A Co-MOF@PDA / GRE Electrochemical Sensor and Its Preparation Method and Application

Abstract

The present invention discloses a Co-MOF@PDA / GRE electrochemical sensor, its preparation method and application. The method includes steps such as preparing the Co-MOF@PDA composite material, preparing the Co-MOF@PDA composite film modified electrode, and detecting L-tryptophan. The method of the present invention can quickly detect the concentration of L-tryptophan, and has high sensitivity and high accuracy. The present invention constructs a related sensing interface as a three-electrode system sensor for detection, uses potential for qualitative analysis of tryptophan configuration, and uses the peak current value for quantification, improving the sensitivity and accuracy of detecting L-tryptophan, and can detect L-tryptophan as low as 4.14×10-7 mol / L at least. Experiments using living plants show that the MOF-modified sensor has biological safety for cells and can detect L-Trp in living plants. The method of the present invention greatly reduces the detection cost and is simple and convenient to operate.

Description

Technical Field

[0001] The present invention belongs to the field of electroanalytical chemistry, and particularly relates to the development and application of an electrochemical sensor, specifically a preparation method and application of a Co-MOF@PDA / GRE micro-electrochemical sensor. Background Art

[0002] Tryptophan, with the chemical name of α-amino-β-indolepropionic acid, has three isomers: L-type, D-type, and racemic DL-type. Among them, L-tryptophan (L-Trp) plays an important role in promoting plant growth and development, and its important metabolite auxin directly affects the growth and development of plants. Moreover, the nitrogen released during the conversion of tryptophan into secondary metabolites can greatly improve the productivity of plants. Under many stress conditions, tryptophan and its metabolites can act synergistically with other hormones to regulate the plant's ability to resist stress. The application of exogenous tryptophan plays an important role in the development of the agricultural field. Therefore, realizing the in-vivo real-time detection of tryptophan content in plants will play an important role in studying the growth status of plants, realizing precise plant cultivation, and regulating the growth and development of various plant organs. Recently, various methods have been used for the measurement of tryptophan, including spectrophotometry, liquid chromatography, gas chromatography-mass spectrometry, spectral detection, etc. These methods have reliable and effective characteristics and are widely used in bioanalysis. However, there are still some disadvantages in using these methods, such as long detection time, expensive equipment, and complex analysis routes.

[0003] In recent years, compared with the above methods, electrochemical analysis is considered a green, highly sensitive, and low-cost method for detecting small biomolecules. In addition, due to the double bond of indole, tryptophan is easily oxidized to form a carbon-nitrogen double bond through an electrochemical pathway. Therefore, electrochemical methods are usually used for the detection of tryptophan. However, the voltammetric response of L-Trp is not satisfactory because of the slow and non-uniform electron transfer on the electrode. This problem can be overcome by chemically modified electrodes, such as carbon paste electrodes modified with potato juice and electrodes modified with heme. However, the stability and repeatability of these modified electrodes are poor, and the types of modified electrodes used for L-Trp determination are still very limited. Therefore, it is very meaningful to find new electrode materials for L-Trp determination.

[0004] In addition, the key to realizing the in-vivo real-time detection of tryptophan in plants by electrochemical methods lies in the construction of a micro-electrochemical sensing system. The construction of a micro-electrochemical sensing system not only enables the in-vivo detection of small molecules in plants but also provides important technical support for portable electrochemical detection workstations and the detection of trace samples. Based on the above background, the present invention proposes a Co-MOF@PDA-modified micro-electrochemical sensing system and its application to the in-vivo detection of tryptophan content in living plants. Summary of the invention

[0005] The present invention aims to overcome the deficiencies of the prior art and provides a method for detecting the concentration of L-tryptophan in a solution, which can quickly and quantitatively detect the content of L-tryptophan in the solution, and establishes a simple, rapid, sensitive and accurate L-tryptophan detection method.

[0006] Another object of the present invention is to provide a micro electrochemical sensor that can measure the tryptophan concentration in living plants, which can achieve long-term, stable response and high-accuracy in vivo detection of living plants.

[0007] The purpose of the present invention and the technical problem to be solved are achieved by adopting the following technical solutions. According to the preparation method of a Co-MOF@PDA / GRE micro electrochemical sensor proposed in the present invention, the method mainly comprises the following steps:

[0008] (1) Preparation of Co-MOF: Weigh 30-60 mg of cobalt acetate tetrahydrate, 25-55 mg of 2,5-dicarboxylic acid-3,4-ethylenedioxythiophene (H 2 L) and 10-30 μL HNO 3 Mix and stir for 1 to 2 hours to obtain mixture A. Take 10 to 20 ml of N, N-dimethylacetamide (DMA) solution, dissolve mixture A in the DMA solution, and transfer to a 15 mL polytetrafluoroethylene-lined stainless steel reactor, heat at 110 to 150 ° C and autogenous pressure for 12 to 36 hours, and then heat at 5 ° C·h -1 After thorough washing with DMA, the average pore size was finally obtained. Red cobalt metal organic framework crystals;

[0009] (2) Preparation of Co-MOF@PDA composite modified electrode material: Weigh 30-50 mg of the synthesized Co-MOF and disperse it in a Tris buffer solution, and ultrasonicate for 30-60 min. Then, slowly add 2 mg / mL dopamine solution while stirring at room temperature for 1 hour. After filtering, the Co-MOF@PDA composite material is obtained, washed several times with ultrapure water, and dried;

[0010] The above method was used to prepare PDA solid without adding Co-MOF solid as a control, with other conditions unchanged;

[0011] (3) Preparation of Co-MOF@PDA dispersion: Weigh 10-20 mg of Co-MOF@PDA composite modified electrode material, add 5-10 mL of ethanol, and ultrasonically disperse for 20-40 min to obtain a 1-3 mg / mL uniform and stable suspension;

[0012] Prepare the Co-MOF dispersion and the PDA dispersion in the same way;

[0013] (4) Prepare the Co-MOF@PDA / GRE electrochemical sensor: Use a microsampler to pipette 3 - 7 μL of the dispersion obtained in step (3) and evenly drop it onto the center of the treated graphite rod electrode (GRE). After modification, the electrode is dried under an infrared lamp for 10 - 20 min to obtain a strongly adherent, uniform, and stable Co-MOF@PDA modified film on the surface of the graphite rod electrode, thus obtaining the Co-MOF@PDA / GRE electrochemical sensor;

[0014] Prepare the Co-MOF / GRE electrochemical sensor and the PDA / GRE electrochemical sensor in the same way.

[0015] The object of the present invention and the solution to its technical problems can also be further realized by the following technical solutions.

[0016] In the aforementioned preparation method of a Co-MOF@PDA / GRE electrochemical sensor, the obtained Co-MOF@PDA / GRE electrochemical sensor is used to determine tryptophan.

[0017] In the aforementioned preparation method of a Co-MOF@PDA / GRE electrochemical sensor, the obtained Co-MOF@PDA / GRE electrochemical sensor is used to continuously monitor the tryptophan concentration of living plants for a long time.

[0018] In the aforementioned preparation method of a Co-MOF@PDA / GRE electrochemical sensor, when the obtained Co-MOF@PDA / GRE electrochemical sensor is used to determine tryptophan, a phosphate buffer solution with pH = 6.8 is selected as the medium, and the detection limit of tryptophan is 4.14×10 -7 mol / L.

[0019] In the aforementioned preparation method of a Co-MOF@PDA / GRE electrochemical sensor, in step (1), the DMA solution is prepared from DMA and H 2 O with a volume ratio of 1:1.

[0020] In the aforementioned preparation method of a Co-MOF@PDA / GRE electrochemical sensor, in step (2), the concentration of the Tris buffer solution is 0.01 mol / L and pH = 8.5.

[0021] The following further explains the present invention:

[0022] The present invention establishes a new method for in vivo detection of L-tryptophan (L-Trp) in living plants, and constructs a micro electrochemical sensing system by modifying a graphite rod electrode with a Co-MOF@PDA composite material. Among them, the working electrode of the Co-MOF@PDA / GRE system, the counter electrode is a platinum wire (Φ = 0.5 mm), and the reference electrode is an Ag / AgCl wire. Preparation of the reference electrode: The front end of a silver wire (Φ = 0.5 mm) exposes 2 mm as the reaction zone, and the rear end exposes 2 mm to connect to the wire of the corresponding electrode, and the rest is covered by a heat shrinkable tube. The wrapped silver wire is used as the working electrode, the activated Ag / AgCl electrode is used as the reference electrode, and the platinum electrode is used as the counter electrode, and is placed in a saturated KCl solution for activation by cyclic voltammetry. The electrochemical parameters are set as follows: the voltage window is 0 V - 0.5 V, the scanning speed is 10 mV / s, and the number of scanning segments is 25,000 cycles, that is, the reference electrode Ag / AgCl wire is obtained. Based on the Co-MOF@PDA / GRE micro electrochemical sensor for detecting L-Trp, the oxidation peak potential is 0.92 V, and the linear range is 1.5×10 -4 ~6.8×10 -7 mol / L, and the detection limit reaches 4.14×10 -7 mol / L. This method has good repeatability, reproducibility and stability, and because the usage amount of bioactive chemicals is minimized, it can continuously monitor in living plants for 20 days. Using this method to detect tryptophan, the results are consistent with those measured by high performance liquid chromatography, and the measured recovery rate is 94.5% - 102.6%, fully indicating that this method is expected to construct a sensing platform for detecting L-tryptophan in living plants and has potential application value in the field of bioanalysis.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) The present invention performs surface modification through a conductive metal-organic framework (MOF) such as Co-MOF, which has a large surface area, high porosity and adjustable catalytic ability.

[0025] (2) When the present invention uses the oxidation and self-polymerization of dopamine under alkaline conditions, it shows a strong adhesion force to almost any material surface to prepare a binary composite material. Therefore, the modified film of the electrode has specific functions such as excellent hydrophilicity and good biocompatibility.

[0026] (3) The present invention uses a graphite rod electrode with a diameter of 2 mm as the modification object and is used to construct a highly feasible micro electrochemical sensor, which can be used for in vivo real-time detection of tryptophan in living plants.

[0027] (4) The formaldehyde-removing catalyst of the present invention has low cost, long service life, good stability, and can also be well attached to various carriers, thus expanding the scope of application. Description of the Drawings

[0028] Figure 1 It is a scanning electron microscope image of the Co-MOF@PDA composite material prepared according to the present invention. Detailed Embodiments

[0029] In order to further elaborate on the technical means and effects adopted by the present invention to achieve the intended invention purpose, the following will be described in detail in conjunction with the drawings and preferred embodiments. Figure 1 It is a transmission electron microscope image of the Co-MOF@PDA composite modified electrode material. As can be seen from Figure 1 it, the Co-MOF@PDA composite modified electrode material has been successfully synthesized. Co-MOF@PDA has a good core-shell structure and a rough surface. The Co-MOF nanosheets as the core layer have been successfully coated with a PDA shell layer.

[0030] The following will further describe the present invention in more detail in conjunction with specific embodiments.

[0031] Example 1

[0032] Weigh 49.8 mg of cobalt acetate tetrahydrate, 46.04 mg of 2,5-dicarboxylic acid-3,4-ethylenedioxythiophene (H 2 L), and 20 μL of HNO 3 Mix them and stir for 1 h to obtain mixture A. Measure 6 ml of H 2 O and 6 ml of N,N-dimethylacetamide (DMA) respectively, and mix them well to obtain a DMA solution. Dissolve mixture A in the DMA solution and transfer it to a 15 mL stainless steel reaction kettle with a polytetrafluoroethylene lining. Heat it at 120 °C and autogenous pressure for 24 h, and then cool it to room temperature at a rate of 5 °C·h -1 . After thorough washing with DMA, finally obtain red cobalt metal-organic framework crystals Co-MOF;

[0033] Weigh 40 mg of the synthesized Co-MOF and disperse it in Tris buffer solution (pH 8.5, 10 mM), and sonicate it for 30 min. Then, while stirring at room temperature for 1 hour, slowly add a dopamine solution of 2 mg / mL. After filtration, obtain the Co-MOF@PDA composite material, wash it several times with ultrapure water, and dry it;

[0034] Weigh 10 mg of the Co-MOF@PDA composite modified electrode material, add 5 mL of ethanol, and sonicate it for 20 min to obtain a uniform and stable suspension of 2 mg / mL;

[0035] Use a micro-sampler to pipette 3.5 μL of the suspension obtained in the previous step, and evenly drop it onto the center of the treated graphite rod electrode (GRE). After modification, the electrode is dried under an infrared lamp for 10 min, and a Co-MOF@PDA modified film with strong adhesion, uniformity, and stability is obtained on the surface of the graphite rod electrode, thus obtaining the Co-MOF@PDA / GRE electrochemical sensor;

[0036] In a phosphate buffer solution with pH = 6.8, select an enrichment time of 60 s, change the concentration of the L-tryptophan standard solution, and record the sensor response peak current at different concentrations. L-tryptophan has a linear relationship between the peak current and the concentration in the range of 2.6×10 -4 ~8.2×10 -7 mol / L, and the detection limit is 5.3×10 -7 mol / L.

[0037] The standard addition recovery experiment of the L-tryptophan to be measured is carried out by linear sweep voltammetry. The recovery rate of L-tryptophan is between 95.8% and 104.2%. This shows that the electrochemical sensor can be used for the determination of L-tryptophan in actual samples; after treating the commercially available 18AA and 17AA-1 compound amino acid injection (the labeled amount of tryptophan in 18AA is 0.9 g / 1000 mL; the labeled amount of tryptophan in 17AA-1 is 0.43 g / 1000 mL), the Co-MOF@PDA / GRE electrochemical sensor is used for electrochemical measurement. The measured results are 0.905 g / 1000 mL for tryptophan in 18AA and 0.434 g / 1000 mL for tryptophan in 17AA-1, which are consistent with the drug label amounts.

[0038] Example 2

[0039] Weigh 74.7 mg of cobalt acetate tetrahydrate, 64.46 mg of 2,5-dicarboxylic acid-3,4-ethylenedioxythiophene (H 2 L), and 30 μL of HNO 3 Mix them and stir for 1 h to obtain mixture A. Measure 6 ml of H 2 O and 6 ml of N,N-dimethylacetamide (DMA) respectively, and mix them well to obtain a DMA solution. Dissolve mixture A in the DMA solution and transfer it to a 20 mL stainless steel reaction kettle with a PTFE liner. Heat it at 130 °C and autogenous pressure for 36 h, and then cool it to room temperature at a rate of 5 °C·h -1 After thorough washing with DMA, red cobalt metal-organic framework crystals are finally obtained;

[0040] Weigh 60 mg of the synthesized Co-MOF and disperse it in Tris buffer solution (pH 8.5, 10 mM), and ultrasonicate for 40 min. Then, while stirring at room temperature for 1 hour, slowly add the dopamine solution at a concentration of 2 mg / mL. After filtration, the Co-MOF@PDA composite material is obtained, washed several times with ultrapure water, and dried;

[0041] Weigh 15 mg of the Co-MOF@PDA composite modified electrode material, add 5 mL of ethanol, and ultrasonically disperse for 30 min to obtain a uniform and stable suspension with a concentration of 3 mg / mL;

[0042] Use a micro-sampler to pipette 4 μL of the suspension obtained in the previous step and evenly drop it onto the center of the treated graphite rod electrode (GRE). After modification, the electrode is dried under an infrared lamp for 20 min, and a strongly adherent, uniform, and stable Co-MOF@PDA modified film is obtained on the surface of the graphite rod electrode, that is, the Co-MOF@PDA / GRE electrochemical sensor is obtained;

[0043] In a phosphate buffer solution with pH = 6.8, select an enrichment time of 60 s, change the concentration of the L-tryptophan standard solution, and record the sensor response peak current at different concentrations. L-tryptophan shows a linear relationship between the peak current and the concentration in the range of 1.5×10 -4 ~6.8×10 -7 mol / L, and the detection limit is 4.14×10 -7 mol / L.

[0044] The standard addition recovery experiment of the L-tryptophan to be measured is carried out by linear sweep voltammetry, and the recovery rate of L-tryptophan is in the range of 96.3% - 103.7%. This indicates that the electrochemical sensor can be used for the determination of L-tryptophan in actual samples; after treating the commercially available 18AA and 17AA-1 compound amino acid injection (the labeled amount of tryptophan in 18AA is 0.9 g / 1000 mL; the labeled amount of tryptophan in 17AA-1 is 0.43 g / 1000 mL), the Co-MOF@PDA / GRE electrochemical sensor is used for electrochemical measurement, and the measured results are that the tryptophan content in 18AA is 0.911 g / 1000 mL; the tryptophan content in 17AA-1 is 0.433 g / 1000 mL, which is consistent with the drug label.

[0045] Comparative Example 3: Co-MOF / GRE electrochemical sensor

[0046] Weigh 74.7 mg of cobalt acetate tetrahydrate, 64.46 mg of 2,5-dicarboxylic acid-3,4-ethylenedioxythiophene (H 2 L) and 30 μL of HNO 3 Mix and stir for 1 h to obtain mixture A. Measure 6 ml of H 26 ml of N,N-dimethylacetamide (DMA) was added to 0, and the mixture was thoroughly mixed to obtain a DMA solution. The mixture A was dissolved in the DMA solution and transferred to a 20 mL stainless steel reactor lined with polytetrafluoroethylene. It was heated at 130 °C under autogenous pressure for 36 h, and then cooled to room temperature at a rate of 5 °C·h -1 After thorough washing with DMA, red cobalt metal-organic framework crystals were finally obtained;

[0047] 15 mg of Co-MOF modified electrode material was weighed and added to 5 mL of ethanol. It was ultrasonically dispersed for 30 min to obtain a uniform and stable suspension of 3 mg / mL;

[0048] 4 μL of the suspension obtained in the previous step was pipetted with a micro-sampler and evenly dropped onto the center of the treated graphite rod electrode (GRE). The modified electrode was dried under an infrared lamp for 20 min to obtain a strongly adherent, uniform and stable Co-MOF modified film on the surface of the graphite rod electrode, that is, the Co-MOF / GRE electrochemical sensor was obtained;

[0049] In a phosphate buffer solution with pH = 6.8, the enrichment time was selected as 60 s, the concentration of the L-tryptophan standard solution was changed, and the response peak current of the sensor at different concentrations was recorded. L-tryptophan showed a linear relationship between the peak current and the concentration in the range of 1.3×10 -5 ~4.5×10 -6 mol / L, and the detection limit was 6.4×10 -6 mol / L.

[0050] The standard addition recovery experiment of the L-tryptophan to be measured was carried out by linear sweep voltammetry. The recovery rate of L-tryptophan was between 82.1% and 90.3%. After treating the commercially available 18AA and 17AA-1 compound amino acid injection (the labeled amount of tryptophan in 18AA is 0.9 g / 1000 mL; the labeled amount of tryptophan in 17AA-1 is 0.43 g / 1000 mL), the Co-MOF / GRE electrochemical sensor was used for electrochemical measurement. The measured results were 0.915 g / 1000 mL for tryptophan in 18AA; 0.436 g / 1000 mL for tryptophan in 17AA-1, which was in agreement with the drug label.

[0051] Comparative Example 4: PDA / GRE electrochemical sensor

[0052] In a Tris buffer solution (pH 8.5, 10 mM), a dopamine solution of 2 mg / mL was slowly added. After filtration, the PDA composite material was obtained, washed several times with ultrapure water and dried;

[0053] Weigh 15 mg of the PDA-modified electrode material, add 5 mL of ethanol, and ultrasonically disperse it for 30 min to obtain a uniform and stable suspension with a concentration of 3 mg / mL;

[0054] Use a microsampler to pipette 4 μL of the suspension obtained in the previous step and evenly drop it onto the center of the treated graphite rod electrode (GRE). After modification, the electrode is dried under an infrared lamp for 20 min to obtain a strongly adherent, uniform, and stable PDA modification film on the surface of the graphite rod electrode, that is, a PDA / GRE electrochemical sensor is obtained;

[0055] In a phosphate buffer solution with pH = 6.8, select an enrichment time of 60 s, change the concentration of the L-tryptophan standard solution, and record the sensor response peak current at different concentrations. The linear relationship between the obtained peak current and the concentration is poor.

[0056] A standard addition recovery experiment for the L-tryptophan to be measured is carried out by linear sweep voltammetry. The recovery rate of L-tryptophan is between 40.5% and 60.4%. This indicates that this electrochemical sensor is not suitable for the determination of L-tryptophan in actual samples.

[0057] It can be seen from the above experimental examples and comparative examples that the conductive metal organic compound Co-MOF is essential for constructing the Co-MOF@PDA / GRE microelectrochemical sensor. The presence of the conductive metal organic compound improves the charge transfer process of the sensor. The constructed electrochemical sensor has excellent measurement resolution in the electrochemical behavior of tryptophan. Further research on optimizing the composition and structure using MOF can produce devices with higher performance, thereby improving conductivity, measurement sensitivity, and selectivity.

Claims

1. Preparation method of a Co-MOF@PDA / GRE electrochemical sensor, characterized in that, it includes the following steps: (1) Preparation of Co-MOF: Weigh cobalt acetate tetrahydrate, 2,5-dicarboxylic acid-3,4-ethylenedioxythiophene H 2 L and HNO 3 Mix them and stir to obtain mixture A; Measure the DMA solution, dissolve mixture A in the DMA solution, and transfer it to a stainless-steel autoclave with a polytetrafluoroethylene liner. Heat it at 110 - 150 °C under autogenous pressure for 12 - 36 h, and then cool it to room temperature at a rate of 5 °C·h -1 ; After thorough washing with DMA, finally obtain red cobalt metal-organic framework crystals Co-MOF; (2) Preparation of Co-MOF@PDA composite modified electrode material: Weigh the synthesized Co-MOF and disperse it in Tris buffer solution, sonicate, then stir at room temperature, and slowly add dopamine solution; After filtration, obtain the Co-MOF@PDA composite material, and wash it several times with ultrapure water and dry it; (3) Preparation of Co-MOF@PDA dispersion: Weigh the Co-MOF@PDA composite modified electrode material, add ethanol, and sonicate to disperse to obtain a uniform and stable suspension; (4) Preparation of Co-MOF@PDA / GRE electrochemical sensor: Pipette the suspension obtained in step (3) and evenly drop it onto the center of the treated graphite rod electrode GRE. After modification, the electrode is dried under an infrared lamp to obtain a layer of strongly adherent, uniform and stable Co-MOF@PDA modified film on the surface of the graphite rod electrode, that is, the Co-MOF@PDA / GRE electrochemical sensor is obtained; In step (1), the DMA solution is prepared from DMA and H 2 O with a volume ratio of 1:1, and the addition amount of the DMA solution is 10 to 20 ml; The addition amount of cobalt acetate tetrahydrate is 30 to 60 mg; the addition amount of 2,5-dicarboxylic acid-3,4-ethylenedioxythiophene H 2 L is 25 to 55 mg; the addition amount of HNO 3 is 10 to 30 μL; In step (2), the addition amount of the Co-MOF is 30-50 mg; the concentration of the dopamine solution is 2 mg / mL, and the added volume is 50-80 mL; The concentration of the Tris buffer solution is 0.01 mol / L, pH = 8.5; In step (3), the addition amount of the Co-MOF@PDA composite modified electrode material is 10-20 mg; the addition amount of the ethanol is 5-10 mL; In step (4), the pipetted amount of the suspension is 3-7 μL.

2. The preparation method of a Co-MOF@PDA / GRE electrochemical sensor as described in claim 1, wherein, in step (1), the stirring time is 1-2 h.

3. The preparation method of a Co-MOF@PDA / GRE electrochemical sensor as described in claim 1, wherein, in step (2), the sonication time is 30-60 min; the stirring time is 1 hour.

4. The preparation method of a Co-MOF@PDA / GRE electrochemical sensor as described in claim 1, wherein, in step (3), the sonication dispersion time is 20-40 min.

5. The preparation method of a Co-MOF@PDA / GRE electrochemical sensor as described in claim 1, wherein, in step (4), the drying time is 10-20 min.

6. A Co-MOF@PDA / GRE electrochemical sensor prepared by the preparation method described in any one of claims 1-5.

7. The application of the Co-MOF@PDA / GRE electrochemical sensor described in claim 6 in the determination of tryptophan.

8. The application of the Co-MOF@PDA / GRE electrochemical sensor described in claim 6 in the method for measuring the tryptophan concentration in living plants.

9. The application as described in claim 8, wherein, When the obtained Co-MOF@PDA / GRE electrochemical sensor is used to determine tryptophan, a phosphate buffer solution with pH = 6.8 is selected as the medium, and the detection limit of tryptophan is 4.14×10 -7 mol / L.

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