Iron-doped laser-induced graphene sensor, preparation method and application thereof

Abstract

The present invention discloses an iron-doped laser-induced graphene sensor, a preparation method and application thereof, and belongs to the field of sensing and chemical detection technology. The iron-doped laser-induced graphene sensor of the present invention is prepared by the following method: a polyimide film is rinsed with ethanol, air-dried, and then laser engraved to form a black carbonized layer; then an FeCl2 solution is dripped onto the black carbonized layer and dried at room temperature; after drying, laser engraving is performed again on the original black carbonized layer to obtain an iron-doped laser-induced graphene sensor. The iron-doped laser-induced graphene sensor prepared by the present invention has the advantages of flexibility, good adhesion to the plant surface, small size, stable performance and good detection effect. It can realize on-site instant in-situ detection of SA expression levels in living plant tissues and has little effect on plant growth.

Description

Technical Field

[0001] The present invention belongs to the field of sensing and chemical detection technology, and in particular relates to an iron-doped laser-induced graphene sensor, a preparation method thereof, and applications thereof. Background Art

[0002] When plants are under stress, hormones change, regulating physiological and biochemical processes related to stress resistance. Salicylic acid (SA) is an important plant hormone and a typical plant disease resistance substance. It plays a vital role in regulating many physiological processes in plants. Therefore, on-site and timely detection of SA helps monitor the physiological state of plants, allowing people to take timely measures to cope with stress, reduce the impact of stress on plant survival and growth, and improve the yield and quality of plants, especially crops. Therefore, monitoring the qualitative and quantitative changes of SA, a key physiological parameter in living plants, is of great significance to precision agriculture.

[0003] Currently, there are many methods for determining SA, such as high performance liquid chromatography, gas chromatography-mass spectrometry, fluorescence spectroscopy, and flow injection atomic absorption spectrometry. However, these methods usually have the disadvantages of expensive instruments, complex sample processing, and time-consuming. Electrochemical detection methods have unique advantages such as high accuracy, low detection limit, and fast response speed, and are widely used. There are also some reports on the use of electrochemistry to detect SA. For example, in alkaline solutions, SA was detected using a gold electrode film modified with copper nanoparticles (Talanta, 2010, 80: 1277-1281); bare carbon paste electrodes were also used to quantify the concentration of SA in acidic solutions (Talanta, 2004, 62(2): 247-254); and electrodes modified with Pt nanoparticles were also used to measure SA (The Journal of Physical Chemistry B, 2005, 109(46): 21593-601). However, most of these methods can only be performed in the laboratory, making it difficult to achieve on-site, immediate in-situ detection of SA in plants, and the preparation cost is relatively high.

[0004] The application of these traditional methods in agriculture is limited by expensive equipment and cumbersome sample processing, and some detection methods can irreversibly affect plant growth. Furthermore, SA content in plants is low, its stability is low, and its expression level is affected by multiple factors, including the environment, water content, nutrition, and growth period. Therefore, there is an urgent need for a method that can detect SA expression levels on-site, in situ, and in real time. Summary of the Invention

[0005] The present invention uses laser direct writing technology to prepare Fe-doped laser-induced graphene (LIG) on a flexible polyimide film (PI film) and uses it as a flexible sensing electrode interface. Since SA and Fe can coordinate, Fe is used to increase the adsorption of SA by the LIG electrode, thereby improving the detection sensitivity. With the help of portable electrochemical equipment, on-site, immediate and in-situ detection of SA in plants can be achieved.

[0006] The technical solutions of the present invention are as follows:

[0007] A method for preparing an iron-doped laser-induced graphene sensor, comprising the following steps:

[0008] The polyimide film was rinsed with ethanol, air-dried, and then laser engraved to form a black carbonized layer. FeCl2 solution was then added dropwise to the black carbonized layer and dried at room temperature. After drying, laser engraving was performed again on the original black carbonized layer to obtain an iron-doped laser-induced graphene sensor.

[0009] In the above preparation method, the laser engraving is selected from: directly engraving on the polyimide film using a 355nm Nd:YVO4 pulse laser system in room temperature air.

[0010] In the above preparation method, the concentration of the FeCl2 solution is selected from 0.01 to 1M; preferably 0.01M, 0.025M, 0.05M, 0.1M, 0.25M, 0.5M, 1M; more preferably 0.1M.

[0011] The present invention provides an iron-doped laser-induced graphene sensor prepared by the method.

[0012] The present invention provides an application of the iron-doped laser-induced graphene sensor in the qualitative or quantitative detection of salicylic acid in plants.

[0013] A method for detecting salicylic acid in plants, comprising the following steps:

[0014] A hole was punched in the plant leaves using a hole punch to obtain holes for salicylic acid release and detection; the working electrode of the iron-doped laser-induced graphene sensor was tightly connected to the hole using tape; the iron-doped laser-induced graphene was connected to the electronic sensing device via an adapter; PB buffer was then dripped into the hole, and electrochemical measurements were performed to obtain electrochemical signals; the electrochemical signals were imported into the standard curve to obtain the salicylic acid concentration.

[0015] The above standard curve is constructed as follows:

[0016] The test conditions are as follows: FeCl2 concentration is 0.1M, the laser printing power of the electrode is 3.2W, the buffer pH is 7, and the buffer concentration is 0.2M; the concentrations of salicylic acid are set to 10μM, 50μM, 100μM, 200μM, 300μM, and 400μM, respectively; differential pulse voltammetry (DPV) is used to measure the electrochemical signals at various salicylic acid concentrations using a HY-E100X portable electrochemical workstation; a salicylic acid concentration-electrochemical signal standard curve is constructed with the salicylic acid concentration as the horizontal axis and the measured electrochemical signal as the vertical axis.

[0017] The beneficial effects of the present invention are:

[0018] The Fe-LIG sensor prepared in the present invention has the advantages of flexibility, good adhesion to plant surfaces, small size, stable performance, and good detection effect. It can realize on-site instant in situ detection of SA expression levels in living plant tissues and has little impact on plant growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of Fe-LIG portable biosensor;

[0020] Figure 2 This is a physical picture of the Fe-LIG portable biosensor;

[0021] Figure 3 Cyclic voltammetry curves under different experimental conditions. At 0.4 V, the curves from top to bottom are SA+Fe-LIG, SA+LIG, and PB+Fe-LIG.

[0022] Figure 4 is the standard curve of SA concentration and electrochemical signal;

[0023] Figure 5 Specificity analysis of Fe-LIG portable biosensors;

[0024] Figure 6 A photo showing the detection of SA in spider plants using Fe-LIG sensors.

[0025] Figure 7 This is a quantitative detection experiment of SA in Chlorophytum under salt stress. DETAILED DESCRIPTION

[0026] The experimental principle of the present invention is as follows:

[0027] First, a black carbonized layer, laser-induced graphene (LIG), is formed by laser induction. Then, an FeCl2 solution is dripped onto the electrode (i.e., a portion of the black carbonized layer) and dried at room temperature. After drying, the pristine LIG is subjected to in situ laser induction treatment again to obtain an iron-doped Fe-LIG electrode, the iron-doped laser-induced graphene sensor (Fe-LIG sensor). The Fe-LIG sensor is integrated with a portable electrochemical sensing device (which includes an adapter that establishes a simple and reliable electronic interface between the sensing device and the Fe-LIG electrode). Using a mobile app data analysis program (e.g., HY-EW100X.exe), it is used for in situ, real-time, quantitative detection of SA in living plants. In in vivo experiments, a hole punch is used to perforate the leaves of plant samples, creating pores for SA release and detection. The working electrode of the Fe-LIG electrode is tightly attached to the pores with tape. PB buffer is dripped into the pores for electrochemical measurements. SA content changes are affected by the environment, and its synthesis is induced by abiotic stresses. When plants are subjected to abiotic stresses such as drought, high salt, high temperature, mechanical damage, air pollution and photooxidative stress, they will rapidly accumulate SA in their bodies and produce enhanced electrochemical signals. Figure 1 shown.

[0028] The other materials used in the present invention, unless otherwise stated, can be obtained through commercial channels. Unless otherwise specified, other terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below with reference to specific examples and data. The following examples are merely for illustration of the present invention and are not intended to limit the scope of the present invention in any way.

[0029] Example 1

[0030] Preparation of iron-doped laser-induced graphene sensors:

[0031] Design a suitable electrode pattern on a computer drawing software. Rinse the PI film with ethanol and air-dry it. Then use a 355nm Nd:YVO4 pulsed laser system (power: 3.2W, speed: 10%) to directly engrave the pre-designed pattern on the film in room temperature air to form a black carbonized layer (i.e., LIG). Then add 0.1M FeCl2 solution (volume: 6μL) dropwise onto the black carbonized layer and dry it at room temperature for 10 minutes. After drying, perform another laser engraving on the original LIG to dope the iron element, obtaining an iron-doped laser-induced graphene sensor, named Fe-LIG sensor. The actual picture of the Fe-LIG sensor is shown below. Figure 2 shown.

[0032] (1) Feasibility study

[0033] The feasibility of the electrochemical sensor prepared in Example 1 for SA detection was verified by measuring cyclic voltammetry (CV) curves under different conditions, as shown below:

[0034] CV graphs of Fe-LIG sensor in 0.2M PB (black line) and 0.2M PB+400μM SA (red line) solutions, as well as CV graph of LIG electrode in 0.2M PB+400μM SA (blue line) solution. Figure 3 As shown in the figure, first excluding the influence of PB buffer, it was found that the CV current generated by Fe-LIG detection of PB was negligible. Then, the detection of SA using two electrodes (LIG and Fe-LIG) was compared. When Fe-LIG was used, a more obvious redox peak was obtained, with a stronger current signal.

[0035] (2) Construction of standard curve

[0036] Under optimal experimental conditions (FeCl2 concentration of 0.1 M; laser printing power of 3.2 W for the electrodes; buffer pH of 7; buffer concentration of 0.2 M), the electrochemical signal (DPV response) of the Fe-LIG biosensor to different concentrations of SA was measured using differential pulse voltammetry (DPV) on a HY-E100X portable electrochemical workstation. SA concentrations were set to 10 μM, 50 μM, 100 μM, 200 μM, 300 μM, and 400 μM. Based on the measurement results, an electrochemical signal standard curve was constructed with the electrochemical signal as the ordinate and the SA concentration as the abscissa.

[0037] The test results are as follows Figure 4 As shown in Figure 2, the peak current increases with the increase of SA concentration. When the SA concentration is in the range of 0 to 400 μM, the ratio of SA concentration to SA peak current is linear, which can cover the SA concentration range in most plants. The linear equation is fitted as Y = 59.86 + 2.25X, R 2 =0.998. The detection limit was 3.3 μM.

[0038] (III) Specificity analysis

[0039] The present invention detects common interfering substances such as indole-3-acetic acid, abscisic acid and gibberellin to verify the specificity of the electrochemical sensor for SA. Figure 5The figure shows the DPV responses of the Fe-LIG sensor to indole-3-acetic acid, abscisic acid, gibberellin, and salicylic acid (SA), all at 50 μM. In the presence of SA, the electrochemical signal of the sensor is significantly higher than that in the presence of other interfering species. These results demonstrate that the Fe-LIG portable electrochemical biosensor has excellent selectivity for the target compound SA.

[0040] Application Example 1

[0041] SA plays a key role in plant resistance to salt stress. The prepared Fe-LIG sensor is applied to detect the SA concentration in spider plant seedlings under different NaCl stress conditions.

[0042] Chlorophytum comosum was cultured in water for 15 days and then analyzed and tested after roots developed. The samples were randomly divided into two groups (3 in each group). The two groups were irrigated with 0mM and 200mM NaCl solutions, respectively. After 12 hours, the samples were used for electrochemical and HPLC-MS measurements.

[0043] In the in vivo experiment, a hole was punched into the leaves of the plant sample using a hole punch to create holes for SA release and detection. The Fe-LIG working electrode was tightly connected to the hole with tape. PB buffer was then dripped into the hole for electrochemical measurement. For different leaves, the electrodes were placed in the same position as much as possible. Actual image of the Fe-LIG sensor detecting SA in spider plants, as shown in the figure. Figure 6 shown.

[0044] For HPLC-MS (Water Acquity I-Class, Waters Corporation, MA, USA; Thermo Q-Exactive, Thermo Scientific, MA, USA) measurements, 1 g of ground sample was added to 2 mL of 4°C pre-cooled methanol (80%) and extracted at 4°C overnight. The extract was mixed with 1 mL of CHCl3. The solution was then shaken at 900 rpm for 5 min at 4°C. After centrifugation at 14,000 rpm for 5 min at 4°C, the supernatant was collected and dried under nitrogen at room temperature. The prepared sample was dissolved in 1 mL of methanol and filtered through a 0.22 mm microporous membrane before HPLC-MS measurement.

[0045] The test results are as follows Figure 7 As shown:

[0046] Figure 7 A and Figure 7 B shows photos of spider plants before and after stress, respectively. Figure 7 C is the HPLC test result, Figure 7 D is the standard curve under HPLC detection results. Figure 7E is the electrochemical detection result, Figure 7 F is the standard curve under electrochemical detection results.

[0047] according to Figure 7 The concentrations of SA in the leaves of Chlorophytum comosum seedlings under 0 mM and 200 mM salt stress were calculated to be 204.71 μM and 230.78 μM, respectively. Figure 7 Standard curve in F, conversion Figure 7 The electrochemical signals in E obtained SA concentrations were 182.91 μM and 204.61 μM, respectively.

[0048] The results showed that SA levels increased in the leaves of spider plant seedlings under 200 mM salt stress, likely due to its important role in inducing systemic acquired resistance to biotic and abiotic stresses. Electrochemical and HPLC-MS assays showed similar trends, with no significant differences in the results, confirming the reliability of the portable electrochemical sensor developed in this study.

[0049] Compared with traditional technologies, the information obtained by the sensor of the present invention can better reflect the actual situation of the plant. For some traditional detection methods, such as HPLC, plant samples need to undergo complex pretreatment, such as grinding, centrifugation, extraction, etc. This process often causes great damage to the plant and loses some biological information. To use the sensor to detect SA in the body, it is only necessary to open a small hole in the leaves of the plant, and then the sensor can directly detect the SA released by the plant, thereby providing more immediate and accurate SA level information, which is simple, accurate, reliable and practical. The diameter of the hole on the leaf is only 6mm, and the impact on the subsequent plant growth is negligible. Moreover, the leaves of spider plant seedlings are flat, and it is difficult to conduct in vivo studies using traditional needle electrodes or column electrodes. However, the improved Fe-LIG sensor is very suitable for this application.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. Application of iron-doped laser-induced graphene sensors for qualitative or quantitative detection of salicylic acid in plants; The iron-doped laser-induced graphene sensor is prepared by the following method: The polyimide film was rinsed with ethanol, air-dried, and then laser-engraved to form a black carbonized layer. An FeCl2 solution was then added dropwise to the black carbonized layer and dried at room temperature. After drying, the original black carbonized layer was laser-engraved again to obtain an iron-doped laser-induced graphene sensor. The laser engraving is selected from: directly engraving on a polyimide film using a 355nm Nd:YVO4 pulse laser system in room temperature air; the concentration of the FeCl2 solution is selected from 0.01~1M.

2. A method for detecting salicylic acid in plants, characterized in that: Here are the steps: A hole was punched in the plant leaf using a hole punch to obtain a hole for the release and detection of salicylic acid; the working electrode of the iron-doped laser-induced graphene sensor was tightly attached to the hole using tape; the iron-doped laser-induced graphene was connected to the electronic sensing device via an adapter; PB buffer was then dripped into the hole, and electrochemical measurements were performed to obtain electrochemical signals; the electrochemical signals were imported into a standard curve to obtain the salicylic acid concentration; The iron-doped laser-induced graphene sensor is prepared by the following method: The polyimide film was rinsed with ethanol, air-dried, and then laser-engraved to form a black carbonized layer. An FeCl2 solution was then added dropwise to the black carbonized layer and dried at room temperature. After drying, the original black carbonized layer was laser-engraved again to obtain an iron-doped laser-induced graphene sensor. The laser engraving is selected from: directly engraving on a polyimide film using a 355nm Nd:YVO4 pulse laser system in room temperature air; the concentration of the FeCl2 solution is selected from 0.01~1M.

3. The detection method according to claim 2, characterized in that The method for constructing the standard curve is as follows: The test conditions were as follows: FeCl2 concentration of 0.1 M, laser printing power of the electrode of 3.2 W, buffer pH of 7, and buffer concentration of 0.2 M; salicylic acid concentrations were set to 10 μM, 50 μM, 100 μM, 200 μM, 300 μM, and 400 μM, respectively; differential pulse voltammetry was performed using a HY-E100X portable electrochemical workstation to measure the electrochemical signals at various salicylic acid concentrations; a salicylic acid concentration-electrochemical signal standard curve was constructed with salicylic acid concentration as the horizontal axis and the measured electrochemical signal as the vertical axis.

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