A Ce-based 3+ / Ce 4+ A fluorescence and colorimetric dual-mode detection method for simultaneous determination of hydrogen peroxide and organophosphates using a cascade mimic enzyme reaction

Abstract

The present invention discloses a method based on Ce 3+ / Ce 4+ A fluorescence and colorimetric dual-mode detection method for the simultaneous determination of hydrogen peroxide and organophosphorus using a cascade simulated enzyme reaction belongs to the field of fluorescence sensing detection technology. The present invention combines a smartphone color recognition application with a ratiometric fluorescence sensor to achieve real-time visual detection of H2O2 and OPs. The materials used in the present invention are not only low-cost and easy to obtain, but also do not require material synthesis, simplifying the detection process. In addition, the sensor is combined with a smartphone color recognition application to further simplify the detection workflow and achieve real-time data analysis and result feedback. This method provides a simple, efficient and economical solution for monitoring organophosphorus pesticides in agricultural products.

Description

Technical Field

[0001] The present invention belongs to the field of fluorescence sensing technology, and specifically relates to a method based on Ce 3+ / Ce 4+ A dual-mode fluorescence and colorimetric detection method for the determination of organophosphorus pesticides using a cascade mimic enzyme reaction. Background Art

[0002] Organophosphorus pesticides are widely used pesticides, but their residues pose potential risks to human health and the environment. Therefore, accurate detection of organophosphorus pesticide residues has become a top priority. Traditional detection methods usually rely on complex laboratory equipment, which is cumbersome and time-consuming to operate and cannot meet the needs of rapid on-site detection. Fluorescent probes have excellent selectivity and sensitivity and can monitor changes in target substances in real time. They have been widely used in the detection of organophosphorus pesticides. Compared with single-emission fluorescent probes, ratiometric fluorescent probes can quantitatively or qualitatively evaluate the target by measuring the intensity of the fluorescent signal or the ratio between two fluorescent signals. In addition, ratiometric fluorescent probes have a self-calibration function to improve the accuracy of the measurement results. The fluorescent color of the probe changes significantly with the concentration of the target, which facilitates visual analysis.

[0003] At present, some studies have reported systems that use nanomaterials, quantum dots, etc. as fluorescence detectors. These systems have shown good performance in detection sensitivity and selectivity. However, the synthesis process of nanomaterials is usually complicated and costly, which may limit their promotion in practical applications. Therefore, there is an urgent need to develop a new type of fluorescence sensor that can not only efficiently detect organophosphorus pesticides in agricultural products, but also simplify the operation process and improve the convenience and economy of detection. By seeking environmentally friendly and low-cost materials and combining them with portable devices such as smartphones for intelligent identification and real-time feedback, this will greatly promote the advancement of agricultural product safety detection technology and provide more reliable technical support for ensuring food safety. Summary of the Invention

[0004] The present invention addresses the problems existing in the prior art and provides a convenient, economical and rapid method for simultaneously detecting hydrogen peroxide and organophosphorus pesticides.

[0005] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0006] A Ce-based 3+ / Ce 4+ A fluorescence and colorimetric dual-mode detection method for simultaneously determining hydrogen peroxide and organophosphorus by cascade mimicking enzyme reaction comprises the following steps:

[0007] (1) Construction of ratiometric fluorescent probe for H2O2 detection: 4-Methylumbelliferyl phosphate (4-MUP) and Ce were added to Tris-HCl buffer. 3+ , and o-phenylenediamine OPD, followed by the addition of different concentrations of H2O 2， The fluorescence intensity curve of the sample was measured at a wavelength of 365 nm, with the concentration of H2O2 in the sample as the horizontal axis and the fluorescence intensity ratio of 2,3-diaminophenazine DAP and 4-MU as the horizontal axis. 550 / I 450 As the vertical axis, a fluorescence standard curve was established;

[0008] (2) Construction of ratiometric fluorescent probe for detecting organophosphorus Ops: First, acetylcholinesterase AChE was incubated with OPs solution of different concentrations, and then choline oxidase ChOx and acetylcholine chloride ATChCl were added. After further incubation, Tris-HCl buffer, 4-methylumbelliferyl phosphate 4-MUP, Ce 3+ and o-phenylenediamine OPD were added to the mixture, incubated for reaction, and the fluorescence intensity curve of the sample was measured at a wavelength of 365 nm, with the concentration of OPs in the sample as the horizontal axis and the fluorescence intensity ratio of DAP and 4-MU as the horizontal axis. 550 / I 450 As the vertical axis, a fluorescence standard curve was established;

[0009] (3) Constructing a smartphone color recognition platform to detect H2O2: Under 365nm UV light, the reaction mixture (Ce 3+ Fluorescence images of the 4-MUP / OPD / H₂O₂ ( / 4-MUP / OPD / H₂O₂) were obtained. Smartphone color recognition software was used to analyze the RGB values ​​of the images. Origin software was used to fit the linear relationship between the blue / red channel (G / B) ratio and the H₂O₂ concentration in the solution. A H₂O₂ standard curve was established, with H₂O₂ concentration as the horizontal axis and the blue / red channel (G / B) ratio as the vertical axis.

[0010] (4) Constructing a smartphone color recognition platform to detect organophosphorus OPs: Under 365 nm ultraviolet light, the reaction mixture (Ce 3+ The fluorescence images of OPs ( / 4-MUP / OPD / AChE / ChOx / ATChCl / OPs) were collected and analyzed using smartphone color recognition software for RGB values. Origin software was used to fit the linear relationship between the G / B ratio and the OPs concentration in the solution. An OPs standard curve was established with the OPs concentration as the abscissa and the blue channel / red channel ratio G / B as the ordinate.

[0011] Furthermore, in step (1), the concentration of Tris-HCl buffer is 50 mM, the pH is 7.0-10.0; the incubation temperature is 25° C., and the incubation reaction time is 10-90 min; Ce 3+ The concentration of is 50-450 μM; the concentration of o-phenylenediamine OPD is 10-90 mM; the quantitative concentration of 4-methylumbelliferyl phosphate 4-MUP is 0.1 μM.

[0012] Furthermore, the pH of the Tris-HCl buffer is 8.0; the incubation reaction time is 60 min; Ce 3+ The concentration of is 300 μM; the concentration of o-phenylenediamine OPD is 60 mM.

[0013] Furthermore, in step (2), the concentration of Tris-HCl buffer is 50 mM, and the pH is 7.0-10.0; the incubation temperature of acetylcholinesterase AChE and OPs solution is 37°C, and the incubation time is 30 minutes; the mixed solution of choline oxidase ChOx, acetylcholine chloride ATChCl, organophosphorus OPs and acetylcholinesterase AChE is incubated at 37°C for 30 minutes; the Tris-HCl buffer, 4-MUP, Ce 3+ and OPD were added to the mixture, the incubation temperature was 25℃, the reaction time was 1h; the concentration of acetylcholinesterase AChE was 0-0.042U / mL; the concentration of choline oxidase ChOx was 0-0.049U / mL; the concentration of acetylcholine chloride ATChCl was 0-7mM; Ce 3+ The concentration of is 50-450 μM; the concentration of o-phenylenediamine OPD is 0-90 mM; the concentration of 4-methylumbelliferyl phosphate 4-MUP is 0.1 μM.

[0014] Furthermore, in step (2), the pH of the Tris-HCl buffer is 8; the concentration of acetylcholinesterase AChE is 0.021U / mL; the concentration of choline oxidase ChOx is 0.028U / mL; the concentration of acetylcholine chloride ATChCl is 4mM; Ce 3+ The concentration of is 300 μM; the concentration of o-phenylenediamine OPD is 60 mM.

[0015] Furthermore, in actual sample detection, the fluorescence intensity ratio of the sample to be tested with unknown H2O2 concentration measured according to step (1) is substituted into the H2O2 fluorescence standard curve of step (1) to obtain the H2O2 concentration in the sample to be tested; the fluorescence intensity ratio of the sample to be tested with unknown OPs concentration measured according to step (2) is substituted into the OPs fluorescence standard curve of step (2) to obtain the OPs concentration in the sample to be tested.

[0016] Furthermore, in actual sample detection, step (3) measures the RGB value of the sample to be tested with unknown H2O2 concentration, obtains the ratio of green channel / blue channel G / B, and substitutes it into the H2O2 standard curve of step (3) to obtain the H2O2 concentration in the sample to be tested; according to step (4), the RGB value of the sample to be tested with unknown OPs concentration is measured, obtains the ratio of green channel / blue channel G / B, and substitutes it into the OPs standard curve of step (4) to obtain the OPs concentration in the sample to be tested.

[0017] Detection mechanism of the present invention: Ce 3+ It can not only dephosphorylate 4-MUP to generate 4-MU with blue fluorescence, but also react with H2O2 to generate Ce. 4+ and ·OH, both of which exhibit peroxidase-like activity. These two substances can oxidize the colorless substrate OPD into a yellow fluorescent product DAP. Due to the inner filter effect, the generated DAP weakens the blue luminescence of 4-MU. Therefore, as the concentration of H2O2 increases, the blue fluorescence of 4-MU weakens and the yellow fluorescence of DAP increases. Based on Ce 3+ The ternary system of 4-MU and 4-OPD was used to establish a ratiometric fluorescence sensor for H₂O₂ detection, with the solution's fluorescence color changing from blue to yellow. OPs inhibit AChE activity, preventing AChE and ChOx from catalyzing ATChCl to produce H₂O₂, thereby reducing DAP formation and restoring the blue luminescence of 4-MU, enabling the detection of OPs.

[0018] Beneficial effects:

[0019] (1) Ce with enzyme catalytic activity 3+ and Ce 4+ It can be directly used to construct sensors without involving the complicated nanozyme preparation process, which simplifies the construction process and improves the overall efficiency and simplicity;

[0020] (2) Through Ce 3+ and Ce 4+ The enzyme catalytic amplification effect improves the sensitivity and selectivity of the probe;

[0021] (3) Combining a smartphone color recognition application with a ratiometric fluorescence sensor can achieve real-time visual detection of H2O2 and OPs;

[0022] (4) The materials used in this invention are not only low-cost and readily available, but also require no material synthesis, simplifying the detection process. Furthermore, the sensor, combined with a smartphone color recognition application, further streamlines the detection workflow and enables real-time data analysis and result feedback, providing a simple, efficient, and economical solution for monitoring organophosphorus pesticides in agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the method for constructing the ratiometric fluorescence / smartphone dual-mode detection of hydrogen peroxide and organophosphorus according to the present invention;

[0024] Figure 2 The conditions for ratiometric fluorescence detection of hydrogen peroxide constructed in Example 1 of the present invention are optimized, wherein (A) and (B) are the emission spectra of 4-MU and DAP under different pH conditions; (C) and (D) are Ce 3+ and the ratio of OPD concentration to fluorescence intensity (F 555 / F 435 ) Effect of reaction time on the detection of H2O2 by probe; (E) Effect of reaction time on the detection of H2O2 by probe;

[0025] Figure 3 The fluorescence spectra and standard curve of the ratiometric fluorescence detection of hydrogen peroxide constructed in Example 2 of the present invention, wherein (A) is the fluorescence spectra of 4-MU and OPD after incubation with different H2O2 concentrations (0, 10, 20, 40, 80, 100, 200 and 300 μM, 338 respectively); (B) is the fluorescence intensity ratio of the sensing system (F 555 / F 435 ) and H2O2 concentration calibration curve;

[0026] Figure 4 This is a comparison chart of the selectivity of the detection system constructed in Example 3 for hydrogen peroxide;

[0027] Figure 5 The conditions for ratiometric fluorescence detection of organophosphorus constructed in Example 4 are optimized, where (A), (B), and (C) are the ratios of the concentrations of AChE, ChOx, and ATChCl to the fluorescence intensity (F 555 / F 435 ); (D), (E), and (F) are the effects of AChE, ATChCl, and OPs reaction time on the fluorescence intensity ratio (F 555 / F 435 )

[0028] Figure 6 The fluorescence spectra and standard curves for the ratiometric fluorescence detection of organophosphorus constructed in Example 5, where (A) is the fluorescence spectra of 4-MU and OPD after incubation with different concentrations of OPs (0, 10, 30, 50, 70, 100, 125, 150, 175, and 200 ng / mL); (B) is the fluorescence intensity ratio of the sensing system (F 555 / F 435 ) and OPs concentration calibration curve;

[0029] Figure 7This is a comparison chart of the selectivity of the detection system constructed in Example 6 for organophosphorus;

[0030] Figure 8 The colorimetric card and standard curve for detecting hydrogen peroxide using a smartphone constructed in Example 7, wherein (A) is the colorimetric card and (B) is the standard curve.

[0031] Figure 9 The colorimetric card and standard curve for detecting organophosphorus using a smartphone constructed in Example 8, wherein (A) is the colorimetric card and (B) is the standard curve. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be further described below with reference to specific embodiments, but is not limited thereto.

[0033] Example 1

[0034] Optimization of ratiometric fluorescence detection conditions for H2O2:

[0035] In order to improve the sensitivity of the probe in detecting H2O2, Ce 3+ The influencing factors such as concentration, OPD concentration, reaction time, and pH value were studied. Figure 2 A shows that the fluorescence intensity of DAP does not change much in the pH range of 7.5-8.5. However, in an alkaline environment, OH - The higher the concentration, the more favorable the dephosphorylation reaction of 4-MUP. The fluorescence intensity of 4-MU reaches its peak at pH 8.0. In the pH range of 6.5-9.5, the fluorescence intensity of 4-MU reaches its peak. Figure 2 B. This may be due to Ce 3+ Under strong alkaline conditions, it can react with OH- to form cerium hydroxide Ce(OH)3, thereby affecting the formation of 4-MU. In order to ensure that DAP and 4-MU in the detection system are in the best state and to achieve more accurate H2O2 detection, the pH value of 8.0 is the best. Figure 2 C, Ce 3+ The presence of Ce significantly affected the production of 4-MU and DAP. 3+ When the concentration of α-glucose was increased from 50 μM to 300 μM, the fluorescence intensity ratio (F 550 / F 450 ) gradually increases. 3+ When the concentration was 300 μM, F 550 / F 450 Reach the peak value and do not change with Ce 3+ This shows that at this concentration, Ce 3+ The catalytic effect of Ce has reached saturation, and further increasing the concentration will not significantly improve the performance of the probe. 3+The concentration was kept at 300 μM. Figure 2 As shown in D, with the increase of OPD concentration, the fluorescence intensity ratio shows a steady upward trend. When the concentration reaches 60mM, the growth rate of the fluorescence intensity ratio slows down and approaches a stable state. This is because when the OPD concentration increases, the OPD molecules react with OH and Ce. 4+ The probability of collision increases, thereby increasing the reaction rate. Therefore, the optimal concentration of OPD is set at 60mM. Figure 2 As can be seen in Figure D, after the addition of H2O2, the fluorescence intensity ratio gradually increases and stabilizes after about 60 minutes of reaction time. Therefore, the optimal reaction time is 60 minutes.

[0036] Example 2

[0037] Ratio fluorescence detection method for H2O2:

[0038] First, 30 μL of 4-MUP (0.1 μM), 90 μL of Ce were added to 500 μL of Tris-HCl buffer (50 mM, pH = 8.0). 3+ (300μM) and 360μL of OPD (60mM). Subsequently, H2O2 solutions of different concentrations were added, and finally pure water was added to 1.5mL, and the reaction was carried out at 25℃ for 1h. The excitation and emission slits were set to 10nm and 5nm respectively, and the fluorescence intensity was measured at a wavelength of 365nm. The concentration in the sample was used as the horizontal axis, and the fluorescence intensity ratio of DAP and 4-MU was used as the value I. 550 / I 450 The fluorescence standard curve was established with y as the vertical axis. Figure 3 As shown in Figure A, as the H2O2 concentration increased from 0μM to 300μM, the fluorescence peak intensity of 4-MU at 450nm gradually decreased, while the fluorescence peak intensity of DAP at 550nm gradually increased. Figure 3 As shown in Figure 2, the fluorescence intensity ratio gradually increases with the increase of H2O2 concentration, and a good linear relationship is established with the H2O2 concentration. The linear regression equation is Y = 0.02381 × X + 0.05436, and the detection limit is 0.03 μM.

[0039] illustrate:

[0040] (1) Ce 3+ Dephosphorylates 4-MUP to generate 4-MU, which has blue fluorescence.

[0041] (2) Ce 3+ Oxidized by H2O2 to generate oxidizing Ce 4+ and ·OH, which can further oxidize OPD to generate yellow fluorescent DAP and quench the blue fluorescence of 4-MU through the inner filter effect.

[0042] Example 3

[0043] H2O2 selectivity experiment:

[0044] In order to investigate the selectivity of the probe, the possible interfering substances Na in milk, beverages and tap water were studied. + , Ca 2+ Mg 2+ , K + 、SO4 2- 、Cl - , cystine, glutathione, glucose, glycine and salicylic acid. The concentration of the interfering substance is 1500μM and the concentration of H2O2 is 300μM.

[0045] The experimental results are as follows Figure 4 The experimental results show that, in addition to significantly increasing the fluorescence intensity ratio, the effects of other ions on the probe are almost negligible. Therefore, the ratiometric fluorescent probe designed in this invention has good selectivity and anti-interference ability.

[0046] Example 4

[0047] Optimization of conditions for ratiometric fluorescence detection of OPs:

[0048] In order to achieve the best effect of detecting OPs using H2O2 as an intermediate, key experimental parameters such as AChE concentration, AChE concentration, AChE concentration, and reaction time were systematically optimized. The enzyme activities of AChE and ChOx are optimal at pH 8, and the optimal pH condition for 4-MU is also 8, so subsequent experiments were carried out at pH 8. Figure 5 As shown in A, when the AChE concentration reaches 0.021U / mL, F 550 / F 450 The ratio of ChOx to F tends to be stable. This is mainly because the reaction between acetylcholinesterase and ATChCl is complete and is completely consumed. Similarly, the concentrations of ChOx and ATChCl will also affect the 550 / F 450 The ratio of . Figure 5 As shown in B and 5C, when the concentrations of ChOx and ATChCl reached 0.021U / mL and 4mM, respectively, F 550 / F 450 The ratio of α and β tends to be stable, proving that the reaction is complete. In addition, the effect of enzymatic reaction time on its performance was studied, such as Figure 5 D, 5E, 5F, after AChE and OPs reacted at room temperature for 15 minutes, the fluorescence intensity ratio tended to be stable. Then ATChCl and ChOx were added and incubated at 37℃ for 40 minutes, and the reaction was basically completed. Finally, Ce was added at 25℃. 3+, OPD and 4-MUP were mixed. After 60 minutes, the fluorescence intensity ratio stabilized at a constant level.

[0049] Example 5

[0050] Constructing a method for ratiometric fluorescence detection of OPs:

[0051] First, 30 μL of acetylcholinesterase (0.021 U / mL) was incubated with OPs solutions of different concentrations at 37°C for 30 minutes, and then 40 μL of ChOx (0.028 U / mL) and 40 μL of ATChCl (3 mM) were added and reacted at the same temperature for 30 minutes. Finally, 500 μL of Tris-HCl buffer (50 mM, pH = 8.0), 30 μL of 4-MUP (0.1 μM), and 90 μL of Ce 3+ (300 μM) and 360 μL OPD (60 mM) were added to the mixture and reacted at 25°C for 1 hour. The fluorescence signal was recorded at an excitation wavelength of 365 nm, with the concentration in the sample as the horizontal axis and the fluorescence intensity ratio of 4-MU and DAP as the value I 550 / I 450 The fluorescence standard curve was established with the excitation and emission slits set to 10 nm and 5 nm, respectively; Figure 6 A shows that with the increase of OPs concentration, the fluorescence peak intensity of 4-MU at 450 nm gradually increased, and the fluorescence peak intensity of DAP at 550 nm gradually decreased. Figure 6 B shows the relationship between the concentration of OPs and I 550 / I 450 There is a strong linear relationship. Y = -0.00617 × X + 1.58552 is the linear regression equation, and the detection limit is 0.59 ng / mL.

[0052] illustrate:

[0053] (1) AChE and Ch Ox catalyze the conversion of ATChCl to produce H2O2.

[0054] (2) OPs inhibit AChE activity and reduce the production of H2O2, thereby preventing OPD oxidation and restoring the fluorescence of 4-MU.

[0055] Example 6

[0056] OPs' optional experiments:

[0057] Considering the complexity of the actual samples, the selectivity of the sensor was evaluated. + , Ca 2+ Mg 2+ , K +, dichlorvos, methyl parathion, 2,4-dichlorophenoxyacetic acid, and fipronil were used as interfering substances. The concentration of the interfering substances was 1000 ng / mL, and the concentration of OPs was 200 ng / mL.

[0058] like Figure 7 The experimental results showed that only OPs pesticides such as dichlorvos, methyl parathion, and trichlorfon significantly changed the fluorescence intensity ratio, while the probe's response to other interfering substances and non-OPs pesticides was negligible. This indicates that the sensor has excellent selectivity for OPs.

[0059] Example 7

[0060] Build a smartphone color recognition platform to detect H2O2:

[0061] Under 365nm UV light, the reaction mixture of Example 1 was captured using a smartphone (Ce 3+ Fluorescence images of the 4-MUP / OPD / H2O2 ( / 4-MUP / OPD / H2O2) were obtained. Smartphone color recognition software was used to analyze the RGB values ​​of the images, and Origin software was used to fit a linear relationship between the G / B ratio and the H2O2 concentration in the solution. A H2O2 standard curve was constructed with H2O2 concentration as the horizontal axis and the blue / red channel ratio (G / B) as the vertical axis.

[0062] like Figure 8 As shown in A, under 365nm ultraviolet light, the fluorescence of the solution gradually changes from blue to yellow. Figure 8 B shows that there is a robust linear relationship between the G / B ratio and the H2O2 concentration. The obtained linear regression equation is Y = 0.00509 × X + 0.49102, and the detection limit is 9.7 μM.

[0063] Example 8

[0064] Smartphone color recognition platform detects OPs:

[0065] Under 365nm UV light, the reaction mixture of Example 3 (Ce 3+ Fluorescence images of OPs ( / 4-MUP / OPD / AChE / ChOx / ATChCl / OPs) were obtained. The RGB values ​​of the images were analyzed using smartphone color recognition software, and Origin software was used to fit a linear relationship between the G / B ratio and the OPs concentration in the solution. An OPs standard curve was constructed with OPs concentration as the horizontal axis and the blue / red channel ratio (G / B) as the vertical axis.

[0066] like Figure 9 As shown in A, under 365nm ultraviolet light, the fluorescence of the solution gradually changes from yellow to blue. Figure 9B shows that there is a robust linear relationship between the G / B ratio and the OPs concentration. The resulting linear regression equation is Y = -0.00292*X + 1.07186, and the detection limit is 19.6 ng / mL.

[0067] Example 9

[0068] In order to verify the 3+ The feasibility of detecting H2O2 using the 4-MUP-OPD ratiometric fluorescent probe was investigated, and the H2O2 residues in milk, beverages, and tap water were analyzed.

[0069] Sample preparation method: After mixing 10mL of milk with an equal volume of water, 5mL of 10% chloroacetic acid was added to precipitate the protein in the milk. The resulting mixture was ultrasonicated for 15 minutes, and then centrifuged at 10,000 rpm for 15 minutes to remove the precipitate. The liquid supernatant was filtered through a 0.22μM filter membrane and then diluted 10 times for use; beverages and tap water were centrifuged at 10,000 rpm for 15 minutes to remove the precipitate, and the liquid supernatant was filtered through a 0.22μM filter membrane, and then the supernatant was diluted 10 times for use. 500μL of the filtrate of milk, beverage and tap water were taken, and then 30μL of 4-MUP (0.1μM), 90μL of Cephalexin (0.1μM), and 100μL of dapoxetine (0.1μM) were added to the three groups respectively. 3+ (300μM), 360μL OPD (60mM), and 500μL Tris-HCl buffer (50mM, pH 8.0). Different concentrations of H2O2 (0, 5, 30, and 60μM) were then added to the sample and mixed for 1 hour. The accuracy of the ratiometric fluorescence method and the smartphone sensor platform analysis method was tested using the standard addition method. The method is as follows:

[0070] (1) First, the ratiometric fluorescence method was used to detect the H2O2 concentration in milk, beverages, and tap water, and no H2O2 was detected in the samples.

[0071] (2) Milk, beverage, and tap water samples were spiked with H₂O₂ at three different concentrations: 5, 30, and 60 μmol / L. The three H₂O₂ samples were analyzed using a fluorescence spectrometer. The spike recovery rates were calculated, and the results are shown in Table 1.

[0072] Table 1 Test results of various data

[0073]

[0074]

[0075] The spiked experiments showed that the fluorescence detection method of the present invention has high accuracy and precision and can sensitively detect H2O2 in samples.

[0076] Example 10

[0077] Using spinach and tomato as actual samples, the Ce 3+ -Feasibility of 4-MUP-OPD ratiometric fluorescent probe.

[0078] Sample preparation method: Tomato or spinach (20g) was crushed and added with 20mL of acetonitrile. The mixture was ultrasonically treated for 30 minutes, then filtered through a 0.22μM filter membrane, and the resulting liquid supernatant was diluted tenfold for later use. Take 500μL of the filtrate of spinach and tomato, and add 30μL AChE (0.021U / mL), 40μL ChOx (0.028U / mL), 40μL ATCh (3mM), 500μL Tris-HCl buffer (50mM, pH 8.0), and 30μL 4-MUP (0.1μM) in sequence according to the fluorescence detection steps of OPs. 90μL Ce was added by standard addition method. 3+ 300 μL of OPs (300 μM) and 360 μL of OPD (60 mM) were added to samples containing different concentrations of OPs (0, 10, 30, and 50 ng / mL). Finally, the spiked samples were analyzed by ratiometric fluorescence analysis and a smartphone sensing platform, and the spike recovery was calculated. The method is as follows:

[0079] (1) First, the ratiometric fluorescence method was used to detect the concentration of OPs in spinach and tomatoes, and no OPs were detected in the samples.

[0080] (2) Spinach and tomato samples were spiked with OPs at three different concentrations: 5, 30, and 60 ng / mL. The samples were analyzed using a fluorescence spectrometer. The spike recovery rates were calculated. The results are shown in Table 2 below.

[0081] Table 2 Test results

[0082]

[0083]

[0084] It should be noted that the above embodiments are only some of the preferred embodiments of the present invention, and not all of them. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

Claims

1. A Ce-based 3+ / Ce 4+ A fluorescence and colorimetric dual-mode detection method for simultaneously determining hydrogen peroxide and organophosphorus by cascade mimicking enzyme reaction, characterized in that: The following steps are involved: (1) Construction of ratiometric fluorescent probe for H2O2 detection: 4-Methylumbelliferyl phosphate (4-MUP) and Ce were added to Tris-HCl buffer. 3+ and o-phenylenediamine OPD, then different concentrations of H2O2 were added, the reaction was incubated, and the fluorescence intensity curve of the sample was measured at a wavelength of 365 nm. The concentration of H2O2 in the sample was used as the horizontal axis, and the fluorescence intensity ratio of the product 2,3-diaminophenazine DAP and 4-methylumbelliferyl phosphate 4-MU was used as the horizontal axis. 550 / I 450 As the vertical axis, a fluorescence standard curve was established; (2) Construction of ratiometric fluorescent probe for detecting organophosphorus Ops: First, acetylcholinesterase AChE was incubated with OPs solution of different concentrations, and then choline oxidase ChOx and acetylcholine chloride ATChCl were added. After further incubation, Tris-HCl buffer, 4-methylumbelliferyl phosphate 4-MUP, Ce 3+ and o-phenylenediamine OPD were added to the mixture, incubated for reaction, and the fluorescence intensity curve of the sample was measured at a wavelength of 365 nm, with the concentration of OPs in the sample as the horizontal axis and the fluorescence intensity ratio of 2,3-diaminophenazine DAP and 4-methylumbelliferyl phosphate 4-MU as the horizontal axis. 550 / I 450 As the vertical axis, a fluorescence standard curve was established; (3) Constructing a smartphone color recognition platform to detect H2O2: Under 365nm ultraviolet light, a smartphone was used to capture the fluorescence image of the reaction mixture in step (1), and the RGB value of the image was analyzed using smartphone color recognition software. The linear relationship between the blue channel / red channel ratio (G / B) and the H2O2 concentration in the solution was fitted using Origin software. A H2O2 standard curve was established with the H2O2 concentration as the horizontal axis and the blue channel / red channel ratio (G / B) as the vertical axis. (4) Constructing a smartphone color recognition platform for detecting organophosphorus OPs: Under 365 nm ultraviolet light, a smartphone was used to capture the fluorescence image of the reaction mixture in step (2). The RGB values ​​of the image were analyzed using smartphone color recognition software. The linear relationship between the G / B ratio and the OPs concentration in the solution was fitted using Origin software. The OPs standard curve was established with the OPs concentration as the horizontal axis and the blue channel / red channel ratio G / B as the vertical axis.

2. According to claim 1, based on Ce 3+ / Ce 4+ A fluorescence and colorimetric dual-mode detection method for simultaneously determining hydrogen peroxide and organophosphorus by cascade mimicking enzyme reaction, characterized in that: Step (1) The concentration of Tris-HCl buffer is 50mM, the pH is 7.0-10.0; the incubation temperature is 25°C, and the incubation reaction time is 10-90min; Ce 3+ The concentration of is 50-450 μM; the concentration of o-phenylenediamine OPD is 10-90 mM; the concentration of 4-methylumbelliferyl phosphate 4-MUP is 0.1 μM.

3. The Ce-based method according to claim 1 or 2. 3+ / Ce 4+ A fluorescence and colorimetric dual-mode detection method for simultaneously determining hydrogen peroxide and organophosphorus by cascade mimicking enzyme reaction, characterized in that: The pH of the Tris-HCl buffer was 8.0; the incubation reaction time was 60 min; Ce 3+ The concentration of is 300 μM; the concentration of o-phenylenediamine OPD is 60 mM.

4. According to claim 1, based on Ce 3+ / Ce 4+ A fluorescence and colorimetric dual-mode detection method for simultaneously determining hydrogen peroxide and organophosphorus by cascade mimicking enzyme reaction, characterized in that: In step (2), the concentration of Tris-HCl buffer is 50 mM and the pH is 7.0-10.0; the incubation temperature of acetylcholinesterase AChE and OPs solution is 37°C and the incubation time is 30 minutes; the mixed solution of choline oxidase ChOx, acetylcholine chloride ATChCl, organophosphorus OPs and acetylcholinesterase AChE is incubated at 37°C for 30 minutes; the Tris-HCl buffer, 4-MUP, Ce 3+ and OPD were added to the mixture, and the incubation temperature was 25°C for 1 h; the concentration of acetylcholinesterase (AChE) was 0.007-0.042 U / mL; The concentration of choline oxidase ChOx is 0.007-0.049U / mL; the concentration of acetylcholine chloride ATChCl is 1-7mM; Ce 3+ The concentration of is 50-450 μM; the concentration of o-phenylenediamine OPD is 10-90 mM; the concentration of 4-methylumbelliferyl phosphate 4-MUP is 0.1 μM.

5. The Ce-based method according to claim 1 or 4. 3+ / Ce 4+ A fluorescence and colorimetric dual-mode detection method for simultaneously determining hydrogen peroxide and organophosphorus by cascade mimicking enzyme reaction, characterized in that: In step (2), the pH of the Tris-HCl buffer is 8; the concentration of acetylcholinesterase AChE is 0.021U / mL; the concentration of choline oxidase ChOx is 0.028U / mL; the concentration of acetylcholine chloride ATChCl is 4mM; Ce 3+ The concentration of is 300 μM; the concentration of o-phenylenediamine OPD is 60 mM.

6. The Ce-based method according to claim 1 3+ / Ce 4+ A fluorescence and colorimetric dual-mode detection method for simultaneously determining hydrogen peroxide and organophosphorus by cascade mimicking enzyme reaction, characterized in that: In actual sample detection, the fluorescence intensity ratio of the sample to be tested with unknown H2O2 concentration is measured according to step (1), and the ratio is substituted into the H2O2 fluorescence standard curve of step (1) to obtain the H2O2 concentration in the sample to be tested; the fluorescence intensity ratio of the sample to be tested with unknown OPs concentration is measured according to step (2), and the ratio is substituted into the OPs fluorescence standard curve of step (2) to obtain the OPs concentration in the sample to be tested.

7. The Ce-based method according to claim 1 3+ / Ce 4+ A fluorescence and colorimetric dual-mode detection method for simultaneously determining hydrogen peroxide and organophosphorus by cascade mimicking enzyme reaction, characterized in that: In actual sample detection, step (3) measures the RGB value of the sample to be tested with unknown H2O2 concentration, obtains the ratio of green channel / blue channel G / B, and substitutes it into the H2O2 standard curve of step (3) to obtain the H2O2 concentration in the sample to be tested; according to step (4), the RGB value of the sample to be tested with unknown OPs concentration is measured, obtains the ratio of green channel / blue channel G / B, and substitutes it into the OPs standard curve of step (4) to obtain the OPs concentration in the sample to be tested.

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