Fluorescent probe for detecting BPO based on and logic gate, detection method and application thereof

Abstract

The application discloses a fluorescent probe for detecting benzoyl peroxide based on an AND logic gate, a detection method and application, and belongs to the technical field of food safety detection. In view of the problems that existing benzoyl peroxide fluorescent probes rely on single chemical input, are easily interfered by coexisting oxidants to produce false positives, and have insufficient adaptability to on-site rapid detection, the application proposes a dual-factor synergistic triggering anti-interference detection scheme. The scheme takes a fluorescent probe containing a naphthalimide fluorophore and a sulfide recognition group as the core, requires the simultaneous existence of benzoyl peroxide chemical input and 365nm ultraviolet light physical input, and can trigger fluorescence quenching at 546nm to construct an AND logic gate detection mechanism. The detection method comprises the steps of sample extraction, system mixing, ultraviolet irradiation and fluorescence detection, and can be combined with a smart phone to build a portable platform. The application fundamentally avoids false positives, improves detection specificity and reliability, is simple to operate, and is suitable for on-site screening of benzoyl peroxide in flour products.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically the application of benzoyl peroxide fluorescent probe based on AND logic gates in the rapid detection of food residues, which is particularly suitable for the high-specificity on-site screening of benzoyl peroxide in flour products. Background Technology

[0002] Benzoyl peroxide (BPO) is a strong oxidizing white crystalline powder with a slightly bitter almond odor. It was once widely used as a flour whitening agent, ripening agent, and preservative. By oxidizing natural pigments such as carotenoids in flour, it significantly improves the whiteness of flour, promotes gluten formation, and enhances baking performance.

[0003] However, the strong oxidizing properties of BPO can also destroy nutrients in flour such as vitamin E and beta-carotene, and its residues may decompose during the heating process of pasta to produce toxic byproducts such as benzene and phenol. Long-term consumption of foods containing BPO can irritate the digestive tract mucosa, causing chronic gastritis, peptic ulcers, and even posing a potential risk of cancer.

[0004] Given its health hazards, my country banned the addition of BPO to flour production on May 1, 2011. Despite the clear regulations, driven by economic interests, illegal addition continues unabated, seriously threatening food safety and public health. Therefore, developing a method for accurately, rapidly, and reliably detecting BPO residues in food is of great practical significance for market supervision, risk warning, and consumer protection.

[0005] To address this need, various BPO detection methods have been developed in existing technologies.

[0006] While high-performance liquid chromatography (HPLC), capillary electrophoresis, Raman spectroscopy, and colorimetric methods have good quantitative reliability and can meet laboratory testing standards, they generally have drawbacks such as long testing cycles (usually several hours), expensive equipment (requiring professional instruments), complex operation (requiring professional personnel), and poor portability, making them unsuitable for on-site rapid testing scenarios such as farmers' markets and processing plants.

[0007] Although chemiluminescence and electrochemical analysis methods have high sensitivity, they often rely on cumbersome electrode modification or BPO derivative synthesis steps, which have high operational thresholds and limit their promotion and application in grassroots supervision.

[0008] In contrast, fluorescent probe technology has become a research hotspot for BPO detection in recent years due to its high sensitivity, ease of operation, rapid response (usually <1 minute), and visualization potential.

[0009] Currently reported BPO fluorescent probes employ a "single-signal response" model, where recognition groups such as thioethers and alkenes are introduced into the probe molecule. When BPO is present, these groups are oxidized, leading to a change in the electronic structure of the fluorophore and thus a change in fluorescence intensity or wavelength. The design logic of such probes is essentially a passive discrimination model of "presence if there is a reaction."

[0010] However, this passive discrimination mode faces serious challenges in complex food matrices: H2O2, ClO － Common strong oxidizing agents can also oxidize recognition groups such as sulfides, triggering similar fluorescence responses and leading to frequent false positives. This defect severely weakens the reliability and practicality of fluorescent probes in real samples (such as flour and noodles), making it difficult for them to move from the laboratory to practical applications.

[0011] In the development of BPO fluorescent probes, the technical approach in this field follows the traditional "single recognition, single response" sensing paradigm. Performance is improved by optimizing the selectivity of the recognition group or the brightness of the fluorophore, but the mindset of "single chemical input triggering signal output" has never been broken.

[0012] Existing technologies focus on improving the chemical affinity or reaction rate of the recognition group for BPO, but neglect the cross-reaction problem caused by coexisting oxidants in complex matrices. Their sensing mechanisms themselves lack the inherent ability to repel interference sources, easily leading to false positive results. Correspondingly, this patent application establishes a novel technical route for "two-factor verification" through "AND logic gates," using the chemical presence of BPO and a specific physical stimulus (365nm ultraviolet light) as necessary conditions for signal output, fundamentally reconstructing the discrimination logic and improving detection accuracy.

[0013] Furthermore, molecular logic gates, as an advanced analytical strategy simulating computer logic operations, are primarily applied in high-end research scenarios such as molecular computing, information encryption, and cell imaging. Among them, the "AND" logic gate requires all input signals to be present simultaneously to generate a corresponding output signal. Theoretically, this can be used to construct probes with advanced recognition capabilities, solving the problem of selective interference in complex matrices.

[0014] However, those skilled in the art generally consider molecular logic gates to fall under the category of "complex molecular computation," whose design goal is to achieve multivariate logical operations. These gates typically involve complex molecular structures and operational procedures, which are significantly incompatible with the "simplicity, speed, and low cost" requirements emphasized in rapid food safety testing. Therefore, technical personnel lack the motivation and technical inspiration to introduce AND logic gates into the field of BPO detection.

[0015] Throughout its development, the application of molecular logic gate technology has long been limited to fields such as molecular computing and cell imaging. Those skilled in the art have developed a ingrained perception that "molecular logic gates are only suitable for complex signal processing," failing to recognize its value in addressing false positives in rapid detection. In existing technologies, molecular logic gates are mismatched with the technical goals of rapid food safety detection; the former emphasizes complex signal processing capabilities, while the latter emphasizes simplicity, speed, and low cost. Accordingly, this patent application introduces and implements AND logic gate technology in the field of BPO rapid detection.

[0016] Meanwhile, existing fluorescent probe detection systems require organic-aqueous mixed solvents (such as ethanol / water, DMSO / water) to maintain probe solubility and stability. This not only increases the complexity of the preparation process but also reduces compatibility with food aqueous matrices, hindering on-site operation. Furthermore, although smartphone-assisted detection platforms have been used in various colorimetric / fluorescence analyses, their application in BPO detection is still limited by the insufficient anti-interference capability of the probe itself, failing to provide reliable on-site screening results.

[0017] Early fluorescence sensing systems often employed organic-inorganic mixtures to improve probe solubility. Subsequent BPO detection probe development continued this design approach, failing to consider the need for system simplicity in rapid on-site detection. The component ratio of organic-inorganic mixtures significantly impacts detection performance, requiring precise control, and the preparation process is cumbersome, making it difficult to adapt to the operational requirements of rapid on-site detection. This patent application uses 100% HEPES buffer (10mM, pH 7.4) as the detection medium and combines it with smartphone RGB analysis, simplifying the system preparation and signal reading process, ensuring the stability and practicality of detection performance.

[0018] In summary, existing BPO detection technologies, whether traditional instrumental analysis methods or fluorescent probe detection technologies, all have their own insurmountable shortcomings: traditional methods cannot meet the timeliness and portability requirements of on-site rapid detection; single-signal fluorescent probes lack reliability due to the lack of inherent anti-interference mechanisms; and although molecular logic gates have theoretical advantages, they have not been introduced into this field. Therefore, a completely new BPO detection strategy is urgently needed, which should be able to: (1) fundamentally avoid H2O2 and ClO － (1) It eliminates false positives caused by interferences; (2) It works stably in a simple, aqueous buffer system; (3) It produces clear and quantifiable signal outputs, which are easy to integrate with portable devices such as smartphones to achieve on-site visualization and quantitative detection. This invention is proposed to solve the above-mentioned technical problems. By introducing an AND logic gate design of "chemical input (BPO) + physical input (365nm ultraviolet light)", a "two-factor verification" intelligent discrimination mode is constructed, thereby achieving high specificity and high reliability of rapid BPO screening in complex food matrices. Summary of the Invention

[0019] The purpose of this invention is to provide a fluorescent probe for detecting benzoyl peroxide based on an AND logic gate, so as to overcome the defects of the prior art, which rely on a single chemical input to trigger the fluorescence response and thus suffer from false positive results due to interference from non-target strong oxidants in complex food matrices.

[0020] To achieve the above objectives, the present invention provides a fluorescent probe for detecting benzoyl peroxide based on an AND logic gate. The fluorescent probe comprises a fluorophore and a recognition group. Only when benzoyl peroxide and 365nm ultraviolet light are simultaneously present does the recognition group react with benzoyl peroxide, and the fluorophore is excited by ultraviolet light. The synergistic effect of these two reactions leads to a detectable change in the fluorescence signal. No change in the fluorescence signal occurs when only benzoyl peroxide, only 365nm ultraviolet light, or neither is present. The chemical structural formula of the fluorescent probe is: .

[0021] The recognition group is a thioether group, which reacts with benzoyl peroxide to generate a sulfone group. The sulfone group, as a strong electron-withdrawing group, triggers a photoinduced electron transfer process after the fluorophore is excited by 365nm ultraviolet light, resulting in fluorescence quenching. The fluorophore is a naphthalimide group.

[0022] The chemical structure of the fluorescent probe is C 28 H 24 N2O5S2.

[0023] The present invention also provides a method for preparing the fluorescent probe, wherein the 4-bromo-1,8-naphthioimide derivative is referred to as compound 1, and the p-methoxythiophenol is referred to as compound 2. Compound 1 and compound 2 are reacted under alkaline conditions in an organic solvent by heating and reflux, and the fluorescent probe is obtained after purification.

[0024] The molar ratio of compound 1 to compound 2 is 1:2, and the molar ratio of compound 1 to potassium carbonate is 1:2.

[0025] The present invention also provides a method for detecting benzoyl peroxide using the fluorescent probe described above, comprising the following steps: (1) The food sample to be tested is extracted to obtain the test solution; (2) The test solution and the fluorescent probe are mixed in a buffer solution to form a detection system; (3) Irradiate the detection system with 365nm ultraviolet light; (4) Detect the fluorescence intensity of the detection system at 546 nm and compare it with the fluorescence intensity of the blank control system; if the fluorescence intensity is significantly reduced, it is determined that the food sample to be tested contains benzoyl peroxide.

[0026] The 4-bromo-1,8-naphthioimide derivative is referred to as Compound 1, and p-methoxythiophenol is referred to as Compound 2. The food sample to be tested is flour, noodles, or dough. The buffer solution is a 10 mM HEPES buffer solution with a pH of 7.4. The concentration of the fluorescent probe in the detection system is 10 μM. The blank control system consists of the buffer solution, the probe, and an equal volume of solvent to replace the test solution.

[0027] The present invention also provides the application of the fluorescent probe in the detection of benzoyl peroxide residues in food. The sample extract to be tested is mixed with the fluorescent probe in a buffer solution, and irradiated with 365nm ultraviolet light. The change in fluorescence intensity at 546nm is detected. The sample is determined to contain benzoyl peroxide only when the fluorescence intensity decreases significantly.

[0028] Fluorescence images are captured using a smartphone camera, and a quantitative relationship between the R+G value and the concentration of benzoyl peroxide is established through RGB color analysis, enabling visualized quantitative detection.

[0029] This invention also provides the application of the fluorescent probe in constructing an AND logic gate molecular sensor. The sensor uses BPO and 365nm ultraviolet light as two input signals and fluorescence intensity at 546nm as the output signal. It strictly follows the truth table of an AND logic gate with two inputs to realize the logical judgment function of "two keys and lock".

[0030] The present invention has the following advantages: This invention integrates analytical chemistry, molecular fluorescence sensing, and the construction of an intelligent rapid detection platform. The characteristics of the fluorescent probe in this invention support using both chemical input (BPO) and physical input (365nm UV) as necessary conditions for an AND logic gate, fundamentally avoiding the use of H2O2 and ClO. － It can eliminate false positives caused by interfering substances and significantly improve the detection specificity in complex food matrices.

[0031] The fluorescent probe in this invention supports the "dual-key and lock" strategy constructed in this invention. This strategy regards the BPO itself as the first "chemical key," the 365nm ultraviolet light as the second "physical key," and the fluorescent probe of this invention constitutes a special "lock." Only when both keys are present and active can the lock be "opened," triggering a detectable change in fluorescence signal (manifested as fluorescence quenching at 546nm). If either key is missing (only BPO, only ultraviolet light, or neither), the lock remains "closed" and no effective signal is generated.

[0032] This design fundamentally differs from traditional single-signal response probes, effectively eliminating the possibility of H2O2 and ClO through two-factor collaborative verification. － Interference from non-target strong oxidants has been eliminated, solving the problem of false positive detection in complex food matrices and achieving highly reliable, highly selective, and rapid screening of BPO. 28 H 24 The structure of N2O5S2 combines high reactivity, good aqueous stability, and well-defined AND logic behavior. The fluorescent probe can be synthesized in a one-step process, which is simple and suitable for large-scale production.

[0033] The detection method of the present invention has the following advantages: ① High anti-interference ability: effectively eliminates H2O2 and ClO － This method is characterized by several advantages: ① It eliminates interference from non-target oxidants, thus resolving false positives; ② High specificity: Two-factor validation ensures a response only to BPO; ③ Simple operation: No complex pretreatment or specialized instruments are required, making it suitable for rapid on-site testing; ④ Reliable results: Interpretation is objective and clear through comparison with a blank control. This detection method has been validated with real flour samples. The recovery rate on a smartphone platform is 93.41%–108.23%, with an RSD of <4.39%; the recovery rate on a fluorescence spectrophotometer is 87.04%–113.15%, with an RSD of <8.64%.

[0034] The fluorescent probe in this invention is used to detect benzoyl peroxide (BPO) in the food testing field, enabling highly specific on-site screening and avoiding false positives. The above operating procedures are standardized, and the results are clearly interpreted. The test sample is an aqueous or ethanol extract, requiring simple pretreatment and suitable for real samples. The application method of this invention does not require specialized instruments, achieving low-cost and portable detection. The visual quantitative detection in this invention utilizes the significant change in fluorescence color from yellow to colorless caused by the described "dual-key and lock" strategy. RGB values ​​are collected and analyzed via a smartphone, enabling on-site visual and quantitative detection of BPO. Attached Figure Description

[0035] Figure 1 The image shows the proton NMR spectrum of the FLZ fluorescent probe prepared in Example 1.

[0036] Figure 2 The image shows the carbon NMR spectrum of the FLZ fluorescent probe prepared in Example 1.

[0037] Figure 3 The mass spectrum of the FLZ fluorescent probe prepared in Example 1 is shown.

[0038] Figure 4 The UV absorption spectrum of the FLZ fluorescent probe prepared in Example 1 for recognizing BPO is shown in Example 2.

[0039] Figure 5The fluorescence spectrum of the FLZ fluorescent probe prepared in Example 1 recognizing BPO is shown. See Example 2.

[0040] Figure 6 The titration fluorescence spectrum of the FLZ fluorescent probe prepared in Example 1 recognizing BPO. See Example 3.

[0041] Figure 7 The linear fit graph shows the recognition of BPO by the FLZ fluorescent probe prepared in Example 1. See Example 3.

[0042] Figure 8 The graph shows the selectivity test results of the FLZ fluorescent probe prepared in Example 1 for recognizing other ions of BPO. See Example 4.

[0043] Figure 9 The image shows a test pattern of the FLZ fluorescent probe prepared in Example 1 recognizing the interference of BPO with other ions. See Example 5.

[0044] Figure 10 The response time diagram for the FLZ fluorescent probe prepared in Example 1 to recognize BPO is shown. See Example 6.

[0045] Figure 11 The fluorescence changes of the FLZ fluorescent probe prepared in Example 1 at different pH values ​​for recognizing BPO are shown in Example 7.

[0046] Figure 12 The symbol diagram and truth table of the FLZ fluorescent probe "AND" logic gate prepared for Example 1, as well as the fluorescence intensity under different input conditions. See Example 8.

[0047] Figure 13 This is an application flowchart for detecting BPO based on a smartphone-assisted platform. See Example 9.

[0048] Figure 14 For the standard curve established based on the smartphone-assisted platform, see Example 9.

[0049] Figure 15 This diagram illustrates the logical response of the probe molecules FLZ and BPO in this invention, along with the mass spectrometry analysis of the reaction products, to support Example 1.

[0050] Figure 16 This is a process roadmap for the synthesis of the FLZ fluorescent probe in this invention, used to support Example 1. (Attached) Figure 16 Compound 2 in the sample is p-methoxythiophenol, which is commercially available. Detailed Implementation

[0051] like Figures 1 to 16As shown, this invention provides a fluorescent probe for detecting benzoyl peroxide based on an AND logic gate. The fluorescent probe comprises a fluorophore and a recognition group. Only when benzoyl peroxide and 365nm ultraviolet light are simultaneously present will the recognition group react with benzoyl peroxide, and the fluorophore be excited by ultraviolet light. The synergistic effect of the two leads to a detectable change in the fluorescence signal. Under conditions where only benzoyl peroxide, only 365nm ultraviolet light, or neither is present, no change in the fluorescence signal will occur. The chemical structural formula of this fluorescent probe is: .

[0052] The fluorophore is a naphthalimide group, and the recognition group is a thioether group; the fluorescence signal change is fluorescence quenching at 546 nm.

[0053] The "lock" structure includes a naphthalimide fluorophore and a thioether recognition group; the thioether recognition group serves as the specific activation site of the first "key" BPO and can be oxidized to a sulfone group; the naphthalimide fluorophore serves as the energy receiving site for the second "key" 365nm ultraviolet light; the synergistic effect of the two keys leads to fluorescence shutdown.

[0054] (3) Additional notes: This probe works stably in 10 mM HEPES buffer (pH 7.4) without the need for organic co-solvents.

[0055] (4) Parameter and formula support explanation: 365nm (nanometer) is the ultraviolet light wavelength, which is the design parameter selected based on the excitation characteristics of naphthalimide; 546nm (nanometer) is the characteristic emission wavelength, which is determined by fluorescence spectroscopy experiments.

[0056] (5) Other supporting content: The AND logic behavior is verified by a truth table (see Figure 12 ), see PET mechanism Figure 15 .

[0057] The recognition group is a thioether group, which reacts with benzoyl peroxide to generate a sulfone group. The sulfone group, as a strong electron-withdrawing group, triggers a photoinduced electron transfer process after the fluorophore is excited by 365nm ultraviolet light, resulting in fluorescence quenching. The fluorophore is a naphthalimide group.

[0058] Thioether oxidation requires no catalyst and can be completed in an aqueous buffer solution. The recognition group is a thioether group, clearly defining the reaction mechanism and ensuring the reliability of the physicochemical basis of the AND logic. The chemical structure of the fluorescent probe is C0. 28 H 24 N₂O₅S₂. C 28 H 24 The structure of N2O5S2 combines high reactivity, good aqueous stability, and clear AND logic behavior.

[0059] The 4-bromo-1,8-naphthylimide derivative is referred to as compound 1 (shown in...). Figure 16 In the Chinese text, p-methoxythiophenol is referred to as compound 2. (shown in...) Figure 16 Compounds 1 and 2 were reacted under alkaline conditions in an organic solvent by heating, and the resulting fluorescent probe, which has a "dual key and lock" recognition function, was obtained after purification.

[0060] Specifically, in a 50 mL round-bottom flask, compound 1 (472 mg, 1 mmol), compound 2 (280 mg, 2 mmol), and potassium carbonate (276 mg, 2 mmol) were added to a reaction flask containing 10 mL of DMF. The mixture was heated to reflux at 120 °C for 8 h. After the reaction was complete, it was cooled to room temperature, and the reaction mixture was added dropwise to ice water. The mixture was then filtered to obtain the crude product. The crude product was separated by dichloromethane column chromatography to obtain probe FLZ (63% yield).

[0061] The alkaline conditions were provided by potassium carbonate, the organic solvent was N,N-dimethylformamide, the reaction temperature was 120℃, and the reaction time was 8 hours. The fluorescent probe was synthesized in one step, which is simple to operate and suitable for large-scale production. The molar ratio of compound 1 to compound 2 was 1:2, and the molar ratio of compound 1 to potassium carbonate was 1:2.

[0062] This invention also discloses a method for detecting benzoyl peroxide using the above-mentioned fluorescent probe, which is carried out according to the following steps: (1) Extract the food sample to be tested to obtain the test solution; (2) Mix the test solution with the fluorescent probe in a buffer solution to form a detection system; (3) Apply 365nm ultraviolet light to the detection system; (4) Detect the fluorescence intensity of the detection system at 546nm and compare it with the fluorescence intensity of the blank control system; if the fluorescence intensity is significantly reduced, it is determined that the food sample to be tested contains benzoyl peroxide.

[0063] "Significantly reduced" means that if the fluorescence intensity of the detection system is less than 60% of that of the blank control system, the sample is considered to contain benzoyl peroxide. In the experiment, when the sample contained benzoyl peroxide (BPO) and 365nm ultraviolet light was applied, the fluorescence intensity of the detection system decreased to approximately 40% of that of the blank control group (i.e., quenched by approximately 60%). This is the measured result, representing a typical positive response level.

[0064] The 60% criterion is a higher-level value, not directly limited to "below 40%", but relaxed to "below 60%" (meaning that a maximum of 60% of the fluorescence intensity is allowed to remain, which implies at least 40% quenching). This "60%" is an upper limit of the judgment threshold set after generalizing the measured value of 40%. On the one hand, it improves the fault tolerance performance, and on the other hand, it avoids narrowing the protection scope by directly writing 40%.

[0065] The technical considerations for setting the 60% cutoff criterion are as follows: In the experiment, the fluorescence intensity of the negative control (without BPO or without UV) was >90%; the positive sample was stable at ~40%. Therefore, "60%" is used as a cutoff point, with a significant gap between positive and negative (40% vs >90%), which is technically distinguishable. This value is not arbitrarily increased, but is designed based on the safety margin of the experimental data distribution, which ensures coverage of all real positive scenarios, leaves reasonable tolerance, and avoids the protection range being too narrow.

[0066] The food sample to be tested is flour, noodles, or dough; the buffer solution is a 10 mM HEPES buffer (containing 0.01 mol of HEPES solute per liter) with a pH of 7.4; the concentration of the fluorescent probe in the detection system is 10 μM (i.e., containing 10 × 10⁻⁶ mol of HEPES solute per liter of detection system). －6 (a fluorescent probe of mole); the blank control system consists of a buffer solution, a probe, and an equal volume of solvent to replace the test solution.

[0067] In this context, "equal volume" means that the volume of solvent added to the blank control system is exactly the same as the volume of the sample extract added to the experimental group. The solvent refers to the solvent used to prepare the sample extract; in the context of this invention's detection method, this solvent is anhydrous ethanol (the sample extract is prepared by "sample + anhydrous ethanol ultrasonic extraction"). The technical objective is to ensure that the solvent environment of the blank control system is identical to that of the experimental group, eliminating the potential influence of anhydrous ethanol itself on the performance of the fluorescent probe and avoiding interference with the accuracy of experimental results due to solvent differences.

[0068] The principle of the detection method of the present invention is as follows: the method is based on the AND logic gate mechanism - only when benzoyl peroxide (chemical input) is present in the sample and 365nm ultraviolet light (physical input) is applied, the thioether group of the probe is oxidized to a sulfone group, which triggers photoinduced electron transfer (PET) in the excited state, resulting in 546nm fluorescence quenching; a single condition cannot trigger the response.

[0069] The BPO content can be further quantified by analyzing the degree of fluorescence quenching and a standard curve; it can also be visualized by combining RGB analysis with a smartphone. 365nm (ultraviolet wavelength) and 546nm (emission wavelength) are the design parameters, determined based on the probe's spectral characteristics; the pH 7.4 of the HEPES buffer is a known buffer condition simulating a physiological environment. This detection method was validated with real flour samples, showing a recovery rate of 93.41%–108.23% and an RSD < 8.64%.

[0070] The present invention also discloses the application of the fluorescent probe in the detection of benzoyl peroxide residues in food. The sample extract to be tested is mixed with the fluorescent probe in a buffer solution, and irradiated with 365nm ultraviolet light. The change in fluorescence intensity at 546nm is detected. The sample is determined to contain benzoyl peroxide only when the fluorescence intensity decreases significantly.

[0071] The food products include flour, noodles, and dough sheets. The buffer solution is 10 mM HEPES (pH 7.4); "significantly reduced" is already defined. The test sample is an aqueous or ethanol extract of flour, noodles, or dough sheets. The extraction method is as follows: take 1 g of sample, add 4 mL of anhydrous ethanol, ultrasonically extract for 20 minutes, centrifuge at 3500 r / min for 5 minutes, and filter the supernatant through a 0.22 μm organic phase filter membrane for later use. The fluorescent probe in this invention is used in the field of food detection to detect benzoyl peroxide, enabling highly specific on-site screening and avoiding false positives. The above operating procedures are standardized, and the results are clearly interpreted. The test sample is an aqueous or ethanol extract, the pretreatment is simple, and it is suitable for real samples.

[0072] Fluorescence images were acquired using a smartphone camera, and a quantitative relationship between R+G values ​​and benzoyl peroxide concentration was established through RGB color analysis, enabling visualized quantitative detection. The relationship between R+G channel values ​​(matching the 546 nm emission peak) and benzoyl peroxide concentration was established using RGB color analysis. This indicator showed a good linear relationship with concentration in the range of 10–50 μM (R... 2 =0.9961), which meets the on-site quantitative requirements.

[0073] The application method of this invention requires no specialized instruments, enabling low-cost and portable detection. The visual quantitative detection method of this invention utilizes the significant change in fluorescence color from yellow to colorless caused by the "dual-key and lock" strategy. RGB values ​​are collected and analyzed via a smartphone, achieving on-site visualization and quantitative detection of BPO.

[0074] This invention also discloses the application of the fluorescent probe in constructing an AND logic gate molecular sensor. The sensor uses BPO and 365nm ultraviolet light as two input signals and fluorescence intensity at 546nm as the output signal. It strictly follows the truth table of an AND logic gate with two inputs to realize the logical judgment function of "two keys and lock". Example 1

[0075] The synthesis route of the above fluorescent probes is shown in the appendix. Figure 16 As shown, proceed with the following steps: Take a 50 mL round-bottom flask and add the 1,8-naphthalimide derivative (472 mg, 1 mmol, as compound 1) containing a bromine substituent and an N-side chain (ethylene-p-toluenesulfonamide), along with p-methoxythiophenol (280 mg, 2 mmol, as compound 2) and potassium carbonate (276 mg, 2 mmol), to a reaction flask containing 10 mL of DMF. Add compound 1 (472 mg, 1 mmol), compound 2 (280 mg, 2 mmol), and potassium carbonate (276 mg, 2 mmol) to the same reaction flask. Heat at 120 °C for 8 h. Determine the completeness of the reaction by TLC. After the reaction is complete, cool to room temperature and add the reaction mixture dropwise into ice water. Filter to obtain the crude product. Separate the crude product by dichloromethane column chromatography to obtain probe FLZ (63% yield). A certain amount of FLZ fluorescent probe was prepared by the above method and dissolved in dimethyl sulfoxide (DMSO) to prepare FLZ fluorescent probe stock solution.

[0076] The results of NMR, CMR and high-resolution mass spectrometry analyses are as follows: Figures 1 to 3 As shown, The proton NMR spectrum is as follows: 1 HNMR (400MHz, CDCl3) δ 8.57(d,J=8.4Hz,1H),8.48(d,J=7.2Hz,1H),8.14(d,J=8.0Hz,1H),7.73(t,J=8.0Hz,1H),7.52(dd,J=16.4,8.4Hz,4H),7.06(d,J=8.8Hz,2 H),6.98(d,J=8.0Hz,1H),6.70(d,J=8.0Hz,2H),5.36(t,J=4.8Hz,1H),4.22(t,J=4.8Hz,2H),3.90(s,3H),3.47–3.43(m,2H),1.95(s,3H). Carbon NMR spectrum, results as follows Figure 2 As shown: 13 CNMR (100MHz, CDCl3) δ 164.3,164.2,161.3,148.2,142.5,137.3,137.0,131.6,131.0,129.9,129.1,128.1,126.6,123.2,122.4,119.1,118.3,115.9,55.5,42.5,39.1,21.1. High-resolution mass spectrometry determination, results as follows Figure 3 As shown: HR-MS: m / z [M+H] + calcd for[C 28 H 24 N2O5S2+H ] + :533.1205.Found:533.1205. Analysis confirms that the synthesized product is a fluorescent probe FLZ, i.e., it possesses the following properties: Figure 16 The synthetic route is shown.

[0077] NMR, C1000 spectroscopy, and high-resolution mass spectrometry confirmed that the synthesized product was N-(4-methoxyphenylthio)-1,8-naphthalimide (molecular formula C1000). 28 H 24 N2O5S2), which is the target fluorescent probe FLZ of this invention.

[0078] For compound 1, binding Figure 16 Based on the disclosure of this invention, those skilled in the art, using common knowledge of naphthalimide chemistry, can reasonably select commercially available naphthalimide derivatives containing halogenated or leaving groups to carry out this reaction. This synthetic route is a conventional design.

[0079] This reaction is a nucleophilic aromatic substitution reaction (SNAr). The bromine atom in the 4-bromo-1,8-naphthylimide derivative is located at position 4 of the naphthalene ring. Activated by the electron-withdrawing effect of the ortho-carbonyl group, it is readily attacked by the thioanion of p-methoxythiophenol, undergoing a substitution reaction to form a C–S bond and simultaneously releasing a bromide ion (Br). － Therefore, although the starting material contains bromine, the final product FLZ does not contain bromine atoms in its molecular structure, and its molecular formula is C. 28 H 24 N₂O₅S₂, and high-resolution mass spectrometry results ([M+H) + =533.1205) are completely consistent. Example 2

[0080] Example 2: Changes in UV absorption and fluorescence emission spectra when a fluorescent probe interacts with BPO.

[0081] The probe prepared in Example 1 was dissolved in DMSO to prepare a 1 mM probe stock solution. An appropriate amount of BPO was dissolved in anhydrous ethanol to prepare a 10 mM BPO standard solution. Subsequently, 30 μL of the probe stock solution and different volumes of BPO standard solution were added to 3 mL of HEPES solution (10 mM, pH=7.4) to prepare a test solution for measuring fluorescence spectra.

[0082] Shake the test solution thoroughly and immediately irradiate it with a 65W UV LED lamp with a center wavelength of 365 nm for 1 min to trigger the AND logic response between the probe and benzoyl peroxide. Then, transfer the solution to a standard quartz cuvette and measure its fluorescence emission spectrum using a fluorescence spectrophotometer. Each experiment was repeated three times. During spectral characterization, the excitation wavelength was set to 400 nm, and both the excitation and emission slits were set to 10 nm. A characteristic emission peak was observed at 546 nm.

[0083] It should be noted that 365nm ultraviolet light is the necessary physical input signal for the AND logic gate of this invention, used to activate the response state of the probe during detection; while 400nm is only used as the excitation wavelength for laboratory spectral characterization, used for high-sensitivity reading of fluorescence intensity changes at 546nm, and is an analytical auxiliary means, not involved in the logical judgment process. Therefore, the use of 400nm does not affect the specificity of the "dual key and lock" mechanism of this invention, nor is it a technical element for solving the false positive problem; its purpose is only to accurately calibrate and quantify the fluorescence quenching state formed after 365nm irradiation.

[0084] To verify the AND logic behavior, four control experiments were simultaneously set up: (a) HEPES buffer only; (b) BPO added but no UV irradiation; (c) irradiation with 365nm UV but no BPO; (d) BPO added and UV irradiation. The results showed that only groups (a), (b), (c), and (d) maintained strong yellow fluorescence, and only the group with both BPO and 365nm UV showed fluorescence quenching. This indicates that the probe's response to BPO strictly follows the AND logic gate rule (see [link to relevant documentation]). Figure 12 Truth table).

[0085] like Figure 4 , Figure 5 As shown, the results indicate that the free probe exhibits strong yellow fluorescence due to the action of naphthalimide, and FLZ shows a strong absorption band at 410 nm. After adding BPO and applying UV (365 nm), the absorbance at 410 nm decreases, and a new absorption peak appears at 350 nm. During this process, the solution color changes from pale yellow to colorless. Figure 4 ).

[0086] Under 400 nm excitation, the fluorescence intensity at 546 nm decreased to 38.5 ± 2.1% of the blank control group in the BPO+UV group (n=3), which is below the 60% judgment threshold and meets the standard of "significant reduction". The fluorescence color changed from yellow to colorless. Figure 5 ). Example 3

[0087] This embodiment focuses on a fluorescent titration experiment for recognizing BPO using a fluorescent probe.

[0088] Take 30 μL of the probe stock solution prepared in Example 2 and add it to 3 mL of HEPES buffer solution (10 mM, pH 7.4) to make the final concentration of the fluorescent probe 10 μM; then add different volumes of BPO standard solution to make the final concentration of BPO 0–100 μM. Irradiate each test solution with a UV LED lamp (65W) with a center wavelength of 365 nm for 1 min, and then measure the fluorescence intensity at 546 nm under 400 nm excitation. Figure 6 As shown, only after irradiation at 365 nm, the fluorescence intensity at 546 nm gradually decreased with increasing BPO concentration, and the degree of fluorescence quenching at 546 nm was related to the BPO concentration in the range of 10-50 μM (R0). 2 It shows a good linear relationship within the range of (=0.9970). Figure 7 ).

[0089] After irradiation with 365nm ultraviolet light, the linear relationship between BPO concentration and 546nm fluorescence quenching rate was established according to the (3σ / k) method recommended by IUPAC. Figure 7 The detection limit of the probe for BPO was determined to be 14.7 μM. It should be emphasized that this value is a theoretical value in a pure solution system.

[0090] In practical application to flour samples, the following calculation is performed: 1g of sample is extracted with 4mL of ethanol, and 30μL is added to 3mL of the detection system. The dilution factor during sample extraction is approximately (4000μL / 30μL) ≈ 133 times. During the detection system construction phase, 30μL of the extract is added to 3mL of HEPES buffer and diluted 100 times again. The total dilution factor is approximately 13300 times. Therefore, the detection limit of 14.7μM in the detection system corresponds to a detection limit of approximately 1.424g / kg in the sample. Although this value is higher than the regulatory limit, it is suitable for rapid screening of illegally added flour (BPO content often reaches hundreds to thousands of mg / kg), rather than trace confirmation. It is very practical for rapid on-site investigation of illegal additives. Example 4

[0091] This embodiment focuses on selective experiments for the recognition of BPO by fluorescent probes.

[0092] Take 30 μL of the probe stock solution prepared in Example 2 and add it to 3 mL of HEPES buffer solution (10 mM, pH 7.4) to make the final concentration of the fluorescent probe 10 μM; then add the following interfering substances to make their final concentrations as follows: For Na + K + Cl － For common ions and bio-thiols such as Cys and GSH, the final concentration is 1 mM; for strong oxidants such as H2O2 and HClO, the final concentration is 100 μM.

[0093] Another group was added with 100 μM BPO as a positive control.

[0094] After irradiating all test solutions with a 65W UV LED lamp with a center wavelength of 365nm for 1 minute, their spectral changes were detected using a UV-Vis spectrophotometer and a fluorescence spectrophotometer. Figure 8 As shown in Figure a, after irradiation at 365 nm, the UV absorption peak at 410 nm and the fluorescence emission peak at 546 nm of the test solutions containing various interfering substances showed no significant changes; however, only the BPO group showed a blue shift of the maximum UV absorption peak from 410 nm to 350 nm after irradiation at 365 nm, and a significant decrease in fluorescence intensity at 546 nm. Figure 8 b).

[0095] In particular, even under 365nm ultraviolet light irradiation, strong oxidants such as H2O2 and HClO did not cause fluorescence quenching at 546nm, indicating that they could not simulate the two-factor effect of BPO, thus effectively avoiding false positives.

[0096] The results show that this probe has high selectivity for BPO under the AND logic gate mechanism, and can effectively distinguish strong oxidizing interfering substances such as H2O2 and HClO. Example 5

[0097] This embodiment aims to verify the anti-interference performance of the fluorescent probe of the present invention for the recognition of benzoyl peroxide (BPO) and to eliminate the influence of common interfering substances in food matrices on the detection results. The specific steps are as follows: 1. Reagent preparation: Prepare a 1mM fluorescent probe stock solution (DMSO solvent), a 10mM pH 7.4 HEPES buffer, and a series of interfering substance stock solutions (including Na+). + K + Cl － Ion stock solutions, Cys, GSH and other bio-thiols stock solutions, H2O2 and HClO strong oxidant stock solutions); 2. Construction of detection system: Take several cuvettes, add 3 mL of HEPES buffer to each, then add 30 μL of probe stock solution, mix well and make the final probe concentration 10 μM; 3. Grouping Setup: Divide the above system into three groups and process them separately: Experimental group: BPO solution (BPO + interfering agent + UV) was added to make the final concentration of BPO 100 μM, and then the following interfering agents were added respectively: For Na + K + Cl － For common ions and biothiols such as Cys and GSH, the final concentration was set to 1 mM; for strong oxidants such as H2O2 and HClO, the final concentration was set to 100 μM to simulate their potential high-concentration interference in complex matrices. Blank control group: No BPO or interfering substances were added (only probe + HEPES), and the other components were the same as the above system; Interference control group (interfering agent + UV, no BPO): No BPO was added, only the corresponding interfering agent was added according to the concentration of the interfering agent in the experimental group, and the other components were the same. Positive control group: BPO solution (BPO+UV) was added to make the final concentration of BPO 100μM. No interfering substances were added, and the other components were the same.

[0098] 4. Signal excitation and detection: All three systems were irradiated with 365nm ultraviolet light for 1 minute to allow the system reaction to reach equilibrium, triggering the AND logic response of the probe. Then, the fluorescence intensity of each system at 546nm was detected. 5. Result Interpretation: Compare the fluorescence intensity of the four systems at 546 nm: There was no significant difference in fluorescence intensity between the blank control group (probe + HEPES only) and the interference control group (probe + interfering substance + HEPES + 365nm UV) (relative deviation <5%), indicating that various interfering substances do not induce fluorescence quenching under 365nm UV irradiation. The fluorescence intensity of the positive control group (probe + 100 μM BPO + HEPES + 365 nm UV) was significantly reduced, confirming that BPO can effectively trigger the AND logic response under dual-input conditions. The fluorescence intensity of the experimental group (probe + 100 μMBPO + interferon + HEPES + 365 nm UV) was similar to that of the positive control group, with a relative deviation of no more than 5%, and was significantly lower than that of the blank control group and the interference control group.

[0099] The above results demonstrate that in a simulated food matrix containing high concentrations of common ions, biothiols, and excessive strong oxidants (such as H2O2 and HClO), the fluorescent probe of this invention does not exhibit significant interference in its recognition response to BPO, demonstrating excellent specificity and anti-interference capabilities.

[0100] Experimental conclusion: The fluorescent probe of this invention still has the ability to specifically recognize BPO in the presence of common interfering substances in complex matrices (high concentration of ions, biothiols, and excessive strong oxidants), which can effectively avoid interference from non-target substances and support its practicality in food sample detection. Example 6

[0101] This embodiment aims to verify the kinetic characteristics of the fluorescent probe of the present invention in response to benzoyl peroxide (BPO), clarify the response speed and the synergistic specificity of the "dual key and lock" (AND logic), and the specific steps are as follows: 1. Preparation of reagents and instruments: Using the probe stock solution prepared in Example 2, prepare a 1mM fluorescent probe FLZ stock solution (solvent: DMSO), a 10mM pH 7.4 HEPES buffer, and a 10mM BPO standard stock solution; the ultraviolet light source used is an ultraviolet LED lamp (65W) with a center wavelength of 365nm, and the detection instrument is a fluorescence spectrophotometer, set with an excitation wavelength of 400nm and an emission wavelength of 546nm.

[0102] 2. Construction of parallel systems: Take four identical cuvettes, add 3 mL of HEPES buffer to each, then add 30 μL of FLZ stock solution, mix well, and stabilize the probe concentration at 10 μM. Construct four parallel systems and perform the following treatments: Group (i) (probe only): No BPO added, no 365nm LED irradiation, used as a blank baseline reference; Group (ii) (probe + BPO + UV): Add BPO standard stock solution to a final concentration of 100 μM, and immediately irradiate with a UV LED lamp (65W) with a center wavelength of 365 nm to construct a dual-input system.

[0103] 3. Kinetic monitoring: The system was simultaneously irradiated with ultraviolet light at t=0. For groups (i) and (ii), the fluorescence intensity changes at 546 nm were continuously monitored over 5 minutes using a fluorescence spectrophotometer. The fluorescence intensity response curve over time was recorded (e.g., ...). Figure 10 (As shown).

[0104] 4. Results analysis: The fluorescence intensity of group (i) remained stable within 5 minutes with a fluctuation range of less than 3%, indicating that the probe itself has good stability; the fluorescence intensity of group (ii) decreased rapidly after UV irradiation and reached the response plateau within 60 seconds, with a fluorescence quenching rate of 92%, indicating that the probe can make a rapid and complete response to BPO only under the synergistic effect of dual inputs.

[0105] Experimental conclusion: The fluorescent probe of this invention has excellent response kinetics performance, and can complete the full response to BPO within 1 minute. Moreover, the response behavior strictly follows the AND logic, and only the two inputs are coordinated to trigger fluorescence quenching, which further verifies the specificity of the "dual key and lock" strategy and is suitable for the needs of rapid on-site detection. Example 7

[0106] This embodiment aims to verify the optical stability of the fluorescent probe FLZ under different pH conditions and its response stability to benzoyl peroxide (BPO), ensuring its suitability for complex food and environmental samples within a wide pH range. The specific steps are as follows: 1. Buffer System and Reagent Preparation: Prepare a series of HEPES buffer solutions with a concentration of 10 mM and a pH range covering 3.0 to 10.0. High-purity HEPES powder was dissolved in ultrapure water, and hydrochloric acid (HCl) and sodium hydroxide (NaOH) solutions were used as acid-base adjusters. Under precise pH meter calibration and monitoring, the pH of each solution was adjusted to the target value. Prepare 1 mM FLZ probe stock solution (solvent: DMSO) and 10 mM MBPO standard solution. The ultraviolet light source used was a 65W ultraviolet LED lamp with a center wavelength of 365 nm, and the detection instrument was a fluorescence spectrophotometer.

[0107] 2. Test system construction: Take several cuvettes and group them according to different pH values. Add 3 mL of the corresponding buffer solution to each group, then add 30 μL of FLZ stock solution. Mix well to stabilize the probe concentration at 10 μM. Each group has two parallel subgroups. Subgroup (a) (without BPO): contains only the probe and the corresponding pH buffer, without adding BPO; Subgroup (b) (BPO+UV group): Add an appropriate amount of BPO standard solution to make the final concentration of BPO 100 μM and construct a dual-input reaction system.

[0108] 3. Irradiation and Detection: All test systems (including the BPO-free group) were irradiated for 1 minute with a 365nm UV LED lamp (65W) to ensure the physical input conditions of the AND logic were met; subsequently, the fluorescence intensity of each group at 546nm was measured using 400nm as the excitation wavelength, and the fluorescence intensity and quenching status of the two groups at different pH values ​​were recorded (e.g., Figure 11 (As shown).

[0109] 4. Results Analysis: Subgroup (a) showed fluorescence intensity fluctuations of less than 5% within the pH range of 3.0–10.0, indicating that the FLZ probe itself has stable optical properties and is minimally affected by pH. Subgroup (b) exhibited significant fluorescence quenching within the same pH range, with quenching rates maintained at 85%–92%, and the degree of quenching showed no significant pH dependence. Among these, the probe response stability was optimal within the pH range of typical food samples (4.0–7.0) and environmental water samples (6.0–9.0), with fluctuations <3%.

[0110] Experimental conclusions: The fluorescent probe FLZ of this invention exhibits excellent optical stability over a wide pH range (3.0–10.0), and its AND logic response under dual-input conditions is unaffected by pH. It is particularly well-suited to the pH range of typical food and environmental samples, providing a solid experimental foundation for the rapid and reliable detection of BPO in real samples. Example 8

[0111] This embodiment aims to construct an AND-type molecular logic gate based on the fluorescent probe FLZ of this invention, using benzoyl peroxide (BPO) and 365nm ultraviolet light as dual inputs, to verify its logic response characteristics and ensure the core logic of the "dual key and lock" strategy is valid. The specific steps are as follows: 1. Preparation of reagents and instruments: Prepare 1 mM FLZ probe stock solution (solvent is DMSO), 10 mM pH 7.4 HEPES buffer solution, and 10 mM MBPO standard stock solution; the ultraviolet light source used is an ultraviolet LED lamp (65W) with a center wavelength of 365 nm, and the detection instrument is a fluorescence spectrophotometer, set with an excitation wavelength of 400 nm and an emission wavelength of 546 nm.

[0112] 2. Logic signal definition: With BPO as input signal 1 (A) and 365nm ultraviolet light as input signal 2 (B), the input states are defined as: 1 = present / applied, 0 = absent / not applied; with whether the probe undergoes significant fluorescence quenching at 546nm as the output signal (Y), the output states are defined as: 1 = significant quenching occurs (fluorescence intensity drops below 60% of the blank control), 0 = no significant quenching.

[0113] 3. Construction and processing of four parallel systems: Take four cuvettes, add 3 mL of HEPES buffer solution to each, then add 30 μL of FLZ stock solution, mix well, and the final probe concentration is 10 μM. Process according to the following input combinations: Group (0,0): A=0, B=0, no BPO added, no 365nm ultraviolet light irradiation; Group (1,0): A=1, B=0, add BPO standard stock solution to a final concentration of 100μM, do not irradiate with 365nm ultraviolet light; Group (0,1): A=0, B=1, no BPO added, irradiated with 365nm UV LED lamp for 1 min; Group (1,1): A=1, B=1, BPO was added to a final concentration of 100μM, and the system was irradiated with a 365nm UV LED lamp for 1 min to construct a dual-input system.

[0114] 4. Fluorescence Detection and Truth Table Construction: After each group was processed, the fluorescence intensity at 546 nm was measured using a fluorescence spectrophotometer. Based on the output state definition, a truth table was constructed as shown in Table 1 (the output is 1 only when both inputs exist simultaneously):

[0115] 5. Results and Mechanism Analysis: No significant fluorescence quenching was observed in groups (0,0), (1,0), and (0,1) (Y=0), indicating that the PET process of the probe could not be triggered when there was a single input or no input. Only in group (1,1) when two inputs coexisted, the sulfone group of the BPO oxidized probe was sulfone group, and the naphthalimide fluorophore was excited by 365nm ultraviolet light. The two synergistically triggered photoinduced electron transfer, resulting in significant fluorescence quenching (Y=1).

[0116] Experimental Conclusion: This invention successfully constructed an AND-type molecular logic gate with BPO and 365nm ultraviolet light as dual inputs and fluorescence quenching as the output. Its response behavior strictly follows the AND logic rule. This design effectively avoids the triggering signal of a single interfering factor by synergistically regulating the PET process through dual inputs, providing core logical support for suppressing false positive detection, and further verifying the innovation and reliability of the "dual key and lock" strategy of this invention. Example 9

[0117] This embodiment aims to construct a portable fluorescence detection platform based on a smartphone, verify the applicability and quantitative accuracy of the fluorescent probe FLZ of this invention in detecting BPO in actual food samples, and demonstrate the application value of the "dual key and lock" strategy in semi-on-site rapid testing. The specific steps are as follows: 1. Reagents, Instruments and Platform Setup: Prepare 1 mM FLZ probe stock solution (solvent: DMSO), 10 mM pH 7.4 HEPES buffer, and 10 mM MBPO standard stock solution; the UV light source used is a 65W UV LED lamp with a center wavelength of 365 nm; the portable detection platform consists of a smartphone, a customized dark box (used to shield ambient light and ensure accurate capture of fluorescence signals), and color picker software; a fluorescence spectrophotometer (excitation wavelength 400 nm, emission wavelength 546 nm) is used as a control.

[0118] 2. Standard curve establishment (under AND logic conditions): Take several cuvettes, add 3 mL of HEPES buffer to each, then add 30 μL of FLZ probe stock solution, mix well, and the final probe concentration is 10 μM; add different volumes of BPO standard stock solution to cover the final BPO concentration from 0 to 50 μM. Irradiate each standard system with a 365 nm UV LED lamp for 1 min to trigger the AND logic response, and then immediately place it in a dark chamber within 30 seconds. Capture fluorescence images with a smartphone camera, and simultaneously measure the fluorescence intensity using a fluorescence spectrophotometer.

[0119] RGB values ​​of the images were extracted using colorimetric software, and luminance (R+G) was selected as a proxy index for fluorescence intensity. A standard curve was plotted comparing the index value with BPO concentration. All standard solutions showed rapid fluorescence signal response after UV irradiation, reaching a quantifiable stable range within 30 seconds and remaining stable without significant fluctuations for 2 minutes. Therefore, detection within 30 seconds can balance timeliness and signal consistency. Figure 14 As shown, within the range of 10–50 μM, the index value and BPO concentration exhibit a good linear relationship, with a linear correlation coefficient R0. 2 =0.9961, which meets the requirements for quantitative detection.

[0120] 3. Sample Pretreatment and Detection (Spike Recovery Experiment): Take 1g of flour sample, add 4mL of anhydrous ethanol, extract ultrasonically for 20 minutes, centrifuge at 3500r / min for 5 minutes, and filter the supernatant through a 0.22μm organic phase filter membrane to obtain the sample extract. Set up two parallel experiments to ensure the consistency of the detection system (each group consists of 3mL HEPES buffer + 30μL FLZ stock solution): Background detection group: Add 30 μL of sample extraction solution to verify the interference of the sample matrix itself on the fluorescence signal; Spiked recovery group: 30 μL of sample extract was added, along with BPO standard solution, to bring the final BPO concentration in the system to 25 μM and 50 μM, respectively, to verify the accuracy of quantification.

[0121] 4. Dual-platform simultaneous detection and result comparison: After all sample systems were irradiated with a 365nm UV LED lamp for 1 minute, dual-platform detection was completed within 30 seconds—a smartphone acquired fluorescence images and extracted RGB indicators in a dark chamber, and a fluorescence spectrophotometer measured the fluorescence intensity at 546nm. Based on the standard curve established in step 2, the measured BPO concentration, spiked recovery rate, and relative standard deviation (RSD) of the spiked recovery group were calculated respectively.

[0122] The results are shown in Table 2, showing the spiked recovery results of BPO in different flour products (unit: μM):

[0123] SmartphoneColorimetry represents the results from a smartphone platform, FluorescenceSpectrophotometry represents the results from a fluorescence spectrophotometer; Recovery (%) represents the spiked recovery rate, and RSD (%) represents the relative standard deviation (n=3).

[0124] 5. Results Analysis: The fluorescence signal in the background detection group showed no significant change (fluctuation <3%), indicating no interference from the sample matrix. The recovery rate in the spiked recovery group was 87.04%–113.15%, with an RSD <8.64%. The RSD of the Wheat flour-1 sample was slightly higher due to its complex matrix, but still within acceptable limits. The results from the smartphone platform and the fluorescence spectrophotometer showed good consistency, with errors within acceptable ranges.

[0125] Experimental Conclusion: This invention successfully constructed a smartphone-assisted fluorescence detection platform based on AND logic gates. Through the synergistic triggering response of 365nm UV and BPO dual inputs, it effectively avoids matrix interference and false positive signals. This platform transforms professional fluorescence detection into a low-cost, portable solution suitable for grassroots regulatory sites or mobile detection platforms. It retains the high specificity of the "dual-key and lock" strategy while achieving rapid quantitative detection of BPO, significantly outperforming traditional single-factor rapid detection methods and demonstrating promising practical application prospects.

Claims

1. A fluorescent probe for detecting benzoyl peroxide based on an AND logic gate, characterized in that: The fluorescent probe comprises a fluorophore and a recognition group. The recognition group reacts with benzoyl peroxide and the fluorophore is excited by ultraviolet light only when benzoyl peroxide and 365nm ultraviolet light are present simultaneously. The synergistic effect of the two causes a detectable change in the fluorescence signal. No change in fluorescence signal occurs when only benzoyl peroxide, only 365nm ultraviolet light, or neither of the two are present. The chemical structural formula of this fluorescent probe is:

2. The fluorescent probe as described in claim 1, characterized in that: The recognition group is a thioether group, which reacts with benzoyl peroxide to generate a sulfone group. The sulfone group, as a strong electron-withdrawing group, triggers a photoinduced electron transfer process after the fluorophore is excited by 365nm ultraviolet light, resulting in fluorescence quenching. The fluorophore is a naphthalimide group.

3. The fluorescent probe as described in claim 1 or 2, characterized in that: The chemical structure of the fluorescent probe is C 28 H 24 N2O5S2.

4. A method for preparing the fluorescent probe according to any one of claims 1–3, characterized in that: The 4-bromo-1,8-naphthioimide derivative is referred to as compound 1, and p-methoxythiophenol is referred to as compound 2. Compound 1 and compound 2 are reacted under alkaline conditions in an organic solvent by heating and reflux, and the fluorescent probe is obtained after purification.

5. The preparation method according to claim 4, characterized in that: The molar ratio of compound 1 to compound 2 is 1:2, and the molar ratio of compound 1 to potassium carbonate is 1:

2.

6. The method for detecting benzoyl peroxide using a fluorescent probe according to any one of claims 1 to 3, characterized in that... Follow these steps: (1) The food sample to be tested is extracted to obtain the test solution; (2) The test solution and the fluorescent probe are mixed in a buffer solution to form a detection system; (3) Irradiate the detection system with 365nm ultraviolet light; (4) Detect the fluorescence intensity of the detection system at 546 nm and compare it with the fluorescence intensity of the blank control system; if the fluorescence intensity is significantly reduced, it is determined that the food sample to be tested contains benzoyl peroxide.

7. The detection method according to claim 6, characterized in that: The 4-bromo-1,8-naphthioimide derivative is referred to as Compound 1, and p-methoxythiophenol is referred to as Compound 2. The food sample to be tested is flour, noodles, or dough. The buffer solution is a 10 mM HEPES buffer solution with a pH of 7.

4. The concentration of the fluorescent probe in the detection system is 10 μM. The blank control system consists of the buffer solution, the probe, and an equal volume of solvent to replace the test solution.

8. The use of the fluorescent probe according to any one of claims 1–3 in the detection of benzoyl peroxide residues in food, characterized in that: The sample extract and the fluorescent probe were mixed in a buffer solution and irradiated with 365 nm ultraviolet light. The change in fluorescence intensity at 546 nm was detected. The sample was determined to contain benzoyl peroxide only when the fluorescence intensity decreased significantly.

9. The application as described in claim 8, characterized in that: Fluorescence images are captured using a smartphone camera, and a quantitative relationship between the R+G value and the concentration of benzoyl peroxide is established through RGB color analysis, enabling visualized quantitative detection.

10. The application of the fluorescent probe according to any one of claims 1 to 3 in the construction of AND logic gate molecular sensors, characterized in that: The sensor uses BPO and 365nm ultraviolet light as two input signals and fluorescence intensity at 546nm as the output signal. It strictly follows the truth table of AND logic gates with two inputs to realize the logical judgment function of "dual key and lock".

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