In-situ extraction and detection system and method for plant nutrients based on microchip electrophoresis

Abstract

The application discloses a plant nutrient in-situ extraction and detection system and method based on microchip electrophoresis. The method first extracts nutrient ions from a plant to be detected to obtain a sample solution. The plant leaf nutrient extraction includes sampling, in-situ burning and in-situ extraction. The plant stem tissue nutrient extraction needs to include the cooperation of a capillary, a valve, a micro camera, an operation table and a micro air pressure pump. The plant rhizosphere soil nutrient extraction includes sampling, grinding, screening and extraction. Then, a control module controls a voltage module to generate a sample injection and separation high voltage, and the sample to be detected is introduced into a microchip and separated. Next, a signal module generates an excitation and reference signal, and a detection module detects the ions separated from the microchip. Different charged ions have different charge numbers, ion radii and masses, so that their migration speeds in a constant electric field are different. The ion types can be distinguished according to the time of arrival, and the concentration of the ions can be determined according to the obtained peak area.

Description

Technical Field

[0001] This invention relates to the field of new technologies and equipment for smart agriculture, and in particular to a system and method for in-situ extraction and detection of plant nutrients based on microchip electrophoresis. Background Technology

[0002] Timely acquisition of nutrient status during crop growth is crucial for maintaining normal growth and development and ensuring precise nutrient supply. Furthermore, crop nutrient diagnosis helps avoid unnecessary nutrient application, reduces costs, minimizes soil nutrient depletion, and promotes sustainable soil use, aligning with current standards for smart agriculture.

[0003] At present, crop nutrient diagnosis mainly relies on laboratory analysis, which usually depends on large-scale testing equipment. This equipment has problems such as expensive instruments, large size, difficult operation, low accuracy, high testing cost, slow testing rate, complex pretreatment and inability to monitor in real time. It is difficult to meet the needs of in-situ detection of crop nutrients and cannot understand the nutrient level of crops in real time and quickly.

[0004] In recent years, microchip electrophoresis-C 4 The capacitively coupled contactless conductivity detection (D) method has been developed. Based on the principle of microchip electrophoresis, different charged ions, due to differences in their charge number, ionic radius, and mass, migrate at different speeds under a constant electric field, resulting in electrophoretic separation. The capacitively coupled contactless conductivity method is used to detect these ions. Capacitive coupling refers to the formation of a capacitance between the detection electrode and the receiving electrode after an excitation signal is input, as well as between the electrode and the test solution. This capacitance physically exists and hinders signal transmission in the form of capacitive reactance. Contactless detection means that the detection and receiving electrodes do not have direct physical contact with the test solution, allowing for the detection of the separated ion signal intensity. Microchip electrophoresis can distinguish ion types based on the peak position time (ion migration time) of the detected ion spectrum and quantify the ion concentration based on the peak height (conductivity signal intensity) to obtain the amount of the analyte ion.

[0005] Microchip electrophoresis-C 4 Method D is highly applicable, easy to miniaturize and integrate, and consumes less sample, making it possible for minimally invasive and high-frequency detection of crops. Its portability, rapid detection, and low cost meet the requirements for in-situ and large-scale monitoring of crop nutrients. Its simultaneous detection of multiple nutrient ions further reduces the sampling volume required, minimizes damage to crops, and improves efficiency.

[0006] However, microchip electrophoresis-C has not yet been observed. 4The D method is applied to the in-situ detection of nutrient ions in crops; however, its many advantages have great application prospects in the field of in-situ detection of crop nutrients, and can provide a new approach for in-situ monitoring of crop nutrients. At present, the following technical bottlenecks still exist in this field and need to be further overcome: (1) How to collect and extract (leach) ion sample solutions from crop stems, leaves and rhizosphere soil in a small amount, in situ and efficiently; (2) How to input the collected / leached ion solution samples into a microchip to achieve automatic detection; (3) How to overcome many interference factors under complex working conditions, improve anti-interference performance, and output stable, reliable and accurate nutrient information. Summary of the Invention

[0007] The problem this invention aims to solve is to provide a system and method for in-situ extraction and detection of plant nutrients based on microchip electrophoresis, which extracts nutrient molecules from plant leaf tissues, stems, and rhizosphere soil, and then performs electrophoresis with C... 4 By combining D-type electrophoresis as a method for plant sample introduction and separation, in-situ detection of plant nutrient ions can be achieved.

[0008] The present invention adopts the following technical solution: a plant nutrient in situ extraction and detection system based on microchip electrophoresis, comprising a control module, a voltage module, a signal module, a microchip module, a detection module, and a plant nutrient molecule extraction module;

[0009] The control module controls the voltage module to generate voltage and controls the signal module to generate excitation signals, which are then provided to the excitation electrodes in the detection module.

[0010] The plant nutrient ion extraction module extracts nutrient ions from the leaves, stem tissues, and rhizosphere soil of the plant to be tested, respectively, to obtain a test solution. The test solution enters the microchip module, where ions undergo electrophoresis under high pressure, and the separated ions reach the detection module.

[0011] The separated ions are detected in situ in the detection module. The ion types are distinguished according to the peak position time of the ion spectrum, and the ion concentration is quantified according to the peak shape height of the ion spectrum, so as to obtain the ion types and concentrations of the test solution.

[0012] Furthermore, the voltage module is a high-voltage power supply, and the control module drives the voltage module to generate sample injection high voltage and separation high voltage, which respectively control the sample ion injection and separation; the control module also controls the signal module to generate a sinusoidal signal, and adjusts the signal intensity of ion detection by controlling the amplitude and phase of the sinusoidal signal.

[0013] Furthermore, the microchip module includes: a plastic tube, a PMMA membrane, a microchannel, an injection port, an injection channel, an injection waste liquid port, a separation port, a separation channel, and a separation waste liquid port; the radii of the injection port, injection waste liquid port, separation port, and separation waste liquid port are the same as the width of the injection channel and the separation channel, and the microchannel cation exchange membrane is fabricated using a printed circuit board; the test solution is injected into the injection port, an injection voltage is applied, and ions in the test solution enter the microchip channel under the action of an electric field; a separation voltage is applied, and different charged ions undergo electrophoresis and separate in the separation channel, the separated ions reach the separation waste liquid port, and the ion spectrum is obtained from the detection module, indicating the types and concentrations of ions in the detected solution.

[0014] Furthermore, the electrodes in the detection module are fabricated on a printed circuit board, including excitation electrodes, detection electrodes, and shielding electrodes. The excitation electrodes generate excitation signals, the detection electrodes acquire the final detection signals for processing, and the shielding electrodes are used to reduce the influence of stray capacitance on the detection signals.

[0015] Furthermore, the plant nutrient molecule extraction module includes: a capillary tube, a valve, a miniature camera, an operating table, and a miniature air pump. The capillary tube is vertically inserted into the stem of the plant to be tested. The operating table controls the miniature air pump to change the air pressure inside the capillary tube to introduce the juice into the capillary tube. The image transmitted from the miniature camera to the operating table is observed. After the juice reaches the designated position, the introduction of juice is stopped, and air is injected into the capillary tube to introduce the juice into the sample introduction channel of the microchip module.

[0016] Furthermore, the plant nutrient in-situ extraction and detection system based on microchip electrophoresis also includes a micro in-situ burner, which consists of a ceramic container and a heating electrode, with the heating electrode wound and fixed in the ceramic container in a loop shape.

[0017] The heating electrode is made of metal, semiconductor or metal ceramic. The working voltage is applied to both ends of the heating electrode to heat or burn the sample in the ceramic container.

[0018] The present invention also provides a method for in-situ extraction and detection of plant nutrients based on microchip electrophoresis, comprising the following steps:

[0019] S1. Extract the nutrient molecules from the plant to be tested to obtain the test solution; the extraction of nutrient molecules is as follows:

[0020] S1.1 Extraction of leaf nutrient ions from the plant to be tested, including: perforation sampling, in-situ ignition, grinding and sieving, concentrated acid cell wall disruption, heating digestion and water addition for sample fixation;

[0021] S1.2 Extraction of nutrient ions from the stem tissue of the plant to be tested, including: opening the tube, taking the juice, introducing it into the capillary tube, controlling the amount of juice, and introducing it into the microchip;

[0022] S1.3 Extraction of rhizosphere soil nutrients from the plants to be tested, including: soil sampling, drying, grinding, sample dissolution, ultrasonic extraction and centrifugation;

[0023] S2. The sample solution obtained after extraction is input from the microchip module inlet. Under high pressure, the ions are separated by electrophoresis and then reach the detection module.

[0024] S3. Ion detection of the separated ions: Different ions arrive at the detection module at different times, and different ion spectra are obtained in the detection module. The ion types are distinguished according to the peak position time of the ion spectra, and the ion concentration is quantified according to the peak height of the ion spectra, so as to obtain the ion types and concentrations of the test solution.

[0025] Specifically, step S1 involves the in-situ extraction and detection of nutrients from plant leaf tissue, stem tissue, and rhizosphere soil, such as... Figure 1 As shown:

[0026] A represents the extraction and detection of nutrients from plant leaves. The extraction process includes: extracting leaf tissue, igniting the leaf tissue in a micro in-situ igniter, grinding and sieving the ignited ash sample, adding concentrated sulfuric acid and hydrogen peroxide for in-situ extraction, and then adding deionized water to a final volume of 2 mL to obtain the nutrient extract. The extracted nutrient extract is then injected into an electrophoresis microchip using a pipette for subsequent ion detection.

[0027] B represents the extraction and detection of nutrients in the plant stem tissue. The specific operation is as follows: an opening is made in the stem tissue with a blade, the end of the capillary glass tube is inserted into the opening of the stem tissue, and the operating table is adjusted to control the micro air pressure pump to regulate the air pressure in the tube to extract the sap from the stem tissue. At the same time, the real-time image transmitted by the micro camera is observed. When the sap reaches the dotted line in the micro camera image, the air pressure pump is controlled to stop extracting the sap, the valve connected to the capillary tube is opened, and a syringe is inserted into the valve to force the sap extracted to the microchip sample inlet into the microchip sample inlet. After the sap reaches the microchip sample inlet, subsequent detection can be performed.

[0028] C represents the extraction and detection of nutrients in the soil around the corn plant roots. The specific procedure for nutrient ion extraction is as follows: Take 100g of soil sample within 10cm of the plant roots, then dry, grind, and filter it. Next, take 0.5g of the soil sample, add 10mL of deionized water, and after shaking, centrifugation, and standing, the supernatant is the nutrient extract. Use a pipette to inject a small amount of the extract into the microchip inlet for subsequent nutrient ion detection.

[0029] Compared with the prior art, the present invention, employing the above technical solution, has the following technical effects:

[0030] (1) This invention combines in-situ extraction of plant nutrient molecules with microchip electrophoresis detection, effectively avoiding problems such as the introduction of other impurities, complex operation and many uncertainties that may occur in traditional extraction and detection methods.

[0031] (2) The method of the present invention extracts and detects nutrient molecules from plant leaf tissue, stem and rhizosphere soil respectively, and explains the extraction and detection methods in detail, providing new ideas for nutrient detection of other crops and plants.

[0032] (3) The present invention designs a plant stem juice extraction device that combines a channel, a miniature camera, a miniature air pump and an operating table. It can accurately control the amount of juice extracted and control the air pressure to inject the extracted juice into the microchip, which simplifies the experimental operation and improves the detection accuracy to a certain extent.

[0033] (4) This invention adopts an in-situ high-temperature calcination method, uses a micro in-situ calciner, and uses alumina ceramic to make the container. It has good thermal conductivity and can absorb the heat from the heating electrode to heat and calcify the blades inside the container. It also proposes a method for manufacturing the heating electrode and a method for connecting it to the ceramic container. Copper is selected as the electrode to fully heat it. It has a high melting point and good flexibility, making it easy to operate. It can quickly heat up to 600°C while meeting the requirements of low power consumption, making it suitable for field operations.

[0034] (5) This invention combines electrophoresis with C 4 The D-combination method uses electrophoresis as a sample introduction and separation method for plants, which saves a lot of pretreatment time compared to traditional detection methods. In addition, electrophoresis does not require any extra operations on the solution; it is directly injected into the channel of the microchip, and different ions can be separated by applying voltage. Compared with the traditional method of adding other solutions to separate ions, it avoids the introduction of other irrelevant ions, which improves the reliability and accuracy of the detection results to a certain extent.

[0035] (6) This invention uses C 4 The D electrode, as the detection electrode, avoids direct contact between the test solution and the detection electrode, thus protecting the detection electrode to some extent. At the same time, non-contact detection can reduce the influence of liquid residue from the previous experiment on subsequent experiments. In addition, the introduction of a shielding electrode reduces the influence of stray capacitance to some extent, improving the accuracy of the final detection results. Attached Figure Description

[0036] Figure 1 This invention relates to a method for detecting nutrient ions in maize plants using a microchip electrophoresis-based in-situ plant nutrient detection system.

[0037] Figure 2 This is the overall scheme of the plant nutrient in situ detection system based on microchip electrophoresis of the present invention;

[0038] Figure 3 This is a schematic diagram of the miniature in-situ burner in the plant nutrient in-situ detection system based on microchip electrophoresis of the present invention;

[0039] Figure 4 This is a dimensional diagram of the ceramic container of the miniature burner in this invention;

[0040] Figure 5 This is a diagram showing the microchip dimensions in the microchip-based in-situ plant nutrient detection system of the present invention.

[0041] Figure 6 This is a side-section image of the microchip in the in-situ plant nutrient detection system based on microchip electrophoresis of the present invention;

[0042] Figure 7 For the present invention C 4 D-electrode dimension diagram;

[0043] Figure 8 The detection module C of this invention 4 Schematic diagram of electrode D;

[0044] Figure 9 This is a schematic diagram of the microchannel fabrication process of the present invention;

[0045] Figure 10 This is a schematic diagram of the microchip assembly of the present invention;

[0046] Figure 11 This is a schematic diagram illustrating the sample introduction and separation principle of the present invention;

[0047] Figure 12 This is a schematic diagram of nutrient ion extraction from plant leaves according to the present invention;

[0048] Figure 13 This is a schematic diagram of nutrient ion extraction from the plant stems of the present invention;

[0049] Figure 14 This is a schematic diagram of nutrient ion extraction from the rhizosphere soil of the plant according to the present invention. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the application will be further described in detail below with reference to the accompanying drawings. The described embodiments are only a part of the embodiments involved in this invention. All non-innovative embodiments based on these embodiments by other researchers in the art are within the protection scope of this invention. Furthermore, the step numbers in the embodiments of this invention are only set for ease of explanation and do not limit the order of the steps. The execution order of each step in the embodiments can be adaptively adjusted according to the understanding of those skilled in the art.

[0051] This invention discloses a system for in-situ extraction and detection of plant nutrients based on microchip electrophoresis, such as... Figure 2 As shown, it includes: control module 1, voltage module 2, signal module 3, microchip module 4, and detection module 5.

[0052] The control module 1 is a PC-based module that mainly controls the voltage module 2 and the signal module 3. It drives the voltage module 2 to generate the injection voltage and separation voltage, thereby controlling the injection and separation of sample ions. The control signal module 3 generates a sinusoidal signal and controls the amplitude and phase of the sinusoidal signal to adjust the signal intensity of subsequent ion detection.

[0053] Voltage module 2 is a high-voltage power supply. The PC directly controls the voltage module to generate a 500V injection high voltage and a 1000V separation high voltage, which controls the liquid to be tested to enter the microchip and separate ions therein to reach the detection module 5.

[0054] The signal module 3 is controlled by the control module 1 to generate a sinusoidal signal and provide it to the excitation electrode in the detection module 5.

[0055] Microchip module 4, such as Figure 10 As shown, it includes: plastic tube 401, PMMA membrane 402, microchannel 403, sample inlet 404, sample inlet channel 405, sample waste liquid inlet 406, separation hole 407, separation channel 408, and separation waste liquid inlet 409.

[0056] The radii of the inlet port 404, the waste inlet port 406, the separation port 407, and the waste separation port 409 are the same as the width of the inlet channel 405 and the separation channel 408. A microchannel cation exchange membrane is fabricated using a printed circuit board. The test solution is injected into the inlet port, and an injection voltage is applied. Ions in the test solution enter the microchip channel 405 under the action of an electric field. A separation voltage is applied, and different charged ions undergo electrophoresis and are separated in the separation channel 408. The separated ions reach the waste separation port 409, and the ion spectrum is obtained from the detection module 5 to detect the types and concentrations of ions in the solution.

[0057] In the microchip electrophoresis system for in-situ detection of plant nutrients, the microchip size is as follows: Figure 5 As shown, the lateral section diagram is as follows Figure 6 As shown, it consists of cross-shaped channels and reservoirs. Due to the small volume of the channels themselves, only microliters of solution are needed to detect the ions in them. The test solution enters the microchip channel, and under high pressure, the ions undergo different trajectories. After detection and conversion, the concentration of different ions can be measured.

[0058] Detection module electrode dimensions, such as Figure 7 As shown: 1.8 mm is the overlap distance between electrode 1 and electrode 2, the distance between the two ends is 28.2 mm, and the width between the two electrodes is 2 mm.

[0059] In detection module 5, electrodes are designed on a printed circuit board using engineering software, and the printed circuit board forms a whole, such as... Figure 8 As shown, the circuit board includes two antiparallel electrodes, namely excitation electrode 501 and detection electrode 502, and shielding electrode 503. The excitation electrode generates an excitation signal, the detection electrode acquires the final detection signal for processing, and the shielding electrode can reduce the influence of stray capacitance on the final detection signal, thereby improving the detection accuracy.

[0060] In one embodiment of the present invention, the method for fabricating and assembling an electrophoretic microchip is as follows:

[0061] All microchannels can be fabricated using precision machining, 3D printing, and photolithography techniques. In this embodiment, printed circuit boards are preferably used to fabricate the microchannel anodizing membrane.

[0062] The microchannels consist of four equal-sized holes with a radius of 1 mm, and both the injection channel and separation channel are 1 mm wide. The injection channel is 17 mm long, and the separation channel is 56 mm long. All channels are 100 μm deep. The microchannels are fabricated using polymethyl methacrylate (PMMA) and a hot-pressing process. PMMA material is pressed onto a pre-made male mold at a specific temperature to create the desired microchannels. The fabrication method is as follows: Figure 9 As shown:

[0063] (1) The microchannel structure was designed using software;

[0064] (2) Custom-designed printed circuit boards with microchannel structures;

[0065] (3) Use chemical corrosion to remove unwanted parts of the metal to obtain a positive mold;

[0066] (4) Fix baffles around the base so that the PMMA poured in the next step will not flow out;

[0067] (5) Pour PMMA and pre-curing agent into the baffle at a volume ratio of 10:1, and bake at 70°C for 1 hour;

[0068] (6) After the PMMA cools down, it is peeled off from the male mold to obtain the microfluidic channel.

[0069] After obtaining the microfluidic channels, four holes are drilled in the PMMA membrane, such as... Figure 10 As shown in Figure 402, the dimensions are the same as the circular holes on the microchip. The PMMA board with microchannels was then cleaned three times with pure water using an ultrasonic cleaner, each time for 10 minutes.

[0070] Next, the channeled PMMA plate and PMMA film were treated with high-power (30W) air plasma at 700 mTorr for 5 minutes; the PMMA plate with microchannels was aligned with the PMMA film and hot-pressed at 105°C and 0.3 MPa for 15 minutes to strongly bond the two together. Then, a plastic base was attached to the top of the reservoir on the PMMA plate to connect the microchip to external devices. Finally, the microchip was integrated with the detection module.

[0071] It is important to note that only channels with hydrophilic properties (i.e., a water contact angle less than 90°) can allow liquids to move spontaneously under capillary forces; while some organic materials are hydrophobic (i.e., a water contact angle greater than 90°), preventing spontaneous liquid movement within the microchannels. Therefore, hydrophilic treatment of the microchannels is necessary. Microfluidic channels prepared from PMMA materials possess a certain degree of hydrophilicity; if needed, a layer of polyvinyl alcohol can be coated onto the inner wall to further enhance the channel's hydrophilicity.

[0072] In this embodiment, the principle of microchip electrophoresis detection is as follows: Figure 11 As shown, the details are as follows:

[0073] First, the sample is introduced into the chip channel, the test solution is injected into the microchip's injection port 404, and a 500V injection voltage is applied. Ions in the solution enter the microchip channel 405 under the action of the electric field.

[0074] Next, a separation voltage of 1000V is applied. Under the action of the separation voltage, different charged ions, due to their different charges, ionic radii, and masses, migrate at different speeds under a constant electric field, thus undergoing electrophoresis and separating in separation channel 408. The separated ions reach two C atoms. 4 D electrode 409.

[0075] Finally, different ions arrive at different times, resulting in different ion spectra. The ion types can be distinguished based on the peak position time (ion migration time) of the detected ion spectra. At the same time, the ion concentration can be quantified based on the peak height (conductivity signal intensity), ultimately yielding the ion types and concentrations of the solution to be tested.

[0076] In another embodiment of the present invention, the plant nutrient in-situ extraction and detection system based on microchip electrophoresis further includes a micro in-situ burner, such as... Figure 3 As shown, it mainly consists of two parts: a miniature ceramic container 6 and a heating wire 7 wound in a spiral shape around the container.

[0077] The heating wire can be made of one of the following materials with good thermal conductivity and high melting point: metal, semiconductor, and metal ceramic. In this embodiment, copper is preferred because it has good thermal conductivity, high melting point, and relatively low cost.

[0078] Alumina ceramics are preferred for manufacturing ceramic containers due to their high mechanical strength, high thermal conductivity, wear resistance, excellent insulation performance at high temperatures and high frequencies, and stable chemical and physical properties. Alumina ceramics are machined using CNC machine tools, and the dimensions of the ceramic containers are as follows... Figure 4 As shown, the container has an outer diameter of 25mm, an inner diameter of 23mm, and a height of 40mm. A conical container made according to these dimensions serves as the container for the miniature in-situ cauterization device. Copper wire is wound in a loop around the heating container six times and then secured.

[0079] The working principle of heating wire heating is as follows: a working voltage is applied across the heating electrode, continuously generating heat. After the heating electrode generates heat, it is transferred to the ceramic container. The heat then comes into contact with the air, resulting in heat transfer between the two media. Eventually, the heat of the entire ceramic container reaches a dynamic equilibrium, known as thermal steady state. The entire micro in-situ calciner operates at a relatively stable temperature within this thermal steady state, thus enabling continuous and stable heating or calcination of the sample inside the container.

[0080] This invention also provides a method for in-situ extraction and detection of plant nutrients based on microchip electrophoresis, the steps of which are as follows:

[0081] S1. Extract nutrient molecules from the plant to be tested to obtain the test solution; the extraction of nutrient molecules includes:

[0082] S1.1 Extraction of leaf nutrient ions from the plant to be tested, including: perforation sampling, in-situ ignition, grinding and sieving, concentrated acid cell wall disruption, heating digestion and water addition for sample fixation;

[0083] S1.2 Extraction of nutrient ions from the stem tissue of the plant to be tested, including: opening the tube, taking the juice, introducing it into the capillary tube, controlling the amount of juice, and introducing it into the microchip;

[0084] S1.3 Extraction of rhizosphere soil nutrients from the plants to be tested, including: soil sampling, drying, grinding, sample dissolution, ultrasonic extraction and centrifugation;

[0085] S2. The sample solution obtained after extraction is input from the microchip module inlet. Under high pressure, the ions are separated by electrophoresis and then reach the detection module.

[0086] S3. Ion detection of the separated ions: Different ions arrive at the detection module at different times, and different ion spectra are obtained in the detection module. The ion types are distinguished according to the peak position time of the ion spectra, and the ion concentration is quantified according to the peak height of the ion spectra, so as to obtain the ion types and concentrations of the test solution.

[0087] In embodiments of the present invention, the extraction of nutrient ions includes: extraction of nutrient ions from plant leaves, extraction of nutrient ions from plant stem tissues, and extraction of nutrient ions from plant rhizosphere soil, as detailed below:

[0088] A. Plant Leaves: Specific procedures for sampling and extracting nutrients from plant leaves, such as... Figure 12 As shown:

[0089] (1) Perforation sampling: Leaf samples were collected by perforation (as shown in ① in the figure). Only 5 mg of leaf tissue was needed for each nutrient analysis. The corresponding leaf area was 0.5-1 cm². 2 The perforation area can be adjusted according to the growth pattern of corn leaves.

[0090] (2) In-situ burning: The obtained leaf tissue was placed in a miniature in-situ burner 6 (as shown in ② in the figure), and a voltage of 15V was applied to the heating electrode 7 to rapidly heat it to 600℃, burning the leaf tissue into an ash sample (as shown in ③ in the figure).

[0091] (3) Grinding and sieving: Pour the ash sample formed by ignition into a grinding dish and grind for 5 minutes (as shown in ④ in the figure). After grinding, sieve through a 60-mesh sieve (as shown in ⑤ in the figure). Pour the sieved ash sample into a micro igniter.

[0092] (4) Cell wall disruption with concentrated acid. Add 100 μL of concentrated sulfuric acid to the ash sample and digest for 5 min, then add 200 μL of 30% hydrogen peroxide solution.

[0093] (5) Heating and digestion: Apply 10V voltage to the heating electrode to rapidly raise the temperature of the micro burner to 300℃ and continue heating for 60 minutes to completely digest it (as shown in ⑥ in the figure).

[0094] (6) Add water to fix the sample: Let it stand for a period of time to cool down, and then add deionized water to 2 mL, which is the final nutrient extract (as shown in ⑦ in the figure).

[0095] (7) Extraction and detection: Use a pipette to take the nutrient extract (as shown in ⑧ in the figure) and inject it into the microchip injection port for subsequent detection.

[0096] B. Plant stem: A device for sampling plant stem tissue, such as... Figure 13 As shown, it includes: capillary tube 9, valve 10, miniature pneumatic pump 13, miniature camera 11, and operating table 12.

[0097] Microneedle sampling was used to extract vascular sap from stem tissue, requiring only 5 μL of sample to complete a single nutrient analysis.

[0098] In this embodiment, the capillary glass tube 9 has an inner diameter of 0.5 mm to extract sap from the stem. A miniature air pump 13 is connected to the right side of the capillary glass tube 9 to control the air pressure inside the tube. By adjusting the air pressure, the sap from the stem is extracted using pressure. A miniature camera 11 is vertically aligned with the capillary tube and sends the image to the operating table 12 for real-time monitoring of the amount of sap extracted. The operating table 12 is connected to the miniature air pump 13 and the miniature camera 11, displaying the camera image and controlling the air pump 13 to adjust the air pressure.

[0099] The specific steps for extracting nutrients from plant stems are as follows:

[0100] (1) Opening: Use a blade to make an opening in the stem tissue of the plant;

[0101] (2) Extracting juice: Insert the capillary glass tube vertically into the corn stalk to extract juice;

[0102] (3) Introducing into the capillary tube: The control panel adjusts the micro air pressure pump to change the air pressure inside the tube and introduce the sap of the corn plant stem into the capillary glass tube.

[0103] (4) Control the amount of juice: Observe the image transmitted from the miniature camera to the operating table. When the juice reaches the position where the camera is facing the dotted line, control the operating table to adjust the miniature air pump to change the air pressure in the tube and stop the introduction of juice.

[0104] (5) Importing the microchip: Open the valve above the microchip injection port, use a syringe to inject air into the capillary, and introduce the juice in the injection port into the microchip channel.

[0105] After the stem sap to be tested is introduced into the microchip injection port, subsequent ion detection can be performed.

[0106] C. Peripheral soil of plants: Most of the soil nutrients that plants can absorb are soluble nutrient molecules. Therefore, direct extraction is used to extract soil nutrient molecules. Specific procedures for sampling and extracting nutrients from the peripheral soil of plants are as follows: Figure 14 As shown:

[0107] (1) Soil sampling: Take 100g of soil around the roots about 10cm away from the plant stem.

[0108] (2) Drying: Pour the collected soil sample into a micro burner, apply a 5V voltage to the heating electrode to rapidly heat it to 60°C, and continue drying for 24 hours to ensure that the moisture in the soil sample is completely evaporated.

[0109] (3) Grinding: Pour the dried soil sample into a grinding dish and grind for 5 minutes (as shown in ② in the figure). Next, sieve the ground soil sample through a 60-mesh analytical sieve (as shown in ③ in the figure).

[0110] (4) Sampling and dissolving: Weigh 0.5g of the sieved soil sample and add 10mL of deionized water to soak for 5min (as shown in ④ in the figure).

[0111] (5) Ultrasonic extraction: After soaking, stir the sample for 5 minutes. Then use an ultrasonic extractor to extract for 5 minutes (as shown in ⑤ in the figure) to allow excess large particles to settle at the bottom.

[0112] (6) Centrifugation: Take the supernatant solution after ultrasonic extraction and centrifuge it using a centrifuge (as shown in ⑥ in the figure) at 6000 r / min for 5 min. Then let it stand for 5 min. The supernatant is the root zone soil nutrient solution (as shown in ⑦ in the figure).

[0113] After obtaining the nutrient solution, use a pipette to extract the soil nutrient solution and input it through the microchip injection port for subsequent ion detection.

[0114] In summary, this invention first extracts nutrients from the plant to be tested to obtain a test solution. Nutrient extraction from plant leaves includes sampling, in-situ ignition, and in-situ extraction. Nutrient extraction from plant stem tissue requires the coordinated action of a capillary, valve, miniature camera, operating platform, and miniature pneumatic pump. Nutrient extraction from plant rhizosphere soil includes sampling, grinding, sieving, and extraction. Then, a control module controls a voltage module to generate high voltage for sample introduction and separation, passing the sample into the microchip and separating it. Next, a signal module generates excitation and reference signals, and a detection module detects the ions separated from the microchip. Different charged ions have different migration speeds under a constant electric field due to variations in their charge number, ionic radius, and mass. Ion types can be distinguished based on their arrival time, and ion concentrations can be determined based on the obtained peak area.

[0115] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A system for in-situ extraction and detection of plant nutrients based on microchip electrophoresis, characterized in that, It includes a control module (1), a voltage module (2), a signal module (3), a microchip module (4), a detection module (5), and a plant nutrient molecule extraction module; The control module (1) controls the voltage module (2) to generate voltage and controls the signal module (3) to generate an excitation signal, which is provided to the excitation electrode in the detection module (5); the detection module (5) uses C 4 The D electrode is used as the detection electrode; The plant nutrient ion extraction module extracts nutrient ions from the leaves, stem tissues, and rhizosphere soil of the plant to be tested, respectively, to obtain a test solution. The test solution enters the microchip module (4), where ions undergo electrophoresis under high pressure and are separated to reach the detection module (5). In the detection module (5), the separated ions are detected in situ. The types of ions are distinguished according to the peak position time of the ion spectrum, and the ion concentration is quantified according to the peak shape height of the ion spectrum to obtain the types and concentrations of ions in the test solution. The microchip module (4) includes: a plastic tube (401), a PMMA membrane (402), a microchannel (403), an injection port (404), an injection channel (405), an injection waste liquid port (406), a separation port (407), a separation channel (408), and a separation waste liquid port (409). The radii of the injection port, the injection waste liquid port, the separation port, and the separation waste liquid port are the same as the width of the injection channel (405) and the separation channel (408). The microchannel cation exchange membrane is made using a printed circuit board. The test solution is injected into the injection port, and an injection voltage is applied. Ions in the test solution enter the microchip channel (405) under the action of an electric field. A separation voltage is applied, and different charged ions undergo electrophoresis and separate in the separation channel (408). The separated ions reach the separation waste liquid port (409). An ion spectrum is obtained from the detection module (5) to detect the types and concentrations of ions in the solution. The plant nutrient ion extraction module includes: a capillary tube (9), a valve (10), a miniature camera (11), an operating table (12), and a miniature air pump (13). The capillary tube (9) is vertically inserted into the stem of the plant to be tested. The operating table (12) is controlled to adjust the miniature air pump (13) to change the air pressure in the capillary tube to introduce the juice into the capillary tube. The image transmitted from the miniature camera (11) to the operating table (12) is observed. After the juice reaches the designated position, the introduction of juice is stopped, and air is injected into the capillary tube (9) to introduce the juice into the microchip module sample inlet channel (405).

2. The plant nutrient in-situ extraction and detection system based on microchip electrophoresis according to claim 1, characterized in that, The voltage module (2) is a high-voltage power supply. The control module (1) drives the voltage module (2) to generate high injection voltage and high separation voltage, respectively controlling the injection and separation of sample ions. The control module (1) also controls the signal module (3) to generate a sinusoidal signal, and adjusts the signal intensity of ion detection by controlling the amplitude and phase of the sinusoidal signal.

3. The plant nutrient in-situ extraction and detection system based on microchip electrophoresis according to claim 1, characterized in that, The electrodes in the detection module (5) are fabricated on a printed circuit board and include two antiparallel electrodes, namely the excitation electrode (501) and the detection electrode (502). The detection module (5) also includes a shielding electrode (503). The excitation electrode generates an excitation signal, the detection electrode obtains the final detection signal for processing, and the shielding electrode is used to reduce the influence of stray capacitance on the detection signal.

4. The plant nutrient in-situ extraction and detection system based on microchip electrophoresis according to claim 1, characterized in that, It also includes a miniature in-situ burner, which consists of a ceramic container (6) and a heating electrode (7). The heating electrode (7) is wound and fixed in the ceramic container (6). The heating electrode (7) is made of metal, semiconductor or metal ceramic. The working voltage is applied to both ends of the heating electrode (7) to heat or burn the sample in the ceramic container (6).

5. A method for in-situ extraction and detection of plant nutrients based on microchip electrophoresis, wherein the system described in any one of claims 1-4 is used for in-situ extraction and detection of nutrients in the plant under test, characterized in that, The steps include the following: S1. Extract the nutrient molecules from the plant to be tested to obtain the test solution; the extraction of nutrient molecules is as follows: S1.1 Extraction of leaf nutrient ions from the plant to be tested, including: perforation sampling, in-situ ignition, grinding and sieving, concentrated acid cell wall disruption, heating digestion and water addition for sample fixation; S1.2 Extraction of nutrient ions from the stem tissue of the plant to be tested, including: opening the tube, taking the juice, introducing it into the capillary tube, controlling the amount of juice, and introducing it into the microchip; S1.3 Extraction of rhizosphere soil nutrients from the plants to be tested, including: soil sampling, drying, grinding, sample dissolution, ultrasonic extraction and centrifugation; S2. The sample solution obtained after extraction is input from the microchip module inlet. Under high pressure, the ions are separated by electrophoresis and then reach the detection module. S3. Ion detection of the separated ions: Different ions arrive at the detection module at different times, resulting in different ion spectra. The types of ions are distinguished based on the peak position time of the ion spectra, and the ion concentration is quantified based on the peak height of the ion spectra, thus obtaining the types and concentrations of ions in the test solution.

6. The method for in-situ extraction and detection of plant nutrients based on microchip electrophoresis according to claim 5, characterized in that, The extraction of nutrient ions from the leaves of the plant to be tested in step S1.1 is performed as follows: Take 5 mg of leaf tissue from the plant to be tested and place it in a miniature in-situ cauterization device. Apply a voltage of 15V to the heating electrode and cauterize at 600℃. The ash sample formed by ignition was ground and passed through a 60-mesh analytical sieve. 100 μL of concentrated sulfuric acid was added to the ash sample for digestion, followed by 200 μL of 30% hydrogen peroxide solution. Apply a 10V voltage to the heating electrode, heat to 300℃ for a period of time, let it stand and cool, then add deionized water to 2mL to complete the extraction.

7. The method for in-situ extraction and detection of plant nutrients based on microchip electrophoresis according to claim 5, characterized in that, The extraction of nutrient ions from the stem tissue of the plant to be tested in step S1.2 is performed as follows: Make an opening in the stem tissue of the plant to be tested, and insert the capillary end vertically into the stem of the plant to be tested; The control panel adjusts the micro air pump to change the air pressure inside the tube, introducing the juice into the capillary, while simultaneously observing the image transmitted to the control panel by the micro camera. Once the juice reaches the dotted line position, stop introducing the juice and open the valve above the microchip injection port; Air is injected into the capillary tube to guide the juice in the injection port into the microchip channel, thus completing the extraction.

8. The method for in-situ extraction and detection of plant nutrients based on microchip electrophoresis according to claim 5, characterized in that, The extraction of nutrient ions from the rhizosphere soil of the plant to be tested in step S1.3 is performed as follows: Take 100g of soil around the roots at a distance of 10cm from the stem of the plant to be tested and pour it into a micro cauterization device. Apply a 5V voltage to the heating electrode, heat to 60℃, and continue drying for 24 hours; After grinding the dried soil sample, it was sieved through a 60-mesh analytical sieve. Weigh 0.5g of sieved soil sample, add 10mL of deionized water to soak, stir, and then centrifuge at 6000r / min; After standing, the clear liquid at the top becomes the root zone soil nutrient solution, thus completing the extraction process.

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