A flexible sensor for real-time in vivo monitoring of solanine content in plants, its preparation method and application

Abstract

The present invention belongs to the field of micro-motor biosensing technology, and particularly relates to a flexible sensor for real-time monitoring of solanine content in living plants, a preparation method thereof, and an application thereof. The flexible sensor includes a flexible substrate and a working electrode, a reference electrode, and a counter electrode disposed on the flexible substrate; the working electrode is a laser-induced graphene electrode LIG with a surface sequentially modified with niobium-doped titanium disulfide crystals, dumbbell-shaped Au-PdAg, and PEDOT:PSS–Nafion composite materials. The flexible sensor can realize on-line detection and analysis of solanine in plant tissues at different times. Since it is a patch type that adapts to the plant surface, it does not cause essential damage to the detected material, and the material can continue to grow until it matures. At the same time, through the modification of niobium-doped titanium disulfide, Au-PdAg, PEDOT:PSS, and Nafion by drop coating, highly sensitive and highly accurate detection of solanine is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of micro-motor biosensors, and particularly relates to a flexible sensor for real-time in-vivo monitoring of solanine content in plants, a preparation method thereof, and an application thereof. Background Art

[0002] Solanine is a class of toxic steroid glycoside alkaloids and an important secondary metabolite in the growth process of Solanaceae plants. Solanine plays an important role in combating plant diseases and pests. It is mainly produced in large quantities when Solanaceae plants are affected by diseases and pests, and kills insects by inhibiting the cholinesterase activity in insects. In addition, solanine also inhibits the growth of bacteria and fungi in Solanaceae plants by changing the cell membrane permeability of the bacteria and destroying the integrity of the cell membrane. At the same time, solanine is also an important factor affecting the food safety of Solanaceae plants such as potatoes, and is mostly found in the young and damaged parts of tuber tissues that have germinated, turned green, become moldy, rotted, or been mechanically damaged. Therefore, detecting the solanine content in Solanaceae plants is particularly important for their screening, cultivation, and food safety.

[0003] Currently, the widely used solanine detection methods are mainly divided into three categories: chemical methods such as titration method, colorimetry, and ultraviolet spectrophotometry; imaging techniques such as mass spectrometry imaging technology and spectral imaging technology; and biological techniques such as enzyme-linked immunosorbent assay and radioimmunoassay. However, these detection methods are relatively cumbersome, often equipped with large-scale instruments, and all belong to ex vivo detection. Samples need to be collected and pretreated. The collection process is likely to cause damage to plants, and the pretreatment process is complex and time-consuming. Compared with other technologies, the electrochemical method has the advantages of simple operation, fast response time, time-saving, high sensitivity, easy integration, etc., and can meet the need for in-situ detection of plant in-vivo. However, traditional sensors based on rigid electrodes (such as glassy carbon electrodes, gold electrodes, etc.) cannot effectively fit the plant surface, reducing the reliability and accuracy of the detection results. On the other hand, it is difficult to fix on the plant surface for long-term detection. For example, environmental factors such as wind, rain, lightning, etc. in the outdoor environment are likely to cause deformation of plant samples, resulting in additional damage to plants by rigid electrodes. Therefore, traditional rigid sensors are difficult to meet the needs of long-term in-situ, fixed-point, and continuous monitoring of plant physiology. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a flexible sensor for real-time in-vivo monitoring of solanine content in plants, a preparation method thereof, and an application thereof. By realizing the in-vivo in-situ detection of solanine in plants, the solanine content can be obtained in a timely and rapid manner, and long-term, stable, and reliable monitoring of solanine in plant in-vivo can be achieved. In terms of breeding and genetic engineering, it can provide theoretical reference and technical guidance for cultivating Solanaceae plants with low solanine content.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] A flexible sensor, comprising a flexible substrate and a working electrode, a reference electrode and a counter electrode disposed on the flexible substrate;

[0007] The working electrode is a laser-induced graphene electrode LIG with a surface successively modified with titanium disulfide crystal doped with niobium (Ti 1-x Nb x S2, x = 0.05, 0.1 or 0.2), Au-PdAg with a dumbbell structure, and a PEDOT:PSS–Nafion composite material;

[0008] The reference electrode is a laser-induced graphene electrode LIG with a surface modified with Ag / AgCl;

[0009] The counter electrode is a laser-induced graphene electrode LIG.

[0010] The present invention uses a flexible substrate, and the pattern of the electrode on the flexible substrate is laser-printed graphene (LIG). The obtained flexible electrode has a soft, bendable, stretchable appearance and a bio-adaptive ability that better fits the shape of the leaf, and can well adapt to the irregular surfaces of crops, such as leaves and fruits, overcoming the defects of destroying plant samples and only being able to perform in vitro static detection in the prior art, and realizing long-term, stable and reliable monitoring of living plants. The present invention uses a laser-induced graphene electrode LIG with a surface successively modified with titanium disulfide crystal doped with niobium (Ti 1-x Nb x S2), Au-PdAg with a dumbbell structure, and a PEDOT:PSS–Nafion composite material as the working electrode to monitor solanine in plants. In the present invention, a dopant Nb is added to TiS2, and the conductivity is enhanced by adjusting the structure and properties of TiS2; at the same time, it is found that the three-dimensional dumbbell structure Au-PdAg exhibits obvious electrocatalytic activity and stability towards the oxidation of solanine, and this remarkable high performance can be attributed to their large specific surface area and unique porous morphology. The present invention utilizes the synergistic effect of the above-mentioned various low-dimensional nanomaterials to improve the in-situ detection performance of the flexible sensor device for solanine in vivo.

[0011] Preferably, the shapes of the working electrode, the reference electrode and the counter electrode are all balloon-shaped. The balloon-shaped electrode has the characteristics of a large electrode area, beauty, simple structure and small integration. At the same time, this shape of the electrode can adopt a serpentine structure to achieve deformations such as bending and twisting, and the electrical properties can still remain stable under these deformation conditions.

[0012] More preferably, the size of the balloon shape is 45 - 50 mm in length and 20 - 25 mm in width.

[0013] Preferably, the material of the flexible substrate is polydimethylsiloxane (PDMS).

[0014] The present invention also provides a method for preparing the above flexible sensor, including:

[0015] Preparing a counter electrode: preparing a laser-induced graphene electrode LIG on a polyimide film (PI film), and transferring the laser-induced graphene electrode LIG to a polydimethylsiloxane flexible substrate through a polydimethylsiloxane (PDMS) transfer strategy to form a counter electrode;

[0016] Preparing a reference electrode: preparing a laser-induced graphene electrode LIG on a polyimide film (PI film), transferring the laser-induced graphene electrode LIG to a polydimethylsiloxane flexible substrate through a polydimethylsiloxane (PDMS) transfer strategy, and then coating Ag / AgCl silver paste on the laser-induced graphene electrode LIG, and curing it to form a reference electrode;

[0017] Preparing a working electrode: preparing a laser-induced graphene electrode LIG on a polyimide film (PI film), transferring the laser-induced graphene electrode LIG to a polydimethylsiloxane flexible substrate through a polydimethylsiloxane (PDMS) transfer strategy, and then successively coating titanium disulfide crystal doped with niobium (Ti 1-x Nb x S2), Au-PdAg with a dumbbell structure, and PEDOT:PSS–Nafion composite material on the laser-induced graphene electrode LIG to form a working electrode.

[0018] Preferably, the successive coating of titanium disulfide crystal doped with niobium (Ti 1-x Nb x S2), Au-PdAg with a dumbbell structure, and PEDOT:PSS–Nafion composite material on the laser-induced graphene electrode LIG specifically includes:

[0019] Drop-coating a Ti 1-x Nb x S2 suspension on the laser-induced graphene electrode LIG, and drying to obtain a Ti 1-x Nb x S2 / LIG electrode;

[0020] Drop-coating an Au-PdAg dispersion on the Ti 1-x Nb x S2 / LIG electrode, and drying to obtain an Au-PdAg / Ti 1- x Nb x S2 / LIG electrode;

[0021] Drop-coating a PEDOT:PSS–Nafion composite material on the Au-PdAg / Ti 1-x Nb xThe mixture of PEDOT:PSS aqueous dispersion and Nafion solution is drop-coated on the S2 / LIG electrode and obtained after drying.

[0022] Further preferably, the Ti 1-x Nb x The concentration of the S2 suspension is 4 - 6 mg / mL;

[0023] The concentration of the Au-PdAg dispersion is 0.9 - 1.1 mg / mL;

[0024] The volume ratio of the PEDOT:PSS aqueous dispersion to the Nafion solution is 1:0.8 - 1.2. The mass fraction of PEDOT:PSS in the PEDOT:PSS aqueous dispersion is 1.0 - 1.5%, and the mass fraction of Nafion in the Nafion solution is 4 - 6%. The above preferred concentrations can ensure the uniformity of the film obtained after drying and further enhance the electrochemical signal.

[0025] Further preferably, the mixture of the PEDOT:PSS aqueous dispersion and the Nafion solution is obtained by continuously stirring the PEDOT:PSS aqueous dispersion and the Nafion solution for 24 - 26 h after mixing.

[0026] The present invention also provides the application of the above flexible sensor in the in-vivo real-time monitoring of solanine content in plants.

[0027] The present invention also provides a method for real-time monitoring of solanine content in plants, including:

[0028] 1) Prepare a series of solanine-phosphate buffer solutions with different concentrations respectively, and use the above flexible sensor for differential pulse voltammetry detection to obtain a set of relationship curves between the concentration and the peak current after subtracting the background current, and make a working curve;

[0029] 2) Attach the above flexible sensor to the part to be measured of the plant to be measured, connect an electrochemical workstation, perform differential pulse voltammetry scanning, and calculate the instantaneous concentration of solanine in the part to be measured of the plant to be measured through the working curve for the obtained current signal.

[0030] In the above method, the part to be measured can be tissues such as the roots, stems, leaves, and fruits of plants, and can be applied to different growth periods and different environments of plants. There is no ex vivo or minimally invasive damage to the part of the plant to be detected.

[0031] The beneficial effects of the present invention are as follows:

[0032] The flexible sensor for real-time in-vivo monitoring of solanine content in plants provided by the present invention can perform online detection and analysis of solanine in plant tissues at different stages. Since it is a patch type that adapts to the plant surface, it does not cause substantial damage to the detected material, and the material can continue to grow until maturity. At the same time, by drop-coating the modification of Ti 1-x Nb x S2, Au-PdAg, PEDOT:PSS, and Nafion nanomaterials, highly sensitive detection of solanine is achieved, and the detection results are highly accurate. Description of the Drawings

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the prior art in the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0034] Figure 1 It is a schematic diagram of the electrode of the flexible sensor described in the present invention.

[0035] Figure 2 It is a schematic diagram of the preparation of the working electrode of the flexible sensor described in the present invention.

[0036] Figure 3 It is the cyclic voltammogram of the flexible electrode described in the present invention in 1 mM [Fe(CN)6] 4- solution. Detailed Embodiments

[0037] The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps, or conditions of the present invention belongs to the scope of the present invention.

[0038] In the following embodiments, the information of each reagent and instrument used is shown in Table 1.

[0039] Table 1 Information Table of Reagents Used in the Embodiment

[0040]

[0041] Embodiment 1 Flexible Sensor and Its Preparation

[0042] Partially refer to Figure 1 , Figure 2 , this embodiment provides a flexible sensor, including a PDMS flexible substrate and a balloon-shaped working electrode, a balloon-shaped reference electrode, and a balloon-shaped counter electrode arranged on the PDMS flexible substrate, with a length of about 45 mm and a width of about 20 mm;

[0043] The balloon-shaped working electrode is a laser-induced graphene electrode LIG with a surface successively modified with niobium-doped titanium disulfide crystals (Ti 1-x Nb x S2), Au-PdAg with a dumbbell-shaped structure, and a PEDOT:PSS–Nafion composite material;

[0044] The balloon-shaped reference electrode is a laser-induced graphene electrode LIG with a surface modified with Ag / AgCl;

[0045] The balloon-shaped counter electrode is a laser-induced graphene electrode LIG.

[0046] For the flexible sensor provided in this embodiment, the preparation method is as follows:

[0047] (1) Stick a polyimide tape (PI) on the glass, and then clamp it with a polymer polymethyl methacrylate (PMMA) mold. Wash the PI tape with distilled water and ethanol. Then, use a computer-controlled laser to pattern the PI tape (laser-induced graphene technology) to obtain three balloon-shaped LIG electrodes (corresponding to the counter electrode, working electrode, and reference electrode from left to right).

[0048] (2) Use a spin coater to uniformly cover the patterned PI tape with liquid polydimethylsiloxane (PDMS) (primer A and curing agent B, mixed and stirred at a ratio of 10:1 for 10 min) at 100 rpm for 60 s, and then heat it in a vacuum drying oven at 100 °C for 10 h. Then, peel off one side of the PDMS / LIG from the PI film to obtain a flexible and stretchable balloon-shaped LIG electrode. The curved conductive region in the middle of the balloon-shaped LIG electrode is insulated by a liquid PDMS coating and the same heat treatment.

[0049] (3) The balloon-shaped LIG electrode on the left is the counter electrode. Coat Ag / AgCl silver paste on the balloon-shaped LIG electrode on the right and heat it until it cures to make an Ag / AgCl reference electrode. Subsequently, place the microelectrode in a 0.5 M dilute sulfuric acid solution and perform cyclic voltammetry scanning (0 - 1.5 V) to obtain a typical cyclic voltammogram to ensure that the electrode surface is clean.

[0050] (4) Ultrasonically treat niobium-doped titanium disulfide crystals (Ti 1-x Nb x S2) with a concentration of 5.0 mg / mL suspended in ultrapure water for three hours to obtain a uniform and well-dispersed suspension. Subsequently, drop 10 μL of the suspension onto the exposed working electrode in the middle, and after drying, obtain a Ti 1-x Nb x S2 / LIG electrode.

[0051] (5) Prepare a uniformly dispersed solution of dumbbell-structured Au-PdAg nanomaterials with a concentration of 1 mg / mL. Take 10 μL and drop-coat it on the Ti 1-x Nb x S2 / LIG electrode to obtain the Au-PdAg / Ti 1-x Nb x S2 / LIG electrode.

[0052] (6) Stir the commercially available PEDOT:PSS aqueous dispersion (mass fraction 1.3%) at room temperature for 48 h to make it evenly distributed. Continuously stir the PEDOT:PSS aqueous dispersion and a 5% Nafion solution by volume ratio of 1:1 at room temperature for 24 h to obtain the PEDOT:PSS–Nafion mixed solution. By directly dropping 5 μL of the PEDOT:PSS–Nafion mixed solution on the Au-PdAg / Ti 1-x Nb x S2 / LIG electrode, and then drying it in a clean environment at room temperature, the working electrode is obtained.

[0053] Example 2 Application of the flexible sensor

[0054] Select potatoes as the experimental material, and use the potatoes at the tuber-forming stage as the detection object.

[0055] (1) Prepare solanine-phosphate buffer (pH = 4.5) solutions with concentrations of 0, 0.1, 0.5, 1, 5, 10, 50, 100, and 1000 μM respectively. Use the flexible sensor prepared in Example 1 for differential pulse voltammetry detection (potential 0 - 0.4 V, potential increase 0.004 V, amplitude 0.05 V, pulse width 0.02 s, pulse period 0.5 s, rest time 20 s) to obtain a set of relationship curves between concentration and the peak potential after subtracting the background current, and make the standard curve of the solanine flexible sensor. The linear equation is I p (μA) = 32.531 + 0.810C (μM), and the linear range can reach 0.5 - 100 μM.

[0056] (2) After the electrode of the flexible sensor is cleaned, first detect three standard concentration (5, 50, 70 μM) solanine solutions for electrochemical calibration respectively. If the deviation of the slope between the working curve and the standard curve is within 15%, it is considered that the electrode can work normally.

[0057] (3) After calibration, attach the flexible sensor to the potato, connect the electrochemical workstation, and perform differential pulse voltammetry scanning on the sample (potential 0 - 0.4V, potential increment 0.004V, amplitude 0.05V, pulse width 0.02s, pulse period 0.5s, rest time 20s). After stable testing for 5 min, the obtained current signal is used to calculate the instantaneous concentration of the tested sample through the calibrated working curve.

[0058] Comparison of experimental results

[0059] Select potato plants of two different varieties, Lv Potatoes No. 1 and Zhongshu No. 2, in the same period, detect their solanine content by high performance liquid chromatography, and compare with the instantaneous detection results of the electrochemical method of the present invention, as shown in the following table. The results show that the detection results of the flexible electrode sensing method are reliable.

[0060] Table 2 Comparison of test results

[0061]

[0062] Comparative example 1

[0063] The difference from Example 1 is only that: the drop coating modification of Au - PdAg is omitted.

[0064] The results characterized the preparation process of the sensor by cyclic voltammetry (CV). The CV scan was carried out in 1 mM [Fe(CN)6] 4- solution (containing 0.1 mol KCL). The current response of the CV scan is as Figure 3 shown. Compared with PEDOT:PSS - Nafion / Ti 1-x Nb x S2 / LIG of Comparative Example 1, when the Au - PdAg material is modified onto Ti 1-x Nb x S2 / LIG, the redox peak current of the obtained PEDOT:PSS - Nafion / Au - PdAg / Ti 1-x Nb x S2 / LIG increases, and the peak - to - peak potential difference also decreases. This is because of the high conductivity and catalytic performance of Au - PdAg3, which improves the electron transfer rate and enhances the reversibility. It shows that only the combination of Ti 1-x Nb x S2, Au - PdAg, and PEDOT:PSS–Nafion composite materials can be used to prepare a flexible sensor with high sensitivity and high accuracy for solanine detection.

[0065] The above embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various modifications and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A flexible sensor, characterized in that, It includes a flexible substrate and a working electrode, a reference electrode, and a counter electrode disposed on the flexible substrate; The working electrode is a laser-induced graphene electrode LIG with a titanium disulfide crystal doped with niobium, a dumbbell-shaped Au-PdAg, and a PEDOT:PSS–Nafion composite material sequentially modified on its surface; The reference electrode is a laser-induced graphene electrode LIG with Ag / AgCl modified on its surface; The counter electrode is a laser-induced graphene electrode LIG.

2. The flexible sensor according to claim 1, characterized in that, The shapes of the working electrode, the reference electrode, and the counter electrode are all balloon-shaped.

3. The flexible sensor according to claim 1 or 2, characterized in that The material of the flexible substrate is polydimethylsiloxane.

4. The preparation method of the flexible sensor according to any one of claims 1-3, characterized in that, It includes: Preparing the counter electrode: Preparing a laser-induced graphene electrode LIG on a polyimide film, and transferring the laser-induced graphene electrode LIG to a polydimethylsiloxane flexible substrate through a polydimethylsiloxane transfer strategy to make the counter electrode; Preparing the reference electrode: Preparing a laser-induced graphene electrode LIG on a polyimide film, transferring the laser-induced graphene electrode LIG to a polydimethylsiloxane flexible substrate through a polydimethylsiloxane transfer strategy, and then coating Ag / AgCl silver paste on the laser-induced graphene electrode LIG and curing it to make the reference electrode; Preparing the working electrode: Preparing a laser-induced graphene electrode LIG on a polyimide film, transferring the laser-induced graphene electrode LIG to a polydimethylsiloxane flexible substrate through a polydimethylsiloxane transfer strategy, and then sequentially coating a titanium disulfide crystal doped with niobium, a dumbbell-shaped Au-PdAg, and a PEDOT:PSS–Nafion composite material on the laser-induced graphene electrode LIG to make the working electrode.

5. The preparation method according to claim 4, characterized in that, The sequential coating of the titanium disulfide crystal doped with niobium, the dumbbell-shaped Au-PdAg, and the PEDOT:PSS–Nafion composite material on the laser-induced graphene electrode LIG specifically includes: Drop Ti on the laser-induced graphene electrode LIG 1-x Nb x S2 suspension, and after drying, obtain the Ti 1-x Nb x S2 / LIG electrode; On the Ti 1-x Nb x S2 / LIG electrode, dropwise coat an Au-PdAg dispersion liquid, and obtain an Au-PdAg / Ti 1-x Nb x S2 / LIG electrode after drying; On the Au-PdAg / Ti 1-x Nb x It is obtained by dropwise coating a mixture of PEDOT:PSS aqueous dispersion and Nafion solution on the S2 / LIG electrode and drying.

6. The preparation method according to claim 5, characterized in that, The Ti 1-x Nb x concentration of the S2 suspension is 4 to 6 mg / mL; The concentration of the Au-PdAg dispersion is 0.9~1.1 mg / mL; The volume ratio of the PEDOT:PSS aqueous dispersion to the Nafion solution is 1:0.8~1.

2. The mass fraction of PEDOT:PSS in the PEDOT:PSS aqueous dispersion is 1.0~1.5%, and the mass fraction of Nafion in the Nafion solution is 4~6%.

7. The preparation method according to claim 5 or 6, characterized in that, By mixing the PEDOT:PSS aqueous dispersion and the Nafion solution and continuously stirring for 24~26 h, the mixed solution of the PEDOT:PSS aqueous dispersion and the Nafion solution is obtained.

8. Application of the flexible sensor according to any one of claims 1-3 or the flexible sensor prepared by the preparation method according to any one of claims 4-7 in real-time in vivo monitoring of solanine content in plants.

9. A method for real-time monitoring of solanine content in plants, characterized in that, It includes: 1) Prepare a series of solanine-phosphate buffer solutions with different concentrations respectively, and use the flexible sensor according to any one of claims 1-3 or the flexible sensor prepared by the preparation method according to any one of claims 4-7 to perform differential pulse voltammetry detection to obtain a set of relationship curves between the concentration and the peak current after deducting the background current, and make a working curve; 2) Attach the flexible sensor described in any one of claims 1-3 or the flexible sensor prepared by the preparation method described in any one of claims 4-7 to the part to be measured of the plant to be measured, connect an electrochemical workstation, perform differential pulse voltammetry scanning, and calculate the instantaneous concentration of solanine in the part to be measured of the plant to be measured through the working curve based on the obtained current signal.

Images