A method for detecting malachite green residue in food based on titanium carbide quantum dot-biomass carbon quantum dot ratio fluorescent paper-based sensor

Abstract

This invention discloses a method for detecting malachite green residues in food based on a ratiometric fluorescent paper-based sensor using titanium carbide quantum dots and biomass carbon quantum dots. First, titanium carbide quantum dots and biomass carbon quantum dots are prepared. Then, a Ti3C2MXene / biomass quantum dot ratiometric fluorescent probe is prepared. Based on this, a Ti3C2MXene / biomass quantum dot ratiometric fluorescent paper-based sensor (filter paper chip) is constructed. A linear equation for the working curve of malachite green concentration is established. Finally, the pretreated sample solution is dropped onto the ratiometric fluorescent filter paper chip. After drying, the paper chip is photographed using a mobile phone under a 365 nm UV analyzer. The RGB values ​​of the paper chip are extracted using smartphone color recognition software. The obtained R value is substituted into the linear equation of the working curve to obtain the malachite green concentration in the sample. The titanium carbide quantum dot / biomass carbon quantum dot ratiometric fluorescent paper-based sensor and malachite green concentration detection method disclosed in this invention are sensitive, accurate, and rapid.

Description

Technical Field

[0001] This invention belongs to the field of rapid food safety detection technology, specifically relating to a method for detecting malachite green residue in food based on a ratiometric fluorescent paper-based sensor of titanium carbide quantum dots and biomass carbon quantum dots. Background Technology

[0002] Food is the paramount necessity of the people, and food quality and safety are crucial to safeguarding people's lives and health. Employing rapid, convenient, low-cost, safe, and efficient testing methods is of great significance for monitoring food safety issues. Currently, conventional analytical methods for food testing mainly include high-performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography-mass spectrometry (LC-MS), electrochemical methods, fluorescence spectrophotometry, and colorimetry. However, these instrumental analytical methods suffer from drawbacks such as long testing times, lack of portability, and the need for professional personnel to operate them in the field. Compared to instrumental analysis methods, rapid testing is gradually becoming an important tool for government regulatory departments to conduct supervision and management and collect evidence on-site. Ratio fluorescence paper-based sensors, based on fluorescence analysis and paper-based sensors, are easy to operate, have significant effects, and are suitable for rapid on-site visual analysis, showing great potential and development prospects in the fields of food testing and portable sensing.

[0003] Based on the fluorescence emission method, ratiometric fluorescence analysis can be divided into single-emission fluorescence analysis and ratiometric fluorescence analysis. However, single-emission fluorescence signal detection is often affected by various conditions, such as concentration changes, instrument efficiency, and complex environmental conditions, all of which can affect the accurate detection of analytes. Ratiometric fluorescence analysis detects the concentration of the analyte by simultaneously measuring the fluorescence intensity generated by two or more different emission wavelengths and using the ratio of their fluorescence intensities. Compared with single-emission fluorescence methods, ratiometric fluorescence methods have a built-in self-calibration function, which can effectively avoid interference from factors unrelated to the target, thereby improving the accuracy and sensitivity of detection. Ratiometric fluorescence sensors based on dual-emission or multi-emission can compensate for the shortcomings of single-wavelength fluorescence detection, eliminate background signals and environmental interference, and can easily realize fluorescence color changes, resulting in better visual detection effects. Therefore, they show great advantages and are currently one of the research hotspots.

[0004] Fluorescent paper-based sensors detect analytes by visually observing changes in fluorescence intensity and color under ultraviolet light. Ratio-fluorescent paper-based sensors, however, exhibit more pronounced and easily observable color changes, enabling faster, more visual, and quantitative detection. Compared to traditional paper sensors, fluorescent paper-based sensors utilize a wide variety of novel fluorescent nanomaterials, prepared through methods such as impregnation and printing. Therefore, their functionality largely depends on the performance of the selected fluorescent nanomaterials. The key to achieving visual analysis with ratio-fluorescent paper-based sensors lies in designing suitable ratio-fluorescent probes that produce highly sensitive responses to the target analyte, resulting in noticeable signal changes, such as changes in the fluorescence color of the sensing system. Therefore, synthesizing high-performance fluorescent nanomaterials and designing efficient ratio-fluorescent probes are crucial for the visual analysis of ratio-fluorescent paper-based sensors.

[0005] Malachite green (MG), a food additive, is a chemical preparation and a banned veterinary drug. Consuming food containing malachite green is carcinogenic, and its addition during food processing is strictly prohibited by the state. However, some unscrupulous vendors still use it in fish and canned products to achieve effects such as prolonging life and preservation. Therefore, it is essential to detect the presence of malachite green in food. Currently, malachite green detection technology has reached a certain level of development, with methods including enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). While these methods can accurately detect malachite green concentrations, they suffer from drawbacks such as high cost, long processing time, and cumbersome operation. Rapid food safety testing technologies not only benefit regulatory personnel in conducting on-site sampling and testing during routine hygiene supervision but also play a crucial role in public health and emergency response during large-scale events. Currently, accuracy, sensitivity, and speed are the key directions for the development of rapid testing technologies.

[0006] To achieve faster, more visual, and quantitative detection of malachite green residues in food, this patent leverages the advantages of ratiometric fluorescence's high selectivity and sensitivity, combined with the convenience and ease of use of paper-based sensors. Ti3C2MXene quantum dots and biomass-based carbon quantum dot fluorescent materials are prepared to construct a rapid and visually-based ratiometric fluorescence paper-based sensor method for detecting the harmful substance malachite green in food. This invention has positive significance in promoting the practical application of fluorescent paper-based sensor visualization technology in food safety. Summary of the Invention

[0007] Purpose of the invention:

[0008] Due to the complexity of food sample composition, paper-based sensors often exhibit low sensitivity and accuracy in rapid on-site food safety testing. This invention addresses this deficiency by designing and developing a ratiometric fluorescence paper-based sensor based on titanium carbide quantum dots and biomass carbon quantum dots. Leveraging the advantages of ratiometric fluorescence detection, it overcomes the shortcomings of ordinary fluorescent paper-based sensors in rapid detection, establishing a new method for sensitive, accurate, and rapid detection of malachite green residues in food samples.

[0009] To achieve the above-mentioned objectives, the following technical solution is proposed:

[0010] 1. Preparation of titanium carbide quantum dots and biomass carbon quantum dots

[0011] (1) Preparation method of titanium carbide quantum dots

[0012] 40 mg of Ti3C2 powder was dispersed in 10 mL of 5 mol / L nitric acid, and then heated in an oil bath to 100℃ for 24 h. After cooling to room temperature, the solution was poured into 30 mL of ice water, and concentrated NaOH solution was added dropwise while stirring to adjust the pH to approximately 7. The mixture was allowed to stand and separate into layers. The supernatant was discarded, and the lower precipitate was dried in a vacuum drying oven to obtain Ti3C2MXene precursor powder. 0.2 g of the powder was weighed, and 2.5 mL of ethylenediamine and 10 mL of ethanol were added. The mixture was sonicated for 5 min to ensure uniform dispersion. The mixture was reacted in a synthesis reactor at 180℃ for 12 h, then filtered through 0.22 μm microporous filter paper. Finally, the filtrate was dialyzed through a 1000 Da dialysis bag for one day. The dialyzed solution was evaporated to dryness, weighed, and then ethanol was added to prepare a 0.5 mg / mL Ti3C2MXene quantum dot solution. The solution was sealed and stored in a refrigerator for later use.

[0013] (2) Biomass carbon quantum dot preparation method

[0014] Using fresh broad bean shells as the carbon source, the broad bean shells were first repeatedly washed with deionized water, dried, chopped, and extracted with ethanol solution with stirring for 24 h. The ethanol in the solution was then removed by rotary evaporation, followed by freeze-drying to remove moisture. 0.01 g of the resulting chlorophyll powder was weighed and dissolved in 10 mL of anhydrous ethanol. The mixture was reacted at 180 ℃ for 6 h in a synthesis reactor, filtered through 0.22 μm microporous filter paper, and finally dialyzed through a 3500 Da dialysis bag for one day. The dialyzed solution was evaporated to dryness, weighed, and then ethanol was added to prepare a 0.5 mg / mL biomass carbon quantum dot solution, which was then sealed and stored in a refrigerator for later use.

[0015] 2. Fabrication method of ratiometric fluorescent paper-based sensor

[0016] (1) Preparation method of Ti3C2MXene / biomass dot ratio fluorescent probe

[0017] A quantum dot mixture was prepared by adding 110 μL of Ti3C2MXene quantum dots to 30 μL of biomass carbon quantum dots into 3 mL of ethanol and shaking thoroughly for 2 h. A ratio fluorescent probe with a fluorescence intensity of 2:1 for Ti3C2MXene quantum dots to biomass carbon quantum dots was obtained.

[0018] (2) Preparation method of Ti3C2MXene / biomass quantum dot ratio fluorescent paper-based sensor

[0019] Organic microporous filter paper (50 mm in diameter) was selected as the paper-based chip. The chip was filtered with a prepared ratiometric fluorescent probe solution, 10 times on each side, to ensure the probe quantum dots were fully and uniformly adsorbed onto the paper-based chip. After drying, the paper was cut into several circular filter paper chips with a diameter of 14 mm using a tablet press and stored for later use.

[0020] 3. Ratio-type fluorescent paper-based sensor for the detection of malachite green residues in food samples

[0021] (1) Method for establishing working curves

[0022] RGB values ​​of paper chips impregnated with malachite green + ratiometric fluorescent probe solution were extracted to establish a working curve. A series of malachite green solutions with concentration gradients (0-140 μmol / L) were prepared. 800 μL of each malachite green solution was added dropwise to filter paper chips containing ratiometric fluorescent quantum dots using a pipette. After drying, the paper chips were photographed using a mobile phone under a 365 nm UV analyzer. The RGB values ​​of the paper chips were extracted using a color recognition app on the mobile phone. Data processing revealed the range of linear relationships between the R value and the malachite green concentration. The linear equation and correlation coefficient were obtained, and the detection limit was calculated.

[0023] (2) Method for detecting malachite green concentration in food samples

[0024] The pretreated sample solution is dropped onto a ratiometric fluorescence filter paper chip. After drying, the paper chip is photographed with a mobile phone under a 365 nm UV analyzer. The RGB values ​​of the paper chip are extracted using the color recognition software of the smartphone. The obtained R value is substituted into the linear equation of the working curve to obtain the concentration of malachite green in the sample.

[0025] Compared with existing methods for detecting malachite green concentration, this invention has the following advantages: (1) By taking advantage of the good selectivity and high sensitivity of ratio fluorescence, the sensor developed in this invention can effectively avoid the defects of low sensitivity and accuracy often caused by the complex composition of food samples. (2) The sensor developed in this invention combines the convenient and easy-to-operate characteristics of paper-based sensors, which can realize the rapid detection of malachite green residue in food samples during on-site supervision, which is conducive to ensuring food safety. Attached Figure Description

[0026] Figure 1(a) and Figure 1(b) are TEM images of the biomass quantum dots and Ti3C2MXene quantum dots in the embodiments of the present invention.

[0027] Figure 2 shows the fluorescence spectrum of the ratiometric fluorescent probe constructed from Ti3C2MXene / biomass sub-dots in an embodiment of the present invention.

[0028] Figure 3 This is a working curve diagram of the ratio fluorescent paper-based sensor in an embodiment of the present invention. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto.

[0030] In the following embodiments, the method for preparing titanium carbide quantum dots includes the following steps:

[0031] (1) Synthesis of Ti3C2: 20 mL of HF was placed in a 100 mL polytetrafluoroethylene beaker, and 1.0 g of Ti3AlC2 was slowly added to the HF while stirring continuously. After sonication for 30 min, the mixture was heated in a water bath at 35 ℃ and stirred for 24 h. After the reaction was completed, the mixture was centrifuged for 5 min, the supernatant was discarded, and a large amount of ultrapure water was added for washing. The mixture was then centrifuged again, and the process was repeated several times until the supernatant was neutral (pH = 6~7). After centrifugation, 200 mL of ultrapure water was added and sonicated for 30 min. The lower precipitate was collected and vacuum dried at 80 ℃ to obtain multilayer Ti3C2 powder. The powder was collected and stored in a drying oven for later use.

[0032] (2) Synthesis of Ti3C2MXene blue quantum dots: 40 mg of Ti3C2 powder was dispersed in 10 mL of 5 mol / L nitric acid, and then heated in an oil bath to 100 ℃ for 24 h. After cooling to room temperature, it was poured into 30 mL of ice water, and concentrated NaOH solution was added dropwise while stirring to adjust the pH to about 7. After standing and separating into layers, the supernatant was discarded, and the lower precipitate was dried in a vacuum drying oven to obtain Ti3C2MXene precursor powder. 0.2 g of powder was weighed, 2.5 mL of ethylenediamine and 10 mL of ethanol were added, and the mixture was sonicated for 5 min to disperse it evenly. The mixture was reacted in a synthesis reactor at 180 ℃ for 12 h, and then filtered with 0.22 μm microporous filter paper. Finally, the filtrate was dialyzed through a 1000 Da dialysis bag for one day. The dialyzed solution was evaporated to dryness and weighed, and ethanol was added to prepare a 0.5 mg / mL Ti3C2MXene quantum dot solution (its transmission electron microscopy image is attached). Figure 1 (b) Seal and store in the refrigerator for later use.

[0033] In the following embodiments, the method for preparing biological mass sub-dots includes the following steps:

[0034] Red-light carbon quantum dots were prepared using biomass materials containing chlorophyll. Fresh broad bean shells were used as the carbon source. First, the broad bean shells were repeatedly washed with deionized water, dried, chopped, and extracted with ethanol solution with stirring for 24 h. The ethanol in the solution was then removed by rotary evaporation, followed by freeze-drying to remove moisture. 0.01 g of the obtained chlorophyll powder was weighed and dissolved in 10 mL of anhydrous ethanol. The mixture was reacted at 180 ℃ for 6 h in a synthesis reactor, filtered through 0.22 μm microporous filter paper, and finally dialyzed through a 3500 Da dialysis bag for one day. The dialyzed solution was evaporated to dryness, weighed, and then ethanol was added to prepare a 0.5 mg / mL biomass carbon quantum dot solution (its transmission electron microscopy image is attached). Figure 1 (a) Seal and store in the refrigerator for later use.

[0035] The following examples illustrate the method for fabricating the fluorescence spectrum of the Ti3C2MXene / biomass dot ratio fluorescent probe:

[0036] Adjusting the fluorescence intensity ratio F of the two quantum dots 434 / F 673 A ratio of 2:1 (110 μL Ti3C2MXene quantum dots + 30 μL biomass carbon quantum dots + 3 mL ethanol) was used to prepare a ratiometric fluorescent probe solution, and the fluorescence spectrum of the ratiometric fluorescent probe was measured (see [link to solution]). Figure 2 ).

[0037] The following embodiments illustrate a method for establishing a visual detection working curve for malachite green:

[0038] 800 μL of MG solutions of different concentrations were added dropwise to a fluorescent paper-based sensor. After drying, images were taken under a 365 nm UV analyzer, and the RGB values ​​of the fluorescent paper-based sensor were extracted using a mobile phone software-based color recognition tool. A calibration curve was established with R (representing chromatic aluminosity) on the ordinate and MG concentration on the abscissa. As the concentration of malachite green increased, the color of the paper-based sensor changed from red to light red, then to blue, and gradually darkened, thus achieving rapid and visual detection of malachite green concentration. The results showed a linear relationship between malachite green concentration and R value in the range of 0-140 μmol / L (R... 2 =0.9969), the linear equation is R = -1.1068C MG +234.34, detection limit is 17.1 μmol / L. Data processing and plotting yielded... Figure 3 .

[0039] Example 1. Method for detecting malachite green residue in canned fish.

[0040] (1) Sample pretreatment. Take 6.00 g of canned fish sample, crush it, add 60 mL of acetonitrile, and sonicate the solution for 30 min, followed by stirring for another 30 min. After filtering with organic filter paper (0.22 μm), evaporate the extract at 50 °C by rotary evaporation until only yellow oil droplets that are not easily evaporated remain. Finally, dissolve the yellow oil droplets in ethanol to prepare a 60 mL sample solution.

[0041] (2) Spike recovery experiment. A certain amount of 0.001 mol / L malachite green standard solution was added to 5.0 mL of sample solution to prepare spiked solutions with concentrations of 1.00 μmol / L, 20.00 μmol / L and 40.00 μmol / L respectively.

[0042] (3) Spiked Recovery Results. Malachite green solutions of different concentrations were individually dropped onto the prepared ratiometric fluorescent paper-based chip. After drying, the chips were photographed using a 365 nm UV analyzer. The RGB values ​​of the paper chips were extracted using mobile software, and calculations were performed based on the linear equation between the R value and the malachite green concentration. The results are shown in Table 1. Table 1 shows that the recovery rate of the ratiometric fluorescent paper-based sensor prepared in this paper for canned fish samples was 92.4%–121.2%, indicating that this ratiometric fluorescent paper-based sensor can be used for actual sample determination. The results are shown in Table 1.

[0043] Table 1. Results of the fish canned food sample recovery experiment

[0044]

[0045] Example 2. Method for detecting malachite green residue in fresh fish meat.

[0046] (1) Sample pretreatment. Take 6.00 g of fresh fish meat, crush it, add 60 mL of acetonitrile, and sonicate the solution for 30 min, followed by stirring for another 30 min. Filter the solution through organic filter paper (0.22 μm) and then evaporate the extract at 50 °C by rotary evaporation until only yellow oil droplets that are not easily evaporated remain. Finally, dissolve the yellow oil droplets in ethanol to prepare a 60 mL sample solution.

[0047] (2) Spike recovery experiment. A certain amount of 0.001 mol / L malachite green standard solution was added to 5.0 mL of sample solution to prepare spiked solutions with concentrations of 1.00 μmol / L, 20.00 μmol / L and 40.00 μmol / L respectively.

[0048] (3) Spiked Recovery Results. Malachite green solutions of different concentrations were individually dropped onto the prepared ratiometric fluorescent paper-based chip. After drying, the chips were photographed using a 365 nm UV analyzer. The RGB values ​​of the paper chips were extracted using mobile software, and calculations were performed based on the linear equation between the R value and the malachite green concentration. The results are shown in Table 2. Table 2 shows that the recovery rate of the ratiometric fluorescent paper-based sensor prepared in this paper for fresh fish samples was 98.6%–121.2%, indicating that this ratiometric fluorescent paper-based sensor can be used for actual sample determination. The results are shown in Table 2.

[0049] Table 2. Results of the fresh fish sample recovery experiment

[0050]

Claims

1. A method for fabricating a ratiometric fluorescent paper-based sensor based on titanium carbide quantum dots and biomass carbon quantum dots, characterized in that, Includes the following steps: (1) Preparation of titanium carbide quantum dots: 40 mg of Ti3C2 powder was dispersed in 10 mL of 5 mol / L nitric acid, and then heated in an oil bath to 100 ℃ for 24 h. After cooling to room temperature, it was poured into 30 mL of ice water, and concentrated NaOH solution was added dropwise while stirring to adjust the pH to about 7. After standing and separating into layers, the supernatant was discarded, and the lower precipitate was dried in a vacuum drying oven to obtain Ti3C2MXene precursor powder. 0.2 g of powder was weighed, 2.5 mL of ethylenediamine and 10 mL of ethanol were added, and the mixture was sonicated for 5 min to disperse it evenly. The mixture was reacted in a synthesis reactor at 180 ℃ for 12 h, and then filtered with 0.22 μm microporous filter paper. Finally, the filtrate was dialyzed with a 1000 Da dialysis bag for one day. After the dialyzed solution was evaporated to dryness, it was weighed and ethanol was added to prepare a 0.5 mg / mL Ti3C2MXene quantum dot solution. The solution was sealed and stored in a refrigerator for later use. (2) Preparation of biomass carbon quantum dots: Using fresh broad bean shells as carbon source, the shelled broad beans were first washed repeatedly with deionized water, dried, chopped, and extracted with ethanol solution and stirred for 24 h. The ethanol in the solution was then removed by rotary evaporation, and the water was removed by freeze drying. 0.01 g of the chlorophyll powder obtained after evaporation was weighed and dissolved in 10 mL of anhydrous ethanol. The mixture was reacted at 180 °C for 6 h in a synthesis reactor, filtered with 0.22 μm microporous filter paper, and finally dialyzed with a 3500 Da dialysis bag for one day. The dialyzed solution was evaporated to dryness and weighed. Ethanol was added to prepare a 0.5 mg / mL biomass carbon quantum dot solution, which was then sealed and stored in a refrigerator for later use. (3) Preparation of Ti3C2MXene / biomass carbon quantum dot ratio fluorescent probe: Take 110 μL of 0.5 mg / mL Ti3C2MXene quantum dot ethanol solution prepared in step (1) above, take 30 μL of 0.5 mg / mL biomass carbon quantum dot ethanol solution prepared in step (2) above, add to 3 mL of ethanol, prepare quantum dot mixture, shake well for 2 h, and obtain a ratio fluorescent probe with Ti3C2MXene quantum dot:biomass carbon quantum dot fluorescence intensity of 2:1; (4) Preparation of Ti3C2MXene / bioquantum dot ratio fluorescent paper-based sensor: Organic microporous filter paper with a diameter of 50 mm was selected as the paper-based chip. The ratio fluorescent probe solution obtained in step (3) above was filtered through it 10 times on each side to ensure that the probe quantum dots were fully and evenly adsorbed on the paper-based chip. After drying, it was cut into several circular filter paper chips with a diameter of 14 mm using a tablet press and stored for later use.

2. The method for detecting malachite green concentration in food samples using a ratiometric fluorescent paper-based sensor based on titanium carbide quantum dots and biomass carbon quantum dots prepared according to claim 1, characterized in that... Includes the following steps: (S1) Establishment of working curve: First, prepare a series of malachite green solutions with concentration gradients, with the concentration range controlled between 0-140 μmol / L. Take 800 μL of malachite green solution and drop it onto the Ti3C2MXene / biomass dot ratio fluorescent paper-based sensor filter paper chip obtained in step (4) above. After drying, take a photo of the paper chip with a mobile phone under a 365 nm ultraviolet analyzer. Extract the RGB value of the paper chip through the color recognition software of the smartphone. After data processing, the range of linear relationship between R value and malachite green concentration can be obtained, and the linear equation of working curve can be obtained. (S2) Detection of malachite green concentration in food samples: The pretreated sample solution is dropped onto the Ti3C2MXene / biomass dot ratio fluorescent paper-based sensor filter paper chip obtained in step (4) above. After drying, the paper chip is photographed with a mobile phone under a 365 nm ultraviolet analyzer. The RGB value of the paper chip is extracted using the color recognition software of the smartphone. The obtained R value is substituted into the linear equation of the working curve obtained in (S1) above to obtain the concentration of malachite green in the sample.

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