As (V) double-signal detection method for enhancing activity of octahedral trimanganese tetraoxide simulated laccase

Abstract

The patent of the invention discloses an As (V) double-signal detection method for enhancing the activity of octahedral trimanganese tetraoxide simulated laccase, and used reagents mainly comprise Mn3O4, 2, 4-DP, 4-AAP, an HEPES buffer solution and a CDs solution. As (V) can enhance the activity of simulated laccase of octahedral Mn3O4 and catalyze more 2, 4-DP and 4-AAP to generate a red product quinoneimine, so that a sensing solution is red and has a characteristic absorption peak at 510nm. Meanwhile, the generated quinoneimine can be used for weakening a fluorescence signal of a sensing solution through an inner filtering effect and dynamic quenching of CDs fluorescence. At the moment, the absorbance change amplitude and the fluorescence intensity change amplitude in the sensing system are in direct proportion to the concentration of the target As (V). The method has the advantages of simplicity and convenience in operation, higher accuracy of a detection result, higher sensitivity and specificity in detection of As (V), short detection time, capability of being distinguished by naked eyes and the like, and can be widely applied to field detection.

Description

Technical Field

[0001] The invention relates to the field of As(V) residual detection in water samples, and in particular to an As(V) dual-signal detection method for enhancing the activity of octahedral manganese tetraoxide (Mn3O4) simulating laccase. Background Art

[0002] As(V) is a heavy metal that is commonly found in water, soil, and food in organic and inorganic forms. Long-term exposure to As(V) can lead to many health problems, including cancer, cardiovascular disease, diabetes, and hypertension. The International Agency for Research on Cancer and the U.S. Centers for Disease Control classify As(V) as carcinogenic to humans. According to the U.S. Environmental Protection Agency and the World Health Organization, As(V) contamination has been identified as one of the major challenges to public health, affecting 140 million people in at least 50 countries. It must be effectively treated to meet relevant national quality standards. The World Health Organization (WHO) maximum limit for As(V) in water is 10 μg·L-1. Therefore, it is particularly important to develop a method for rapid detection of As(V).

[0003] Traditional As(V) detection methods such as atomic absorption spectroscopy, atomic fluorescence spectroscopy and inductively coupled plasma mass spectrometry have high selectivity and sensitivity, but require the use of complex and expensive instruments and equipment, are time-consuming and cannot be measured on site. At present, in order to solve the above drawbacks, colorimetry, SERS, fluorescence and electrochemical methods have been widely used in the detection of As(V) in water. However, single signal detection is easily affected by the surrounding environment or human factors, resulting in inaccurate detection results. Dual-signal sensors that combine colorimetry and fluorescence can largely eliminate instrumental and human errors, making the sensor more reliable. In recent years, more and more researchers have developed sensing strategies with colorimetric and fluorescence dual modes for applications in cell imaging, drug monitoring and food testing. However, there are few reports on dual-signal sensors for the detection of As(V). Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an As(V) dual-signal detection method for enhancing the activity of octahedral manganese tetraoxide (Mn3O4) simulating laccase, which has the advantages of being identifiable by the naked eye, high sensitivity, convenient operation, and rapid detection.

[0005] In order to solve the above technical problems, the technical solution of the present invention is:

[0006] A dual-signal detection method for As(V) for enhancing the activity of octahedral manganese tetraoxide (Mn3O4) simulating laccase, comprising the following steps:

[0007] S1: a certain concentration of octahedral Mn3O4 nanoparticle solution is mixed with a As(V) sample of a test concentration and incubated for a certain period of time to prepare an actual test sample solution;

[0008] S2: The actual sample solution to be tested was mixed with a HEPES buffer solution of pH 7.0 and a concentration of 10 mM, and then a mixture of 2,4-DP, 4-AAP and CDs of a certain concentration was added and mixed;

[0009] S3: Determine the concentration of As(V) by measuring the change in absorbance of the mixed solution at 510 nm;

[0010] S4: The concentration of As(V) is determined by measuring the change in the fluorescence intensity value corresponding to the emission peak of the mixed solution at 432 nm.

[0011] Further, the concentration of the octahedral Mn3O4 nanoparticle solution was 25 μg·mL-1, the concentration of 2,4-DP was 10 mg·mL-1, the concentration of 4-AAP was 5 mg·mL-1, and the concentration of HEPES buffer was 10 mM.

[0012] Furthermore, in step S1, a 1.5 mL centrifuge tube was taken, and 10 μL of a 25 μg·mL-1 Mn3O4 solution and 10 μL of an As(V) sample of the concentration to be tested were added, mixed thoroughly, and then incubated at 40°C for 1 hour.

[0013] Furthermore, in step S2, 450 μL of 10 mM HEPES buffer with a pH of 7.0 was added to the actual sample solution, and finally 10 μL of 10 mg·mL-1 2,4-DP, 10 μL of 5 mg·mL-1 4-AAP and 10 μL LCDs were added and mixed thoroughly.

[0014] Furthermore, step S3 specifically includes:

[0015] (1) using As(V) solutions of different known concentrations ranging from 0 to 2000 μg·L-1, preparing standard sample solutions according to the method of step S1, and processing according to step S2;

[0016] (2) Using 10 μL of double distilled water instead of the As(V) solution, prepare a blank control system solution according to the method of step S1, and process according to step S2;

[0017] (3) taking 200 μL of the standard sample solution and the blank control system solution prepared in step (1) and step (2) respectively, and placing them in a 96-well ELISA plate, and scanning with an ELISA reader to measure the absorption peak at 510 nm; the full wavelength scanning range is 300-800 nm, and the absorption spectrum thereof is obtained;

[0018] (4) Plot different concentrations of As(V) against the measured ΔA to draw a standard curve, where ΔA = absorbance of blank sample - absorbance of the sample to be tested;

[0019] (5) After the actual sample solution prepared in step S1 is processed in step S2, its absorption peak at 510 nm is measured;

[0020] (6) Based on the absorbance values ​​of the samples measured in the HEPES buffer system, the standard curve was checked to determine the As(V) content in different samples.

[0021] Furthermore, step S4 specifically includes:

[0022] (1) using As(V) solutions of different known concentrations ranging from 0 to 2000 μg·L-1, preparing standard sample solutions according to the method of step S1, and processing according to step S2;

[0023] (2) Using 10 μL of double distilled water instead of the As(V) solution, prepare a blank control system solution according to the method of step S1, and process according to step S2;

[0024] (3) taking 200 μL of the standard sample solution and the blank control system solution prepared in step (1) and step (2), respectively, scanning with a fluorescence photometer to measure the fluorescence intensity corresponding to the emission peak of the solution at 432 nm, and obtaining its fluorescence emission spectrum;

[0025] (4) Plotting different concentrations of As(V) against the measured ΔF to draw a standard curve, where ΔF = fluorescence intensity of blank sample - fluorescence intensity of the sample to be tested;

[0026] (5) After the actual sample solution prepared in step S1 is processed in step S2, the fluorescence intensity value corresponding to the emission peak at 432 nm is measured;

[0027] (6) Based on the fluorescence intensity values ​​of the samples measured in the HEPES buffer system, the standard curve was checked to determine the As(V) content in different samples.

[0028] The advantages of the present invention are:

[0029] The detection method provided by the present invention can output signals through a dual-mode of fluorescence and colorimetry. Compared with a single-signal detection method, its detection results have better reliability and accuracy. The detection of As(V) has the advantages of high sensitivity and specificity, short detection time, and can be distinguished by the naked eye. Moreover, it does not require reliance on large-scale instruments and equipment and can be widely used in on-site detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1The color change, absorbance change, fluorescence change and fluorescence emission peak change caused by As(V);

[0031] Figure 2 Schematic diagram for detecting As(V);

[0032] Figure 3 The corresponding relationship between different concentrations of As (V) and the changes in absorbance value (ΔA) and fluorescence intensity value (ΔF);

[0033] Figure 4 The influence of other interfering substances on the detection of As(V);

[0034] Figure 5 The results are from actual sample testing. DETAILED DESCRIPTION

[0035] The specific embodiments of the present invention are further described below in conjunction with the accompanying drawings. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0036] Technical solution of the patent of this invention:

[0037] 1. Verification of the basic principle of the present invention

[0038] 1. Verification of As(V)-enhanced laccase-mimicking activity of octahedral Mn3O4 nanoparticles:

[0039] (1) Preparation of octahedral Mn3O4 nanoparticles: 0.0525 g potassium permanganate powder was dissolved in 15 mL distilled water under a magnetic stirrer. Then 15 mL polyethylene glycol was added to the above solution. After the solution was homogenized, the mixture was transferred to a polytetrafluoroethylene liner and placed in a 60 mL stainless steel autoclave, which was sealed and kept at 120°C for 8 h. After the reaction was completed, it was naturally cooled to room temperature, the precipitate was filtered out, washed several times with distilled water and anhydrous ethanol, respectively, and centrifuged to obtain a black precipitate, which was vacuum dried at 60°C for 12 h to obtain a powder.

[0040] (2) Take three centrifuge tubes and add 2,4-DP and 4-AAP; Mn3O4, 2,4-DP and 4-AAP; Mn3O4, 2,4-DP, 4-AAP and As(V) solutions respectively. Then add HEPES buffer to the centrifuge tubes to obtain a total volume of 500 μL. Record the color changes of different reaction solutions, and then use an ELISA reader to measure the absorption spectrum of the solution in the range of 300nm-700nm.

[0041] 2. Verification of the effect of enzyme catalysis products on CDs fluorescence

[0042] (1): Preparation of CDs solution: 108 mg of o-phenylenediamine and 192 mg of citric acid were dissolved in 10 mL of distilled water and ultrasonicated for 10 min. Then, the solution was heated at 120°C for 6 h, and after cooling, the solution was dialyzed (500 MW) for 6 h. Finally, the resulting solution was filtered through a filter membrane (0.22 μm) and stored at 4°C for further use.

[0043] (2): Take 4 centrifuge tubes and add CDs; CDs, 2,4-DP and 4-AAP; CDs, Mn3O4, 2,4-DP and 4-AAP; CDs, Mn3O4, 2,4-DP, 4-AAP and As(V) solution respectively. Then add HEPES buffer to the centrifuge tube to obtain a total volume of 500 μL. Record the fluorescence changes of different reaction solutions, and then use a fluorescence spectrophotometer to measure the fluorescence emission spectrum of the solution in the range of 400nm-600nm.

[0044] 3. Verification of CDs fluorescence quenching mechanism

[0045] (1): Take a centrifuge tube and add Mn3O4, 2,4-DP, 4-AAP and As(V) solutions, then add pH7.0, 10mM HEPES buffer to the centrifuge tube to obtain a total volume of 500μL. Record the absorption spectrum of the reaction solution in the range of 300nm-700nm. Then take a centrifuge tube and add CDs solution, then add HEPES buffer to obtain a total volume of 500μL. Then use a fluorescence photometer to scan and measure the excitation spectrum and emission spectrum of the CDs solution. Finally, the measured absorption spectrum is integrated with the excitation spectrum and emission spectrum through Origin to explore whether the absorption spectrum of the enzyme-catalyzed product overlaps with the excitation and emission spectra of CDs.

[0046] (2): Take 3 centrifuge tubes and add CDs solution and HEPES buffer respectively, react at 24℃, 37℃ and 45℃ for 10 minutes, and use a fluorescence spectrophotometer to measure the fluorescence intensity value corresponding to the emission peak of the solution at 432nm. Then take 3 centrifuge tubes and add As(V) solution of different concentrations (20μg·mL-1, 150μg·mL-1 and 400μg·mL-1), add Mn3O4, 2,4-DP, 4-AAP and CDs to the solution. Then add HEPES buffer to get a total volume of 500μL, react at 24℃ for 10 minutes, and use a fluorescence spectrophotometer to measure the fluorescence intensity value corresponding to the emission peak of the solution at 432nm. Then, three centrifuge tubes were added with As(V) solutions of different concentrations (20μg·mL-1, 150μg·mL-1 and 400μg·mL-1), and Mn3O4, 2,4-DP, 4-AAP and CDs were added to the solution. HEPES buffer was added to obtain a total volume of 500μL, and the reaction was carried out at 37℃ for 10 minutes. The fluorescence intensity value corresponding to the emission peak of the solution at 432nm was measured by a fluorescence spectrophotometer. Then, three centrifuge tubes were added with As(V) solutions of different concentrations (20μg·mL-1, 150μg·mL-1 and 400μg·mL-1), and Mn3O4, 2,4-DP, 4-AAP and CDs were added to the solution. HEPES buffer was added to obtain a total volume of 500μL, and the reaction was carried out at 45℃ for 10 minutes. The fluorescence intensity value corresponding to the emission peak of the solution at 432nm was measured by a fluorescence spectrophotometer. The obtained data were used to study the quenching constants at 24°C, 37°C and 45°C according to the Stern-Volmer equation: F0 / F=1+Ksv[Q]. Ksv and [Q] are the Stern-Volmer quenching constant and the concentration of As(V), respectively. F0 represents the fluorescence intensity value when there is no quencher, and F represents the fluorescence intensity value when there is a quencher. Finally, the relationship between the quenching constant and temperature was obtained by integrating the data in Origin.

[0047] (3): 100μL CDs solution and 4.9mL HEPES buffer were mixed and added to a cuvette and the fluorescence lifetime was measured using a transient fluorescence spectrometer. Then 100μL Mn3O4, 100μL 2,4-DP, 100μL 4-AAP, 100μL As(V) solution and 100μL CDs solution were added to 4.5mL HEPES buffer and mixed and added to a cuvette. After 10 minutes of reaction, the fluorescence lifetime was measured using a transient fluorescence spectrometer. The obtained data were integrated by Origin to determine whether the CDs fluorescence lifetime changed in the presence of the enzyme catalytic product.

[0048] 2. Based on the above experimental phenomena, a dual-signal detection method based on As(V)-enhanced octahedral Mn3O4 laccase simulation activity was constructed, including the following steps:

[0049] S1: Preparation of the actual test sample solution: take a 1.5 mL centrifuge tube, add 10 μL of 25 μg mL-1 octahedral Mn3O4 nanoparticle solution and 10 μL of the As(V) sample of the test concentration, mix thoroughly and incubate at 40°C for 1 hour.

[0050] S2: Add 450 μL of 10 mM HEPES buffer at pH 7.0 to the actual sample solution, and finally add 10 μL of 10 mg·mL-1 2,4-DP, 10 μL of 5 mg·mL-1 4-AAP and 10 μL CDs and mix thoroughly.

[0051] S3: Determine the concentration of As(V) by measuring the change in absorbance of the mixed solution at 510 nm. The specific steps are as follows:

[0052] (1) using As(V) solutions of different known concentrations ranging from 0 to 2000 μg·L-1, preparing standard sample solutions according to the method of step S1, and processing according to step S2;

[0053] (2) Using 10 μL of double distilled water instead of the As(V) solution, prepare a blank control system solution according to the method of step S1, and process according to step S2;

[0054] (3) taking 200 μL of the standard sample solution and the blank control system solution prepared in step (1) and step (2) respectively, and placing them in a 96-well ELISA plate, and scanning with an ELISA reader to measure the absorption peak at 510 nm; the full wavelength scanning range is 300-800 nm, and the absorption spectrum thereof is obtained;

[0055] (4) Plot different concentrations of As(V) against the measured ΔA to draw a standard curve, where ΔA = absorbance of blank sample - absorbance of the sample to be tested;

[0056] (5) After the actual sample solution prepared in step S1 is processed in step S2, its absorption peak at 510 nm is measured;

[0057] (6) Based on the absorbance values ​​of the samples measured in the HEPES buffer system, the standard curve was checked to determine the As(V) content in different samples.

[0058] S4: The concentration of As(V) is determined by measuring the change in the fluorescence intensity value corresponding to the emission peak of the mixed solution at 432 nm. The specific steps are as follows.

[0059] (1) using As(V) solutions of different known concentrations ranging from 0 to 2000 μg·L-1, preparing standard sample solutions according to the method of step S1, and processing according to step S2;

[0060] (2) Using 10 μL of double distilled water instead of the As(V) solution, prepare a blank control system solution according to the method of step S1, and process according to step S2;

[0061] (3) taking 200 μL of the standard sample solution and the blank control system solution prepared in step (1) and step (2), respectively, scanning with a fluorescence photometer to measure the fluorescence intensity corresponding to the emission peak of the solution at 432 nm, and obtaining its fluorescence emission spectrum;

[0062] (4) Plotting different concentrations of As(V) against the measured ΔF to draw a standard curve, where ΔF = fluorescence intensity of blank sample - fluorescence intensity of the sample to be tested;

[0063] (5) After the actual sample solution prepared in step S1 is processed in step S2, the fluorescence intensity value corresponding to the emission peak at 432 nm is measured;

[0064] (6) Based on the fluorescence intensity values ​​of the samples measured in the HEPES buffer system, the standard curve was checked to determine the As(V) content in different samples.

[0065] 3: The detection principle of this method is: As(V) can enhance the laccase mimicking activity of octahedral Mn3O4 nanoparticles, oxidize more 2,4-DP and 4-AAP to generate the red product quinoneimine. Quinoneimine has a significant characteristic absorption peak at 510nm, and the change amplitude of the absorbance value at 510nm is proportional to the concentration of As(V). In addition, the generated quinoneimine can reduce the fluorescence peak of the system at 432nm through the inner filtration effect and dynamic quenching of the blue fluorescence of CDs, and the change amplitude of the fluorescence intensity at 432nm is proportional to the concentration of As(V). Therefore, this method can be used for the detection of As(V) through the colorimetric fluorescence dual signal mode. This method has a good detection effect on As(V) in actual samples of tap water, lake water and river water. Figure 5As shown, in the colorimetric signal, the average detection recovery of As(V) was 89.38%-98.41%, and the average relative standard deviation (RSD) was 1.21%-5.71%. In the fluorescence signal, the average detection recovery of As(V) was 86.40%-98.92%, and the average relative standard deviation (RSD) was 1.07%-6.96%. In addition, the method can specifically detect As(V) in the presence of interfering substances such as Fe(II), Mg(II), Fe(III), Hg(II), K(I), Zn(II), Ca(II), Na(I), Cr(VI), Co(II), Ag(I), and NO3-. The method of the present invention is used to measure tap water, lake water and river water samples, and 40, 100 and 1000 μg·L-1 of As(V) are added to the samples, and the recovery rate obtained on the colorimetric signal is 89.38%-98.41%, and the average relative standard deviation (RSD) is 1.21%-5.71%. The recovery rate obtained on the fluorescence signal is 86.40%-98.92%, and the average relative standard deviation (RSD) is 1.07%-6.96%.

[0066] The minimum detection limit of As(V) determined by the present invention in the colorimetric method is 6.96 μg·L-1 respectively, and the minimum detection limit of As(V) determined by the fluorescence method is 2.56 μg·L-1 respectively.

[0067] IV: Experiment Icon Description

[0068] Attached Figure 1 In A, sample 1: CDs + Mn3O4 + 2,4-DP + 4-AAP; sample 2: sample 1 + 20μg·L-1As(V); sample 3: sample 1 + 100μg·L-1As(V); sample 4: sample 1 + 400μg·L-1As(V). Figure 1 In B, sample 1: CDs+Mn3O4+2,4-DP+4-AAP; sample 2: sample 1+20μg·L-1As(V); sample 3: sample 2+100μg·L-1As(V); sample 4: sample 2+400μg·L-1As(V).

[0069] Attached Figure 1 and attached Figure 2 This is a feasibility study and principle speculation of the invention, which shows that the method of the invention can be used for As(V) detection.

[0070] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the scope of protection of the present invention.

Claims

1. A dual-signal detection method for As(V) to enhance the activity of octahedral manganese tetraoxide-mimicking laccase, characterized in that: The following steps are involved: S1: a certain concentration of octahedral Mn3O4 nanoparticle solution is mixed with a As(V) sample of a test concentration and incubated for a certain period of time to prepare an actual test sample solution; S2: The actual sample solution to be tested was mixed with a HEPES buffer solution of pH 7.0 and a concentration of 10 mM, and then a mixture of 2,4-DP, 4-AAP and CDs of a certain concentration was added and mixed; S3: Determine the concentration of As(V) by measuring the change in absorbance of the mixed solution at 510 nm; S4: The concentration of As(V) is determined by measuring the change in the fluorescence intensity value corresponding to the emission peak of the mixed solution at 432 nm.

2. The As(V) dual signal detection method for enhancing the activity of laccase simulated by octahedral manganese tetraoxide according to claim 1, characterized in that: The concentration of the octahedral Mn3O4 nanoparticle solution is 25 μg·mL-1, the concentration of 2,4-DP is 10 mg·mL-1, the concentration of 4-AAP is 5 mg·mL-1, and the concentration of HEPES buffer is 10 mM.

3. The As(V) dual signal detection method for enhancing the activity of octahedral manganese tetraoxide simulating laccase according to claim 2, characterized in that: In step S1, take a 1.5 mL centrifuge tube, add 10 μL of octahedral Mn3O4 nanoparticle solution with a concentration of 25 μg·mL-1 and 10 μL of As(V) sample with the concentration to be tested, mix thoroughly and incubate at 40°C for 1 hour.

4. The As(V) dual signal detection method for enhancing the activity of octahedral manganese tetraoxide simulating laccase according to claim 3, characterized in that: In step S2, the actual sample solution is added with 450 μL of 10 mM HEPES buffer at pH 7.0, and finally 10 μL of 10 mg·mL-1 2,4-DP, 10 μL of 5 mg·mL-1 4-AAP and 10 μL Cds are added and mixed thoroughly.

5. The As(V) dual signal detection method for enhancing the activity of octahedral manganese tetraoxide simulating laccase according to claim 4, characterized in that: Step S3 specifically includes: (1) using As(V) solutions of different known concentrations ranging from 0 to 2000 μg·L-1, preparing standard sample solutions according to the method of step S1, and processing according to step S2; (2) Using 10 μL of double distilled water instead of the As(V) solution, prepare a blank control system solution according to the method of step S1, and process according to step S2; (3) taking 200 μL of the standard sample solution and the blank control system solution prepared in step (1) and step (2) respectively, and placing them in a 96-well ELISA plate, and scanning with an ELISA reader to measure the absorption peak at 510 nm; the full wavelength scanning range is 300-800 nm, and the absorption spectrum thereof is obtained; (4) Plot different concentrations of As(V) against the measured ΔA to draw a standard curve, where ΔA = absorbance of blank sample - absorbance of the sample to be tested; (5) After the actual sample solution prepared in step S1 is processed in step S2, its absorption peak at 510 nm is measured; (6) Based on the absorbance values ​​of the samples measured in the HEPES buffer system, the standard curve was checked to determine the As(V) content in different samples.

6. The As(V) dual signal detection method for enhancing the activity of octahedral manganese tetraoxide simulating laccase according to claim 4, characterized in that: Step S4 specifically includes: (1) using As(V) solutions of different known concentrations ranging from 0 to 2000 μg·L-1, preparing standard sample solutions according to the method of step S1, and processing according to step S2; (2) Using 10 μL of double distilled water instead of the As(V) solution, prepare a blank control system solution according to the method of step S1, and process according to step S2; (3) taking 200 μL of the standard sample solution and the blank control system solution prepared in step (1) and step (2), respectively, scanning with a fluorescence photometer to measure the fluorescence intensity corresponding to the emission peak of the solution at 432 nm, and obtaining its fluorescence emission spectrum; (4) Plotting different concentrations of As(V) against the measured ΔF to draw a standard curve, where ΔF = fluorescence intensity of blank sample - fluorescence intensity of the sample to be tested; (5) After the actual sample solution prepared in step S1 is processed in step S2, the fluorescence intensity value corresponding to the emission peak at 432 nm is measured; (6) Based on the fluorescence intensity values ​​of the samples measured in the HEPES buffer system, the standard curve was checked to determine the As(V) content in different samples.

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