Colorimetric detection method for carbosulfan based on smartphone-assisted photoresponse nano-enzyme functionalized paper base

Abstract

The invention discloses a colorimetric detection method for carbosulfan based on a smartphone-assisted photoresponse nano-enzyme functionalized paper base, and the method comprises the following steps: firstly preparing a carbon quantum dot / graphite phase carbon nitride (C-dots / g-C3N4) nano-enzyme, under a visible light illumination condition, catalyzing 3, 3 ', 4', 5 '-tetramethyl-3, 4'-tetramethyl-3, 4 '-tetramethyl-3, 4'-tetramethyl-3, 4 '-tetramethyl-3, 4'-tetramethyl-3, 4 '-tetramethyl-3, 4'-tetramethyl-3, 4 '-tetramethyl-3, 4'-tetramethyl-3, 4 '-tetramethyl-3, 4 According to the invention, TMB (oxTMB) in a blue oxidation state is generated through oxidation of 3, 3 ', 5, 5'-tetramethyl benzidine (TMB), and the TMB has a strong absorption peak at 652 nm; carbosulfan is hydrolyzed under an acidic condition to generate a sulfur-containing compound, and the chromogenic reaction is inhibited, so that the absorbance of the system at 652 nm is reduced. Based on the change of the absorbance of the system at 652 nm along with the concentration of carbosulfan, the carbosulfan can be detected. The catalytic activity of the C-dots / g-C3N4 photoresponse nano-enzyme is obviously improved compared with that of g-C3N4, and guidance is provided for preparation of the g-C3N4-based photoresponse nano-enzyme with high catalytic activity. According to the method, the photoresponse nano-enzyme is adopted and can be accurately regulated and controlled by switching on and off a light source, so that the detection result is more accurate, large instruments are not needed, the operation is simple and convenient, and the phenomenon is intuitive.

Description

Technical Field

[0001] The invention belongs to the field of pesticide detection, and in particular relates to a colorimetric detection method for carbosulfan based on a paper substrate functionalized with light-responsive nanozymes assisted by a smartphone. Background Art

[0002] Carbosulfan is a highly effective carbamate pesticide that can effectively control pests of a variety of crops. However, carbosulfan can be easily decomposed into furadan, which is extremely toxic to humans. Therefore, the development of highly sensitive methods for detecting carbosulfan is crucial to protecting human health. Currently, chromatography, as the main method for detecting carbosulfan, has high sensitivity and good reproducibility, but is limited by expensive equipment and complex and time-consuming operation steps. Colorimetry, with its intuitive phenomenon, simple operation, and no reliance on large and expensive instruments, provides a new idea for the detection of carbosulfan.

[0003] Nanozymes, especially peroxidase nanozymes and oxidase nanozymes, provide a new opportunity for the colorimetric detection of carbosulfan. Most of them are based on the color development reaction catalyzed by peroxidase nanozymes or oxidase nanozymes, while the reduced sulfur-containing compounds generated by the hydrolysis of carbosulfan inhibit the color development reaction. Among them, the color development reaction catalyzed by the photoresponsive oxidase nanozymes does not require the participation of hydrogen peroxide, which is easier to operate; and most of them can be precisely controlled by switching the light source, making the detection results more accurate. However, there are few types of photoresponsive oxidase nanozymes reported so far, and they generally have disadvantages such as high cost and complex synthesis. Graphitic carbon nitride (g-C3N4) has the advantages of low cost, simple preparation method and high stability. Researchers have found that g-C3N4 nanosheets have photoresponsive oxidase-like activity, but its activity is low. Summary of the invention

[0004] To address the above problems, we loaded carbon quantum dots on g-C3N4 nanosheets to prepare carbon quantum dots stone / ink phase carbon nitride (C-dots / g-C3N4) nanozymes with high catalytic activity, and used their light-responsive oxidase-like activity to detect carbosulfan. The detection mechanism is as follows: Under visible light conditions, C-dots / g-C3N4 light-responsive nanozymes catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate blue oxidized TMB (oxTMB), which has a strong absorption peak at 652 nm; carbosulfan hydrolyzes under acidic conditions to generate sulfur-containing compounds, which inhibit the color development reaction and cause the absorbance of the system at 652 nm to decrease. Based on the change of the absorbance of the system at 652 nm with the concentration of carbosulfan, the detection of carbosulfan can be achieved. Based on the above detection mechanism, a paper substrate functionalized with C-dots / g-C3N4 photoresponsive nanozyme was prepared, and the colorimetric detection of butyrate was achieved using a smartphone.

[0005] The present invention is achieved through the following technical solutions: Preparation and characterization of C-dots / g-C3N4 photoresponsive nanozymes: 20 g urea and 10-100 mg citric acid were added to a mortar and ground evenly. The resulting powder was then transferred to an alumina crucible and heated in a muffle furnace at 0.5 o C / min and calcined at 600 °C for 3 h. Afterwards, the calcined solid was ground into powder and 0.3 g was added to 30 mL 5 MHNO3 solution. o C refluxed for 24 h. Finally, it was washed with ultrapure water until the pH of the supernatant was close to neutral. Finally, the samples were named 0.05% C-dots / g-C3N4, 0.2% C-dots / g-C3N4, and 0.5% C-dots / g-C3N4 due to the different amounts of citric acid added. Among them, 0.2% C-dots / g-C3N4 had the best catalytic activity. The preparation of g-C3N4 nanosheets is similar to that of C-dots / g-C3N4 photoresponsive nanozymes, except that citric acid does not need to be added. The morphology, structure and catalytic activity of the prepared C-dots / g-C3N4 photoresponsive nanozymes were characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction spectroscopy, kinetics and specific activity tests.

[0006] Determination of carbosulfan: 100 μL of 0.2% C-dots / g-C3N4 (0.2 mg / mL), 100 μL of TMB (5 mM), and 50 μL of carbosulfan solutions of different concentrations (0-100 μM) were added to 750 μL of NaAc-HAc buffer solution (10 mM, pH=2.8) in sequence. After mixing evenly, the mixture was irradiated with a xenon lamp for 3 min. The solution was filtered and its UV-visible absorption spectrum was recorded. Smartphone-assisted paper-based colorimetric detection of carbosulfan: First, filter paper was cut into multiple circular paper discs with a diameter of 10 mm using a laser cutter. 20 μL of 0.2% C-dots / g-C3N4 solution (0.2 mg / mL) was evenly applied on the paper disc and vacuum dried. Subsequently, 20 μL of NaAc-HAc buffer solution (pH=2.8) containing TMB (5 mM) and 20 μL of carbosulfan solutions of different concentrations (5-100 μM) were evenly dripped onto the paper disc. After irradiation with a xenon lamp for 3 min, the RGB values ​​of the paper disc were extracted using a smartphone color picker and carbosulfan was quantitatively analyzed.

[0007] Compared with the prior art, the present invention has the following beneficial effects: (1) This method uses light-responsive nanozymes, which can be precisely controlled by switching the light source on and off, making the detection results more accurate.

[0008] (2) The method of detecting carbosulfan based on smartphone-assisted paper-based colorimetric analysis does not rely on large instruments and is easy to operate and the phenomenon is intuitive.

[0009] (3) The catalytic activity of C-dots / g-C3N4 photoresponsive nanozymes was significantly improved compared with that of g-C3N4, which provided guidance for the preparation of g-C3N4-based photoresponsive nanozymes with high catalytic activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 TEM images of g-C3N4 (A) and C-dots / g-C3N4 (B, C, D), XRD (E) and Fourier transform infrared spectra (F) of g-C3N4 and C-dots / g-C3N4.

[0011] Figure 2 (A) UV-visible absorption spectra of g-C3N4-TMB and C-dots / g-C3N4-TMB under illumination or without illumination, and TMB under illumination. (B) Light-controlled oxidase activity of C-dots / g-C3N4.

[0012] Figure 3 Specific activity of C-dots / g-C3N4 (A) and g-C3N4 (B). Steady-state kinetics of C-dots / g-C3N4 (C) and g-C3N4 (D). Insets are the corresponding Lineweaver-Burk plots of C-dots / g-C3N4 and g-C3N4.

[0013] Figure 4 (A) UV-visible absorption spectra of C-dots / g-C3N4-TMB system when different concentrations of carbosulfan are added. (B) Relationship between ∆A and carbosulfan concentration. (C) Linear calibration graph of carbosulfan concentration. (D) Absorbance of the detection system at 652 nm when other pesticides are added alone or simultaneously with carbosulfan.

[0014] Figure 5 (A) Schematic diagram of the C-dots / g-C3N4-TMB system combined with smartphone-assisted colorimetry for the detection of carbosulfan. (B) Relationship between R / B after colorimetry and carbosulfan concentration. (C) Linear calibration diagram of R / B after colorimetry and carbosulfan concentration. DETAILED DESCRIPTION

[0015] The following is combined with Figure 1 -Attached Figure 5The technical scheme of the present invention is further described in the following embodiments. Unless otherwise specified, the technical means used in the specific embodiments of the present invention are methods known to those skilled in the art. If no specific technology or conditions are specified in the embodiments, they are all conventional methods or carried out according to the technology or conditions described in the literature of the field, or according to the product instructions. If no manufacturer is specified, they are all conventional products that can be purchased through regular channels.

[0016] Example 1 (1) Preparation of g-C3N4 and C-dots / g-C3N4 photoresponsive nanozymes: 20 g of urea and citric acid were added to a mortar and ground evenly. The resulting powder was then transferred to an alumina crucible and heated in a muffle furnace at 0.5 o C / min heating rate at 600 °C for 3 h. Afterwards, the calcined solid was ground into powder and 0.3 g was added to 30 mL 5 M HNO3 solution. o C was refluxed for 24 h. Finally, it was washed with ultrapure water until the pH of the supernatant was close to neutral. Finally, due to the different amounts of citric acid (10 mg, 40 mg, 100 mg) added, the samples were named 0.05% C-dots / g-C3N4, 0.2% C-dots / g-C3N4, and 0.5% C-dots / g-C3N4. Among them, 0.2% C-dots / g-C3N4 had the best catalytic activity. The preparation of g-C3N4 nanosheets is similar to that of C-dots / g-C3N4 photoresponsive nanozymes, except that citric acid does not need to be added.

[0017] (2) Characterization of the morphology and structural composition of g-C3N4 and C-dots / g-C3N4 photoresponsive nanozymes: The morphology of g-C3N4 and C-dots / g-C3N4 was characterized by transmission electron microscopy. g-C3N4 exhibits a typical flake structure ( Figure 1 A). C-dots / g-C3N4 presents a sheet-like structure similar to that of g-C3N4 ( Figure 1 B), and after magnification, it was found that the sheet structure was loaded with carbon quantum dots with an average size of 5.28 nm ( Figure 1 C and 1D). Then, the crystal structures of these samples were investigated by X-ray diffraction (XRD) ( Figure 1 E). All samples have an obvious characteristic peak at 27.8°, corresponding to the interlayer stacking of the conjugated aromatic system of g-C3N4. With the increase of carbon quantum dot loading, the peak becomes significantly broader and weaker, which is caused by the interaction between the loading of C-dots and the surrounding lattice during the formation of C-dots / g-C3N4. The chemical structure of the samples was further studied by Fourier transform infrared spectroscopy ( Figure 1F). At 1200-1700 cm -1 The multiple prominent vibration peaks in the range belong to the stretching and bending modes of the CN heterocyclic ring, among which the peaks at 1238, 1317, and 1407 cm -1 The peaks at 1580 and 1640 cm -1 The peak is C=N stretching vibration. -1 The absorption band centered at 3250 cm belongs to the typical triazine unit bending model. -1 The broad absorption band near the surface corresponds to the stretching vibration mode of the NH group. The characteristic peaks of C-dots / g-C3N4 and g-C3N4 are basically consistent, indicating that the loading of carbon quantum dots has no significant change on the basic structural composition of g-C3N4. The above results indicate that C-dots / g-C3N4 was successfully prepared.

[0018] (3) Evaluation of the photoresponsive nanozyme catalytic activity of g-C3N4 and C-dots / g-C3N4: TMB was used as the oxidase substrate to evaluate the photoresponsive oxidase catalytic activity of g-C3N4 and C-dots / g-C3N4. Figure 2 A). Under visible light irradiation, C-dots / g-C3N4 catalyzes the oxidation of colorless TMB to dark blue oxidized TMB (oxTMB), which has a strong absorption peak at 652 nm. Although g-C3N4 can also catalyze the oxidation of TMB under visible light conditions, the absorbance of oxTMB is lower, proving that the light-responsive oxidase-like activity of g-C3N4 is low. Conversely, without light irradiation, blue oxTMB cannot be generated. In addition, in the absence of light irradiation of C-dots / g-C3N4, TMB is hardly oxidized. These results indicate that C-dots / g-C3N4 has light-responsive oxidase-like activity, and its catalytic activity is significantly enhanced compared to g-C3N4. Next, the visible light switch response experiment of the oxidase-like activity of C-dots / g-C3N4 was investigated ( Figure 2 B), proved that the light-responsive oxidase activity of C-dots / g-C3N4 can be precisely regulated by switching the light source. Then, the specific activity of C-dots / g-C3N4 and g-C3N4 was compared ( Figure 3 A and 3B). The specific activities of C-dots / g-C3N4 and g-C3N4 were 0.83 U / mg and 0.039 U / mg, respectively, which further confirmed that C-dots / g-C3N4 had higher photoresponsive oxidase catalytic activity. Its catalytic activity was studied in depth using steady-state kinetics. Compared with g-C3N4, C-dots / g-C3N4 had a lower Michaelis constant (Km) and a higher maximum reaction rate ( V max )( Figure 3 C and 3D), indicating that C-dots / g-C3N4 have better affinity for TMB and higher light-responsive oxidase catalytic activity.

[0019] (4) Detection of carbosulfan: 100 μL of 0.2% C-dots / g-C3N4 (0.2 mg / mL), 100 μL of TMB (5 mM), and 50 μL of carbosulfan solution of different concentrations (0-100 μM) were added to 750 μL of NaAc-HAc buffer solution (10 mM, pH=2.8) in sequence. After mixing evenly, the mixture was irradiated with a xenon lamp for 3 min. The solution was filtered and the UV-visible absorption spectrum was recorded ( Figure 4 A). Linear fitting was performed using ∆A and carbosulfan concentration ( Figure 4 B and 4C), the linear range of ∆A and carbosulfan concentration was 5-70 μM, and the detection limit was 2.11 μM. In addition, the effects of other pesticides on the detection system were determined, and the results showed that these pesticides were added alone or simultaneously with carbosulfan ( Figure 4 D) has little effect on the detection system, proving that this method has high selectivity and good anti-interference ability.

[0020] (5) Smartphone-assisted paper-based colorimetric detection of carbosulfan: First, filter paper was cut into multiple circular paper discs with a diameter of 10 mm using a laser cutter. 20 μL of 0.2% C-dots / g-C3N4 solution (0.2 mg / mL) was evenly applied on the paper disc and vacuum dried. Subsequently, 20 μL of NaAc-HAc buffer solution (pH=2.8) containing TMB (5 mM) and 20 μL of carbosulfan solutions of different concentrations (5-100 μM) were evenly dripped onto the paper disc. After irradiation with a xenon lamp for 3 min, the RGB value of the paper disc was extracted using a smartphone color picker and carbosulfan was quantitatively analyzed ( Figure 5 A). Linear fitting of R / B and carbosulfan concentration after color picking using a smartphone ( Figure 5 C), the linear range of R / B and carbosulfan concentration was 5-60 μM, and the detection limit was 2.52 μM.

Claims

1. A method for preparing carbon quantum dots / graphite phase carbon nitride (C-dots / g-C3N4) nanozyme, characterized in that: The following steps are involved: The urea and citric acid are evenly ground, calcined, refluxed and washed to obtain C-dots / g-C3N4 nanozyme.

2. The method for preparing a C-dots / g-C3N4 nanozyme according to claim 1, characterized in that: The added amounts of urea and citric acid are 20 g and 10-100 mg, respectively.

3. The method for preparing a C-dots / g-C3N4 nanozyme according to claim 1, characterized in that: Calcination is 0.5 o The calcinations were carried out at 600 °C for 3 h at a heating rate of 0.147 W / min.

4. The method for preparing a C-dots / g-C3N4 nanozyme according to claim 1, characterized in that: The specific operation steps of reflux are as follows: take 0.3 g sample, add it to 30 mL of 5 M HNO3 solution, o C and reflux for 24 h.

5. A colorimetric detection method for carbosulfan using a light-responsive nanozyme, characterized in that: The method comprises the following steps: adding C-dots / g-C3N4 prepared by the preparation method of claim 1 and TMB to a buffer solution, adding a carbosulfan solution, irradiating with a xenon lamp, and recording a UV-visible absorption spectrum after filtering.

6. The method for colorimetric detection of carbosulfan using a light-responsive nanozyme according to claim 5, characterized in that: The added amounts of C-dots / g-C3N4, TMB, and carbosulfan were 100 μL, 100 μL, and 50 μL, respectively. The concentration of C-dots / g-C3N4 was 0.2 mg / mL, the concentration of TMB was 5 mM, and the concentration of carbosulfan was 0-100 μM.

7. A colorimetric detection method for carbosulfan based on a smartphone-assisted light-responsive nanozyme-functionalized paper substrate, characterized in that: The colorimetric detection method of claim 5 also includes using a smartphone color picker to extract the RGB value of the paper plate and perform a quantitative analysis of carbosulfan.

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