solution (2.5 mM) with 1600 μL of phosphate buffer solutions of varying pH values ​​(pH = 10.0, 10.5, 11.0, 11.5, and 12.0) and 200 μL of a hydrophilic upconversion nanoparticle solution (10 mg / mL). Then, 100 μL of 4HR aqueous solution (50 μM) was added. Under 980 nm laser excitation, fluorescence coherence tomography (UCL) spectra were recorded from 500 to 600 nm using a Fluorolog-3 fluorescence spectrometer, and absorbance spectra were recorded from 400 to 700 nm using a Shimadzu UV-2550 spectrophotometer. The results showed that as the pH of the phosphate buffer increased from 10.0 to 11.5, the absorbance at 485 nm increased continuously, reaching a maximum at pH = 11.5, while the absorbance decreased at pH = 12.0. Therefore, pH 11.5 was selected as the optimal pH for the phosphate buffer.

[0042] Example 4

[0043] To the probe solution prepared in Example 1, 100 μL of a 50 μM 4HR aqueous solution or 100 μL of ultrapure water was added and mixed. Under 980 nm laser excitation, the UCL spectrum in the 500–600 nm range was recorded using a Fluorolog-3 fluorescence spectrometer, and the absorbance spectrum in the 400–700 nm range was recorded using a Shimadzu UV-2550 spectrophotometer. The results showed that without the addition of 4HR, the probe solution had a UCL emission peak at 540 nm, and the absorbance in the 400–700 nm range decreased, with no absorption peak. After the addition of 4HR, the probe solution's UCL emission peak at 540 nm decreased, and an absorption peak at 485 nm overlapped with the UCL emission peak.

[0044] Example 5

[0045] To the probe solution prepared in Example 1, 100 μL of 4HR aqueous solution of different concentrations (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μM) was added and mixed. The UCL spectra in the range of 500-600 nm were recorded using a Fluorolog-3 fluorescence spectrometer under 980 nm laser excitation ( Figure 3 A). The results showed that as the 4HR concentration gradually increased, the UCL intensity gradually decreased, and the UCL color changed from green to red ( Figure 3 A illustration). The luminescence intensity I at 540 nm 540As a detection signal, its intensity showed a strong linear relationship with the 4HR concentration (0-50 μM) (R 2 =0.999, Figure 3 B). According to the formula LOD = 3δ / S, the detection limit of the luminescence mode was 96 nM (δ is the standard deviation of 10 blank experiments, S is the slope).

[0046] Example 6

[0047] To the probe solution prepared in Example 1, 100 μL of 4HR aqueous solution (50 μM) or interfering substance aqueous solution (250 μM, interfering substances were Na + 、Cd 2+ , K + , Ca 2+ 、Fe 3+ Mg 2+ 、Cl - 、SO4 2- Mix the solution with 4% paraformaldehyde (5%, 0.05% paraformaldehyde, 0.06 ...

[0048] To the probe solution prepared in Example 1, 100 μL of 4HR (50 μM) and interfering substances (250 μM, interfering substances were Na + 、Cd 2+ , K + , Ca 2+ 、Fe 3+ Mg 2+ 、Cl - 、SO4 2- Mix the aqueous solution of 1% dapoxetine (1% dapoxetine, 1% glucose, and 1% glycine) and record the UCL spectra in the 500–600 nm range using a Fluorolog-3 fluorescence spectrometer under 980 nm laser excitation.

[0049] The luminescence intensity when the interfering substance or 4HR was added alone was used as the selectivity index (red), and the luminescence intensity when the interfering substance and 4HR were added together was used as the anti-interference index (blue). The results showed that the luminescence intensity did not change significantly when the interfering substance was added alone, and the UCL color remained green ( Figure 4 A). However, when the interfering substance and 4HR were added simultaneously, the luminescence intensity decreased significantly ( Figure 4 B). The results showed that the probe has a high selective recognition ability for 4HR and can resist the influence of various interferences.

[0050] Example 7

[0051] 5 g of commercially purchased frozen shrimp was crushed, added to 15 mL of acetone, and ultrasonically treated for 30 min. The supernatant was collected by centrifugation. The residue was extracted twice more with 10 mL of acetone, and the supernatants were combined. The supernatant was evaporated to dryness at 40°C, and the resulting precipitate was added to 5 mL of acetonitrile, ultrasonically dissolved, and then the volume was adjusted to 50 mL with ultrapure water. Then, a 4HR aqueous solution with concentrations of 0, 15, 30, and 45 μM was added using the standard addition method to obtain a sample solution. 100 μL of the sample solution was added to the probe solution prepared in Example 1 and mixed thoroughly. The 4HR concentration was analyzed using a Fluorolog-3 fluorescence spectrometer. The results are shown in Table 1.

[0052] Table 1. Recovery test data of spiked samples in real samples

[0053]

[0054] As shown in Table 1, the recoveries measured by fluorescence spectrometry ranged from 98.58% to 105.07%, with relative standard deviations of 1.98 or less. The detected amount was highly consistent with the standard addition amount. This demonstrates that the probes described herein exhibit excellent recognition capabilities in complex biological matrices, with recovery and precision meeting the requirements for food testing.

[0055] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A ratiometric fluorescent probe, characterized in that: including upconversion nanoparticles, Fast Blue B salt, and phosphate buffer; The upconversion nanoparticles are NaYF4:Yb,Er@NaYF4 upconversion nanoparticles with a core-shell structure.

2. The ratiometric fluorescent probe according to claim 1, wherein: The pH value of the phosphate buffer is 11.

5.

3. A method for preparing a ratiometric fluorescent probe, characterized in that: The following steps are involved: (1) NaYF4:Yb,Er upconversion nanoparticles were prepared by solvothermal reaction of yttrium source, ytterbium source, erbium source, sodium source, fluorine source and ligand; (2) NaYF4:Yb,Er upconversion nanoparticles, yttrium source, sodium source, fluorine source and ligand were reacted by solvothermal reaction to prepare NaYF4:Yb,Er@NaYF4 upconversion nanoparticles with core-shell structure; (3) Surface modification of NaYF4:Yb,Er@NaYF4 upconversion nanoparticles to obtain hydrophilic upconversion nanoparticles; (4) The hydrophilic upconversion nanoparticles, Fast Blue B salt, and phosphate buffer are mixed uniformly to obtain a ratiometric fluorescent probe.

4. The preparation method according to claim 3, wherein: The ytterbium source is at least one of ytterbium chloride, ytterbium sulfate, ytterbium nitrate, ytterbium acetate and hydrates thereof; The erbium source is at least one of erbium chloride, erbium sulfate, erbium nitrate, erbium acetate and hydrates thereof; The sodium source is sodium hydroxide; The fluoride source is at least one of ammonium fluoride, sodium fluoride and potassium fluoride; The ligand is at least one of oleic acid and oleylamine; The solvent is at least one of 1-octadecene and cyclohexane.

5. The preparation method according to claim 3, wherein: The solvothermal reaction temperature is 100-300°C and the time is 30-60 min; The solvothermal reaction is carried out in an inert atmosphere.

6. The preparation method according to claim 3, wherein: The surface modification includes acid treatment to remove ligands and surface modification with hydrophilic molecules; The acid treatment uses dilute hydrochloric acid with a pH value of 0.8-1.2; the hydrophilic molecule is selected from at least one of polyacrylic acid and polyethylene glycol.

7. Use of the ratiometric fluorescent probe according to any one of claims 1 to 2 or the ratiometric fluorescent probe prepared by the preparation method according to any one of claims 3 to 6 in the detection of 4-hexylresorcinol.

8. The use according to claim 7, characterized in that: The 4-hexylresorcinol detection is performed by fluorescence detection at an excitation wavelength of 980 nm and an emission wavelength within the range of 500-600 nm.

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