Preparation method and application of fluorescence enhanced sensor based on porphyrin-based carbon dots

Abstract

The invention relates to the technical field of analysis and detection, in particular to a preparation method and application of a fluorescence enhanced sensor based on porphyrin-based carbon dots, and the specific technical scheme is as follows: the preparation method comprises the following steps: (1) dissolving 2-aminoterephthalic acid in DMF; (2) dissolving the TAPP in ultrapure water; (3) mixing the solution obtained in the step (1) and the solution obtained in the step (2), stirring at normal temperature and normal pressure, and then carrying out hydrothermal reaction at 200 DEG C for 2 hours to obtain TAPP-CDs; and (4) filtering the TAPP-CDs solution by using a microfiltration membrane, and dialyzing the filtrate to obtain the porphyrin-based carbon dots. The fluorescence enhanced sensor constructed by the invention has a good enrichment effect on the aflatoxin B2, has high sensitivity, selectivity and anti-interference performance, can accurately detect and analyze the aflatoxin B2 in an actual grain sample, and has the advantages of simple operation, low cost, high sensitivity and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical detection technology, and particularly relates to a preparation method and application of a fluorescence enhancement type sensor based on a porphyrin-based carbon dot, in particular to a fluorescence enhancement type sensor constructed by using a porphyrin-based carbon dot of 5,10,15,20-tetra(4-aminophenyl)porphyrin modified by 2-amino terephthalic acid. BACKGROUND

[0002] Aflatoxin B2 (AFB2) is a toxin produced by Aspergillus flavus, which belongs to aflatoxins. It is a white crystalline solid, insoluble in water, soluble in alcohol and fatty solvents. In addition to the common aflatoxin AFB2, there are also aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1). AFB2 has the characteristics of strong stability, and can remain relatively stable at high temperatures. AFB2 has certain toxicity to humans and animals, and can cause various poisoning symptoms and health problems. Long-term exposure or ingestion of high concentrations of AFB2 can have adverse effects on the liver, immune system and nervous system. Therefore, it is urgent to develop a simple, rapid and accurate analysis method to realize the high sensitivity and strong specificity detection of AFB2 generated by early moldy food.

[0003] Generally, chromatographic analysis, enzyme-linked immunosorbent assay, high-performance liquid chromatography and other traditional methods are used to detect mycotoxins. Although they have the characteristics of high sensitivity and high accuracy, these techniques are time-consuming, high-cost and require high-precision instruments or sample preparation. For example, some people have used aptamer affinity columns to selectively extract AFB2. The amino-modified aptamer for AFB2 is covalently immobilized on CNBr-activated Sepharose. The extraction recovery rate is evaluated by HPLC and fluorescence detection. Under the optimal conditions, the linear response range of the aptamer affinity column to AFB2 is 0.5-80 ng, and the capacity is 84.6 ng. The method uses spiked peanut samples (0.5-50 μg·kg -1 AFB2), the average recovery rate is 80.9%, and the average relative standard deviation is 1.9%. The detection limit is 25 pg·mL -1 However, it is still difficult to meet the rapid, portable and accurate detection of AFB2 in food. SUMMARY

[0004] In view of the deficiencies of the prior art, the application provides a preparation method and application of a fluorescence-enhanced sensor based on porphyrin-based carbon dots, which uses porphyrin-based carbon dots of 2-amino terephthalic acid modified 5,10,15,20-tetrakis(4-aminophenyl) porphyrin to construct a fluorescence-enhanced sensor, and the sensor has high sensitivity, selectivity and anti-interference performance, and can be used for accurate detection and analysis of aflatoxin B2 in actual grain samples (peanuts, corn, dried chili, etc.).

[0005] To achieve the above object, the application is implemented by the following technical solutions:

[0006] The application discloses a preparation method of porphyrin-based carbon dots, comprising the following steps:

[0007] (1) Dissolve 2-amino terephthalic acid in DMF;

[0008] (2) Dissolve 5,10,15,20-tetrakis(4-aminophenyl) porphyrin in ultrapure water;

[0009] (3) Mix the solutions of steps (1) and (2) and stir at normal temperature and pressure, and then perform hydrothermal reaction at 200 DEG C for 2h to obtain 5,10,15,20-tetrakis(4-aminophenyl) porphyrin carbon dots;

[0010] (4) Filter the 5,10,15,20-tetrakis(4-aminophenyl) porphyrin carbon dot solution with a microporous filter membrane, and then dialyze the filtrate to obtain porphyrin-based carbon dots.

[0011] Preferably, the mass ratio of the 2-amino terephthalic acid to the 5,10,15,20-tetrakis(4-aminophenyl) porphyrin is 1:10-20.

[0012] Preferably, the mass-volume ratio of the 2-amino terephthalic acid to DMF is 1:1900-2100, and the mass-volume ratio of the 5,10,15,20-tetrakis(4-aminophenyl) porphyrin to ultrapure water is 1:200-300.

[0013] Correspondingly, the porphyrin-based carbon dots prepared by the preparation method have a spherical morphology, a particle size of 3-5nm, and emit blue fluorescence.

[0014] Correspondingly, a fluorescence-enhanced sensor constructed by using the porphyrin-based carbon dots.

[0015] Correspondingly, the application of the fluorescence-enhanced sensor constructed by using the porphyrin-based carbon dots in detection of aflatoxin B2.

[0016] Preferably, the application of the fluorescence-enhanced sensor in detection of aflatoxin B2 in grains.

[0017] Preferably, the detection limit of aflatoxin B2 is 1x10 -11 mol / L.

[0018] Preferably, the volume ratio of aflatoxin B2 to the fluorescence-enhanced sensor is 1:3-3:1.

[0019] Correspondingly, a visual paper-based sensor is constructed by loading the fluorescence-enhanced sensor on a portable paper-based chip.

[0020] The present application has the following beneficial effects:

[0021] The present application uses a fluorescence-enhanced sensor constructed by 2-amino terephthalic acid modified 5,10,15,20-tetrakis(4-aminophenyl) porphyrin porphyrin-based carbon dots, thereby having a good enrichment effect on aflatoxin B2, and the detection limit is as low as 1x10 -11 mol / L. The fluorescence sensor has high sensitivity and selectivity and anti-interference performance. Compared with traditional sensors, it has many advantages such as easy availability of raw materials, low cost, high selectivity, and can be used for qualitative and quantitative analysis of aflatoxin B2, and can accurately detect aflatoxin B2 in actual grain samples (peanuts, corn, dried chili, etc.), and has the advantages of simple operation, low cost, high sensitivity, etc. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 A fluorescence-enhanced sensor constructed by 2-amino terephthalic acid modified 5,10,15,20-tetrakis(4-aminophenyl) porphyrin porphyrin-based carbon dots for detecting aflatoxin B2 method mechanism diagram;

[0023] Figure 2 TEM diagram of porphyrin-based carbon dots (TAPP-CDs);

[0024] Figure 3 Fluorescence-enhanced sensor after adding AFB2 fluorescence enhancement effect diagram;

[0025] Figure 4 Fluorescence-enhanced sensor for accurate quantitative analysis of AFB2 diagram; (A) fluorescence enhancement diagram after adding TAPP-CDs to different concentrations of AFB2; (B) AFB2 quantitative curve with a concentration of 1x10 -11 -10 -8 mol / L; (C) AFB2 quantitative curve with a concentration of 1x10 -8 -3x10 -7 mol / L;

[0026] Figure 5(A) pH stability test chart of the fluorescence sensor at pH 6-12 range; (B) anti-interference test chart of the sensor with common metal ions and amino acids at high concentration (1 x 10 - 7 Common metal ions and amino acids at high concentration (1 x 10

[0027] Figure 6 To verify the selectivity of the fluorescence-enhanced sensor to AFB2, AFB1, AFG1, T-2 toxin, zearalenone, ochratoxin and deoxynivalenol were added to the detection system to test the fluorescence response of the sensor

[0028] Figure 7 The fluorescence comparison bar chart of the sensor for actual sample grains (peanuts, corn, dried chili, raisins, wheat); the sensor has good stability and can realize accurate detection of AFB2 in complex food;

[0029] Figure 8 The 96-well plate chart of the fluorescence-enhanced sensor for visualizing AFB2 under ultraviolet light; (A) AFB2 different concentration gradient chart; (B) selectivity chart for other types of mycotoxins; the fluorescence sensor has excellent fluorescence visualization performance;

[0030] Figure 9 The performance test chart of the visualized paper-based sensor. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0032] If not specifically indicated, the technical means used in the examples is the conventional means known to those skilled in the art.

[0033] The detection mechanism of the fluorescence-enhanced sensor constructed by the porphyrin-based carbon dots modified by 2-amino terephthalic acid is that: the porphyrin-based carbon dots are synthesized by a simple aqueous phase synthesis method, and AFB2 can form hydrogen bonds with the amino groups on the surface of the porphyrin-based carbon dots, so that the porphyrin-based carbon dots are aggregated on the surface of the carbon dots, and the fluorescence intensity of the porphyryl carbon dots is enhanced. Finally, a strategy with high sensitivity and selectivity is realized, and AFB2 in food is detected by constructing a fluorescence-enhanced sensor. At the same time, this strategy is successfully applied to it which can be attached to paper for visualization. The fluorescence-enhanced sensor constructed in the application combines the large pi conjugated system of porphyrin and the nano characteristics of carbon dots, and is modified by covalent bond or coordination, to form a hybrid system with multiple functional groups.

[0034] Specific schemes are as follows:

[0035] 1. The application provides a preparation method of porphyrin-based carbon dots, comprising the following steps:

[0036] (1) dissolving 2-amino terephthalic acid in DMF;

[0037] (2) dissolving 5,10,15,20-tetra(4-aminophenyl) porphyrin in ultrapure water;

[0038] (3) mixing the solutions of steps (1) and (2) and stirring at normal temperature and pressure, and then hydrothermally reacting at 200 DEG C for 2h to obtain 5,10,15,20-tetra(4-aminophenyl) porphyrin carbon dots;

[0039] (4) filtering the 5,10,15,20-tetra(4-aminophenyl) porphyrin carbon dot solution with a microporous filter membrane, and then dialyzing the filtrate to obtain the porphyrin-based carbon dots.

[0040] In the application, the mass ratio of the 2-amino terephthalic acid to the 5,10,15,20-tetra(4-aminophenyl) porphyrin is 1:10-20, the mass-volume ratio of the 2-amino terephthalic acid to DMF is 1:1900-2100, and the mass-volume ratio of the 5,10,15,20-tetra(4-aminophenyl) porphyrin to ultrapure water is 1:200-300.

[0041] 2. The porphyrin-based carbon dots prepared by the preparation method have a spherical shape, a particle size of 3-5nm, and blue fluorescence.

[0042] 3. The application provides a fluorescence-enhanced sensor constructed by the porphyrin-based carbon dots.

[0043] 4.The application provides application of the fluorescent enhanced sensor constructed by the porphyrin-based carbon dots in detection of aflatoxin B2, and a mechanism diagram is shown in Figure 1 Especially, the application in detection of aflatoxin B2 in grains. The grains include but are not limited to peanuts, corns, dried peppers, raisins and wheat. In the detection of aflatoxin B2, the detection limit is 1x10 -11 mol / L. In the use of the fluorescent enhanced sensor, the fluorescent enhanced sensor needs to be diluted, the concentration of the porphyrin-based carbon dots can be appropriately adjusted according to the concentration range of the aflatoxin B2 to be detected, and the volume ratio of the aflatoxin B2 to the fluorescent enhanced sensor after dilution of the fluorescent enhanced sensor is 1:3-3:1.

[0044] 5.The application provides a visual paper-based sensor, which is constructed by loading the fluorescent enhanced sensor on a portable paper-based chip.

[0045] The technical solutions of the application are further described in detail below by taking aflatoxin B2, a mycotoxin small molecule pure substance and a small molecule compound in a grain substrate as examples in combination with the drawings.

[0046] The reagents used in the following examples are shown in Table 1.

[0047] Table 1 Experimental reagents

[0048]

[0049]

[0050] Example 1

[0051] The steps of synthesizing the fluorescent enhanced sensor of the porphyrin-based carbon dots constructed by 2-amino terephthalic acid modified 5,10,15,20-tetrakis(4-aminophenyl) porphyrin are as follows:

[0052] (1) Synthesis of porphyrin-based carbon dots by a hydrothermal method

[0053] 0.005g of 2-amino terephthalic acid is dissolved in 10ml of DMF; 0.08g of 5,10,15,20-tetrakis(4-aminophenyl) porphyrin (TAPP) is dissolved in 20ml of ultrapure water, the two solutions are mixed and stirred at normal temperature and pressure for 15min, and then quickly placed in a reaction kettle, and hydrothermal reaction is carried out at 200℃ for 2h to obtain a brownish-brown solution, i.e., porphyrin-based carbon dots (TAPP-CDs), and a TEM diagram is shown in Figure 2 .

[0054] (2) The TAPP-CDs solution was filtered by a 0.22 μm microporous filter, and then the filtrate was transferred to a dialysis bag with a molecular weight of 3500 Da for dialysis. The water was changed every 6 h, and the dialysis lasted for 24 h. The obtained TAPP-CDs solution was diluted 100 times with ultrapure water as a stock solution and stored in a refrigerator at 4°C.

[0055] Example 2

[0056] Example 1 uses a fluorescent enhanced sensor constructed by porphyrin-based carbon dots for the identification of AFB2.

[0057] (I) Detection of AFB2

[0058] It should be specifically pointed out that: the first group: water (900 μL), TAPP-CDs (100 μL); the second group: water (870 μL), TAPP-CDs (100 μL) and AFB2 (30 μL, 1×10 -6 mol / L).

[0059] After adding the last reaction material, the timing started, and the fluorescence emission spectrum of the system was recorded after 5 min. By extracting the fluorescence peaks between 350 nm and 500 nm in the above fluorescence spectrum, the effect diagram of the porphyrin-based carbon dots after adding AFB2 is as shown in Figure 3 , the black curve is the original fluorescence intensity of TAPP-CDs carbon dots without AFB2, and the red curve is the fluorescence intensity after adding AFB2, which enhances the fluorescence effect of porphyrin-based carbon dots.

[0060] Then, different concentrations (1×10 -11 , 1×10 -10 , 1×10 -9 , 1×10 -8 , 3×10 -8 , 5×10 -8 , 7×10 -8 , 9×10 -8 , 1×10 -7 , 2×10 -7 , 3×10 -7 mol / L) of AFB2 were added for detection, as shown in Figure 4 (A), and the data were linearly fitted and analyzed. The quantitative curve of the concentration and the fluorescence response signal of the porphyrin-based carbon dots is shown in Figure 4 (B-C). There was a fluorescence response at a concentration of 1×10 -11 to 1×10 -8 mol / L, and the fluorescence intensity showed a linear relationship with the logarithm of aflatoxin B2, while the fluorescence intensity showed a linear relationship with the logarithm of aflatoxin B2 at a concentration of 1×10 -8 to 3×10 -7Within the mol / L concentration range, a clear linear relationship was observed between fluorescence intensity and aflatoxin B2 concentration.

[0061] (II) Stability and anti-interference performance of fluorescence-enhanced sensors

[0062] To ensure the stability of the fluorescence-enhanced sensor, this invention first tested the sensor's fluorescence response under different pH conditions. A buffer solution (NaOH-HCl mixture) with pH 6-12 was prepared, namely the third group: pH buffer (800 μL), TAPP-CDs (100 μL), and AFB2 (100 μL, 1×10⁻⁶). -7 (mol / L). The results are as follows: Figure 5 As shown in (A), the porphyrin-based carbon dot fluorescence-enhanced sensor can maintain good fluorescence response stability in the pH range of 6-12.

[0063] Subsequently, to test the anti-interference performance of the fluorescence-enhanced sensor for AFB2 detection, a high concentration (1×10⁻⁶) was prepared. - 7 Common metal ions and amino acids (Mg) at concentrations of mol / L 2+ Na + K + Ca 2+ Fe 3+ (Gly, Glu, Ala) were used as interfering components added to the detection system. For example... Figure 5 As shown in (B), even with the addition of high concentrations of interfering components to the detection system, the sensor can still produce a stable fluorescence response to AFB2.

[0064] (III) Selectivity of Fluorescence-Enhanced Sensors

[0065] The control experimental group consisted of water (800 μL), TAPP-CDs (100 μL), and AFB2 (100 μL, 1×10⁻⁶). -6 mol / L). In 100 μL, 1×10 -6 Based on mol / L AFB2, add 100 μL, 1×10 -6 Other mycotoxins at the same concentration (mol / L). The mycotoxins identified in this example include: AFB1, AFG1, T-2 toxin, zearalenone (ZEA), ochratoxin (OTA), and deoxynivalenol (DON), all prepared at a concentration of 1×10⁻⁶ mol / L. -6 Used at mol / L. Other mycotoxin selectivity comparison bar charts are shown below. Figure 6 As shown, even similar AFB1 and AFG1 do not significantly enhance fluorescence, indicating that the fluorescence-enhanced sensor constructed in this invention has good selectivity.

[0066] Example 3

[0067] The fluorescence-enhanced sensor based on the porphyrin-based carbon dots constructed in the present application can realize the identification of AFB2 small molecule compounds in 10 -11 mol / L of the grain matrix. In the above step (I) of Example 2, the different concentrations of AFB2 are replaced with peanuts, corn, dried chili, raisins, and wheat containing AFB2. The grains are ground and soaked, and are respectively prepared into a concentration of 10 -9 mol / L, 10 -10 mol / L, and 10 -11 mol / L.

[0068] Similarly, after adding the last reaction raw material, the timing starts, and the fluorescence emission spectrum of the system is recorded after 5 min. All data are repeated 3 times. The above fluorescence comparison bar chart is shown in Figure 7 Table 1. By adding different concentrations of AFB2 in the actual sample (peanuts, corn, dried chili, raisins, and wheat), the spiked sample is prepared, and the recovery rate of AFB2 is used as an evaluation index to determine the accuracy of quantitative analysis. The results are shown in Table 2. Even in real food samples, the sensor can still achieve accurate detection of AFB2, and the recovery rate is between 98.44% and 101.42%.

[0069] Table 2 Recovery rate of porphyrin-based carbon dot fluorescence sensor for detection of AFB2 in real food samples

[0070]

[0071] Example 4

[0072] The fluorescence-enhanced sensor constructed in the present application is used to drop the sensor on the 96-well plate after the sensor is added to different concentrations (1×10 -12 , 1×10 -11 , 1×10 -10 , 1×10 -9 , 1×10 -8 , 3×10 -8 , 5×10 -8 , 7×10 -8 , 9×10 -8 , 1×10 -7 , 2×10 -7 , 3×10 -7 mol / L) of AFB2 and other kinds of mycotoxins (mycotoxins disclosed in step (III) of Example 2), and is placed under the irradiation of the ultraviolet lamp. The visual color development result chart is shown in Figure 8 Figure 8 ​In A, the constructed fluorescence-enhanced sensor based on porphyrin-based carbon dots has good sensitivity, Figure 8 In B, the sensor has good selectivity to other kinds of mycotoxins, and the results show that the brightness of AFB2 is the highest. After the fluorescence-enhanced sensor based on porphyrin-based carbon dots is dropped on a 96-well plate, visual detection of AFB2 can be realized.

[0073] Example 5

[0074] The fluorescence-enhanced sensor constructed in the application has excellent fluorescence visualization performance, so the visual detection performance of the sensor on AFB2 is further studied. The sensor is loaded on a portable paper-based chip (which can be understood as a paper sheet) to construct a visual paper-based sensor, and the visual color development result of the sensor on AFB2 is as shown in Figure 9 After being loaded on the paper-based chip, the fluorescence sensor still has good selectivity and sensitivity. When the concentration of AFB2 added is 1x10 -12 mol / L, obvious color change phenomenon can be produced. After the porphyrin-based carbon dot fluorescence sensor is loaded on the portable paper-based chip, visual detection of AFB2 can be realized.

[0075] It can be seen that the fluorescence-enhanced sensor constructed by using the porphyrin-based carbon dots of 2-amino terephthalic acid modified 5,10,15,20-tetra(4-aminophenyl) porphyrin can realize recognition of AFB2 in food, and has high sensitivity and high selectivity.

[0076] The above-described examples only describe the preferred modes of the application, and do not limit the scope of the application. Without departing from the design spirit of the application, various modifications and improvements to the technical solutions of the application made by those skilled in the art shall fall within the protection scope determined by the claims of the application.

Claims

1. A method for preparing porphyrin-based carbon dots, characterized in that: The method comprises the following steps: (1) dissolving 2-amino terephthalic acid in DMF; (2) dissolving 5,10,15,20-tetrakis(4-aminophenyl) porphyrin in ultrapure water; (3) mixing the solutions of step (1) and step (2) and stirring at normal temperature and pressure, and then hydrothermally reacting at 200℃ for 2h to obtain 5,10,15,20-tetrakis(4-aminophenyl) porphyrin carbon dots; (4) filtering the solution of 5,10,15,20-tetrakis(4-aminophenyl) porphyrin carbon dots with a microporous filter membrane, and then dialyzing the filtrate to obtain porphyrin-based carbon dots.

2. The method of claim 1, wherein: The mass ratio of the 2-amino terephthalic acid to the 5,10,15,20-tetrakis(4-aminophenyl) porphyrin is 1:10-20.

3. The method of manufacturing according to claim 2 or 3, characterized in that: The mass-volume ratio of the 2-amino terephthalic acid to DMF is 1:1900-2100, and the mass-volume ratio of the 5,10,15,20-tetrakis(4-aminophenyl) porphyrin to ultrapure water is 1:200-300.

4. The porphyrin-based carbon dots prepared by the method of any one of claims 1-3, characterized in that: The porphyrin-based carbon dots have a spherical morphology, a particle size of 3-5nm, and emit blue fluorescence.

5. A fluorescence-enhanced sensor constructed by using the porphyrin-based carbon dots of claim 4.

6. Application of the fluorescence-enhanced sensor constructed by using the porphyrin-based carbon dots of claim 5 in detection of aflatoxin B2.

7. Use according to claim 6, characterized in that: Application of the fluorescence-enhanced sensor in detection of aflatoxin B2 in grains.

8. Use according to claim 6 or 7, characterized in that: The detection limit for aflatoxin B2 was 1 x 10 -11 mol / L.

9. Use according to claim 8, characterized in that: The volume ratio of the aflatoxin B2 to the fluorescence-enhanced sensor is 1:3-3:

1.

10. A visual paper-based sensor characterized by: A visual paper-based sensor is constructed by loading the fluorescence-enhanced sensor of claim 5 on a portable paper-based chip.

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