A biosensor for real-time online monitoring of chemical components in living plants

Abstract

This invention belongs to the field of biosensor technology, specifically a biosensor for real-time online monitoring of chemical components within plants. The biosensor of this invention mounts the working electrode and reference electrode within a liquid cavity between two substrates, preventing damage during electrode insertion and removal from the plant and improving sensor durability. It utilizes microfluidic technology to draw liquid from within the plant into the liquid cavity through microchannels, enabling real-time monitoring of chemical components within the plant. This ensures consistent calibration and testing environments, improving measurement accuracy. Employing multiple microfluidic channels eliminates the need for micron-level precise positioning of the working / reference electrodes at the measurement site within the plant, improving measurement efficiency and field applicability. This biosensor enables online monitoring of chemical components within living plants without causing inherent damage to the sample. The obtained data accurately and dynamically reflects the chemical composition information within the plant in real time. Its simple operation facilitates widespread application in plant cultivation.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to a biosensor for real-time online monitoring of chemical components in living plants. Background Technology

[0002] Real-time online monitoring of changes in chemical components within plants plays a crucial role in their normal growth and development. Currently, in plant physiology research, traditional methods for detecting chemical components (such as various ions, proteins, and hormones) in plants primarily employ in vitro static analysis techniques such as photochemically induced fluorescence, high-performance liquid chromatography (HPLC), and chromatography-mass spectrometry. These methods require in vitro sampling of plant material and inferring the content within the plant by detecting the concentration of chemical substances in the extract. Therefore, complex sample pretreatment is necessary, which is time-consuming and requires high sample purity. Furthermore, this in vitro detection only reflects the static concentration or cumulative effect within the plant at a specific moment, and cannot provide long-term, real-time dynamic analysis. As research progresses, there is a desire to obtain real-time dynamic information on the changes in chemical components within plants during growth, development, and environmental adaptation. This would allow for a more comprehensive and clear understanding of plant physiological activities, ultimately guiding agricultural production. Therefore, it is necessary to invent and design novel, reliable, and field-compatible detection instruments to achieve in-situ real-time monitoring of chemical substances within plants during their growth process.

[0003] Currently, microelectrode biosensing technology is widely used for in-situ real-time monitoring of various chemical components within plants. Microelectrode sensors have a three-electrode system, including a reference electrode, a counter electrode, and a working electrode. These electrodes are integrated into the very small tip of the microelectrode (tens of micrometers), resulting in a very small sensing area. This necessitates the use of more advanced instruments (such as electrochemical workstations) to amplify the measured electrical signal, increasing instrument costs and hindering large-scale application in agricultural planting. Furthermore, during detection, the tip of the microelectrode must be precisely inserted into the measurement site on the plant. For example, measuring the ion concentration of vascular fluid in plants using microelectrode biosensors requires precise insertion of the microelectrode sensing portion (working, reference, and counter electrodes) into the plant's vascular system. Without the assistance of other equipment (such as microscopes or micromanipulators), achieving this manually is extremely difficult. If the electrode deviates from the vascular system, its surface may come into contact with other substances (such as plant cells), resulting in data that does not accurately reflect the ion concentration in the vascular system. This also leads to differences between the calibration environment (no electrode contact) and the testing environment (electrode contact with an object), resulting in measurement errors and reducing practicality in agricultural fields. Furthermore, repeated insertion and removal of the microelectrode can cause frictional damage and detachment of the sensitive membrane of the working electrode and the metal membrane of the reference electrode, reducing the sensor's durability. Therefore, developing a field-compatible and durable biosensor for real-time, in-vivo monitoring of chemical components within plants is of great significance to plant agriculture production. Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this invention is to provide a biosensor for real-time online monitoring of chemical components in living plants.

[0005] The present invention provides a biosensor for real-time online monitoring of chemical components in plants, comprising a working electrode and a reference electrode, and a pair of substrates designed according to the size of the electrodes;

[0006] The width of the pair of substrates is greater than that of the electrodes, and the length is selected according to the depth of the plant measurement site.

[0007] The pair of substrates each have grooves on their flat surfaces for mounting a working electrode and a reference electrode, respectively. The electrode-mounted surfaces of the two substrates are bonded together (the electrode portions can be aligned or misaligned, but misalignment is recommended to avoid short circuits). Alternatively, one substrate has a groove for mounting the working electrode and reference electrode, while the other substrate does not have a groove. The surfaces of the two substrates are bonded together. A cavity (e.g., square) is formed between the two substrates, called a liquid cavity. The working electrode and reference electrode are led out to the outside of the biosensor via leads. The two substrates are aligned, placing the electrodes within the central cavity, which also protects the electrodes from damage during insertion or removal from the plant. This ensures that the calibration and testing environments remain unchanged, improving testing accuracy.

[0008] The liquid chamber has multiple microfluidic channels on both sides (i.e., the left and right sides of the substrate) leading to the electrodes. Using microfluidic technology, the liquid inside the plant is drawn into the liquid chamber through the fluid channels, and the electrodes monitor the chemical composition inside the plant in real time.

[0009] The number of working electrodes and reference electrodes can be one or more, and their shapes can be circular, elliptical, triangular, square, or polygonal; the shape of the pair of substrates can be square, triangular, circular, elliptical, or polygonal; in order not to cause essential damage to the sample being tested, the substrate size should be as small as possible, but it is necessary to accommodate all electrodes in the liquid cavity of the substrate.

[0010] To promote microfluid flow without increasing manufacturing difficulty or cost, the cross-sectional area of ​​the microfluidic channel should be as small as possible, and the number of channels on each side should be selected based on the actual measured area.

[0011] The size range of the liquid chamber is selected based on the number and size of the electrodes.

[0012] Specifically:

[0013] In this invention, the diameter of the microfluidic channels ranges from 0.01 to 1 mm, and the number of channels per side ranges from 1 to 20. Preferably, the diameter ranges from 0.1 to 0.5 mm, and the number of channels per side ranges from 5 to 10.

[0014] In this invention, the diameter of the working electrode and the reference electrode ranges from 0.1 to 10 mm, and the size of the substrate ranges from 1 (width) x 2 (length) to 20 (width) x 40 (length) mm. The preferred electrode diameter range is 1 to 5 mm, and the preferred substrate size range is 2 (width) x 5 (length) mm to 10 (width) x 20 (length) mm.

[0015] In this invention, the size of the liquid cavity ranges from 1 (width) x 1 (length) to 20 (width) x 20 (length) millimeters. Preferably, the size of the liquid cavity ranges from 2 (width) x 2 (length) millimeters to 10 (width) x 10 (length) millimeters.

[0016] In this invention, the substrate material can be plastic, ceramic, metal, or composite material, etc.

[0017] The operation process of the biosensor of this invention is as follows: The biosensor is vertically cut into the measuring part (such as the vascular bundle) of the plant stem, allowing the microfluidic channel of the electrode to enter the measuring part of the plant, drawing liquid into the liquid cavity, and the electrode pairs monitor the chemical composition within the plant in real time (see reference). Figure 3 ).

[0018] Using the biosensor designed in this invention, there is no need for micron-level precise positioning of the working / reference electrode at the measurement site within the plant.

[0019] The biosensor of this invention enables online monitoring of chemical components within living plants without causing any intrinsic damage to the sample being tested. The obtained data results can accurately and dynamically reflect information about the chemical components within the plant in real time. The actual operation is simple and does not require additional equipment, which is conducive to its widespread application in plant cultivation sites.

[0020] The biosensor designed in this invention solves both the durability problem of previous microelectrode technologies and the challenge of precise positioning of commonly used electrodes within plants, thus improving its practicality in the field. It also ensures that the calibration and testing environments remain unchanged, enhancing testing accuracy. This invention is applicable to in-situ online real-time monitoring of plant roots, stems, fruits, or leaves. Attached Figure Description

[0021] Figure 1 This is a structural diagram of the biosensor of the present invention.

[0022] Figure 2 This is a structural diagram of the multi-channel biosensor of the present invention.

[0023] Figure 3 Illustration of application scenarios for measuring the chemical components (ions, proteins, hormones, etc.) of plant vascular tissue.

[0024] The following are the labels in the diagram: 1 is the working electrode, 2 is the substrate, 3 is the electrode lead, 4 is the reference electrode, 5 is the liquid chamber, 6 is the microfluidic channel, 7 is the working electrode, 8 is the second working electrode, 9 is the third working electrode, 10 is the fourth working electrode, 11 is the biosensor, 12 is the vascular bundle, 13 is the flow direction of the vascular liquid, and 14 is the plant outer skin. Implementation

[0025] The invention will be further described below with reference to specific implementation details and accompanying drawings.

[0026] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. Example

[0027] like Figure 1 As shown, a single-channel biosensor for real-time online monitoring of potassium ion concentration in plants is disclosed. The microfluidic channels have a diameter of 0.2 mm and six channels on each side. The working electrode and reference electrode each have a diameter of 1 mm, and the substrate measures 2 (width) x 5 mm. The liquid chamber measures 2 (width) x 3 (length) mm. The working electrode is a potassium-selective electrode, and the reference electrode is a silver / silver chloride electrode. This single-channel biosensor is connected to a Bluetooth wireless module circuit, enabling real-time data transmission to a mobile phone or computer. The single-channel biosensor is vertically cut into the vascular bundle of the plant stem, allowing the microfluidic channels of the electrodes to enter the vascular bundle and draw liquid into the liquid chamber. The electrodes then monitor the potassium ion concentration in the plant in real time, transmitting the data to a mobile phone or personal computer for analysis and monitoring of the plant's growth and health status (see reference). Figure 3 ). Example

[0028] like Figure 2 As shown, a multi-channel biosensor for real-time online monitoring of pH / potassium / calcium / sodium ion concentrations in plants is disclosed. The microfluidic channels have a diameter of 0.4 mm and six channels on each side. The working electrodes are each 1 mm in diameter, the reference electrode is 2 mm in diameter, and the substrate measures 4 (width) x 10 (length) mm. The liquid chamber measures 4 (width) x 6 (length) mm. The working electrodes are a pH electrode (7), a potassium ion selective electrode (8), a calcium ion selective electrode (9), and a sodium ion selective electrode (10). The reference electrode is a silver / silver chloride electrode. This multi-channel biosensor is connected to a Bluetooth wireless module circuit, enabling real-time data transmission to a mobile phone or computer. The multi-channel biosensor is vertically cut into the vascular bundle of the plant stem, allowing the microfluidic channels of the electrodes to enter the vascular bundle and draw liquid into the liquid chamber. The electrodes then monitor the pH / potassium / calcium / sodium ion concentrations in the plant in real time, transmitting the data to a mobile phone or personal computer for comprehensive analysis and monitoring of the plant's growth and health status.

[0029] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A biosensor for in vivo online real-time monitoring of chemical components in a plant body, characterized by, The pair of substrates includes a working electrode and a reference electrode, and is designed according to the size of the electrodes; The pair of substrates has a width greater than that of the electrodes and a length selected according to the depth of the plant measurement site; The pair of substrates has recesses on their planes for mounting the working electrode and the reference electrode respectively; the surfaces of the two substrates on which the electrodes are mounted are bonded together, or a recess for mounting the working electrode and the reference electrode respectively is formed on one of the substrates, and the other substrate is not provided with a recess; the surfaces of the two substrates are bonded together; a cavity is formed between the two substrates, which is referred to as a liquid chamber; the working electrode and the reference electrode are led out of the biosensor through leads; the two substrates are closed to place the electrodes in the cavity in the middle, which also protects the electrodes from being damaged when the electrodes are inserted into and pulled out of the plant; The liquid chamber is provided with a plurality of micro liquid flow channels on its two sides, which lead to the electrodes; the liquid in the plant is sucked into the liquid chamber through the liquid flow channels by using micro tube flow technology, and the chemical components in the plant are monitored in real time by the electrodes; The diameters of the working electrode and the reference electrode range from 0.1 mm to 10 mm, and the size of the substrate ranges from 1 X 2 mm to 20 X 40 mm; The diameters of the micro liquid flow channels range from 0.01 mm to 1 mm, and the number of channels on each side ranges from 1 to 20; The size of the liquid chamber ranges from 1 X 1 mm to 20 X 20 mm.

2. The biosensor of claim 1, wherein, The number of the working electrode and the reference electrode is one or more, and the size of the substrate is as small as possible under the condition that all the electrodes are accommodated in the liquid chamber of the substrate.

3. The biosensor of claim 1, wherein, The cross-sectional area of the micro liquid flow channel is as small as possible under the condition that the manufacturing difficulty and cost are not increased; and the number of channels on each side is selected according to the actual measurement area.

4. The biosensor of claim 1, wherein, The size of the liquid chamber is selected according to the number and size of the electrodes.

5. The biosensor of claim 1, wherein, The substrate material is plastic, ceramic, metal or composite material.

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