Semiconductor polymer dot-based rapid quantitative detection method for benzoyl peroxide based on ratio fluorescence

Abstract

This invention discloses a rapid quantitative detection method for benzoyl peroxide based on ratiometric fluorescence using semiconductor polymer dots, belonging to the field of fluorescence chemical sensing and analysis technology. This invention uses PFBT as the fluorescent donor polymer and PFO-DBT as the fluorescent acceptor polymer, and co-assembles highly stable semiconductor polymer dots using a nanoprecipitation method in the presence of a surfactant. This invention applies semiconductor polymer dots to the rapid quantitative detection of benzoyl peroxide based on ratiometric fluorescence, demonstrating advantages such as high sensitivity, high selectivity, anti-interference, accuracy, and reliability. The ratio signal of this invention originates from the coupling regulation of fluorescence energy transfer between polymers and free radical-triggered photoinduced electron transfer, avoiding the synthetic complexity and stability problems introduced by small molecule probes. It eliminates the need for small molecule reaction probes and achieves portability and visualization, showing broad prospects for the quantitative detection of benzoyl peroxide.

Description

Technical Field

[0001] This invention relates to the field of fluorescence chemical sensing and analysis technology, and in particular to a rapid quantitative detection method for benzoyl peroxide ratio fluorescence based on semiconductor polymer dots. Background Technology

[0002] Benzoyl peroxide (BPO) has been widely used for decades in polymer manufacturing (free radical initiator / crosslinking agent), topical acne treatment formulations, food processing additives, and fabric bleaching due to its efficient free radical generation and strong oxidizing properties. Benzoyl peroxide can decompose under heat or light to generate reactive species such as benzoyloxy radicals. Further reactions may produce decomposition products such as benzoic acid, phenyl benzoate, and biphenyl, and may even generate benzene, which has carcinogenic risks. Therefore, it poses potential safety hazards in applications such as food, pharmaceuticals, and daily chemical products.

[0003] Regulatory requirements for benzoyl peroxide vary across different countries and regions: the Codex Alimentarius Commission allows a maximum usage of 60 mg / kg as a wheat flour additive, but the EU and China have banned its use as a flour bleaching agent; for dermal applications, benzoyl peroxide is typically limited to below 10% and is permitted only for topical use. Although there is currently no direct evidence that benzoyl peroxide is carcinogenic, studies have suggested that it may promote cell proliferation or have a promoting effect on existing tumors under certain conditions. These regulatory inconsistencies and safety concerns make sensitive and reliable quantitative monitoring of benzoyl peroxide in various products and complex matrices an urgent need.

[0004] Existing methods for benzoyl peroxide analysis mainly include high-performance liquid chromatography (HPLC), chemiluminescence, and electrochemical detection. Instrumental methods such as HPLC typically require expensive equipment, have complex and time-consuming procedures, and are difficult to implement for rapid on-site screening. Chemiluminescence and electrochemical methods are susceptible to matrix interference in real samples, and some systems suffer from insufficient sensitivity or stability. In recent years, fluorescent probes have attracted attention due to their high sensitivity, fast response, and relatively simplified sample pretreatment. However, most reports are based on small-molecule organic probes, which often suffer from cumbersome synthesis steps, susceptibility to interference from benzoyl peroxide analogs or other reactive oxygen species, and insufficient stability in aqueous phases. Some fluorescent nanomaterials (such as metal-organic frameworks, MOFs) are also often limited by poor solubility and insufficient structural / luminescent stability, thus affecting quantitative accuracy and large-scale applications.

[0005] Semiconducting polymer dots (Pdots) possess advantages such as high brightness, excellent photostability, and good biocompatibility, showing great potential in fluorescence sensing. Most existing Pdot sensing strategies rely on introducing small-molecule reactive probes (covalent modification or physical encapsulation), inevitably inheriting the inherent limitations of small-molecule probes. In contrast, ratiometric fluorescence modulation based entirely on the conjugated polymer bulk can provide dual-channel self-calibrating signals and reduce the influence of external fluctuations; however, existing technologies lack relevant reports, and the mechanism is not clearly understood, failing to guide practical applications. Therefore, there is an urgent need to develop a novel strategy for benzoyl peroxide detection driven by intrinsic photophysical processes of semiconductor polymer dots, enabling both visualization and quantification. Summary of the Invention

[0006] In view of the above-mentioned deficiencies of the prior art, the purpose of this invention is to address the problems of equipment dependence, insufficient anti-interference, complex probe synthesis, and limited stability in the detection of benzoyl peroxide in the prior art, and to design and provide: 1) Stable fluorescence resonance energy transfer (FRET) is achieved by constructing a donor-acceptor system within a single particle through the co-assembly of two semiconductor polymers. 2) Based on the photoinduced electron transfer (PET) between the benzoyl oxygen free radical generated by the decomposition of benzoyl peroxide and the donor polymer, the fluorescence energy transfer efficiency is modulated, thereby generating a "red-green" dual-channel visualized and quantifiable ratio fluorescence response; 3) Further, a portable detection platform is provided to fix semiconductor polymer dots in sodium alginate hydrogel microspheres, enabling RGB ratio reading and rapid on-site detection on portable platforms such as mobile phones.

[0007] To achieve the above objectives, the technical solution provided by the present invention is as follows: In a first aspect of the present invention, a method for preparing semiconductor polymer dots is provided, comprising the following steps: Semiconductor polymer dots were co-assembled using PFBT (poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazol-4,8-diyl)], Poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazol-4,8-diyl)]) as fluorescent donor polymers and PFO-DBT (poly[2,7-(9,9-dioctylfluorenyl)-alt-4,7-bis(thiophen-2-yl)benzo-2,1,3-thiadiazole], Poly[2,7-(9,9-dioctylfluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2,1,3-thiadiazole]) as fluorescent acceptor polymers via nanoprecipitation in the presence of surfactants.

[0008] Preferably, the mass ratio of PFBT to PFO-DBT is 4:1 to 1:4.

[0009] The objectives of this invention can be achieved by controlling the ratio of the fluorescent donor polymer and the fluorescent acceptor polymer within a preferred range. Specifically, a significant dual-channel ratio response can be obtained when the mass ratio of PFBT to PFO-DBT is 4:1; other ratios, such as 1:1 and 1:4, can be used for comparison and screening. Those skilled in the art can adjust the ratio flexibly according to actual needs.

[0010] Preferably, the surfactant includes at least one of Pluronic F-127, phospholipid-polyethylene glycol copolymer (DSPE-PEG), and polystyrene-polyethylene glycol copolymer (PS-PEG); the mass ratio of surfactant to PFBT is 1:1 to 1:2.

[0011] Surfactants play a role in this invention by promoting dispersion and enhancing system stability. Those skilled in the art can select appropriate surfactant types and commercial products according to actual conditions or needs, such as the preferred types mentioned above. Among them, Pluronic F-127 is a nonionic triblock copolymer that, in addition to fulfilling the basic functions of surfactants, has a cost advantage compared to other types, making it a particularly suitable choice for this invention.

[0012] Preferably, the preparation of semiconductor polymer dots by the nanoprecipitation method includes the following steps: (1) Dissolve PFBT, PFO-DBT and surfactant in an organic solvent and mix well to obtain a mixture; (2) The mixture was injected into ultrapure water and subjected to ultrasonic treatment to obtain a nanoparticle dispersion; (3) The organic solvent in the nanoparticle dispersion was removed under an inert gas flow, and then large particle impurities were removed by filtration and semiconductor polymer dots were collected.

[0013] More preferably, in step (1), the organic solvent includes at least one of tetrahydrofuran (THF), acetonitrile, ethanol, and acetone.

[0014] Nanoprecipitation is a conventional method for preparing nanoparticles in this field. It involves rapidly mixing two solutions, causing a solute in one solution to precipitate quickly in the other, thereby forming nanoparticles. In this invention, PFBT, PFO-DBT, and a surfactant are dissolved in an organic solvent, then added to ultrapure water, and the resulting dispersion of the target nanoparticles is formed under ultrasonication. Given the raw material selection and proportions of this invention, those skilled in the art can choose appropriate solvent amounts based on actual conditions or needs, for example, by adding solvent to control the PFBT content in the system to be 0.1~10 mg / mL.

[0015] In a second aspect of the invention, a semiconductor polymer dot is provided, which is prepared using the preparation method of the first aspect of the invention.

[0016] In a third aspect of the invention, applications of the semiconductor polymer dots of the second aspect of the invention are provided, including: rapid quantitative detection of benzoyl peroxide ratio fluorescence.

[0017] Preferably, the method for rapid quantitative detection of benzoyl peroxide ratio fluorescence includes a solution detection method, which includes the following steps: Semiconductor polymer dots are diluted with phosphate buffer saline (PBS) solution to form a semiconductor polymer dot detection system. At the excitation wavelength, the sample to be tested or standard benzoyl peroxide solution is added to the semiconductor polymer dot detection system, mixed, and the fluorescence emission spectrum is recorded. Quantification is performed based on the ratio of fluorescence intensity.

[0018] Preferably, the method for rapid quantitative detection of benzoyl peroxide ratio fluorescence includes a hydrogel microsphere detection method, which includes the following steps: Hydrogel microspheres containing semiconductor polymer dots are immersed in an aqueous solution or sample extract containing benzoyl peroxide, photographed under UVA ultraviolet light, and quantified by extracting the G / R ratio.

[0019] More preferably, the method for preparing the hydrogel microspheres containing semiconductor polymer dots includes the following steps: Semiconductor polymers were mixed with sodium alginate aqueous solution to form sodium alginate mixture; the sodium alginate mixture was added dropwise to calcium chloride aqueous solution to crosslink into spheres, the product was collected and purified to obtain hydrogel microspheres containing semiconductor polymer dots.

[0020] The semiconductor polymer dots of this invention exhibit excellent compatibility for benzoyl peroxide detection and are suitable for various benzoyl peroxide detection applications. Under the preferred method described above, those skilled in the art can employ a solution detection method to directly apply the semiconductor polymer dots for benzoyl peroxide detection, or an indirect method to apply hydrogel microspheres containing the semiconductor polymer dots for benzoyl peroxide detection. As presented in one or more embodiments of this invention, when using the direct method, at the excitation wavelength (e.g., 470 nm), based on the fluorescence emission spectrum, the donor emission (e.g., 536 nm) and acceptor emission (e.g., 628 nm) spectra are read, and the ratio is calculated. I536 / I628 The methods involve constructing calibration curves and performing sample calculations to achieve quantification. Alternatively, an indirect method involves adding hydrogel microspheres containing semiconductor polymer dots to the sample, photographing it under UVA (e.g., 365nm) ultraviolet light, and extracting the G / R ratio using a mobile device to conveniently complete quantification. These methods provide preferred detection methods based on convenience and accuracy. Those skilled in the art can also employ other suitable detection methods to use semiconductor polymer dots for rapid quantitative detection of benzoyl peroxide ratio fluorescence, depending on actual conditions or needs.

[0021] Based on the above technical solutions, the design concept and principle of this invention are as follows: This invention designs a target semiconductor polymer dot using PFBT as the fluorescent donor polymer and PFO-DBT as the fluorescent acceptor polymer, for the rapid quantitative detection of benzoyl peroxide. The design concept is that, under the detection conditions, among benzoyl peroxide, benzoyloxy radical, and phenyl radical, the benzoyloxy radical is the only species with a single unoccupied molecular orbital (SUMO) between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of PFBT. Its single occupied molecular orbital (SOMO) is located below the HOMO of PFBT, enabling thermodynamically favorable photoinduced electron transfer from excited-state PFBT to the benzoyloxy radical. This photoinduced electron transfer process manifests as fluorescence quenching of PFBT, reduced fluorescence energy transfer efficiency in the presence of benzoyl peroxide, and a mitigating effect of the radical scavenger. According to the fluorescence energy transfer-photoinduced electron transfer (FRET-PET) modulation design proposed in this invention, the benzoyl oxygen radical generated by benzoyl peroxide partially quenches the fluorescence of PFBT through a photoinduced electron transfer mechanism, while simultaneously disrupting the fluorescence energy transfer pathway from PFBT to PFO-DBT within the semiconductor polymer site. The decrease in donor emission efficiency and the interruption of fluorescence energy transfer together lead to the characteristic red-green fluorescence shift upon exposure to benzoyl peroxide. Furthermore, the design of this invention ensures that only benzoyl peroxide simultaneously satisfies two conditions: sufficient proximity between the analyte and the polymer chain within the semiconductor polymer site, and appropriate molecular orbital arrangement to enable photoexcitation of PFBT to accept electrons. Therefore, this invention overcomes interference from other similar substances and exhibits excellent selectivity for benzoyl peroxide.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1) High sensitivity: The detection limit for benzoyl peroxide in solution system is about 8.2 nM, the detection limit for hydrogel microsphere system is about 13.0 nM, and the detection limit for lyophilized microspheres after rehydration is about 22.5 nM; 2) High selectivity and anti-interference: It exhibits low background response to various structural analogs, common amino acids, inorganic ions and food / pharmaceutical additives and other interfering substances; it can still produce significant ratio changes for benzoyl peroxide under conditions of coexisting interference. 3) High stability: The semiconductor polymer dots exhibit stable luminescence over a wide pH range and under continuous light irradiation; they also remain stable after long-term storage in aqueous solutions (e.g., 30 days). 4) No small molecule reaction probes are required: The ratio signal comes from the coupling regulation of fluorescence energy transfer between polymers and free radical-triggered photoinduced electron transfer, avoiding the synthetic complexity and stability problems introduced by small molecule probes.

[0023] 5) Portable visualization: After hydrogel immobilization, a visible "red-green" color change can be achieved, and RGB ratio analysis can be performed using portable devices such as mobile phones, making it suitable for rapid on-site detection; 6) Accurate and reliable: In the spiked recovery experiment of flour and benzoyl peroxide-containing drug samples, the recovery rate was about 93.5%~101.9%, the relative standard deviation (RSD) was less than 5%, and the results were in good agreement with those of high performance liquid chromatography. Attached Figure Description

[0024] Figure 1 In the diagram, (a) shows the preparation of semiconductor polymer dots and their application in the detection of benzoyl peroxide; (b) shows the preparation of semiconductor polymer dots integrated with hydrogel microspheres based on smartphone colorimetric analysis and the application of the quantitative method for benzoyl peroxide. Figure 2 In the image, (a) is a transmission electron microscope (TEM) image of PFBT / PFO-DBT semiconductor polymer dots (scale bar: 100 nm); (b) is the hydrodynamic particle size distribution and zeta potential of PFBT / PFO-DBT semiconductor polymer dots as determined by dynamic light scattering (DLS). Figure 3 In the table, (a) shows the fluorescence spectra of PFBT semiconductor polymer dots (λex=470 nm), PFO-DBT semiconductor polymer dots (λex=560 nm), and PFBT / PFO-DBT semiconductor polymer dots (λex=470 nm) at the same concentration (10 μg / mL); (b) shows the UV-Vis absorption spectrum of PFO-DBT semiconductor polymer dots and the fluorescence emission spectrum of PFBT semiconductor polymer dots. Figure 4 The fluorescence decay kinetics of PFBT and PFBT / PFO-DBT semiconductor polymer dots at 536 nm; Figure 5The effects of different environmental conditions on the stability of PFBT / PFO-DBT semiconductor polymer dots were investigated. (a) The effect of pH on the fluorescence behavior of PFBT / PFO-DBT semiconductor polymer dots (10 μg / mL) in Britton-Robinson buffer was shown. (b) The photostability of PFBT / PFO-DBT semiconductor polymer dots (10 μg / mL) under continuous irradiation (λex = 470 nm) was shown. (c) The fluorescence stability of PFBT / PFO-DBT semiconductor polymer dots (10 μg / mL) after long-term storage in aqueous solution was shown. (d) The results of adding benzoyl peroxide (40 µM) after long-term storage were also investigated. Figure 6 In the figure, (a) to (c) show the fluorescence intensity of semiconductor polymer dots composed of PFBT and PFO-DBT at ratios of 1:4, 1:1, and 4:1, respectively, measured by fluorescence titration experiments using different concentrations of benzoyl peroxide (λex = 470 nm). The inset shows the fluorescence color changes of the semiconductor polymer dot solution; (d) shows the fluorescence intensity ratio of semiconductor polymer dots with a PFBT / PFO-DBT ratio of 4:1. I536 / I628 The linear relationship between benzoyl peroxide concentration and benzoyl peroxide concentration; Figure 7 The fluorescence intensity ratio of PFBT / PFO-DBT semiconductor polymer dots in the presence of benzoyl peroxide (40 µM) and interfering analytes (200 µM) is ( I536 / I628 ); Figure 8 In the figure, (a) and (b) show the fluorescence decay kinetics of PFBT / PFO-DBT semiconductor polymer dots (10 μg / mL) before and after the addition of benzoyl peroxide (40 μM) at wavelengths of 536 nm and 628 nm, respectively. Figure 9 In the figure, (a) and (b) are the fluorescence spectra of PFBT and PFO-DBT in the presence of different concentrations of benzoyl peroxide, respectively; Figure 10 In the figure, (a) and (b) are the Fourier transform infrared spectrum and hydrogen nuclear magnetic resonance spectrum of PFBT before and after the addition of benzoyl peroxide, respectively; Figure 11 In the figure, (a) and (b) are the fluorescence spectra of PFBT / PFO-DBT semiconductor polymer dots and PFBT semiconductor polymer dots in the presence of benzoyl peroxide (40 μM) and different concentrations of isopropyl alcohol (IPA), respectively. Figure 12The electron paramagnetic resonance (EPR) spectra of PFBT / PFO-DBT semiconductor polymer dots in the presence of benzoyl peroxide (40 μM) and different concentrations of isopropanol are shown. Figure 13 To obtain the model fluorophore units of PFBT and PFO-DBT, as well as the highest occupied molecular orbital and lowest unoccupied molecular orbital energy levels and frontier orbital gaps of benzoyl peroxide and its free radical species, through theoretical calculations using B3LYP-D3 / def2-TZVP. Figure 14 In the image, (a) shows fluorescence images of PFBT / PFO-DBT semiconductor polymer dots embedded in hydrogel microspheres and their lyophilized reference standards under 365 nm ultraviolet light irradiation in the presence of different concentrations of benzoyl peroxide; (b) shows the linear correlation results between the G / R value of the hydrogel microspheres and the concentration of benzoyl peroxide obtained by smartphone image analysis. Figure 15 In the image, (a) shows the fluorescence image of PFBT / PFO-DBT semiconductor polymer dots embedded in hydrogel microspheres after treatment with different interfering analytes (200 μM); (b) shows the fluorescence image of PFBT / PFO-DBT semiconductor polymer dots embedded in hydrogel microspheres after treatment with different interfering analytes (200 μM) and then exposure to benzoyl peroxide (40 µM). Figure 16 The recovery rate of benzoyl peroxide detection in flour and pharmaceutical samples using a solution and hydrogel sensor based on PFBT / PFO-DBT semiconductor polymer dots is compared with the spiked concentration and standard measurement value by high performance liquid chromatography. Among them, (a) is the detection result of flour sample and (b) is the detection result of pharmaceutical sample. Detailed Implementation

[0025] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0026] Example 1 This embodiment provides a PFBT / PFO-DBT semiconductor polymer dot, such as Figure 1 As shown in (a), its preparation method is as follows: (1) Weigh or measure 100 μL of tetrahydrofuran solution of PFBT (1 mg / mL) and 25 μL of tetrahydrofuran solution of PFO-DBT (1 mg / mL), and prepare a mixture of fluorescent donor polymer / fluorescent acceptor polymer at a mass ratio of 4:1. Add 200 μL of tetrahydrofuran solution of Pluronic F-127 (1 mg / mL) as a stabilizer and mix well to obtain the mixture. In addition to the parameters in the above embodiments, within the limited scope, the preparation can actually be carried out by scaling up or down proportionally, which can also achieve the purpose of the present invention; (2) The mixture was rapidly injected into 10 mL of ultrapure water and treated under ultrasonic conditions for 2 min to obtain a nanoparticle dispersion. (3) Remove tetrahydrofuran under nitrogen flow, filter through a filter membrane (pore size 0.22 μm) to remove large particulate impurities, and obtain a stock solution containing PFBT / PFO-DBT semiconductor polymer dots.

[0027] Construction of semiconductor polymer dot sensing system and verification of efficient fluorescence energy transfer mechanism: Fluorescent probes based on PFBT / PFO-DBT semiconductor polymer dots were prepared by co-assembly of PFBT polymer PFO-DBT via nanoprecipitation. The effects of systematically altering the polymer composition on photophysical properties were investigated. The microstructure of the PFBT / PFO-DBT semiconductor polymer dots was observed using transmission electron microscopy, and their hydrodynamic particle size distribution and zeta potential were characterized by dynamic light scattering.

[0028] Figure 2 (a) Transmission electron microscopy image shows that the average diameter of the obtained PFBT / PFO-DBT semiconductor polymer dots is approximately 25.5 nm. Figure 2 (b) Dynamic light scattering measurements show that its hydrodynamic diameter is 50.7 nm and the zeta potential of the PFBT / PFO-DBT semiconductor polymer point is -12.63 mV, indicating that it has sufficient electrostatic and colloidal stability under physiological conditions.

[0029] Using the same method described above, and at the same concentration (10 μg / mL), semiconductor polymer dots prepared solely with PFBT and PFO-DBT were used as comparisons. The fluorescence spectra of PFBT semiconductor polymer dots, PFO-DBT semiconductor polymer dots, and PFBT / PFO-DBT semiconductor polymer dots were measured, along with the UV-Vis absorption spectrum of PFO-DBT semiconductor polymer dots and the fluorescence emission spectrum of PFBT semiconductor polymer dots.

[0030] like Figure 3As shown in (a), the PFBT / PFO-DBT semiconductor polymer dots prepared with a 1:4 PFBT:PFO-DBT ratio exhibit strong red fluorescence, with an emission peak at 647 nm, approximately 85 times stronger than that of pure PFO-DBT semiconductor polymer dots. This significant enhancement stems from the efficient fluorescence energy transfer process from PFBT to PFO-DBT. Figure 3 As shown in (b), the high fluorescence energy transfer efficiency is attributed to the significant spectral overlap between the PFBT emission spectrum and the PFO-DBT absorption spectrum, as well as the tight co-encapsulation of the two polymers in the polymer dot matrix. These results demonstrate the advantages of semiconductor polymer dots, namely, that efficient Foster resonance energy transfer significantly enhances fluorescence output and effectively suppresses aggregation-caused quenching (ACQ) effects.

[0031] Figure 4 The fluorescence decay kinetics of PFBT and PFBT / PFO-DBT semiconductor polymer sites at 536 nm were investigated. By testing and calculating the average fluorescence lifetime at 536 nm in the presence of donor PFBT alone and acceptor PFO-DBT, a fluorescence energy transfer efficiency as high as 78% was obtained.

[0032] Semiconductor polymer dot environmental and long-term stability assessment: To evaluate the stability of PFBT / PFO-DBT semiconductor polymer dots, they were characterized and evaluated under various environmental conditions. For example... Figure 5 As shown in (a), fluorescence emission remained stable over a wide pH range, with only slight decreases observed at pH 11 and 12, likely due to reduced stability of the benzothiadiazole unit under strongly alkaline conditions. The photostability of the semiconductor polymer dots was further evaluated. Figure 5 (b) The fluorescence intensity at 536 nm and 628 nm remains essentially unchanged under sustained excitation, indicating excellent resistance to photobleaching. Furthermore, Figure 5 (c) shows that the PFBT / PFO-DBT semiconductor polymer dots maintain fluorescence intensity at both emission wavelengths. For example... Figure 5 As shown in (d), the consistent ratio fluorescence response to benzoyl peroxide was observed even after long-term storage (30 days) in aqueous solution, demonstrating its durable stability and reliable sensing performance.

[0033] Example 2 This embodiment demonstrates the application of PFBT / PFO-DBT semiconductor polymer dots, using the PFBT / PFO-DBT semiconductor polymer for rapid quantitative detection of benzoyl peroxide ratio fluorescence in a solution system. The steps are as follows: (1) Dilute the stock solution containing PFBT / PFO-DBT semiconductor polymer dots with phosphate buffer (10 mM, pH=7.4) to 10 μg / mL; (2) Prepare benzoyl peroxide standard solution (10 mM, solvent is N,N-dimethylformamide), add different volumes of benzoyl peroxide standard solution to 3.00 mL of the above semiconductor polymer spot detection system and add phosphate buffer solution to constant volume; or add the sample extract to be tested; (3) Excite at 470 nm, record the emission spectrum, read the fluorescence intensity at 536 nm and 628 nm and calculate. I536 / I628 The concentration of benzoyl peroxide in the sample was determined based on the calibration curve.

[0034] The limit of detection (LOD) and the limit of quantitation (LOQ) can be calculated using the σ / k method: LOD = 3σ / k, LOQ = 10σ / k, where k is the slope of the calibration curve and σ is the standard deviation of the blank sample ratio.

[0035] Polymer formulation optimization and benzoyl peroxide ratio response / linear quantification relationship The fluorescence intensity of semiconductor polymer dots composed of PFBT and PFO-DBT was detected by fluorescence titration experiments by varying the ratio of the fluorescent donor polymer / fluorescent acceptor polymer mixtures in the following ratios: (a) 1:4, (b) 1:1, and (c) 4:1, using different concentrations of benzoyl peroxide (λex = 470 nm). The insets show the fluorescence color changes of the semiconductor polymer dot solutions.

[0036] Figure 6 (a) shows that, despite having strong fluorescence properties, the semiconductor polymer dots with a PFBT:PFO-DBT ratio of 1:4 have a weak ratio fluorescence response to benzoyl peroxide; Figure 6 (b) and (c) show that although the red emission weakens after the addition of benzoyl peroxide, the green emission at 536 nm remains almost unchanged, causing the ratio analysis to fail. To improve sensing performance, semiconductor polymer dots with different polymer ratios were optimized. With increasing PFBT ratio, their fluorescence response to benzoyl peroxide is significantly enhanced. In particular, the optimized semiconductor polymer dots with a PFBT:PFO-DBT ratio of 4:1 exhibit unique ratio behavior characteristics. Figure 6 In (c), while the red emission at 628 nm gradually decreases, the green emission at 536 nm simultaneously increases. Correspondingly, Figure 6 (d) indicates that the fluorescence intensity is higher than (I536 / I628 The concentration of benzoyl peroxide showed a good linear relationship with the concentration of benzoyl peroxide. The limit of detection and the limit of quantitation were calculated to be 8.2 nM and 27.1 nM, respectively, both significantly lower than the maximum allowable concentration of benzoyl peroxide in food and pharmaceuticals. This indicates that the semiconductor polymer dots under optimized conditions have high sensitivity and quantitative capability, enabling accurate detection of benzoyl peroxide.

[0037] Selectivity and anti-interference performance of the probe The selectivity and interference immunity of the PFBT / PFO-DBT semiconductor polymer dots were evaluated using 22 potentially coexisting substances. These substances included perbenzoyl structural analogs such as benzaldehyde, benzoic acid, tert-Butyl peroxybenzoate (TBPB), and tert-Butyl hydroperoxide (TBHP), various amino acids (alanine, glycine, glutamic acid, leucine, threonine, aspartic acid, and phenylalanine), and common inorganic ions (Ca). 2+ K + Na + Cl - and NO 3- ), as well as other common additives such as glycerol, glucose, acrylamide, vitamin C (ascorbic acid), azobiscarboxamide (ADA) and sodium formaldehyde sulfoxylate (SFS). Figure 7 The results showed that after adding these potential interfering substances, the fluorescence intensity ratio was ( I536 / I628 The ratio remained at approximately 0.5, comparable to the value when the probe was used alone, indicating that these substances did not affect the probe's fluorescence response. Furthermore, even in the presence of these interfering substances, subsequent addition of benzoyl peroxide induced a significant red-green fluorescence transition, increasing the ratio to nearly 2.5, similar to the probe's response to benzoyl peroxide under interference-free conditions. These results collectively demonstrate the probe's excellent selectivity and strong anti-interference ability, highlighting its potential for precise quantification of benzoyl peroxide in complex samples.

[0038] Example 3 This embodiment studies and verifies the free radical-triggered FRET-PET coupling regulation mechanism of PFBT / PFO-DBT semiconductor polymer dots in applications. The steps are as follows: (1) The changes in the lifetime of PFBT / PFO-DBT semiconductor polymer dots at 628 nm (acceptor emission) and 536 nm (donor emission) before and after the addition of benzoyl peroxide were compared by time-resolved fluorescence measurement to assess the degree of inhibition of fluorescence energy transfer; (2) Add free radical scavengers such as isopropanol, ethanol, methanol, and tetramethylpiperidinooxy (TEMPO) and observe the inhibitory effect of ratio response; and verify that benzoyl peroxide-derived free radicals participate in signal transduction by detecting free radical signals through electron paramagnetic resonance. (3) Density functional theory (DFT) calculations were used to analyze the energy levels of PFBT, PFO-DBT fragments, benzoyl peroxide and its free radicals to explain the thermodynamic feasibility of photoinduced electron transfer.

[0039] Coupled FRET-PET photophysical modulation mechanism Based on the excellent benzoyl peroxide sensing performance of the PFBT / PFO-DBT semiconductor polymer point probe, its mechanism of action was further explored. First, time-resolved fluorescence measurements were used to assess the photophysical changes induced by benzoyl peroxide. For example... Figure 8 As shown in (a) and (b), the 628 nm fluorescence lifetime corresponding to PFO-DBT emission is significantly shortened from 2.645 ns to 1.035 ns after the addition of benzoyl peroxide, indicating that the fluorescence energy transfer process is effectively quenched. Notably, although fluorescence energy transfer is suppressed, the 536 nm fluorescence lifetime attributable to PFBT emission still decreases from 0.834 ns to 0.783 ns. This phenomenon suggests that in the presence of benzoyl peroxide, the excited-state energy of PFBT is partially dissipated through non-radiative pathways.

[0040] To illustrate this phenomenon, the fluorescence response of PFBT and PFO-DBT to benzoyl peroxide was detected. Figure 9 The results showed that the fluorescence intensity of PFBT decreased significantly with the addition of benzoyl peroxide, while the fluorescence change of PFO-DBT was negligible. These results indicate that benzoyl peroxide directly quenches the fluorescence of PFBT and has little effect on PFO-DBT.

[0041] like Figure 10 As shown in (a) and (b), the Fourier transform infrared (FTIR) and proton nuclear magnetic resonance (NMR) spectra of PFBT were tested sequentially before and after the addition of benzoyl peroxide. Analysis showed no significant change in the chemical structure of PFBT, confirming that the fluorescence quenching was not due to polymer degradation or a covalent reaction. Instead, the results indicate a non-covalent intermolecular interaction between PFBT and benzoyl peroxide.

[0042] Benzoyl peroxide is a known free radical initiator capable of generating benzoyloxy and phenyl radicals. The effect of benzoyl peroxide on PFBT / PFO-DBT semiconductor polymer dots and PFBT semiconductor polymer dots in the presence of isopropanol was tested. Figure 11 The results in (a) and (b) show that it can effectively suppress the fluorescence response of semiconductor polymer dots. It is worth noting that this scavenging effect is concentration-dependent, and high concentrations of isopropanol are required to completely suppress fluorescence changes.

[0043] Figure 12 Electron paramagnetic resonance (EPR) spectra of PFBT / PFO-DBT semiconductor polymer dots are shown in the presence of benzoyl peroxide (40 μM) and different concentrations of isopropanol. When the isopropanol content reaches 25 vol.%, the EPR signal intensity almost completely disappears, which is highly consistent with the fluorescence results. These observations strongly demonstrate that the fluorescence response of the PFBT / PFO-DBT semiconductor polymer dots is induced by radical-mediated interactions.

[0044] To further explore their mechanism of action, density functional theory was used to calculate and analyze the HOMO-LUMO energy levels of the fluorophore units in PFBT, PFO-DBT, benzoyl peroxide, and their radical derivatives. For example... Figure 13As shown, the HOMO-LUMO band gap of PFBT (3.05 eV) is larger than that of PFO-DBT (2.32 eV), which is consistent with photophysical data. Among benzoyl peroxide, benzoyloxy radical, and phenyl radical, benzoyloxy radical is the only species with a single unoccupied molecular orbital between the highest occupied molecular orbital and the lowest unoccupied molecular orbital of PFBT. Its single occupied molecular orbital is located below the highest occupied molecular orbital of PFBT, thus enabling thermodynamically favorable photoinduced electron transfer from excited-state PFBT to benzoyloxy radical. This photoinduced electron transfer process explains the PFBT fluorescence quenching phenomenon, the reduced fluorescence energy transfer efficiency in the presence of benzoyl peroxide, and the mitigating effect of radical scavengers. Overall, these results support the proposed FRET-PET modulation design: the benzoyloxy radical generated from benzoyl peroxide partially quenches the fluorescence of PFBT through a photoinduced electron transfer mechanism, while simultaneously disrupting the fluorescence energy transfer pathway from PFBT to PFO-DBT within the semiconductor polymer dot. The decrease in donor emission efficiency and the interruption of fluorescence energy transfer together lead to the characteristic red-green fluorescence shift upon exposure to benzoyl peroxide. Furthermore, given that structure-related peroxides (tert-butyl peroxide and tert-butyl hydrogen peroxide) do not elicit a similar response, effective signal transduction requires two key conditions: (1) sufficient proximity of the analyte to the polymer chains within the semiconductor polymer dot; and (2) appropriate molecular orbital alignment to enable photoexcitation of the PFBT to accept electrons. Of the substances tested, only benzoyl peroxide simultaneously satisfies both conditions, explaining the probe's excellent selectivity.

[0045] Example 4 This embodiment demonstrates the application of PFBT / PFO-DBT semiconductor polymer dots, using the PFBT / PFO-DBT semiconductor polymer in the preparation of sodium alginate hydrogel microspheres and their readout on a mobile device. Figure 1 As shown in (b), the steps are as follows: (1) Dissolve sodium alginate (3 g) in water (50 mL), and add PFBT / PFO-DBT semiconductor polymer dots to make the final concentration 20 μg / mL; (2) Add sodium alginate mixture to saturated calcium chloride aqueous solution to crosslink into spheres, collect and wash to obtain hydrogel microspheres; or freeze-dry to form solid microsphere sensing material, and rehydrate to form hydrogel microspheres when used; (3) Soak the hydrogel microspheres in the benzoyl peroxide solution / sample extract to be tested and incubate (about 5 min for fresh microspheres, about 20 min for lyophilized and rehydrated), and take pictures under 365 nm UV excitation; (4) Use mobile phone RGB analysis software to extract the green and red channel intensities, calculate G / R, and obtain the benzoyl peroxide concentration through calibration curve.

[0046] Visual detection of hydrogel microspheres and quantitative calibration of mobile phone G / R After observing a significant, visually apparent change in red-green fluorescence upon exposure to benzoyl peroxide, the feasibility of using this probe as a portable, in-situ benzoyl peroxide quantification platform was further evaluated. Semiconductor polymer dots were immobilized in sodium alginate hydrogel, and their ratiofluorescence response was detected. Figure 14 As shown in (a), under 365 nm ultraviolet light irradiation, the hydrogel microspheres exhibit a significant shift in red-green fluorescence with increasing benzoyl peroxide concentration. Figure 14 As shown in (b), the corresponding green and red fluorescence intensities were extracted using a smartphone colorimetric method, and the resulting G / R ratio was linearly correlated with the benzoyl peroxide concentration. The detection limit was determined to be 13.0 nM, indicating that the hydrogel platform still maintains the excellent sensing capability of semiconductor polymer dots.

[0047] For portable applications, ease of storage and transportation is crucial. Therefore, this embodiment involves freeze-drying hydrogel microspheres to prepare freeze-dried sensing materials. Figure 14 As shown in (a), when exposed to benzoyl peroxide solution, the lyophilized microspheres still exhibited a similar fluorescence response after reabsorbing water, although the incubation time was extended from 5 min to 20 min. Although Figure 14 (b) shows that the G / R ratio maintains a good linear relationship with the concentration of benzoyl peroxide, but the slope is reduced, indicating a decrease in sensitivity, with a corresponding detection limit of 22.5 nM. However, this detection limit is still far below the threshold required by regulations, confirming that the lyophilized hydrogel probe still has sufficient sensitivity and is very suitable for practical portable benzoyl peroxide detection.

[0048] Visualization of the anti-interference effect and benzoyl peroxide-specific response of the hydrogel platform Similarly, this embodiment investigated the selectivity and anti-interference ability of semiconductor polymer dot-immobilized hydrogels. For example... Figure 15 As shown in (a), the hydrogel did not show any color change after treatment with these interfering substances. Furthermore, as... Figure 15 As shown in (b), the hydrogel still exhibits a good red-green conversion response to benzoyl peroxide in the presence of these interfering substances, indicating that the hydrogel maintains excellent specificity and anti-interference ability in the detection of benzoyl peroxide. Overall, this portable lyophilized sensing platform provides a practical and robust method for the on-site visual quantitative detection of benzoyl peroxide.

[0049] Example 5 This embodiment uses the application method as in Embodiment 2 or Embodiment 4 to apply PFBT / PFO-DBT semiconductor polymer dots to the detection of real food or pharmaceutical samples (including flour and drugs containing benzoyl peroxide). The steps are as follows: (1) Flour sample: Take 5 g of sample, add ethanol, sonicate for 20 min, vortex for 10 min, centrifuge at 7000 rpm for 5 min, take the supernatant and filter (0.45 μm). (2) Drug gel sample: Weigh 242 mg of gel, dilute to 5 mL with ethanol, and obtain the extract by sonication, vortexing, centrifugation and filtration. (3) Spiking recovery: Add different concentrations of benzoyl peroxide standard (e.g., 5 μM, 15 μM, 25 μM) to the extract and detect it according to the method of Example 2 or Example 4; the recovery rate and relative standard deviation are used to evaluate the accuracy and precision and can be compared with the results of high performance liquid chromatography.

[0050] Real sample testing and verification The recovery rates of benzoyl peroxide in flour and pharmaceutical samples were obtained using solution and hydrogel sensors based on PFBT / PFO-DBT semiconductor polymer dots, and compared with spiked concentrations and standard measurements by high-performance liquid chromatography. The recovery rates of benzoyl peroxide in actual samples are shown in Table 1.

[0051] Table 1: Recovery rate of benzoyl peroxide in actual samples

[0052] In the table: ND* represents not detected (below the detection limit).

[0053] To evaluate the practicality of semiconductor polymer-based point probes in real-world sample analysis, spiked recovery experiments were conducted using two representative matrices (flour and a benzoyl peroxide-containing reagent). Known concentrations of benzoyl peroxide (0, 5, 15, and 25 µM) were added to the samples using a gradient spiking method. The benzoyl peroxide content in liquid samples was quantified by fluorescence spectroscopy, while the content in hydrogel microspheres was determined by the G / R fluorescence intensity ratio. The recoveries for both samples ranged from 93.5% to 101.9%, with relative standard deviations below 5% (Table 1), indicating good accuracy and precision under practical conditions. To further verify the reliability of this method in real-world sample analysis, the results were compared with those obtained by high-performance liquid chromatography (HPLC). Figure 16 As shown, no statistically significant differences were observed between the two methods, confirming the accuracy and robustness of the semiconductor polymer dot-based strategy for quantifying benzoyl peroxide in complex real-world samples.

[0054] Based on the above embodiments and test results, it is evident that the semiconductor polymer dots designed and provided by this invention possess high stability, exhibiting stable luminescence over a wide pH range and under continuous illumination, and remaining stable even after long-term storage in aqueous solution (e.g., 30 days). The rapid quantitative detection method for benzoyl peroxide based on semiconductor polymer dots, with an optimized ratio (PFBT:PFO-DBT=4:1), achieves a detection limit of approximately 8.2 nM for the solution system, approximately 13.0 nM for the hydrogel microsphere system, and approximately 22.5 nM for the rehydrated lyophilized microspheres, demonstrating high sensitivity. This invention exhibits low background response to various structural analogs, common amino acids, inorganic ions, and food / pharmaceutical additives, and still produces significant ratio changes for benzoyl peroxide under coexisting interference conditions, showcasing the high selectivity and anti-interference capability of the semiconductor polymer dots under application conditions. Furthermore, this invention eliminates the need for small molecule reaction probes. The ratio signal originates from the coupling regulation of interpolymer fluorescence energy transfer and free radical-triggered photoinduced electron transfer, avoiding the synthetic complexity and stability issues introduced by small molecule probes. Hydrogel immobilization enables a visible red-green color change, and RGB ratio analysis can be performed using a mobile phone, making it suitable for rapid on-site detection and achieving a portable and visualized application. In spiked recovery experiments on flour and benzoyl peroxide-containing samples, the recovery rate was approximately 93.5%–101.9%, with a relative standard deviation of less than 5%, and good consistency with high-performance liquid chromatography results, demonstrating accuracy and reliability in real-world sample detection environments.

[0055] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for preparing semiconductor polymer dots, characterized in that, Includes the following steps: Using PFBT as the fluorescent donor polymer and PFO-DBT as the fluorescent acceptor polymer, semiconductor polymer dots were co-assembled in the presence of surfactants using a nanoprecipitation method; wherein PFBT is poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazole-4,8-diyl)] and PFO-DBT is poly[2,7-(9,9-dioctylfluorenyl)-alt-4,7-bis(thiophene-2-yl)benzo-2,1,3-thiadiazole].

2. The method for preparing semiconductor polymer dots according to claim 1, characterized in that: The mass ratio of PFBT to PFO-DBT is 4:1 to 1:

4.

3. The method for preparing semiconductor polymer dots according to claim 1, characterized in that: The surfactant includes at least one of Pluronic F-127, phospholipid-polyethylene glycol copolymer, and polystyrene-polyethylene glycol copolymer; the mass ratio of surfactant to PFBT is 1:1 to 1:

2.

4. The method for preparing semiconductor polymer dots according to claim 1, characterized in that, The method for preparing semiconductor polymer dots by nanoprecipitation includes the following steps: (1) Dissolve PFBT, PFO-DBT and surfactant in an organic solvent and mix well to obtain a mixture; (2) The mixture was injected into ultrapure water and subjected to ultrasonic treatment to obtain a nanoparticle dispersion; (3) The organic solvent in the nanoparticle dispersion was removed under an inert gas flow, and then large particulate impurities were removed by filtration and the semiconductor polymer was collected.

5. The method for preparing semiconductor polymer dots according to claim 4, characterized in that: In step (1), the organic solvent includes tetrahydrofuran.

6. A semiconductor polymer dot, characterized in that: It is prepared by the preparation method described in any one of claims 1 to 5.

7. An application of the semiconductor polymer dot as described in claim 6, characterized in that, include: For rapid quantitative detection of benzoyl peroxide ratio fluorescence.

8. The application of the semiconductor polymer dot according to claim 7, characterized in that, The method for rapid quantitative detection of benzoyl peroxide ratio fluorescence includes a solution detection method, which includes the following steps: Semiconductor polymer dots are diluted with phosphate buffer solution to form a semiconductor polymer dot detection system. At the excitation wavelength, the sample to be tested or standard benzoyl peroxide solution is added to the semiconductor polymer dot detection system, mixed, and the fluorescence emission spectrum is recorded. Quantification is performed based on the ratio of fluorescence intensity.

9. The application of the semiconductor polymer dot according to claim 7, characterized in that, The method for rapid quantitative detection of benzoyl peroxide ratio fluorescence includes a hydrogel microsphere detection method, which includes the following steps: Hydrogel microspheres containing semiconductor polymer dots are immersed in an aqueous solution or sample extract containing benzoyl peroxide, photographed under UVA ultraviolet light, and quantified by extracting the G / R ratio.

10. The application of the semiconductor polymer dot according to claim 9, characterized in that, The preparation method of the hydrogel microspheres containing semiconductor polymer dots includes the following steps: Semiconductor polymers were mixed with sodium alginate aqueous solution to form sodium alginate mixture; the sodium alginate mixture was added dropwise to calcium chloride aqueous solution to crosslink into spheres, the product was collected and purified to obtain hydrogel microspheres containing semiconductor polymer dots.

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