Fluorescence and colorimetric dual-mode detection method for simultaneously determining hydrogen peroxide and organic phosphorus based on Ce3+ / Ce4 + cascade mimic enzyme reaction

Abstract

The invention discloses a fluorescence and colorimetric dual-mode detection method for simultaneously determining hydrogen peroxide and organic phosphorus based on Ce < 3 + > / Ce < 4 + > cascade mimic enzyme reaction, and belongs to the technical field of fluorescence sensing detection. According to the invention, a smart phone color identification application program is combined with the ratio fluorescence sensor, so that real-time visual detection of H2O2 and OPs can be realized. The used materials are low in cost and easy to obtain, material synthesis is not needed, and the detection process is simplified. In addition, the sensor is combined with a color identification application program of the smart phone, so that the detection work flow is further simplified, and real-time data analysis and result feedback are realized. The 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 technical field of fluorescence sensing detection, and specifically relates to a method based on Ce 3+ / Ce 4+ Fluorescence and colorimetric dual-mode detection method for the determination of organophosphorus pesticides by 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, can monitor changes in target substances in real time, and 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 is convenient for 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, it is urgent 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 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 aims at solving the problems 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+ The fluorescence and colorimetric dual-mode detection method for simultaneously determining hydrogen peroxide and organic phosphorus 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， , incubate the reaction, measure the fluorescence intensity curve of the sample at a wavelength of 365nm, take the concentration of H2O2 in the sample as the horizontal axis, and take the fluorescence intensity ratio of 2,3-diaminophenazine DAP and 4-MU as the horizontal axis. 550 / I 450 As the ordinate, establish a fluorescence standard curve;

[0008] (2) Construction of ratiometric fluorescent probe for detecting organophosphorus Ops: First, acetylcholinesterase AChE was incubated with OPs solutions 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, and Ce 3+ and o-phenylenediamine OPD were added to the mixture, the reaction was incubated, 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 ordinate, establish a fluorescence standard curve;

[0009] (3) Constructing a smartphone color recognition platform to detect H2O2: Under 365nm ultraviolet light, the reaction mixture (Ce 3+ / 4-MUP / OPD / H2O2) fluorescence images. The RGB values ​​of the images were analyzed using smartphone color recognition software, and the linear relationship between the blue channel / red channel ratio G / B ratio and the H2O2 concentration in the solution was fitted using Origin software. The 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.

[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 4-MUP / OPD / AChE / ChOx / ATChCl / OPs were taken and the RGB values ​​of the images 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.

[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 50-450μM; the concentration of o-phenylenediamine OPD is 10-90mM; the concentration of 4-methylumbelliferyl phosphate 4-MUP is quantitatively 0.1μM.

[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 300μM; the concentration of o-phenylenediamine OPD is 60mM.

[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°C, 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 50-450μM; the concentration of o-phenylenediamine OPD is 0-90mM; the concentration of 4-methylumbelliferyl phosphate 4-MUP is 0.1μM.

[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 300μM; the concentration of o-phenylenediamine OPD is 60mM.

[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, substitutes it into the H2O2 standard curve of step (3), and obtains 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, the ratio of green channel / blue channel G / B is obtained, substitutes it into the OPs standard curve of step (4), and obtains 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+ -4-MUP-OPD ternary system established a ratiometric fluorescence sensor for H2O2 detection, and the fluorescence color of the solution changed from blue to yellow. OPs inhibited the activity of AChE, preventing AChE and ChOx from catalyzing ATChCl to produce H2O2, thereby reducing the generation of DAP and restoring the blue luminescence of 4-MU, thereby detecting OPs.

[0018] Beneficial effects:

[0019] (1) Ce with enzyme catalytic activity 3+ and Ce 4+ It is directly used to construct sensors without involving complicated nanozyme preparation processes, which simplifies the construction process and improves 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 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, providing a simple, efficient and economical solution for monitoring organophosphorus pesticides in agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A schematic diagram of the method for constructing the ratio fluorescence / smartphone dual-mode detection of hydrogen peroxide and organophosphorus of 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 ) ; (E) The effect of reaction time on the detection of H2O2 by the probe;

[0025] Figure 3 The fluorescence spectra and standard curves of the ratio 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 A comparison diagram of the selectivity of the detection system constructed in Example 3 to hydrogen peroxide;

[0027] Figure 5 The conditions for ratiometric fluorescence detection of organic phosphorus constructed in Example 4 are optimized, wherein (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),(F) are the effects of reaction time of AChE,ATChCl and OPs on fluorescence intensity ratio (F 555 / F 435 )

[0028] Figure 6 The fluorescence spectra and standard curves of the ratiometric fluorescence detection of organophosphorus constructed in Example 5, wherein (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, respectively); (B) is the fluorescence intensity ratio (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 by a smartphone constructed in Example 7, wherein (A) is the colorimetric card and (B) is the standard curve graph;

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

[0032] The technical solution of the present invention is further described below in conjunction with 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 to detect 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 concentration is high, which helps the dephosphorylation reaction of 4-MUP. The fluorescence intensity of 4-MU reaches a peak at pH 8.0, and in the pH range of 6.5-9.5, such as Figure 2 B. This may be due to Ce 3+ Under strong alkaline conditions, it can react with OH- to generate 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 generation 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 indicates 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 D shows that after adding 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 make the volume 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, with the concentration in the sample as the horizontal axis and the fluorescence intensity ratio of DAP and 4-MU as I 550 / I 450 The fluorescence standard curve was established with y as the ordinate. Figure 3 As shown in A, as the H2O2 concentration increased from 0 μM to 300 μM, the fluorescence peak intensity of 4-MU at 450 nm gradually decreased, while the fluorescence peak intensity of DAP at 550 nm gradually increased. Figure 3 B It can be seen that with the increase of H2O2 concentration, the fluorescence intensity ratio gradually increases, 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 0.03μM.

[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 influence of other ions on the probe is almost negligible. Therefore, the ratio fluorescence probe designed by the present invention has good selectivity and anti-interference ability.

[0046] Example 4

[0047] Optimization of ratiometric fluorescence detection conditions for 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 best 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 of acetylcholinesterase with ATChCl is complete and it is completely consumed. Similarly, the concentrations of ChOx and ATChCl 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 is stable, proving that the reaction is complete. In addition, the effect of enzyme reaction time on its performance was studied. 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 different concentrations of OPs solution 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 horizontal axis. 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 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, indicating that the sensor has excellent selectivity for OPs.

[0059] Example 7

[0060] Building 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+ / 4-MUP / OPD / H2O2) fluorescence images. The RGB values ​​of the images were analyzed using smartphone color recognition software, and the linear relationship between the G / B ratio and the H2O2 concentration in the solution was fitted using Origin software. The 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.

[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 9.7μM.

[0063] Example 8

[0064] Smartphone color recognition platform detects OPs:

[0065] Under 365nm UV light, the reaction mixture of Example 3 was captured by a smartphone (Ce 3+ / 4-MUP / OPD / AChE / ChOx / ATChCl / OPs) fluorescence images. The RGB values ​​of the images were analyzed using smartphone color recognition software, and 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 concentration of OPs as the horizontal axis and the ratio of the blue channel to the red channel G / B as the vertical axis.

[0066] like Fig. 9 As shown in A, under 365nm ultraviolet light, the fluorescence of the solution gradually changes from yellow to blue. Fig. 9B shows that there is a robust linear relationship between the G / B ratio and the OPs concentration. The obtained 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+ -4-MUP-OPD ratiometric fluorescent probe was used to detect H2O2, 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 using a 0.22μM filter membrane, and then the supernatant was diluted 10 times for use. 500μL of the filtrate from milk, beverages, and tap water was taken, and then 30μL 4-MUP (0.1μM), 90μL Ce (0.1μM), and 100μL HCl (0.1μM) were added to the three groups respectively. 3+ (300μM), 360μL OPD (60mM) and 500μL Tris-HCl buffer solution (50mM, pH8.0). Then add different concentrations of H2O2 (0, 5, 30, 60μM) to the sample and mix for 1h. The accuracy of the ratio fluorescence method and the smartphone sensor platform analysis method was tested by 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) The milk, beverage and tap water samples were spiked with H2O2 at three different concentrations of 5, 30 and 60 μmol / L. The three H2O2 samples were tested using a fluorescence spectrometer. The spike recovery was calculated and the test results are shown in Table 1.

[0072] Table 1 Test results of each data

[0073]

[0074]

[0075] It can be seen from the spiked experiment that the fluorescence detection method of the present invention has high accuracy and precision and can sensitively detect H2O2 in the sample.

[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: Crush tomatoes or spinach (20g) and add 20mL acetonitrile. The mixture is ultrasonically treated for 30 minutes, then filtered through a 0.22μM filter membrane, and the resulting liquid supernatant is diluted tenfold for later use. Take 500μL of the filtrate from 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. Use the standard addition method to add 90μL Ce 3+ (300μM) and 360μL OPD (60mM) were added to samples containing different concentrations of OPs (0, 10, 30, 50ng / mL). Finally, the spiked samples were analyzed by ratiometric fluorescence analysis and a smartphone sensing platform to calculate the spiked recovery. 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 of 5, 30, and 60 ng / mL. The samples were tested using a fluorescence spectrometer. The spike recovery was calculated and the test 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 partial embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field 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 organic phosphorus 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), followed by the addition of different concentrations of H2O 2， , incubate the reaction, measure the fluorescence intensity curve of the sample at a wavelength of 365nm, take the concentration of H2O2 in the sample as the horizontal axis, and take the fluorescence intensity ratio of the product 2,3-diaminophenazine DAP and 4-methylumbelliferyl phosphate 4-MU as the horizontal axis. 550 / I 450 As the ordinate, establish a fluorescence standard curve; (2) Construction of ratiometric fluorescent probe for detecting organophosphorus Ops: First, acetylcholinesterase AChE was incubated with OPs solutions 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, and Ce 3+ and o-phenylenediamine OPD were added to the mixture, the reaction was incubated, 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 abscissa and the fluorescence intensity ratio of 2,3-diaminophenazine DAP and 4-methylumbelliferyl phosphate 4-MU as the index. 550 / I 450 As the ordinate, establish a fluorescence standard curve; (3) Constructing a smartphone color recognition platform to detect H2O2: Under 365 nm ultraviolet light, use a smartphone to capture the fluorescence image of the reaction mixture in step (1), use smartphone color recognition software to analyze the RGB value of the image, use Origin software to fit the linear relationship between the blue channel / red channel ratio G / B ratio and the H2O2 concentration in the solution, and establish a H2O2 standard curve 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 irradiation with 365 nm ultraviolet light, the fluorescent image of the reaction mixture in step (2) was captured using a smartphone, and the RGB value of the image was 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, and an 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 organic phosphorus by cascade mimicking enzyme reaction, characterized in that: Step (1) The concentration of Tris-HCl buffer is 50 mM, and 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 50-450μM; the concentration of o-phenylenediamine OPD is 10-90mM; the concentration of 4-methylumbelliferyl phosphate 4-MUP is 0.1μM.

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 organic phosphorus 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 300μM; the concentration of o-phenylenediamine OPD is 60mM.

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 organic phosphorus 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, the incubation temperature was 25°C, the reaction time was 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 50-450μM; the concentration of o-phenylenediamine OPD is 10-90mM; the concentration of 4-methylumbelliferyl phosphate 4-MUP is 0.1μM.

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 organic phosphorus 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 300μM; the concentration of o-phenylenediamine OPD is 60mM.

6. According to claim 1, based on Ce 3+ / Ce 4+ A fluorescence and colorimetric dual-mode detection method for simultaneously determining hydrogen peroxide and organic phosphorus 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 measured in 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 in step (2) 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 organic phosphorus 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, substitutes it into the H2O2 standard curve of step (3), and obtains 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, the ratio of green channel / blue channel G / B is obtained, substitutes it into the OPs standard curve of step (4), and obtains the OPs concentration in the sample to be tested.

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