A colorimetric detection method for carbofuran based on a paper substrate functionalized with smartphone-assisted photoresponsive nanozymes.

Abstract

This invention discloses a colorimetric detection method for carbofuran based on a paper-based nanozyme functionalized with smartphone assistance. The method includes the following steps: First, carbon quantum dot / graphite-phase carbon nitride (C-dots / g-C3N4) nanozymes are prepared. Under visible light irradiation, the C-dots / g-C3N4 photoresponsive nanozyme catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate blue oxidized TMB (oxTMB), which has a strong absorption peak at 652 nm. Carbofuran hydrolyzes under acidic conditions to generate sulfur-containing compounds, inhibiting the colorimetric reaction and causing a decrease in absorbance at 652 nm. Based on the change in absorbance at 652 nm with carbofuran concentration, the detection of carbofuran can be achieved. The catalytic activity of the C-dots / g-C3N4 photoresponsive nanozyme is significantly improved compared to g-C3N4, providing guidance for the preparation of highly catalytically active g-C3N4-based photoresponsive nanozymes. This method uses photoresponsive nanozymes, which can be precisely controlled by switching the light source on and off, making the detection results more accurate. It does not rely on large instruments, is easy to operate, and the phenomena are intuitive.

Description

Technical Field

[0001] This invention belongs to the field of pesticide detection, specifically relating to a colorimetric detection method for carbofuran based on a paper substrate functionalized with smartphone-assisted photoresponsive nanozymes. Background Technology

[0002] Thiophanate-carbofuran is a highly effective carbamate pesticide that can effectively control pests on various crops. However, thiophanate-carbofuran readily decomposes into furadan, which is extremely toxic to humans. Therefore, developing highly sensitive methods for detecting thiophanate-carbofuran is crucial for protecting human health. Currently, chromatography is the primary method for detecting thiophanate-carbofuran, offering high sensitivity and good reproducibility, but it is limited by expensive equipment and complex, time-consuming procedures. Colorimetric methods, due to their intuitive observations, ease of operation, and lack of reliance on large, expensive instruments, offer a completely new approach to the detection of thiophanate-carbofuran.

[0003] Nanozymes, especially peroxide nanozymes and oxidative nanozymes, have provided new opportunities for the colorimetric detection of thiocarbofuran. Most of them are based on the colorimetric reaction catalyzed by peroxide or oxidative nanozymes, while the reducing sulfur-containing compounds generated from the hydrolysis of thiocarbofuran inhibit this colorimetric reaction. Among them, the colorimetric reaction catalyzed by photoresponsive oxidative nanozymes does not require the participation of hydrogen peroxide, making the operation simpler; and most can be precisely controlled by switching the light source on and off, resulting in more accurate detection results. However, currently reported photoresponsive oxidative nanozymes are few in number and generally suffer from drawbacks such as high cost and complex synthesis. Graphitic carbon nitride (g-C3N4) has advantages such as low cost, simple fabrication method, and high stability. Researchers have found that g-C3N4 nanosheets possess photoresponsive oxidase-like activity, but its activity is low. Summary of the Invention

[0004] To address the aforementioned issues, we prepared a highly catalytically active carbon quantum dot / graphite-phase carbon nitride (C-dots / g-C3N4) nanozyme by loading carbon quantum dots onto g-C3N4 nanosheets. We then utilized its photoresponsive oxidase-like activity to detect carbofuran. The detection mechanism is as follows: Under visible light irradiation, the C-dots / g-C3N4 photoresponsive nanozyme catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to form blue oxidized TMB (oxTMB), which exhibits a strong absorption peak at 652 nm. Carbofuran hydrolyzes under acidic conditions to form sulfur-containing compounds, inhibiting this colorimetric reaction and leading to a decrease in absorbance at 652 nm. Based on the change in absorbance at 652 nm with carbofuran concentration, the detection of carbofuran can be achieved. Based on the above detection mechanism, a paper-based material functionalized with C-dots / g-C3N4 photoresponsive nanozyme was prepared, and colorimetric detection of thiocarbamate was achieved using a smartphone.

[0005] This invention is achieved through the following technical solution:

[0006] Preparation and characterization of C-dots / g-C3N4 photoresponsive nanozymes: 20 g of urea and 10-100 mg of citric acid were added to a mortar and ground evenly; then the resulting powder was transferred to an alumina crucible and heated in a muffle furnace at 0.5°C. o The solid was calcined at 600 °C for 3 h at a heating rate of C / min. Afterwards, 0.3 g of the calcined solid was ground into powder and added to 30 mL of 5 MHNO3 solution. o The solution was refluxed at C for 24 h. Finally, it was washed with ultrapure water until the supernatant pH was close to neutral. The samples were named 0.05% C-dots / g-C3N4, 0.2% C-dots / g-C3N4, and 0.5% C-dots / g-C3N4 based on the amount of citric acid added. The 0.2% C-dots / g-C3N4 nanosheets showed the best catalytic activity. The preparation of g-C3N4 nanosheets was similar to that of the C-dots / g-C3N4 photoresponsive nanozyme, except that citric acid was not added. The morphology, structure, and catalytic activity of the prepared C-dots / g-C3N4 photoresponsive nanozyme were characterized using transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction spectroscopy, kinetics, and specific activity tests.

[0007] Determination of carbofuran: 100 μL of 0.2% C-dots / g-C3N4 (0.2 mg / mL), 100 μL of TMB (5 mM), and 50 μL of carbofuran solutions of different concentrations (0-100 μM) were added sequentially to 750 μL NaAc-HAc buffer solution (10 mM, pH=2.8). After mixing thoroughly, the solution was irradiated with a xenon lamp for 3 min. The solution was then filtered, and its UV-Vis absorption spectrum was recorded.

[0008] Smartphone-assisted paper-based colorimetric detection of carbofuran: 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 spread onto the paper discs and vacuum dried. Subsequently, 20 μL of NaAc-HAc buffer solution containing TMB (5 mM) (pH=2.8) and 20 μL of carbofuran solutions of different concentrations (5-100 μM) were evenly dropped onto the paper discs. After irradiation with a xenon lamp for 3 min, the RGB values ​​of the paper discs were extracted using a smartphone colorimeter, and the carbofuran was quantitatively analyzed.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0010] (1) This method uses photoresponsive nanozymes, which can be precisely controlled by switching the light source on and off, making the detection results more accurate.

[0011] (2) The method of detecting carbofuran based on paper-based colorimetric analysis with the assistance of smartphones does not rely on large instruments, is easy to operate, and produces intuitive results.

[0012] (3) The catalytic activity of C-dots / g-C3N4 photoresponsive nanozymes is significantly improved compared with g-C3N4, which provides guidance for the preparation of g-C3N4-based photoresponsive nanozymes with high catalytic activity. Attached Figure Description

[0013] 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.

[0014] Figure 2 (A) UV-Vis absorption spectra of g-C3N4-TMB and C-dots / g-C3N4-TMB under illumination or no illumination, and TMB under illumination. (B) Photo-controlled oxidase activity of C-dots / g-C3N4.

[0015] 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 show the corresponding Lineweaver-Burk plots for C-dots / g-C3N4 and g-C3N4.

[0016] Figure 4 (A) The UV-Vis absorption spectra of the C-dots / g-C3N4-TMB system with different concentrations of carbofuran added. (B) The relationship between ∆A and carbofuran concentration. (C) The linear calibration graph of carbofuran concentration. (D) The absorbance of the system at 652 nm when other pesticides are added alone or simultaneously with carbofuran.

[0017] Figure 5 (A) Schematic diagram of the C-dots / g-C3N4-TMB system combined with smartphone-assisted color sampling for the detection of carbofuran. (B) Relationship between R / B after color sampling and carbofuran concentration. (C) Linear calibration graph of R / B after color sampling and carbofuran concentration. Detailed Implementation

[0018] The following is in conjunction with the appendix Figure 1 - Appendix Figure 5The embodiments further illustrate the technical solutions of the present invention. Unless otherwise specified, the technical means used in the specific embodiments of the present invention are all methods known to those skilled in the art. Where specific techniques or conditions are not specified in the embodiments, they are all conventional methods or performed according to the techniques or conditions described in the literature in the field, or according to the product manual. Where the manufacturer is not specified, they are all conventional products that can be purchased through legitimate channels.

[0019] Example 1

[0020] (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; then the resulting powder was transferred to an alumina crucible and heated in a muffle furnace at 0.5°C. o The solid was calcined at 600 °C for 3 h at a heating rate of C / min. Afterwards, 0.3 g of the calcined solid was ground into powder and added to 30 mL of 5 M HNO3 solution. o Reflux at C for 24 h. Finally, wash with ultrapure water until the supernatant pH is close to neutral. The samples were named 0.05% C-dots / g-C3N4, 0.2% C-dots / g-C3N4, and 0.5% C-dots / g-C3N4, respectively, based on the different amounts of citric acid added (10 mg, 40 mg, and 100 mg). The 0.2% C-dots / g-C3N4 sample showed the best catalytic activity. The preparation of g-C3N4 nanosheets was similar to that of the C-dots / g-C3N4 photoresponsive nanozyme, except that citric acid was not required.

[0021] (2) Morphology and structural composition characterization of g-C3N4 and C-dots / g-C3N4 photoresponsive nanozymes: The morphology of g-C3N4 and C-dots / g-C3N4 was characterized using transmission electron microscopy. g-C3N4 exhibits a typical sheet-like structure ( Figure 1 A). C-dots / g-C3N4 exhibits a similar plate-like structure to g-C3N4 ( Figure 1 B), and upon magnification, it was found that its sheet-like structure was loaded with carbon quantum dots with an average size of 5.28 nm. Figure 1 C and 1D). Then, the crystal structure of these samples was studied by X-ray diffraction (XRD). Figure 1 E). All samples exhibit a distinct characteristic peak at 27.8º, corresponding to the interlayer stacking of the conjugated aromatic system of g-C3N4. This peak broadens and weakens significantly with increasing carbon quantum dot loading, which is attributed to the interaction between the C-dot loading and the surrounding lattice during the formation of C-dots / g-C3N4. The chemical structure of the samples was further investigated using Fourier transform infrared spectroscopy. Figure 1 F). In 1200-1700 cm -1 Several significant vibrational peaks within the range belong to the tensile and bending modes of the CN heterocycle, with peaks located at 1238, 1317, and 1407 cm⁻¹. -1 The peaks are due to CN tensile vibration, while those located at 1580 and 1640 cm⁻¹ are due to CN tensile vibration. -1 The peak represents C=N tensile vibration. (At 810 cm⁻¹) -1 The absorption band centered thereon belongs to a typical triazine unit bending model. 3250 cm -1 The broad absorption band in the vicinity corresponds to the stretching vibration mode of the NH group. The characteristic peaks of C-dots / g-C3N4 are basically consistent with those of g-C3N4, indicating that the loading of carbon quantum dots does not significantly change the basic structural composition of g-C3N4. The above results indicate that C-dots / g-C3N4 was successfully prepared.

[0022] (3) Evaluation of the catalytic activity of photoresponsive nanozymes g-C3N4 and C-dots / g-C3N4: The photoresponsive oxidase catalytic activity of g-C3N4 and C-dots / g-C3N4 was evaluated using TMB as the oxidase substrate. Figure 2 A). Under visible light irradiation, C-dots / g-C3N4 catalyzes the oxidation of colorless TMB to deep blue oxidized TMB (oxTMB), exhibiting a strong absorption peak at 652 nm. Although g-C3N4 can also catalyze TMB oxidation under visible light, the absorbance of oxTMB is lower, indicating that g-C3N4 has lower photoresponsive oxidase-like activity. Conversely, without light irradiation, blue oxTMB cannot be generated. Furthermore, without light irradiation from C-dots / g-C3N4, TMB is hardly oxidized. These results indicate that C-dots / g-C3N4 possesses photoresponsive oxidase-like activity, and its catalytic activity is significantly enhanced compared to g-C3N4. Next, a visible light switching response experiment was conducted to investigate the visible light switching response of the C-dots / g-C3N4 oxidase-like activity. Figure 2 (B) demonstrates that the light-responsive oxidase activity of C-dots / g-C3N4 can be precisely regulated by switching the light source on and off. Next, the specific activities of C-dots / g-C3N4 and g-C3N4 were 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, further confirming that C-dots / g-C3N4 possesses higher photoresponsive oxidase catalytic activity. Its catalytic activity was investigated in detail using steady-state kinetics. Compared with g-C3N4, C-dots / g-C3N4 exhibits a lower Michaelis constant (Km) and a higher maximum reaction rate (…). V max() Figure 3 (C and 3D), indicating that C-dots / g-C3N4 has a better affinity for TMB and higher photoresponsive oxidase catalytic activity.

[0023] (4) Detection of carbofuran: 100 μL of 0.2% C-dots / g-C3N4 (0.2 mg / mL) and 100 μL of TMB (5 mM), and 50 μL of carbofuran solutions of different concentrations (0-100 μM) were added sequentially to 750 μL NaAc-HAc buffer solution (10 mM, pH=2.8). After mixing thoroughly, the solution was irradiated with a xenon lamp for 3 min. The solution was then filtered, and the UV-Vis absorption spectrum was recorded. Figure 4 A). Linear fitting was performed using ∆A and the concentration of carbofuran (butyrate). Figure 4 (B and 4C), the linear range of ∆A with respect to the concentration of carbofuran was found to be 5-70 μM, and the limit of detection was 2.11 μM. In addition, the effects of other pesticides on the detection system were determined, and the results showed that these pesticides, added alone or simultaneously with carbofuran (B and 4C), showed that... Figure 4 D) It has virtually no impact on the detection system, proving that the method has high selectivity and good anti-interference ability.

[0024] (5) Smartphone-assisted paper-based colorimetric detection of carbofuran: First, the 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 coated onto the paper discs and vacuum dried. Subsequently, 20 μL of NaAc-HAc buffer solution containing TMB (5 mM) (pH=2.8) and 20 μL of carbofuran solutions of different concentrations (5-100 μM) were evenly dropped onto the paper discs. After irradiation with a xenon lamp for 3 min, the RGB values ​​of the paper discs were extracted using a smartphone colorimeter, and the carbofuran was quantitatively analyzed. Figure 5 A). Linear fitting was performed using the R / B ratio obtained from color sampling by a smartphone and the concentration of carbofuran (butyrate). Figure 5 (C) The linear range of R / B and thiocarbofuran concentration was found to be 5-60 μM, and the detection limit was 2.52 μM.

Claims

1. A method for preparing a C-dots / g-C 3 N 4 photo-responsive oxidase nanoreactor, characterized in that, The method comprises the following steps: The urea and citric acid are ground, calcined, refluxed and washed to obtain C-dots / g-C3N4 nanoscale enzyme; The added amount of urea and citric acid is 20 g and 10-100 mg respectively; Calcination is at 600 °C for 3 h with a heating rate of 0.5 o C / min.

2. The method according to claim 1, wherein, The specific operation steps of refluxing are as follows: 0.3 g of sample is taken and added into 30 mL of 5 M HNO3 solution, 100 o C refluxing for 24 h.

3. A method for colorimetric detection of carbosulfan using photo-responsive oxidized nanozyme, characterized in that, The method comprises the following steps: adding the C-dots / g-C3N4 photoresponsive oxidizing nanoscale enzyme prepared by the preparation method of claim 1 and TMB in a buffer solution, adding a carbosulfan solution, irradiating with a xenon lamp, filtering and recording the ultraviolet-visible absorption spectrum.

4. The colorimetric detection method of carbosulfan using a light-responsive oxidized nanozyme according to claim 3, characterized in that, The added amount of C-dots / g-C3N4, TMB and carbosulfan is 100 μL, 100 μL and 50 μL respectively, the concentration of C-dots / g-C3N4 is 0.2 mg / mL, the concentration of TMB is 5 mM and the concentration of carbosulfan is 0-100 μM.

5. A smartphone-assisted colorimetric method for the detection of carbosulfan based on photo-responsive oxidized nanozyme functionalized paper, characterized in that, The colorimetric detection method of claim 3 further comprises using a smartphone color picker to extract the RGB value of the paper disc and quantitatively analyzing the carbosulfan.

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