A method for detecting diphenyl phosphate by using a smartphone-assisted molecularly imprinted sensor and application thereof

Abstract

This invention discloses a method and application for constructing a dual-mode sensor for detecting diphenyl phosphate using smartphone-assisted surface molecular imprinting, belonging to the fields of biochemical analysis, functional nanomaterials, and life science technology. In the synthesis process, the solubility, selectivity, and photoinitiation efficiency of the target analyte during polymerization are optimized, and molecularly imprinted nanozymes (MIPs) for diphenyl phosphate are rapidly prepared using molecular imprinting technology. In the detection process, the peroxidase-like activity of Fe,N-CDs catalyzes the colorimetric reaction between H₂O₂ and 3,3,5,5-tetramethylbenzidine (TMB), and the internal filtering effect of oxTMB suppresses the fluorescence signal. Combined with a smartphone, dual-signal cross-validation is achieved, constructing a colorimetric-fluorescence sensor for detecting diphenyl phosphate. This sensor exhibits specific recognition selectivity and binding affinity for diphenyl phosphate, is simple to operate, and low in cost. It can achieve sensitive detection of diphenyl phosphate over a wide linear range, with a detection limit as low as 16 pg / mL, making it suitable for on-site analysis of trace DPH in complex aquatic environments.

Description

Technical Field

[0001] This invention belongs to the technical field of functional nanomaterials and colorimetric-fluorescence chemical sensors, specifically relating to a method and application of a smartphone-assisted molecular imprinting dual-mode sensor for detecting diphenyl phosphate. Background Technology

[0002] Diphenyl phosphate (DPhP) is a common and high-performance organophosphorus flame retardant widely used in plastics, textiles, and electronic devices. However, organophosphorus esters are easily released into environmental media such as the atmosphere, water, soil, and dust. DPhP is a characteristic pollutant from the electronic waste dismantling process. Persistent human exposure to organophosphorus flame retardant contamination, including DPhP acquired through inhalation, digestion, skin, or the food chain, can cause mutagenic, carcinogenic, and neurotoxic effects, and may induce glucose metabolism disorders, posing potential health hazards. Therefore, it is necessary to develop a reliable method to detect the concentration of diphenyl phosphate in wastewater discharged from electronic waste dismantling areas.

[0003] Current methods for detecting DPhP include chromatography, mass spectrometry, and electrochemical sensor analysis, which are typically expensive, time-consuming, and require complex sample pretreatment. Colorimetric-fluorescence sensors, on the other hand, offer advantages such as simple operation, low cost, fast detection speed, no need for expensive equipment, and suitability for on-site analysis. Molecularly imprinted polymers exhibit strong specificity in recognizing template molecules and possess advantages such as ease of preparation, low cost, high stability, and a large number of recognition sites, making them widely used to improve sensor selectivity. However, the uneven distribution of recognition sites in single-functional monomers and the influence of single solvent polarity on polymerization rate and photoinitiation efficiency limit the specificity and stability of molecularly imprinted polymer materials. Compared to natural enzymes, iron-doped carbon nanoparticles simultaneously possess fluorescence, stability, and enzyme activity. To address these challenges, this invention utilizes a methacrylic acid / styrene bifunctional monomer, a methanol / DMF mixed solvent, and Fe,N-CDs as the photocatalytic matrix and dual-mode signal source in the synthesis process. This optimizes the solubility, selectivity, and photoinitiation efficiency of the target analyte during polymerization, enabling the rapid preparation of diphenyl phosphate molecularly imprinted nanozymes (MIPs) using molecular imprinting technology, thus expanding the applicability of molecularly imprinted dual-mode sensors.

[0004] In the sensor analysis, the peroxidase-like activity of Fe,N-CDs catalyzes the colorimetric reaction between H2O2 and 3,3,5,5-tetramethylbenzidine (TMB) (colorimetric channel), and the internal filtering effect of oxTMB suppresses the fluorescence signal (fluorescence channel). Combined with a smartphone for dual-signal cross-validation, a diphenyl phosphate concentration-dependent colorimetric-fluorescence sensor is constructed. Based on molecular imprinting and this technology, this invention constructs a colorimetric-fluorescence sensor capable of specifically recognizing and detecting diphenyl phosphate, exhibiting good detection sensitivity and accuracy. The detection limit for the colorimetric method is 0.016 ng / mL, and the detection limit for the fluorescence method is 0.018 ng / mL, better meeting the needs of on-site analysis of trace DPH in complex aquatic environments. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for constructing and applying a colorimetric-fluorescence sensor for diphenyl phosphate that is simple to operate and has a fast detection speed.

[0006] The technical solution adopted in this invention is:

[0007] A method for constructing a dual-mode sensor using smartphone-assisted molecular imprinting to detect diphenyl phosphate.

[0008] In the synthesis of MMIPs, a bifunctional monomer of methacrylic acid / styrene, a methanol / DMF mixed solvent, and Fe,N-CDs were used as the photocatalytic matrix and a dual-mode signal source. The solubility, selectivity, and photoinitiation efficiency of the polymerization process of the target analyte were optimized. Molecularly imprinted nanozymes (MIPs) of diphenyl phosphate were rapidly prepared using molecular imprinting technology. In the detection, the peroxidase-like activity of Fe,N-CDs was used to catalyze the colorimetric reaction between H2O2 and 3,3,5,5-tetramethylbenzidine (TMB) (colorimetric channel), and the internal filtering effect of oxTMB was used to suppress the fluorescence signal (fluorescence channel). With the help of a smartphone, dual-signal cross-validation was achieved, and a colorimetric-fluorescence sensor for detecting diphenyl phosphate was constructed.

[0009] The target compound and bifunctional monomer prepolymerization is carried out by dissolving 50 mg of diphenyl phosphate (DPhP) (pre-dissolved in 10% acetonitrile) and bifunctional monomers (250 mg styrene and 300 mg methacrylic acid) in a 10 mL solution of methanol / N,N-dimethylformamide (DMF) at a volume ratio of 7:13, and then treating the solution under light at 4°C for 8 h to 12 h to obtain the prepolymer of the target compound DPhP and the bifunctional monomer.

[0010] The synthesis of the diphenyl phosphate molecularly imprinted nanozymes (MIPs) involved mixing 10 mL of a methanol / N,N-dimethylformamide (DMF) mixed solution (7:13 volume ratio) with 50 mg of the functional monomer prepolymer prepared from diphenyl phosphate (DPhP) for every 0.2 mL of Fe,N-CDs. The crosslinking agent was ethylene glycol dimethacrylate (EGDMA), with 1.2 g of EGDMA corresponding to every 0.2 mL of Fe,N-CDs. Fe,N-CDs served as both the matrix and the photocatalyst. Benzoin dimethyl ether (DMPA) was used as the photoinitiator, with 0.03 mg of DMPA corresponding to every 0.2 mL of Fe,N-CDs. The light source was a visible light superimposed with a 365 nm ultraviolet lamp, which was used for stable power irradiation for 20–70 min.

[0011] The rapid polymerization involves Fe,N-CDs catalyzing the generation of carboxyl radicals from methacrylic acid under light irradiation, which in turn synergistically generates methyl radicals from DMPA under ultraviolet irradiation, thus shortening the polymerization time to 30-70 min.

[0012] The eluted DPH molecularly imprinted nanozymes (MIPs) are obtained by repeatedly eluting the template molecules three times with an elution buffer (a mixed solution of methanol and acetic acid, with a volume ratio of methanol to acetic acid of 9:1) until no DPH is detected in the supernatant. The resulting eluted DPH molecularly imprinted nanozymes (MMIPs) are a smartphone-assisted molecularly imprinted dual-mode sensor for detecting diphenyl phosphate.

[0013] The method for constructing a molecularly imprinted dual-mode sensor is as follows: 100 mg of diphenyl phosphate molecularly imprinted nanozymes (MMIPs) are dispersed in 10 mL of pure water and ultrasonically dispersed for 20 min to obtain an MMIPs dispersion. Then, 30 μL of the MIPs dispersion is added to 100 μL of the DPhP solution to be tested, and the mixture is incubated in a shaker at a constant temperature for 20 min for adsorption. Subsequently, 30 μL of the mixture is taken out, and 445 μL of HAc-NaAc buffer (0.05 mol / L, pH=3.8), 5 μL of H2O2 solution with a concentration of 200 mol / L, and 20 μL of 3,3',5,5'-tetramethylbenzidine (TMB) solution with a concentration of 0.02 mol / L are added sequentially. The mixture is mixed evenly and reacted in a 25°C incubator for 10-30 min. The supernatant is collected by centrifugation and the RGB values ​​are obtained by taking a picture with a smartphone COLOR PICKER APP.

[0014] In constructing the dual-mode sensor, the solvent for the DPhP solution to be tested is water, and the solvent for the 3,3',5,5'-tetramethylbenzidine TMB solution is a 0.05 mol / L HAc-NaAc buffer solution with a pH of 3.8, containing 5% dimethyl sulfoxide (DMSO).

[0015] The smartphone-assisted molecular imprinting dual-mode sensor for detecting diphenyl phosphate constructed using the described method is applied to the on-site analysis of trace DPH in complex aquatic environments.

[0016] The constructed smartphone-assisted molecularly imprinted dual-mode sensor has a detection range of 0.05 ng / mL to 25 ng / mL, with a detection limit of 0.016 ng / mL for colorimetric assay and 0.018 ng / mL for fluorescence assay. Furthermore, the test results demonstrate that the constructed smartphone-assisted molecularly imprinted dual-mode sensor exhibits excellent sensitivity, specificity, storage stability, and reusability, making it suitable for trace detection of DPhP in seawater.

[0017] This invention uses Fe,N-CDs carbon quantum dots as biomimetic nanozymes to catalyze the oxidation of H2O2 into a chromogenic substrate. It has both photocatalytic activity and peroxidase catalytic activity, and also has the fluorescence properties of carbon dots. (1) Molecularly imprinted polymers (MMIPs) can be rapidly constructed using Fe,N-CDs carbon quantum dots as a matrix. These polymers possess the catalytic activity of nanozymes and exhibit stable fluorescence signals. (2) The smartphone-assisted molecularly imprinted dual-mode sensor for detecting diphenyl phosphate has the specificity of molecularly imprinted polymer recognition, enabling specific detection of diphenyl phosphate. (3) While constructing the dual-mode sensor for detecting diphenyl phosphate, the detection results are processed using a smartphone, giving the sensor high sensitivity and selectivity while also providing the advantages of simplicity, speed, and on-site detection. (4) RGB value analysis shows that the colorimetric and fluorescence signals of the dual-mode sensor exhibit a wide linear range (0.05 ng / mL ~ 25 ng / mL) for diphenyl phosphate. The detection limit of the colorimetric method is 0.016 ng / mL, and the detection limit of the fluorescence method is 0.018 ng / mL. (5) The constructed dual-mode sensor has a short preparation cycle, simple process, and low cost, and has certain prospects for industrial application. Attached Figure Description

[0018] Figure 1 This is a flowchart of a method for constructing a dual-mode sensor using smartphone-assisted molecular imprinting to detect diphenyl phosphate.

[0019] Figure 2 This is the X-ray photoelectron spectrum of MMIPs.

[0020] Figure 3This is a transmission electron microscope image of MMIPs.

[0021] Figure 4 It is a linear relationship between the RGB colorimetric values ​​obtained from sensor testing and the concentration of DPHp.

[0022] Figure 5 It is a linear relationship between the RGB fluorescence obtained from sensor testing and the concentration of DPHP.

[0023] Figure 6 It is the color change of the solution (under visible light) when the sensor detects different concentrations of diphenyl phosphate.

[0024] Figure 7 The sensor detects the color change of the solution when different concentrations of diphenyl phosphate are measured (under 365 nm light). Detailed Implementation

[0025] Example 1

[0026] Fe,N-CDs carbon quantum dots were synthesized by a solvothermal method. The precursors were citric acid, thiourea, ammonium fluoride, and ferric chloride. The reaction was carried out under vacuum and high pressure at 180℃ for 8 hours. After centrifugation, washing, and drying, Fe,N-CDs carbon quantum dots were obtained. 0.05 g of the template molecule diphenyl phosphate (DPhP) was dissolved in 30 mL of a methanol / DMF (2:3, v / v) mixed solution with 0.25 g of the functional monomers Sty and 0.25 g of methacrylate (MAA). After 8 h, a prepolymer of the functional monomers and template molecules was obtained. 0.2 mL of Fe,N-CDs carbon quantum dots were added to 10 mL of the prepolymer of functional monomers and template molecules prepared in step one, followed by 0.8 g of ethylene glycol dimethacrylate (EGDMA) crosslinking agent and 0.006 mg of benzoin dimethyl ether (DMPA) initiator. The mixture was irradiated with a 365 nm UV lamp for 70 min to obtain fluorescent diphenyl phosphate molecularly imprinted nanozymes (MMIPs). After the reaction was completed, the diphenyl phosphate molecularly imprinted nanozymes (MIPs) were recovered by centrifugation. The template molecules were eluted repeatedly with a methanol / acetic acid (9:1, v / v) mixed elution buffer until no DPH was detected in the supernatant, thus obtaining the eluted molecularly imprinted nanozymes (MMIPs).

[0027] Example 2

[0028] Fe,N-CDs carbon quantum dots were synthesized by a solvothermal method. The precursors were citric acid, thiourea, ammonium fluoride, and ferric chloride. The reaction was carried out under vacuum and high pressure at 180°C for 8 hours. After centrifugation, washing, and drying, Fe,N-CDs carbon quantum dots were obtained. 0.05 g of the template molecule diphenyl phosphate (DPhP) was dissolved in 30 mL of a methanol / DMF (2:3, v / v) mixture with 0.25 g of the functional monomers styrene (Sty) and 0.25 g of methacrylate (MAA). After 12 h, a prepolymer of the functional monomers and template molecule was obtained. 0.2 mL of Fe,N-CDs carbon quantum dots were added to 10 mL of the prepolymer of the functional monomers and template molecule prepared in step one, followed by 1.2 g of ethylene glycol dimethacrylate (EGDMA) crosslinking agent and 0.03 mg of benzoin dimethyl ether (DMPA) initiator. The mixture was irradiated with a 365 nm UV lamp for 20 min to obtain fluorescent diphenyl phosphate molecularly imprinted nanozymes (MMIPs). After the reaction was completed, the diphenyl phosphate molecularly imprinted nanozymes (MIPs) were recovered by centrifugation. The template molecules were eluted repeatedly with a methanol / acetic acid (9:1, v / v) mixed elution buffer until no DPH was detected in the supernatant, thus obtaining the eluted molecularly imprinted nanozymes (MMIPs).

[0029] Example 3

[0030] Fe,N-CDs carbon quantum dots were synthesized by a solvothermal method. The precursors were citric acid, thiourea, ammonium fluoride, and ferric chloride. The reaction was carried out under vacuum and high pressure at 180°C for 8 hours. After centrifugation, washing, and drying, Fe,N-CDs carbon quantum dots were obtained. 0.05 g of the template molecule diphenyl phosphate (DPhP) was dissolved in 30 mL of methanol / DMF (1:2, v / v) mixed solution with 0.25 g of functional monomers (Sty) and 0.25 g of methacrylate (MAA). After 10 h, a prepolymer of functional monomers and template molecules was obtained. 0.2 mL of Fe,N-CDs carbon quantum dots were added to 10 mL of the prepolymer of functional monomers and template molecules prepared in step one, followed by 1.2 g of ethylene glycol dimethacrylate (EGDMA) crosslinking agent and 0.03 mg of benzoin dimethyl ether (DMPA) initiator. The mixture was irradiated with a 365 nm UV lamp for 20 min to obtain fluorescent diphenyl phosphate molecularly imprinted nanozymes (MMIPs). After the reaction was completed, the diphenyl phosphate molecularly imprinted nanozymes (MIPs) were recovered by centrifugation. The template molecules were eluted repeatedly with a methanol / acetic acid (9:1, v / v) mixed elution buffer until no DPH was detected in the supernatant, thus obtaining the eluted molecularly imprinted nanozymes (MMIPs).

[0031] Example 4

[0032] Fe,N-CDs carbon quantum dots were synthesized by a solvothermal method. The precursors were citric acid, thiourea, ammonium fluoride, and ferric chloride. The reaction was carried out under vacuum and high pressure at 180°C for 8 hours. After centrifugation, washing, and drying, Fe,N-CDs carbon quantum dots were obtained. 0.05 g of the template molecule diphenyl phosphate (DPhP), 0.25 g of the functional monomers styrene (Sty), and 0.3 g of methacrylate (MAA) were dissolved in 30 mL of methanol / DMF (2:3, v / v) mixed solution. After 8 h, a prepolymer of the functional monomers and template molecule was obtained. 0.2 mL of Fe,N-CDs carbon quantum dots were added to 10 mL of the prepolymer of the functional monomers and template molecule prepared in step one, followed by 1.2 g of ethylene glycol dimethacrylate (EGDMA) crosslinking agent and 0.03 mg of benzoin dimethyl ether (DMPA) initiator. The mixture was irradiated with a 365 nm UV lamp for 20 min to obtain fluorescent diphenyl phosphate molecularly imprinted nanozymes (MMIPs). After the reaction was completed, the diphenyl phosphate molecularly imprinted nanozymes (MIPs) were recovered by centrifugation. The template molecules were eluted repeatedly with a methanol / acetic acid (9:1, v / v) mixed elution buffer until no DPH was detected in the supernatant, thus obtaining the eluted molecularly imprinted nanozymes (MMIPs).

[0033] Example 5: An application method of a smartphone-assisted molecularly imprinted dual-mode sensor for detecting diphenyl phosphate.

[0034] The process involved dispersing 100 mg of diphenyl phosphate molecularly imprinted nanozymes (MMIPs) prepared in Example 4 into 10 mL of pure water, ultrasonically dispersing for 20 min to obtain an MMIPs dispersion. Then, 30 μL of the MIPs dispersion was added to 100 μL of the DPHP solution to be tested, and the mixture was incubated in a shaker at a constant temperature for 20 min. Subsequently, 30 μL of the mixture was removed, and 445 μL of HAc-NaAc buffer (0.05 mol / L, pH=3.8), 5 μL of 200 mol / L H2O2 solution, and 20 μL of 0.02 mol / L TMB solution were added sequentially. The mixture was thoroughly mixed, reacted in a 25°C incubator for 4 min, centrifuged, and the supernatant was collected in a centrifuge tube. The RGB values ​​were obtained by taking a picture using a smartphone COLOR PICKER APP. Figure 1 As shown, the linear relationship between the RGB values ​​(colorimetric method) obtained from the sensor test and the concentration of DPHp is expressed by the linear equation y = -0.11305 logC. DPhP +0.95404, the linear correlation coefficient is R 2 =0.998, the calculated detection limit is 0.023 ng / mL; Figure 2As shown, the linear relationship between the RGB values ​​(fluorescence method) obtained from the sensor test and the concentration of DPHp is expressed by the equation y = 0.0667 logC. DPhP +0.58202, the linear correlation coefficient is R 2 =0.99068, and the calculated detection limits are 0.016 ng / mL and 0.018 ng / mL, respectively; where Figure 3 and Figure 4 The illustrations show the color changes of the solution under visible light and 365 nm conditions when the sensor detects different concentrations of diphenyl phosphate.

Claims

1. A method for detecting diphenyl phosphate using a smartphone-assisted molecularly imprinted construct dual-mode sensor, characterized in that, After prepolymerization of the target compound and bifunctional monomer, it is combined with iron-nitrogen-doped carbon quantum dots (Fe,N-CDs), crosslinking agent ethylene glycol dimethacrylate (EGDMA), and photoinitiator benzoin dimethyl ether (DMPA) under photo-assisted conditions to form diphenyl phosphate molecularly imprinted nanozymes (MMIPs). Subsequently, the DPhP-eluted molecularly imprinted nanozymes (MIPs), which are then used as dual-mode sensors, are incubated with different concentrations of DPhP in an H2O2 and TMB system for 30 min. The RGB values ​​are then captured by taking pictures with a smartphone to assist in the analysis of colorimetric-fluorescence dual signals.

2. The method for constructing a dual-mode sensor for detecting diphenyl phosphate using a smartphone-assisted molecular imprinting method according to claim 1, characterized in that, The prepolymerization of the target compound and bifunctional monomer was carried out according to the following steps: 50 mg of the target compound, diphenyl phosphate (DPhP), was pre-dissolved in 10% acetonitrile, and then mixed with 250 mg of the bifunctional monomer, styrene, and 300 mg of methacrylic acid, and dissolved in 10 mL of methanol / N,N-dimethylformamide (DMF) at a volume ratio of 7:

13. After being treated under light-shielding conditions at 4°C for 8 h to 12 h, the prepolymer of the target compound DPhP and the bifunctional monomer was obtained.

3. The method for constructing a dual-mode sensor for detecting diphenyl phosphate using a smartphone-assisted molecular imprinting method according to claim 2, characterized in that, The synthesis of diphenyl phosphate molecularly imprinted nanozymes (MMIPs) was carried out according to the following steps: 10 mL of a methanol / N,N-dimethylformamide (DMF) mixed solution with a volume ratio of 7:13 was mixed with 50 mg of the functional monomer prepolymer prepared by diphenyl phosphate (DPhP) for every 0.2 mL of Fe,N-CDs. Fe,N-CDs served as both a matrix and a photocatalyst. 0.03 mg of DMPA was also used for every 0.2 mL of Fe,N-CDs. The light source was a visible light superimposed with a 365 nm ultraviolet lamp, and the ultraviolet lamp was irradiated at a stable power for 20–70 min.

4. The method for constructing a dual-mode sensor for detecting diphenyl phosphate using smartphone-assisted molecular imprinting according to claim 3, characterized in that: Fe,N-CDs catalyze the generation of carboxyl radicals from methacrylic acid under light irradiation, and synergistically generate methyl radicals from DMPA under ultraviolet irradiation, shortening the polymerization time to 30~70 min.

5. The method for constructing a dual-mode sensor for detecting diphenyl phosphate using smartphone-assisted molecular imprinting according to claim 1, characterized in that: The elution of DPH molecularly imprinted nanozymes (MIPs) is carried out according to the following steps: the DPH molecularly imprinted nanozymes (MIPs) are eluted three times with an elution buffer, which is a mixed solution of methanol and acetic acid with a volume ratio of 9:1, until no DPH is detected in the supernatant. The eluted DPH molecularly imprinted nanozymes (MIPs) are then obtained, which are smartphone-assisted molecularly imprinted dual-mode sensors for detecting diphenyl phosphate.

6. The method for constructing a dual-mode sensor for detecting diphenyl phosphate using smartphone-assisted molecular imprinting according to claim 1, characterized in that: 100 mg of diphenyl phosphate molecularly imprinted nanozymes (MIPs) were dispersed in 10 mL of pure water and ultrasonically dispersed for 20 min to obtain a MIPs dispersion. Then, 30 μL of the MIPs dispersion was added to 100 μL of the DPHP solution to be tested, and the mixture was incubated in a shaker at a constant temperature for 20 min for adsorption. Subsequently, 30 μL of the mixture was taken out, and 445 μL of 0.05 mol / L HAc-NaAc buffer (pH=3.8), 5 μL of 200 mol / L H2O2 solution, and 20 μL of 0.02 mol / L 3,3',5,5'-tetramethylbenzidine (TMB) solution were added sequentially. The mixture was mixed thoroughly and reacted in a 25°C incubator for 10–30 min. The supernatant was collected by centrifugation and the RGB values ​​were obtained by taking a picture with a smartphone using the COLOR PICKER APP.

7. The application method of the dual-mode sensor according to claim 5, characterized in that: The solvent for the DPhP solution to be tested was water, and the solvent for the 3,3',5,5'-tetramethylbenzidine TMB solution was a 0.05 mol / L HAc-NaAc buffer solution with a pH of 3.8, containing 5% dimethyl sulfoxide (DMSO).

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