Method for detecting mercury ions and vitamin C

Abstract

The invention discloses a method for detecting mercury ions and vitamin C. According to the method, aqueous solutions of citric acid and urea are used as raw materials, and fluorescent carbon dots prepared by a microwave heating method have an absorption range within 300-400 nm and have the maximum fluorescence intensity under excitation of 340 nm; the fluorescent carbon dots have selective specificity on Hg < 2 + > and Vc respectively. The fluorescent carbon dots are prepared by adopting a microwave method, and the method is simple, good in water solubility, non-toxic, good in fluorescent light stability and high in sensitivity. The fluorescent carbon dots are used for detecting mercury ions and vitamin C on the basis of an'on-off-on 'fluorescent response mode of the fluorescent carbon dots, qualitative / quantitative detection of Hg < 2 + > and / or Vc in the solution to be detected is realized according to the fluorescence intensity change of the fluorescent carbon dot solution, and the method has the advantages of low detection limit, good selectivity, simplicity, rapidness, low cost, no limitation of experimental conditions and the like.

Description

Technical Field

[0001] The invention belongs to the technical field, and in particular relates to a method for detecting mercury ions and vitamin C. Background Art

[0002] With the development of the chemical industry, heavy metal pollution is becoming increasingly serious. Mercury is very harmful to the human body, and rapid quantitative detection of mercury ions in the environment is of great significance to human health and environmental green development.

[0003] Vitamin C (Vc) is a substance that plays a vital role in the metabolic process and has many functions. Appropriate intake of Vc is beneficial to the human body, while insufficient or excessive intake may cause harm to the human body. Therefore, quantitative detection of vitamin C is of great significance in the food and pharmaceutical industries. In addition, multiple hydroxyl groups of Vc can form chelates with heavy metals to play a detoxifying role. This coordination effect provides a theoretical basis for the continuous detection of mercury ions and Vc.

[0004] Traditionally, Hg 2+ The methods for measuring Vc often involve large instruments and require professionals to operate at specific locations. They are also expensive, take a long time to test, require a large amount of samples, have a fixed test environment, and require a small number of people, making them difficult to popularize. Therefore, it is very necessary to develop a simple, fast, low-cost test method with few experimental conditions.

[0005] Carbon dots are a new type of fluorescent nanomaterial with the advantages of good water solubility, non-toxicity, and good photostability. They have broad application prospects in the fields of biological imaging, photosensitive materials, photocatalysis, luminescent materials, anti-counterfeiting technology, and fluorescence analysis. In particular, they can be used as excellent fluorescent probes in the field of fluorescence analysis to achieve high-sensitivity sensing. However, most carbon dots have certain selectivity. For example, in the detection of metal ions, they only react to Fe 3+ It has strong recognition ability; in the detection of acid radical ions, only SCN - It has strong recognition. However, for other metal ions such as Hg 2+ , or acid ions such as Vc, there is still a lack of effective, sensitive and highly selective specific materials.

[0006] On the other hand, in recent years, with the rapid development of computer science, image detection methods have emerged. Image detection has made detection instruments more diversified and miniaturized, greatly improved the convenience and speed of detection, and provided new ideas for simplifying detection methods.

[0007] The present invention combines carbon dots with image detection means to develop a method for detecting mercury ions and vitamin C. Summary of the invention

[0008] Purpose of the invention: In order to overcome the shortcomings of the prior art, the present invention provides a method for detecting mercury ions and vitamin C, specifically a method for continuously detecting mercury ions and vitamin C based on the "on-off-on" fluorescence response mode of carbon dots, that is, first adding Hg to the carbon dot solution 2+ , Hg can be detected by fluorescence quenching 2+ ions; then add Vc into the solution, and the amount of Vc can be detected based on the fluorescence recovery.

[0009] like Figure 1 As shown, the quenching and recovery of carbon dot fluorescence in the present invention is due to Hg 2+ , Vc and carbon dots coordination competition: mercury ions can coordinate with the hydroxyl groups on the surface of the carbon dots, so that the photogenerated electrons jump to the orbit of mercury ions and quench their fluorescence; while vitamin C has a stronger coordination ability with mercury ions than the carbon dots, causing mercury ions to coordinate with vitamin C instead and restore the fluorescence of the carbon dots. This fluorescence response mechanism is called the "on-off-on" mechanism. Through the "on-off-on" phenomenon of fluorescence, the same carbon dot can be used to qualitatively detect two substances; further, through the establishment of a linear relationship between concentration and fluorescence intensity, the same carbon dot can be used to quantitatively detect two substances.

[0010] Technical solution: To achieve the above purpose, the technical solution adopted by the present invention is:

[0011] The first object of the present invention is to provide a fluorescent carbon dot, which has an absorption range of 300 to 400 nm and has a maximum fluorescence intensity under 340 nm excitation; 2+ , Vc have selective specificity respectively.

[0012] Optionally, in some embodiments of the present invention, the fluorescent carbon dots are hollow particles with a particle size of 4 to 10 nm.

[0013] The second object of the present invention is to provide a microwave preparation method of fluorescent carbon dots as described above, comprising the following steps:

[0014] S1, citric acid and urea in molar ratio 柠檬酸 :n 尿素 =1:1~1:4 dissolved in water;

[0015] S2, microwave heating at a power of 500-800 W for 1-7 min, followed by cooling;

[0016] S3, washing with water, centrifuging, taking the supernatant for dialysis, and drying to obtain a black solid, which is the fluorescent carbon dot.

[0017] Optionally, in some embodiments of the present invention, in step S1, the molar ratio of citric acid to urea is n 柠檬酸:n 尿素 =1:2.

[0018] Optionally, in some embodiments of the present invention, in step S1, the concentration of citric acid dissolved in water is 0.02 to 0.08 g / mL.

[0019] Optionally, in some embodiments of the present invention, in step S1, the concentration of citric acid dissolved in water is 0.04 g / mL.

[0020] Optionally, in some embodiments of the present invention, in step S2, microwave heating is performed at a power of 500 to 700 W for 1 to 5 min.

[0021] Optionally, in some embodiments of the present invention, in step S2, microwave heating is performed at a power of 500 W for 1 to 5 minutes, preferably 2 minutes.

[0022] Optionally, in some embodiments of the present invention, in step S3, the drying includes drying at 70-85° C., preferably 80° C. The drying time is not fixed, and the drying time can be as long as the drying is completed.

[0023] The third object of the present invention is to provide a fluorescent carbon dot as described above for detecting Hg 2+ and / or use in Vc.

[0024] Optionally, in some embodiments of the present invention, Hg 2+ The method comprises the following steps: adding a fluorescent carbon dot solution to a test solution, and when the excitation wavelength is 300 to 400 nm, the Hg in the test solution is detected according to the degree of fluorescence intensity reduction of the fluorescent carbon dot solution. 2+ Qualitative / quantitative detection;

[0025] Preferably, the final concentration of the carbon dots in the fluorescent carbon dot solution is 0.03 to 0.06 mg·mL -1 ;

[0026] Preferably, Hg 2+ The detection limit was no higher than 0.15 μM.

[0027] Optionally, in some embodiments of the present invention, the method for detecting Vc comprises the following steps: adding Hg 2+ The solution is quenched, and then the test solution is added, and when the excitation wavelength is 320nm to 420nm, the qualitative / quantitative detection of Vc in the test solution is achieved according to the degree of recovery of the fluorescence intensity of the fluorescent carbon dot solution;

[0028] Preferably, the final concentration of the carbon dots in the fluorescent carbon dot solution is 0.03 to 0.06 mg·mL -1 , Hg 2+The final concentration is 30-70 μM;

[0029] Preferably, the detection limit of Vc is no higher than 0.25 μM.

[0030] Optionally, in some embodiments of the present invention, the Hg content in the solution to be tested is measured according to the change in the fluorescence intensity of the fluorescent carbon dot solution. 2+ And / or qualitative / quantitative detection of Vc, the method for characterizing the fluorescence intensity includes taking a picture and identifying the RGB value of the picture, and converting it into a grayscale value I.

[0031] Optionally, in some embodiments of the present invention, the identifying image RGB value is to directly read the image attributes to obtain the R value, G value, and B value of the image.

[0032] Optionally, in some embodiments of the present invention, the conversion formula between the image RGB value and the grayscale value I is:

[0033] I=0.299R+0.587G+0.114B

[0034] Among them, R, G, and B are the R value, G value, and B value in the RGB value of the image respectively.

[0035] The fluorescence intensity of carbon dots is characterized by grayscale values. Specifically, the camera can capture and identify the RGB value of the image, which can be converted into grayscale value I through the formula I = 0.299R + 0.587G + 0.114B. Through the linear relationship between grayscale value and sample concentration, a grayscale value-concentration standard curve is established, thereby achieving the same experimental purpose as the fluorescence spectrophotometer.

[0036] Beneficial effects: The method for detecting mercury ions and vitamin C provided by the present invention has the following advantages:

[0037] (1) Fluorescent carbon dots are prepared by microwave heating, which is simple, water-soluble, non-toxic, with good fluorescence light stability and high sensitivity. It is combined with photography and image RGB recognition methods to avoid the disadvantages of traditional detection methods, such as the need for large instruments, professionals, and fixed detection sites.

[0038] (2) This method for detecting mercury ions and vitamin C has the advantages of low detection limit, good selectivity, simplicity, rapidity, low cost, and no limitation of experimental conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The reaction mechanism diagram of the present invention is: carbon dots react with Hg 2+ and the “on-off-on” response mechanism of Vc.

[0040] Figure 2 This is a transmission electron microscope image (TEM image) of carbon dots.

[0041] Figure 3 is the UV-visible absorption spectrum of carbon dots.

[0042] Figure 4 It is the fluorescence spectrum of carbon dots.

[0043] Figure 5 It is the Fourier transform infrared spectrum of carbon dots.

[0044] Figure 6 It is composed of carbon dot-intelligent detection system.

[0045] Figure 7 It is the working curve of the carbon dot-intelligent detection system.

[0046] Figure 8 Is the test system for Hg 2+ (A) and Vc (B). DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with the accompanying drawings and Examples. According to the following examples, the present invention can be better understood. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0048] Example 1 Preparation of fluorescent carbon dots

[0049] Specifically, the preparation method of the present invention comprises the following steps:

[0050] S1. Weigh citric acid and urea (n 柠檬酸 :n 尿素 =1:2, m 柠檬酸 =0.40 g), put it into a beaker, add 10 mL of double distilled water, stir and dissolve.

[0051] S2. Place the beaker in a microwave oven, set the power to 500W, and microwave for 2 minutes.

[0052] S3. After the reaction stops and the product cools down, add distilled water to the beaker, stir thoroughly for washing, transfer the solution to a centrifuge tube, centrifuge, and discard the precipitate. The above clear liquid is dialyzed for two hours using a dialysis bag (1000Da) to remove small molecules that have not yet reacted. The obtained liquid is dried in an oven at 80°C to obtain a black solid, which is the fluorescent carbon dots CDs.

[0053] The characterization of fluorescent carbon dots is as follows:

[0054] The prepared fluorescent carbon dots were observed under a transmission electron microscope and it was found that CDs were hollow particles with a particle size of 4 to 10 nm ( Figure 2 ).

[0055] The UV-visible absorption spectrum of fluorescent carbon dots is shown in Figure 3 As shown, the fluorescent carbon dots have a wide absorption range in the range of 300-400nm, which is caused by the electron transition of oxygen-containing groups such as carbonyl on the surface of the fluorescent carbon dots from n→π*.

[0056] From the fluorescence spectrum of fluorescent carbon dots ( Figure 4 ) It can be seen that when the excitation wavelength gradually increases from 300nm to 400nm, the position of the emission peak of the fluorescent carbon dots remains basically unchanged, and the fluorescence intensity shows a trend of first increasing and then decreasing, and the fluorescence intensity is the highest under 340nm excitation.

[0057] From the Fourier transform infrared spectra of fluorescent carbon dots ( Figure 5 ) It can be seen that its surface contains a variety of functional groups, such as C=O, -OH, COC, etc. 3442cm -1 The stretching vibration absorption peak attributed to -OH is 3130 cm -1 The stretching vibration absorption peak of NH is 1717 cm -1 、1615cm -1 、1269cm -1 The absorption peaks at the two positions correspond to the stretching vibrations of the carbonyl group (C=O) and COC, respectively.

[0058] Example 2 Working curve drawing

[0059] The present invention combines picture recognition technology, utilizes camera tools (such as smart phones, or other picture shooting tools) and image recognition technology to form a picture recognition system, converts fluorescent RGB values ​​into grayscale values, and then draws a standard curve of the concentration of the analyte to achieve fast and convenient detection.

[0060] The detection system adopts Figure 6 In the device, a 365nm portable ultraviolet lamp replaces the ultraviolet-visible spectrophotometer, and an image recognition system replaces the fluorescence spectrophotometer. The detection equipment of the present invention is convenient and fast, and gets rid of the disadvantage of having to use large laboratory instruments.

[0061] (1) Draw the mercury ion working curve

[0062] A mixed solution of the fluorescent carbon dots prepared in Example 1 and a series of gradient concentrations of mercuric nitrate was prepared, wherein the final concentration of the fluorescent carbon dots was 0.05 mg·mL -1 , the final concentrations of mercuric nitrate were 0, 10, 15, 20, 25, and 30 μM, respectively.

[0063] Take 10 μL of each of the above series of mixed solutions, and add them dropwise from left to right into six wells in a row of the well plate with increasing mercury ion concentrations ( Figure 6 ). A 365nm portable ultraviolet lamp was used for irradiation, and a smartphone was used to take photos. The photos were read and the RGB values ​​were identified. The gray value I was obtained by the conversion formula I = 0.299R + 0.587G + 0.114B, and a standard working curve of gray value I and mercuric nitrate concentration was drawn.

[0064] Figure 7 (A) and Table 1 are the carbon dots for determination of Hg 2+ (R 2 =0.991) working curve, the linear range and detection limit of this method were determined, and the results are as follows: 2+ The linear range is 10-30μM, and the detection limit is 0.138μM, which is much lower than the standard value (4.985μM) in the relevant national document "Technical Specifications for Safety of Cosmetics".

[0065] Table 1 Hg determination by carbon dots 2+ Working curve

[0066] Detection object Linear range (μM) Correlation coefficient Detection limit (μM) <![CDATA[Hg 2+ ]]> 10.0～30.0 <![CDATA[R 2 =0.991]]> 0.138

[0067] (2) Draw the Vc working curve

[0068] First, prepare carbon dots-Hg 2+ solution, and then added different gradient concentrations of vitamin C solution (carbon dots and Hg 2+ The final concentrations were 0.05 mg mL -1 and 50 μM, the final concentrations of vitamin C were 0, 10, 20, 30, 40, 50 μM, respectively). Take 10 μL of each of the above series of mixed solutions and draw a standard curve using the same method.

[0069] Figure 7 (B) and Table 2 are the carbon dots for determination of Vc(R 2 =0.993) working curve, the linear range and detection limit of the method were determined, and the results were as follows: the linear range of Vc detection was 10-50 μM, and the detection limit was 0.212 μM.

[0070] Table 2 Hg determination by carbon dots 2+ Working curve with Vc

[0071] Detection object Linear range (μM) Correlation coefficient Detection limit (μM) Vc 10.0～50.0 <![CDATA[R 2 =0.993]]> 0.212

[0072] Example 3 Detection system for Hg 2+ Selectivity

[0073] This example is intended to study the detection system of the present invention for Hg 2+ The specific method is as follows:

[0074] The following metal ions (Al 3+ Mg 2+ ,Ca 2+ ,Zn 2+ ,Cd 2+ ,Ba 2+ ,Co 2+ ,Pb 2+ ,Cu 2+ ,Ni 2 + ,Mn 2+ ,Ag + ,Fe 3+ ,Hg 2+ ) were added to a concentration of 0.05 mg mL -1 The fluorescence intensity of the solution was tested in the carbon dot solution and compared with the blank group.

[0075] Test system for Hg 2+ The selective results are as follows Figure 8 As shown in (A), it can be seen from the figure that compared with the control group, Hg 2+ The fluorescence intensity of the carbon dot solution was significantly reduced, while other ions had little effect on the fluorescence intensity of the carbon dot solution. 2+ The fluorescence of carbon dots can be quenched, and the detection system is sensitive to Hg 2+ There is good selectivity.

[0076] Example 4 Selectivity of the detection system for Vc

[0077] This example is intended to study the selectivity of the detection system of the present invention for Vc, and the specific method is as follows:

[0078] In order to observe the above solution (CDs-Hg 2+ ) in the fluorescence recovery of carbon dots in CDs-Hg 2+ Different substances (tyrosine Try, leucine Leu, proline Pro, glutamic acid Glu, glycine Gly, arginine Arg, lysine Lys, methionine Met, glutamine Gln, ascorbic acid Vc) were added to the solution at a concentration of 50 μM, and the fluorescence intensity of the solution was tested.

[0079] The results of the test system's selectivity for Vc are as follows Figure 8 As shown in (B), it can be seen from the figure that only Vc can significantly restore CDs-Hg 2+ The fluorescence of the solution, while other substances 2+ The fluorescence of the system is almost unaffected. This experimental study shows that the detection system has good selectivity for Vc.

[0080] Example 5 Test system for Hg 2+ Spike recovery test.

[0081] This example is intended to study the detection system of the present invention for Hg 2+ The specific method is as follows:

[0082] The different concentrations of Hg 2+ (10 μM, 30 μM) were added to a concentration of 0.05 mg mL -1 The carbon dot solution (CDs solution as blank control) was dropped into the well plate, and then the well plate was placed in a dark box, photographed and identified, and the concentration was calculated according to the standard curve to calculate Hg 2+ The recovery rate.

[0083] The detection system of the present invention is effective for Hg 2+ The results of the spike recovery are shown in Table 3. It can be seen from Table 3 that the system has a good 2+ The recoveries of (10μM, 30μM) were 97.8% and 107.5% respectively, and the relative standard deviations were 1.5% and 0.73% respectively. 2+ The detection has high accuracy and can be used for Hg 2+ Detection.

[0084] Table 3 The detection system of the present invention for Hg 2+ The spike recovery

[0085]

[0086] Example 6 Test system for the recovery rate of spiked Vc.

[0087] This example is intended to study the recovery rate of Vc by the detection system of the present invention, and the specific method is as follows:

[0088] Different concentrations of Vc (20 μM, 50 μM) were added to CDs-Hg 2+ The solution was dropped into the well plate (CDs-Hg 2+ The solution was used as a blank control), and then the well plate was placed in a dark box, photographed and identified, and the concentration was calculated according to the standard curve to calculate the recovery rate of Vc.

[0089] The results of the spike recovery rate of Vc by the detection system of the present invention are shown in Table 4. It can be seen from Table 4 that the spike recovery rates of Vc (20μM, 50μM) by the system are 106.3% and 103.5% respectively, and the relative standard deviations are 0.9% and 0.6% respectively. This experimental study shows that the test system has a high accuracy in the detection of Vc and can be used for the detection of Vc.

[0090] Table 4 The recovery rate of Vc by the detection system of the present invention

[0091]

[0092] Example 7 Continuous Detection of Mercury Ions and Vitamin C by Fluorescent Carbon Dots

[0093] This example is intended to study the use of the fluorescent carbon dots of the present invention for continuous detection of mercury ions and vitamin C. The specific method is as follows:

[0094] Hg 2+ solution (10 μM) was added to the carbon dot solution (0.05 mg mL -1 ), take 10 μL and drop it into the well plate, then place the well plate in a dark box, take a photo, identify it, and calculate Hg according to the standard curve 2+ concentration.

[0095] Add Vc solution (50 μM) to the above solution, take 10 μL and drop it into the well plate, place it in a dark box, take pictures, identify it, and calculate the concentration of Vc according to the standard curve.

[0096] The detection results of mercury ions and Vc by the detection system of the present invention are shown in Table 5, from which it can be seen that the detection system has no significant effect on Hg 2+ The relative errors of mercury ions and vitamin C detection were -2.20% and 3.42% respectively. This experimental study shows that fluorescent carbon dots can realize continuous detection of mercury ions and vitamin C through the "on-off-on" fluorescence response mode.

[0097] Table 5 The detection system of the present invention for Hg 2+ Continuous detection of Vc

[0098] Test samples Sample concentration (μM) Mobile phone detection concentration (n=5,μM) Relative error (%) <![CDATA[Hg 2+ ]]> 10 9.78±0.15 -2.20％ Vc 50 51.71±0.36 3.42％

[0099] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fluorescent carbon dot, characterized in that: The fluorescent carbon dots have an absorption range of 300 to 400 nm, and the fluorescence intensity is the highest under 340 nm excitation; the ... 2+ , Vc have selective specificity respectively.

2. A fluorescent carbon dot according to claim 1, characterized in that: The fluorescent carbon dots are hollow particles with a particle size of 4 to 10 nm.

3. The microwave preparation method of fluorescent carbon dots according to claim 1 or 2, characterized in that: The following steps are involved: S1, citric acid and urea in molar ratio 柠檬酸 :n 尿素 =1:1~1:4 dissolved in water; S2, microwave heating at a power of 500-800 W for 1-7 min, followed by cooling; S3, washing with water, centrifuging, taking the supernatant for dialysis, and drying to obtain a black solid, which is the fluorescent carbon dot.

4. The method according to claim 3, characterized in that In step S1, the concentration of citric acid dissolved in water is 0.02 to 0.08 g / mL.

5. The method according to claim 3, characterized in that: In step S2, microwave heating is performed at a power of 500 to 700 W for 1 to 5 minutes.

6. The method according to claim 3, characterized in that In step S3, the drying includes drying at 70-85° C. until black dry carbon dot solid particles are obtained.

7. A fluorescent carbon dot according to claim 1 or 2 for detecting Hg 2+ and / or use in Vc.

8. The use according to claim 7, characterized in that Detection of Hg 2+ The method comprises the following steps: adding a fluorescent carbon dot solution to a test solution, and when the excitation wavelength is 300 to 400 nm, the Hg in the test solution is detected according to the degree of fluorescence intensity reduction of the fluorescent carbon dot solution. 2+ Qualitative / quantitative detection; Preferably, the final concentration of the carbon dots in the fluorescent carbon dot solution is 0.03 to 0.06 mg·mL -1 ; Preferably, Hg 2+ The detection limit was no higher than 0.15 μM.

9. The use according to claim 7, characterized in that: The method for detecting Vc comprises the following steps: adding Hg 2+ The solution is quenched, and then the test solution is added, and when the excitation wavelength is 300nm to 400nm, the qualitative / quantitative detection of Vc in the test solution is achieved according to the degree of recovery of the fluorescence intensity of the fluorescent carbon dot solution; Preferably, the final concentration of the carbon dots in the fluorescent carbon dot solution is 0.03 to 0.06 mg·mL -1 , Hg 2+ The final concentration is 30-70 μM; Preferably, the detection limit of Vc is no higher than 0.25 μM.

10. The use according to any one of claims 7 to 9, characterized in that: The Hg content in the solution to be tested is determined by the change in the fluorescence intensity of the fluorescent carbon dot solution. 2+ and / or qualitative / quantitative detection of Vc, the fluorescence intensity characterization method includes taking a picture and identifying the RGB value of the picture, and converting it into a grayscale value I; Preferably, the conversion formula between the image RGB value and the grayscale value I is: I=0.299R+0.587G+0.114B Among them, R, G, and B are the R value, G value, and B value in the RGB value of the image respectively.

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