Microelectrodes, probes and devices for electrochemical detection

Abstract

This invention discloses a biomicroelectrode and its use in detecting neurotransmitters. The biomicroelectrode comprises a glass capillary, electrode fibers, and an electrode solution; the electrode solution is encapsulated within the glass capillary; one end of the electrode fiber extends into the electrode solution, and the other end of the electrode fiber is located outside the glass capillary; wherein the electrode solution comprises a liquid polymer or a polyol. This biomicroelectrode has advantages such as repeatability and high detection sensitivity, and its preparation method is simple and low-cost. Therefore, the above-mentioned biomicroelectrode can be used to detect neurotransmitters (especially peripheral neurotransmitters) with high accuracy and high sensitivity.

Description

Technical Field

[0001] This invention relates to the field of electrochemical analysis technology, specifically to a microelectrode, probe, and electrochemical detection device, and more specifically to the use of liquid polymers or polyols in the preparation of microelectrodes, the microelectrodes, probes, and electrochemical detection devices, their uses, and methods for in vitro detection of neurotransmitters. Background Technology

[0002] Neurotransmitters are crucial chemical messengers in the nervous system, playing a vital role in physiology and physical health. Current neurotransmitter research primarily focuses on neurotransmitters in the brain. In vivo in situ detection of neurochemicals in the brain can reveal the material basis of brain activity and brain disease processes, providing the most direct information for research in neuroscience and other fields. Currently, in vivo in situ electrochemical analysis methods, including electrochemical analysis, rapid scanning voltammetry, differential pulse voltammetry, amperometric methods, and potentiometry, are constantly being developed and improved, and are commonly used for the in situ real-time detection of physiologically active substances in the brain, such as vitamin C, catecholamines, pH, and oxygen. Electrochemical analysis methods, in particular, have advantages such as high sensitivity, good selectivity, and high spatiotemporal resolution, making them suitable for in vivo in situ analysis.

[0003] However, no publicly available electrochemical analysis methods for detecting peripheral neurotransmitters are currently available. Therefore, there is an urgent need to develop a microelectrode that can detect both central and peripheral neurotransmitters. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0005] This invention is based on the following discoveries of the inventors:

[0006] Nerve cells consist of two parts: the cell body and the nerve fiber. Previous neurotransmitter signal detection methods have focused on measuring the cell body as recorded by electrophysiological patch-clamp techniques. However, patch-clamp techniques can only directly record (detect) the cell body, or indirectly record signals from nerve axons or dendrites connected to the cell body; they cannot record signals from nerve axons or dendrites detached from the cell body.

[0007] The sympathetic nervous system follows the blood vessels of peripheral tissues throughout the body to all peripheral tissues, even though the cell bodies of these peripheral tissues in the sympathetic ganglia have been severed. However, the inventors unexpectedly discovered that nerve axons or dendrites detached from the cell bodies still secrete signals (such as sympathetic signals), and peripheral nerves can maintain the function of secreting neurotransmitters for up to several days in fluids, enough to detect their secretory signals (e.g., in animal tissues or clinical biopsy samples).

[0008] Based on this, in a first aspect, the present invention proposes the use of liquid polymers or polyols as effective components in electrode solutions. Bioelectrodes prepared using liquid polymers or polyols as effective components of electrode solutions have advantages such as repeatability and high detection sensitivity, and the preparation method of these bioelectrodes is simple and low in cost.

[0009] According to an embodiment of the present invention, the liquid polymer is polyethylene glycol.

[0010] According to an embodiment of the present invention, the average molecular weight of the polyethylene glycol is 50-800 g / mol, preferably 100-600 g / mol.

[0011] According to an embodiment of the present invention, the polyethylene glycol is selected from at least one of PEG-200, PEG-300, PEG-400, and PEG-600.

[0012] According to embodiments of the present invention, the polyol includes at least one of ethylene glycol, glycerol, pentaerythritol, 1,2-propanediol, 1,4-butanediol, 1,6-hexanediol, neopentyl glycol, diethylene glycol, dipropylene glycol, and trimethylolpropane.

[0013] In a second aspect, the present invention proposes the use of liquid polymers or polyols in the preparation of microelectrodes, wherein the electrode solution of the microelectrode comprises a liquid polymer or polyol. According to embodiments of the present invention, the above substances serve as effective components of the electrode solution. Bioelectrodes prepared using this electrode solution have advantages such as repeatability and high detection sensitivity, and the preparation method of this bioelectrode is simple and low in cost.

[0014] According to an embodiment of the present invention, the liquid polymer is polyethylene glycol.

[0015] According to an embodiment of the present invention, the average molecular weight of the polyethylene glycol is 50-800 g / mol, preferably 100-600 g / mol.

[0016] According to an embodiment of the present invention, the polyethylene glycol is selected from at least one of PEG-200, PEG-300, PEG-400, and PEG-600.

[0017] According to embodiments of the present invention, the polyol includes at least one of ethylene glycol, glycerol, pentaerythritol, 1,2-propanediol, 1,4-butanediol, 1,6-hexanediol, neopentyl glycol, diethylene glycol, dipropylene glycol, and trimethylolpropane.

[0018] In a third aspect, the present invention provides a biomicroelectrode. According to an embodiment of the present invention, the biomicroelectrode includes a glass capillary, electrode fibers, and an electrode liquid; the electrode liquid is encapsulated within the glass capillary; one end of the electrode fiber extends into the electrode liquid, and the other end of the electrode fiber is located outside the glass capillary; wherein the electrode liquid comprises a liquid polymer or a polyol. The biomicroelectrode according to the embodiment of the present invention has advantages such as repeatability and high detection sensitivity, and the preparation method of the biomicroelectrode is simple and the preparation cost is low.

[0019] According to an embodiment of the present invention, the liquid polymer or polyol is in a liquid state at room temperature.

[0020] According to an embodiment of the present invention, the liquid polymer is polyethylene glycol.

[0021] According to an embodiment of the present invention, the average molecular weight of the polyethylene glycol is 50-800 g / mol, preferably 100-600 g / mol.

[0022] According to an embodiment of the present invention, the polyethylene glycol is selected from at least one of PEG-200, PEG-300, PEG-400, and PEG-600.

[0023] According to embodiments of the present invention, the polyol includes at least one of ethylene glycol, glycerol, pentaerythritol, 1,2-propanediol, 1,4-butanediol, 1,6-hexanediol, neopentyl glycol, diethylene glycol, dipropylene glycol, and trimethylolpropane.

[0024] According to an embodiment of the present invention, one end of the glass capillary is tapered, and the electrode fiber extends from the tapered end of the glass capillary into the electrode liquid.

[0025] According to an embodiment of the present invention, the electrode fiber is selected from conductive inert material fibers.

[0026] According to an embodiment of the present invention, the conductive inert material fiber is selected from at least one of carbon fiber, tungsten fiber, gold fiber and platinum fiber.

[0027] According to an embodiment of the present invention, the diameter of the electrode fiber is 100 nm to 15 μm.

[0028] According to an embodiment of the present invention, the length of the electrode fiber is 50 mm to 100 mm.

[0029] In a fourth aspect, the present invention provides a probe. According to an embodiment of the present invention, the probe comprises the bio-microelectrode described in the third aspect. As is known from the foregoing, the bio-microelectrode of the third aspect has advantages such as repeatability, high detection sensitivity, simple preparation method, and low preparation cost. Therefore, a probe containing the above-mentioned bio-microelectrode can be used to detect neurotransmitters (especially peripheral neurotransmitters), with advantages such as high detection accuracy and high sensitivity.

[0030] In a fifth aspect, the present invention provides an electrochemical detection device. According to embodiments of the present invention, the electrochemical detection device comprises the biomicroelectrode described in the third aspect or the probe described in the fourth aspect. As is known from the foregoing, the biomicroelectrode of the third aspect has advantages such as repeatability, high detection sensitivity, simple preparation method, and low preparation cost. Therefore, a device containing the aforementioned biomicroelectrode can be used to detect neurotransmitters (especially peripheral neurotransmitters), offering advantages such as high detection accuracy and high sensitivity.

[0031] In a sixth aspect, the invention proposes the use of biomicroelectrodes or products containing biomicroelectrodes in electrochemical detection. Biomicroelectrodes or products containing biomicroelectrodes can be used for electrochemical detection, particularly for neurotransmitter detection.

[0032] According to an embodiment of the present invention, the biomicroelectrode is the biomicroelectrode described in the third aspect.

[0033] According to an embodiment of the present invention, the product comprising a biomicroelectrode is the probe described in the fourth aspect or the device described in the fifth aspect.

[0034] According to an embodiment of the present invention, the electrochemical detection is a neurotransmitter detection.

[0035] According to embodiments of the present invention, the neurotransmitter includes central neurotransmitters or peripheral neurotransmitters.

[0036] According to embodiments of the present invention, the peripheral neurotransmitters originate from the heart, blood vessels, kidneys, lungs, spleen, liver, fat, intestines, pancreas, skeletal muscle, bladder, and skin.

[0037] According to an embodiment of the present invention, the central neurotransmitter originates from the brain.

[0038] According to an embodiment of the present invention, the neurotransmitter detection includes in vitro neurotransmitter detection or in vivo neurotransmitter detection.

[0039] According to an embodiment of the present invention, the electrochemical detection is an in vitro neurotransmitter detection, and the sample for detection is selected from biological samples.

[0040] According to an embodiment of the present invention, the biological sample is selected from slice samples or cell samples.

[0041] According to an embodiment of the present invention, the neurotransmitter is selected from monoamine neurotransmitters or their metabolites, or vitamin C.

[0042] According to embodiments of the present invention, the monoamine neurotransmitters include at least one of dopamine, adrenaline, noradrenaline, histamine, and serotonin.

[0043] According to embodiments of the present invention, the metabolites of the monoamine neurotransmitter include at least one of 3,4-dihydroxyphenylglycol, 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindoleacetic acid.

[0044] In a seventh aspect, the present invention provides a method for in vitro detection of neurotransmitters. According to an embodiment of the invention, the method includes: detecting a sample to be tested using a biomicroelectrode or a product containing a biomicroelectrode. According to the method of the present invention, the biomicroelectrode or a product containing a biomicroelectrode can be used for electrochemical detection, particularly for neurotransmitter detection.

[0045] According to an embodiment of the present invention, the biomicroelectrode is the biomicroelectrode described in the third aspect.

[0046] According to an embodiment of the present invention, the product comprising a biomicroelectrode is the probe described in the fourth aspect or the device described in the fifth aspect.

[0047] According to embodiments of the present invention, the neurotransmitter includes central neurotransmitters or peripheral neurotransmitters.

[0048] According to embodiments of the present invention, the peripheral neurotransmitters originate from the heart, blood vessels, kidneys, lungs, spleen, liver, fat, intestines, pancreas, skeletal muscle, bladder, and skin.

[0049] According to an embodiment of the present invention, the central neurotransmitter originates from the brain.

[0050] According to an embodiment of the present invention, the neurotransmitter detection includes in vitro neurotransmitter detection or in vivo neurotransmitter detection.

[0051] According to an embodiment of the present invention, the electrochemical detection is an in vitro neurotransmitter detection, and the sample for detection is selected from biological samples.

[0052] According to an embodiment of the present invention, the biological sample is selected from slice samples or cell samples.

[0053] According to an embodiment of the present invention, the neurotransmitter is selected from monoamine neurotransmitters or their metabolites, or vitamin C.

[0054] According to embodiments of the present invention, the monoamine neurotransmitters include at least one of dopamine, adrenaline, noradrenaline, histamine, and serotonin.

[0055] According to embodiments of the present invention, the metabolites of the monoamine neurotransmitter include at least one of 3,4-dihydroxyphenylglycol, 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindoleacetic acid.

[0056] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0057] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0058] Figure 1 This is a schematic diagram of a biomicroelectrode in one embodiment of the present invention;

[0059] Figure 2 This is a schematic diagram or detection result of recording the release of the sympathetic neural network on a brain slice in Embodiment 2 of the present invention;

[0060] Figure 3 This is a schematic diagram or detection result of recording the release of the sympathetic neural network on a heart slice in Embodiment 3 of the present invention;

[0061] Figure 4 The results of SEC electrochemical recording of cardiac slices in Example 3 of this invention; wherein, ① when the action potential is reached; ② voltage-dependent Ca 2+ The channel opens, inducing Ca 2+ Influx; ③ Intracellular Ca 2+ Increased concentration; ④ Triggering the release of vesicular catecholamines; ⑤ Oxidizing catecholamines (NE) at the holding potential on the CFE; ⑥ Generating a typical SEC signal (ICFE);

[0062] Figure 5 The results of SEC detection in human heart biopsy in Embodiment 4 of the present invention are as follows:

[0063] Figure 6The results of SEC recording of sympathetic neuron release in peripheral organs (blood vessels, kidneys, lungs) in Example 5 of this invention;

[0064] Figure 7 The results of SEC recording of sympathetic neuron release in peripheral organs (spleen, liver, brown adipose tissue) in Example 5 of this invention;

[0065] Figure 8 The results of SEC recording of sympathetic neuron release in peripheral organs (intestine, skeletal muscle, and bladder) in Example 5 of this invention;

[0066] Figure 9 This is the SEC recording result of sympathetic neuron release in the LPS-induced inflammatory mouse model in Example 6 of the present invention;

[0067] Figure 10 The results of NE release detection in acute LPS-treated cardiac slices in Example 6 of this invention;

[0068] Figure 11 This is the SEC recording result of sympathetic neuron release in the HF mouse model in Example 7 of the present invention;

[0069] Figure 12 This is the result of detecting the sympathetic nerve NE in the mouse heart under in vivo conditions in Example 8 of the present invention. Detailed Implementation

[0070] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0071] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0072] Detailed description of the invention

[0073] Definitions and General Terms

[0074] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0075] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0076] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0077] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0078] This invention proposes the use of liquid polymers or polyols in the preparation of microelectrodes, microelectrodes, probes and electrochemical detection devices, their uses, and methods for in vitro detection of neurotransmitters, which will be described in detail below.

[0079] use

[0080] In a first aspect, the present invention proposes the use of liquid polymers or polyols as effective components in electrode solutions. Bioelectrodes prepared using liquid polymers or polyols as effective components in electrode solutions have advantages such as reproducibility (e.g., stable and repeatable recording in different tissue samples such as the heart, liver, and kidneys), high detection sensitivity, and the preparation method of these bioelectrodes is simple and low in cost.

[0081] In this paper, "electrode fluid" is an important liquid in biomicroelectrodes, which is responsible for providing conductivity and helping to establish a current path between the electrode and the sample.

[0082] In this paper, the term "effective component of electrode fluid" refers to the component that primarily provides electrical conductivity to achieve the function of the electrode fluid.

[0083] It should be noted that the liquid polymer or polyol of the present invention is derived from commercially available sources, wherein the liquid polymer may be selected from polyethylene glycol. In an optional embodiment of the present invention, the polyethylene glycol is analytical grade polyethylene glycol.

[0084] According to embodiments of the present invention, the average molecular weight of the polyethylene glycol is 50 to 800 g / mol, for example, 50 g / mol, 100 g / mol, 200 g / mol, 300 g / mol, 400 g / mol, 500 g / mol, 600 g / mol, 700 g / mol, 800 g / mol, or any two of these values ​​as endpoints, for example, 200 to 600 g / mol.

[0085] According to an embodiment of the present invention, the polyethylene glycol is selected from at least one of PEG-200, PEG-300, PEG-400, and PEG-600.

[0086] According to embodiments of the present invention, the polyol includes at least one of ethylene glycol, glycerol, pentaerythritol, 1,2-propanediol, 1,4-butanediol, 1,6-hexanediol, neopentyl glycol, diethylene glycol, dipropylene glycol, and trimethylolpropane.

[0087] In a second aspect, the present invention proposes the use of liquid polymers or polyols in the preparation of microelectrodes, wherein the electrode solution of the microelectrode comprises a liquid polymer or polyol. According to embodiments of the present invention, the liquid polymer or polyol serves as an effective component of the electrode solution. Bioelectrodes prepared using this electrode solution have advantages such as repeatability and high detection sensitivity, and the preparation method of this bioelectrode is simple and low in cost.

[0088] According to embodiments of the present invention, the average molecular weight of the polyethylene glycol is 50 to 800 g / mol, for example, 50 g / mol, 100 g / mol, 200 g / mol, 300 g / mol, 400 g / mol, 500 g / mol, 600 g / mol, 700 g / mol, 800 g / mol, or any two of these values ​​as endpoints, for example, 200 to 600 g / mol.

[0089] According to an embodiment of the present invention, the polyethylene glycol is selected from at least one of PEG-200, PEG-300, PEG-400, and PEG-600.

[0090] According to embodiments of the present invention, the polyol includes at least one of ethylene glycol, glycerol, pentaerythritol, 1,2-propanediol, 1,4-butanediol, 1,6-hexanediol, neopentyl glycol, diethylene glycol, dipropylene glycol, and trimethylolpropane.

[0091] Biomicroelectrodes, probes and devices

[0092] like Figure 1As shown, in a third aspect of the invention, a bio-microelectrode is provided. According to an embodiment of the invention, the bio-microelectrode includes a glass capillary, electrode fibers, and an electrode solution; the electrode solution is encapsulated within the glass capillary; one end of the electrode fiber extends into the electrode solution, and the other end of the electrode fiber is located outside the glass capillary; wherein the electrode solution comprises a liquid polymer or a polyol.

[0093] The inventors discovered that there are currently no reports on the detection of peripheral neurotransmitters using biomicroelectrodes. This is because existing traditional biomicroelectrodes have low sensitivity and cannot detect secretory signals from nerve axons or dendrites that have detached from the cell body. Furthermore, traditional biomicroelectrodes are expensive, difficult to fabricate, and hard to initiate peripheral tissue recording.

[0094] Based on this, the present invention, through improvements to the electrode solution composition of the bio-microelectrode, unexpectedly discovered that using liquid polymers or polyols as the electrode solution can improve the sensitivity and preparation composition of the bio-microelectrode, thereby enabling effective detection of neurotransmitters, especially peripheral neurotransmitters. Furthermore, the bio-microelectrode of the present invention has advantages such as repeatability and high detection sensitivity, and the preparation method of this bio-microelectrode is simple and low in cost.

[0095] According to an embodiment of the present invention, the liquid polymer or polyol is in a liquid state at room temperature.

[0096] According to an embodiment of the present invention, the liquid polymer is polyethylene glycol.

[0097] According to embodiments of the present invention, the average molecular weight of the polyethylene glycol is 50 to 800 g / mol, for example, 50 g / mol, 100 g / mol, 200 g / mol, 300 g / mol, 400 g / mol, 500 g / mol, 600 g / mol, 700 g / mol, 800 g / mol, or any two of these values ​​as endpoints, for example, 200 to 600 g / mol.

[0098] According to an embodiment of the present invention, the polyethylene glycol is selected from at least one of PEG-200, PEG-300, PEG-400, and PEG-600.

[0099] According to embodiments of the present invention, the polyol includes at least one of ethylene glycol, glycerol, pentaerythritol, 1,2-propanediol, 1,4-butanediol, 1,6-hexanediol, neopentyl glycol, diethylene glycol, dipropylene glycol, and trimethylolpropane.

[0100] According to an embodiment of the present invention, one end of the glass capillary is tapered, and the electrode fiber extends from the tapered end of the glass capillary into the electrode liquid.

[0101] According to an embodiment of the present invention, the electrode fiber is selected from conductive inert material fibers.

[0102] According to embodiments of the present invention, the conductive inert material fiber is selected from at least one of carbon fiber, tungsten fiber, gold fiber, and platinum fiber. This further improves the detection sensitivity of the bio-microelectrode.

[0103] According to an embodiment of the present invention, the diameter of the electrode fiber is 100 nm to 15 μm. This further improves the detection sensitivity of the biomicroelectrode.

[0104] According to an embodiment of the present invention, the length of the electrode fiber is 5 mm to 10 mm. This further improves the detection sensitivity of the bio-microelectrode.

[0105] In a fourth aspect, the present invention provides a probe. According to an embodiment of the present invention, the probe comprises the bio-microelectrode described in the third aspect. As is known from the foregoing, the bio-microelectrode of the third aspect has advantages such as repeatability, high detection sensitivity, simple preparation method, and low preparation cost. Therefore, a probe containing the above-mentioned bio-microelectrode can be used to detect neurotransmitters (especially peripheral neurotransmitters), with advantages such as high detection accuracy and high sensitivity.

[0106] In a fifth aspect, the present invention provides an electrochemical detection device. According to embodiments of the present invention, the electrochemical detection device comprises the biomicroelectrode described in the third aspect or the probe described in the fourth aspect. As is known from the foregoing, the biomicroelectrode of the third aspect has advantages such as repeatability, high detection sensitivity, simple preparation method, and low preparation cost. Therefore, a device containing the aforementioned biomicroelectrode can be used to detect neurotransmitters (especially peripheral neurotransmitters), offering advantages such as high detection accuracy and high sensitivity.

[0107] Applications and methods for in vitro detection of neurotransmitters

[0108] In a sixth aspect, the invention proposes the use of biomicroelectrodes or products containing biomicroelectrodes in electrochemical detection. Biomicroelectrodes or products containing biomicroelectrodes can be used for electrochemical detection, particularly for neurotransmitter detection.

[0109] Currently, it is only possible to directly record signals from the cell body or indirectly record signals from nerve axons or dendrites connected to the cell body; signals from nerve axons or dendrites detached from the cell body cannot be recorded. Furthermore, while sympathetic norepinephrine (NE) has been reported to play an important role in immune and cardiovascular diseases, the correlation and causal relationship of NE secretion during immune and cardiovascular diseases are poorly understood due to the lack of methods for real-time and in situ recording of NE release.

[0110] Although the sympathetic nervous system follows the blood vessels of peripheral tissues throughout the body to all peripheral tissues, even though the cell bodies of these peripheral tissues in the sympathetic ganglia have been severed, the inventors unexpectedly discovered that nerve axons or dendrites detached from the cell bodies still secrete signals (such as sympathetic signals). Peripheral nerves can maintain the function of secreting neurotransmitters for up to several days in fluid, and the secretory signals in animal tissues or clinical biopsy samples can be detected by bioelectrodes.

[0111] In some optional embodiments of the present invention, the biomicroelectrode may also include the following structure: an inert conductive material (e.g., carbon fiber, tungsten fiber, gold fiber, platinum fiber), preferably with an outer layer of insulating varnish covering the inert conductive material. This allows it to be used for detecting neurotransmitters, particularly peripheral neurotransmitters.

[0112] In some optional embodiments of the present invention, the biomicroelectrode is the biomicroelectrode described in the third aspect. As is known from the foregoing, the biomicroelectrode of the third aspect has advantages such as repeatability, high detection sensitivity, simple preparation method, and low preparation cost. Therefore, the above-mentioned biomicroelectrode can be used to detect neurotransmitters (especially peripheral neurotransmitters), with advantages such as high detection accuracy and high sensitivity.

[0113] According to embodiments of the present invention, the product comprising a biomicroelectrode is either the probe described in the fourth aspect or the device described in the fifth aspect. As is known prior, the biomicroelectrode of the third aspect has advantages such as repeatability, high detection sensitivity, simple preparation method, and low preparation cost. Therefore, using a probe or device containing the aforementioned biomicroelectrode can detect neurotransmitters (especially peripheral neurotransmitters), offering advantages such as high detection accuracy and high sensitivity.

[0114] According to an embodiment of the present invention, the electrochemical detection is a neurotransmitter detection.

[0115] According to embodiments of the present invention, the neurotransmitter includes central neurotransmitters or peripheral neurotransmitters.

[0116] According to embodiments of the present invention, the peripheral neurotransmitters originate from the heart, blood vessels, kidneys, lungs, spleen, liver, fat, intestines, pancreas, skeletal muscle, bladder, and skin.

[0117] According to an embodiment of the present invention, the central neurotransmitter originates from the brain.

[0118] According to an embodiment of the present invention, the neurotransmitter detection includes in vitro neurotransmitter detection or in vivo neurotransmitter detection.

[0119] According to an embodiment of the present invention, the electrochemical detection is an in vitro neurotransmitter detection, and the sample for detection is selected from biological samples.

[0120] According to an embodiment of the present invention, the biological sample is selected from slice samples or cell samples.

[0121] According to an embodiment of the present invention, the neurotransmitter is selected from monoamine neurotransmitters or their metabolites, or vitamin C.

[0122] According to embodiments of the present invention, the monoamine neurotransmitters include at least one of dopamine, adrenaline, noradrenaline, histamine, and serotonin.

[0123] According to embodiments of the present invention, the metabolites of the monoamine neurotransmitter include at least one of 3,4-dihydroxyphenylglycol, 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindoleacetic acid.

[0124] In a seventh aspect, the present invention provides a method for in vitro detection of neurotransmitters. According to an embodiment of the invention, the method includes: detecting a sample to be tested using a biomicroelectrode or a product containing a biomicroelectrode. According to the method of the present invention, the biomicroelectrode or a product containing a biomicroelectrode can be used for electrochemical detection, particularly for neurotransmitter detection.

[0125] In some optional embodiments of the present invention, the biomicroelectrode may also include the following structure: an inert conductive material (e.g., carbon fiber, tungsten fiber, gold fiber, platinum fiber), with an outer layer of insulating varnish covering the inert conductive material. This allows it to be used for detecting neurotransmitters, particularly peripheral neurotransmitters.

[0126] According to an embodiment of the present invention, the biomicroelectrode is the biomicroelectrode described in the third aspect. As is known from the foregoing, the biomicroelectrode of the third aspect has advantages such as repeatability, high detection sensitivity, simple preparation method, and low preparation cost. Therefore, the above-described biomicroelectrode can be used to detect neurotransmitters (especially peripheral neurotransmitters), offering advantages such as high detection accuracy and high sensitivity.

[0127] According to embodiments of the present invention, the product comprising a biomicroelectrode is the probe described in the fourth aspect or the device described in the fifth aspect. As is known prior, the biomicroelectrode of the third aspect has advantages such as repeatability, high detection sensitivity, simple preparation method, and low preparation cost. Therefore, using a probe or device containing the aforementioned biomicroelectrode can detect neurotransmitters (especially peripheral neurotransmitters), offering advantages such as high detection accuracy and high sensitivity. According to embodiments of the present invention, the electrochemical detection is neurotransmitter detection.

[0128] According to embodiments of the present invention, the neurotransmitter includes central neurotransmitters or peripheral neurotransmitters.

[0129] According to embodiments of the present invention, the peripheral neurotransmitters originate from the heart, blood vessels, kidneys, lungs, spleen, liver, fat, intestines, pancreas, skeletal muscle, bladder, and skin.

[0130] According to an embodiment of the present invention, the central neurotransmitter originates from the brain.

[0131] According to an embodiment of the present invention, the neurotransmitter detection includes in vitro neurotransmitter detection or in vivo neurotransmitter detection.

[0132] According to an embodiment of the present invention, the electrochemical detection is an in vitro neurotransmitter detection, and the sample for detection is selected from biological samples.

[0133] According to an embodiment of the present invention, the biological sample is selected from slice samples or cell samples.

[0134] According to an embodiment of the present invention, the neurotransmitter is selected from monoamine neurotransmitters or their metabolites, or vitamin C.

[0135] According to embodiments of the present invention, the monoamine neurotransmitters include at least one of dopamine, adrenaline, noradrenaline, histamine, and serotonin.

[0136] According to embodiments of the present invention, the metabolites of the monoamine neurotransmitter include at least one of 3,4-dihydroxyphenylglycol, 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindoleacetic acid.

[0137] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0138] Example 1: Preparation method of bio-microelectrode

[0139] The fabrication method of the microelectrode was based on the published literature (Shi-Rong, Wang, Wei, et al. Role of vesicle pools in action potential pattern-dependent dopamine overflow in ratstriatumin vivo[J]. Journal of Neurochemistry, 2011, 119(2):342-353.DOI:10.1111 / j.1471-4159.2011.07440.x.), and the structure of the bio-microelectrode is as follows. Figure 1 As shown, the only difference lies in the electrode solution of the bio-microelectrode in this embodiment, as detailed in the table below:

[0140] bio-microelectrode name Electrode solution composition bio-microelectrode name Electrode solution composition Sample 1 Polyethylene glycol 200 Sample 6 3M KCl Sample 2 Polyethylene glycol 400 Sample 7 KCl concentration of 0.15M Sample 3 Polyethylene glycol 600 Sample 8 0.15M NaCl Sample 4 Ethylene glycol Sample 5 Glycerol

[0141] Example 2: Microelectrode detection of brain slices

[0142] 1. Preparation of brain slices

[0143] Adult C57BL / 6 (6-8 weeks old) wild-type male mice (1.5 g·kg⁻¹, ip) were anesthetized with polyurethane. The brains were quickly removed, cut into 300 μm horizontal sections on a vibrator, and then incubated for 30 minutes in Tyrode's solution (125 mM NaCl, 2.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 10 mM glucose, pH 7.4) to obtain striatal brain sections.

[0144] 2. The brain slices from step 1 of this example were analyzed using different bio-microelectrodes (samples 1 to 8) prepared in Example 1. The specific detection steps are as follows:

[0145] 1) Insert a pCFE with an electrode tip diameter of 5 or 7 μm into a brain slice;

[0146] 2) The bipolar platinum electrode (150 μm in diameter) is placed within a 50 μm range of the pCFE;

[0147] 3) Set the pCFE potential (V) CFE The voltage was set to 780mV to oxidize the released dopamine, and the corresponding amperometric current (IA) was recorded using a patch-clamp amplifier connected to a CFE. CFE );

[0148] 4) Apply single-pulse electric field stimulation (Estim, 1p) or 70mM KCl (70K +The solution depolarizes dopamine terminals in brain slices and triggers the release of dopamine from neurotransmitter vesicles. Biomicroelectrode samples 1-5 exhibited reproducible I-wave activity on brain slices. CFE However, the biomicroelectrode samples 6-8 prepared using Examples 6-8 were also subjected to brain slice electrochemical detection, and the results showed that their electrochemical signal quality was poor and the signal-to-noise ratio was significantly reduced. The detection results for biomicroelectrode sample 1 are shown below. Figure 2 .

[0149] Example 3: Detection of cardiac slices using microelectrodes

[0150] 1. Preparation of cardiac slices

[0151] Adult C57BL / 6 (6-8 weeks old) wild-type male mice (1.5 g·kg⁻¹, ip) were anesthetized with polyurethane. The hearts were rapidly removed and transferred to Tyrode's solution (see Example 2). The heart was incised, and the mid-left ventricle region was selected for sectioning. The mid-left ventricle region was fixed with 4% low-melting-point agarose (Sigma, A9414), cooled on ice at 4°C, and adhered to a cutting base. Using a vibrating microtome (Leica VT 1200S, Germany), sections were cut into 250 μm thick sections in Tyrode's solution at a speed of 0.05 mm / s and an amplitude of 1 mm. These sections were then transferred to storage bottles containing Tyrode's solution at room temperature to obtain heart sections. Sections could be left to stand for 6-7 hours. The heart sectioning process is described in [reference needed]. Figure 3 a.

[0152] 2. The heart slices from step 1 of this example were analyzed using different bio-microelectrodes (samples 1 to 8) prepared in Example 1. The specific analysis steps are as follows:

[0153] 1) Insert a pCFE with an electrode tip diameter of 5 or 7 μm into a heart slice;

[0154] 2) The bipolar platinum electrode (150 μm in diameter) is placed within a 50 μm range of the pCFE;

[0155] 3) Set the pCFE potential (V) CFE The NE was oxidized to 780mV, and the corresponding ampere current (I) was recorded through a patch-clamp amplifier connected to a CFE. CFE );

[0156] 4) Apply an electric field stimulation pulse (Estim) or 70mM KCl (70K + ) liquid (see Figure 3 b) This depolarizes the sympathetic nervous system in the cardiac slices and triggers the release of neurotransmitter vesicles (NE), which manifests as reproducible I-waves on the cardiac slices.CFE (cSEC) signal. The results showed that bioelectrode samples 1-5 could detect NE release from neurotransmitter vesicles in the heart slices, while the bioelectrode samples 6-8 showed a significantly lower signal-to-noise ratio in the electrochemical signals detected by the bioelectrodes, and the signals could not be recorded stably. This embodiment exemplifies the results for sample 1; see [link to documentation] for details. Figure 3 c~ Figure 3 e and Figure 4 .

[0157] Figure 3 c and Figure 4 middle, I CFE The amplitude reflects the release of NE, the decay phase corresponds to NE reuptake mediated by NE transporters, and the charge represents the release of total NE molecules.

[0158] See results Figure 3 d~ Figure 3 e. Photomicrographs showing SEC electrochemical recordings on cardiac slices. (See also...) Figure 3 d. Figure 3 In image e, the left image shows a representative SEC signal induced by a single electric field stimulation (Estim) pulse (1p) recorded on a cardiac slice when the CFE is maintained at 780mV or 0mV; the right image shows the I... CFE Amplitude statistics (n=7 pieces, 4 mice, paired t-test). Figure 3 f and Figure 3 e is the same, but it is composed of 70K + Induced SEC signals (n=11 slices, from 6 mice, paired t-test).

[0159] Example 4: Application of microelectrodes in human cardiac biopsy

[0160] To investigate the capability of human biopsy, in this embodiment, human cardiac biopsy specimens retrieved from a hospital operating room were preserved in ice-cold Tyrode's solution (see Example 2) and delivered to the laboratory within 30 minutes. Human cardiac slices were prepared and used within 30 minutes following a method similar to that used for mouse heart slices in step 1 of Example 3 (see flowchart for details). Figure 5 a) Then, based on the method in step 2 of Example 3, the biomicroelectrode sample 1 prepared in Example 1 was used to record the SEC electrochemical signal of the above-mentioned human heart slices. The detection results are shown in [reference]. Figure 5 c~ Figure 5 d.

[0161] Immunostaining with tyrosine hydroxylase (TH) on human heart sections revealed sympathetic innervation (see details). Figure 5b). Similar to the results in mice, the SEC signal in human heart slices showed high reproducibility (see details). Figure 5 c~ Figure 5 d) Drug administration can be repeated during the recording process.

[0162] Example 5: Application of microelectrodes in peripheral organs of mice

[0163] Slices of mouse blood vessels (mesenteric artery), kidney, lung, spleen, liver, brown adipose tissue, intestine, skeletal muscle, and bladder were prepared using the method in step 1 of Example 3. Then, based on the method in step 2 of Example 3, the SEC electrochemical signals of the above-mentioned blood vessel (mesenteric artery), kidney, lung, spleen, liver, brown adipose tissue, intestine, skeletal muscle, and bladder slices were recorded using the biomicroelectrode sample 1 prepared in Example 1. The results are shown in [reference needed]. Figures 6-8 .in, Figure 6 a-6c Figure 7 a-7c and Figure 8 a-8c refers to CFE in blood vessels (mesenteric arteries) at 780mV or 0mV. Figure 6 a) Kidneys Figure 6 b) Lungs Figure 6 c) Spleen ( Figure 7 a) Liver ( Figure 7 b) Brown fat ( Figure 7 c) Intestine ( Figure 8 a) Skeletal muscle ( Figure 8 b) and bladder ( Figure 8 c) Typical SEC signals recorded on the slice; Figure 6 d is Figure 6 a-6c (n=4 for blood vessel sections (from 2 mice), n=7 for kidney sections (from 3 mice), n=7 for lung sections (from 5 mice), paired samples t-test) in I CFE Amplitude statistics; Figure 7 d is Figure 7 a-7c (spleen sections n=5 (from 3 mice), liver sections n=4 (from 2 mice), brown adipose tissue sections n=4 (from 2 mice), paired samples t-test) in I CFE Amplitude statistics; Figure 8 d is Figure 8 a-8c (intestinal sections n=3 (from 2 mice), skeletal muscle sections n=2 (from 2 mice), bladder sections n=3 (from 2 mice), paired samples t-test) in I CFE Amplitude statistics; Figure 6 e represents the charge table and corresponding vesicle number for three tissue sections: blood vessels, kidneys, and lungs (data are presented as mean ± sem * P < 0.05, *** P < 0.001). Figure 7 e represents the charge table and corresponding vesicle number of three tissue sections: spleen, liver, and brown adipose tissue (data are presented as mean ± sem * P < 0.05, ** P < 0.01, *** P < 0.001). Figure 8 e represents the charge table and corresponding vesicle number for tissue sections from the intestine, skeletal muscle, and bladder (data are expressed as mean ± sem * P < 0.05).

[0164] The results show that the bio-microelectrode of the present invention can be widely applied to most organs and tissues innervated by the sympathetic nervous system. Therefore, it can be further demonstrated that the bio-microelectrode of the present invention provides a potential tool for studying the sympathetic regulation of peripheral organ blood supply, immune response, metabolism, and other organ functions in health and disease.

[0165] Example 6: Application of microelectrodes in a mouse inflammation model

[0166] Studies have reported that the sympathetic nervous system (NE) plays an important role in immunity. However, due to the lack of methods for real-time and in situ recording of NE release, little is known about the correlation and causal relationship of NE secretion during inflammation. Therefore, this embodiment uses the bioelectrode prepared in Example 1 to detect real-time and in situ sympathetic neural network release in an inflammatory mouse model. The specific steps are as follows:

[0167] A systemic inflammation mouse model induced by lipopolysaccharide (LPS, 0.5 mg / kg, ip) was used. Four hours after LPS injection (see details...),... Figure 9 a) Heart slices from a mouse model were obtained using the method in step 1 of Example 3. Then, based on the method in step 2 of Example 3, the SEC electrochemical signals of the heart slices from the mouse model were recorded using the biomicroelectrode sample 1 prepared in Example 1. The detection results are shown in [reference missing]. Figure 9 b~ Figure 9 g.

[0168] Depend on Figure 9 b and Figure 9 c indicates that a single Estim pulse induced an 80±20% increase in sympathetic neural network release (amplitude, charge, and vesicle number), but the decay constant remained unchanged. From Figure 9 According to e-9g, when a burst of Estim pulse stimulation (5 pulses, 10Hz) is used to induce the release of vesicles from the neural network pool ( Figure 9 At time d), the SEC signal increased significantly by 81 ± 20%, which indicates that the size of the release vesicle pool of NE was increased in LPS-treated mouse heart slices.

[0169] In contrast, acute LPS treatment (direct administration via dosing tube) of normal mouse heart sections had no effect on NE release (see [link to original text]). Figure 10In summary, the SEC record shows a significant increase in NE release pool size and NE release at cardiac sympathetic nerve endings during the cascade reaction, but not in the initial phase.

[0170] Example 7: Application of microelectrodes in a mouse model of heart failure

[0171] Studies have reported impaired sympathetic neuron (NE) release in cardiovascular diseases, particularly heart failure (HF); however, how sympathetic regulation is altered in HF remains controversial. Based on this, this embodiment uses aortic transconstriction (TAC) to create a mouse model of HF. Figure 11 a) Echocardiography was performed 8 weeks post-surgery to assess cardiac function and morphology. (See details below) Figure 10 a. Then observe the gross morphological images of the hearts of sham-operated mice and TAC mice (see details). Figure 11 b); The heart-to-weight ratio of sham and TAC mice was statistically analyzed (5 pairs of mice). See the figure for the statistical results. Figure 11 c; and the ejection fraction, fraction shortening, and diastolic thickness of the left ventricular anterior wall were statistically analyzed in Sham and TAC mice (5 pairs of mice). See the figure for the statistical results. (See also...) Figure 11 d. The results showed that, morphologically, the size of the heart and the ratio of heart weight increased significantly after TAC surgery; compared with the sham-operated group, the ejection fraction and fractional shortening of the heart of mice treated with TAC were significantly reduced, and the thickness of the left ventricular anterior wall during diastole increased, confirming the decompensated period of HF in mice.

[0172] Furthermore, heart slices from the HF mouse model were obtained using the method in step 1 of Example 3. Then, based on the method in step 2 of Example 3, the SEC electrochemical signal within the heart slices of the HF mouse model was recorded using biomicroelectrode sample 1 prepared in Example 1. The results are shown in [reference needed]. Figure 11 e and Figure 11 f.

[0173] The results showed that SEC recordings of heart slices revealed a near 100% increase in NE release in the HF mouse model (see details). Figure 11 e, Figure 11 (Left figure of f). By fitting the curve of the exponential decay function, the reuptake of NE was characterized, and it was found that the NE uptake time constant was prolonged by 31% in the HF mouse model (see details). Figure 11 The right figure (f) shows that the NE transporter is impaired in NE reuptake during HF.

[0174] As can be seen from the above, in addition to NE release, NE reuptake can also lead to sympathetic regulatory dysfunction in heart failure, resulting in excessive cardiac adrenergic stimulation during heart failure.

[0175] Example 7: Application of microelectrodes in the heart of a live mouse

[0176] Examples 1-6 describe the application of microelectrodes in tissue sections. Similarly, under in vivo mouse conditions, microelectrodes can also record the secretion of norepinephrine (NE) in peripheral tissues. This example uses 20 μm diameter gold wire electrodes, and bio-microelectrode samples 1-8 (hereinafter referred to as microelectrodes) were fabricated using Example 1. The specific detection steps are as follows:

[0177] 1) Mice were anesthetized and their hearts were opened to allow for organ cannulation.

[0178] 2) Create a stimulation-recording dual electrode by binding and fixing gold wire electrodes as stimulation electrodes with microelectrodes;

[0179] 3) A dual-electrode array was inserted into the mouse heart, with the stimulating electrode connected to an electrical stimulator and the recording electrode connected to a patch-clamp amplifier. Depolarization stimulation was performed using electrical stimulation to record NE secretion from the cardiac sympathetic nervous system in vivo. Bioelectrode samples 1–5 could all detect NE in the mouse heart under in vivo conditions; however, the bioelectrode samples 6–8 showed a significantly lower signal-to-noise ratio in the electrochemical signals detected from cardiac slices, and the signals could not be recorded stably.

[0180] This embodiment exemplifies the detection results of biomicroelectrode sample 1. See details below. Figure 12 The blue curve represents the SEC signal caused by a single electric field stimulation pulse recorded when the recording electrode is held at 780 mV during the first depolarization stimulation. After three minutes, the recording electrode is held at 0 mV, and the same electric field stimulation is recorded as the black curve, indicating the disappearance of the SEC signal. After three minutes, the stimulation electrode is held at 780 mV again, and the recorded SEC signal is shown as the red curve. The downward-pointing pulse-like signal is caused by the mouse's heartbeat.

[0181] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0182] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of liquid polymers or polyols as active ingredients in electrode solutions.

2. Use of liquid polymers or polyols in the preparation of microelectrodes, wherein the electrode solution of the microelectrode comprises liquid polymers or polyols.

3. The use according to claim 1 or 2, characterized in that, The liquid polymer is polyethylene glycol; Optionally, the average molecular weight of the polyethylene glycol is 50–800 g / mol; Optionally, the polyethylene glycol is selected from at least one of PEG-200, PEG-300, PEG-400, and PEG-600; Optionally, the polyol includes at least one of ethylene glycol, glycerol, pentaerythritol, 1,2-propanediol, 1,4-butanediol, 1,6-hexanediol, neopentyl glycol, diethylene glycol, dipropylene glycol, and trimethylolpropane.

4. A biomicroelectrode, characterized in that, Includes glass capillary tubes, electrode fibers, and electrode solution; The electrode solution is encapsulated within the glass capillary tube; One end of the electrode fiber extends into the electrode liquid, and the other end of the electrode fiber is located outside the glass capillary; wherein the electrode liquid comprises a liquid polymer or a polyol.

5. The biomicroelectrode according to claim 4, characterized in that, The liquid polymer or polyol is in a liquid state at room temperature; The liquid polymer is polyethylene glycol; Optionally, the average molecular weight of the polyethylene glycol is 50–800 g / mol; Optionally, the polyethylene glycol is selected from at least one of PEG-200, PEG-300, PEG-400, and PEG-600; Optionally, the polyol includes at least one of ethylene glycol, glycerol, pentaerythritol, 1,2-propanediol, 1,4-butanediol, 1,6-hexanediol, neopentyl glycol, diethylene glycol, dipropylene glycol, and trimethylolpropane.

6. The biomicroelectrode according to any one of claims 4 to 5, characterized in that, One end of the glass capillary is tapered, and the electrode fiber extends from the tapered end of the glass capillary into the electrode liquid; Optionally, the electrode fibers are selected from conductive inert material fibers; Optionally, the conductive inert material fiber is selected from at least one of carbon fiber, tungsten fiber, gold fiber and platinum fiber; Optionally, the diameter of the electrode fiber is 100 nm to 15 μm; Optionally, the length of the electrode fiber is 5 mm to 10 mm.

7. A probe, characterized in that, Includes the biomicroelectrode as described in any one of claims 4 to 6.

8. An electrochemical detection device, characterized in that, Includes the biomicroelectrode as described in any one of claims 4 to 6 or the probe as described in claim 7.

9. Use of biomicroelectrodes or products containing biomicroelectrodes in electrochemical detection.

10. A method for in vitro detection of neurotransmitters, characterized in that, include: The test sample is tested using a biomicroelectrode or a product containing a biomicroelectrode.

11. The use according to claim 9 or the method according to claim 10, characterized in that, The biomicroelectrode is the biomicroelectrode according to any one of claims 4 to 6; Optionally, the product comprising the biomicroelectrode is the probe of claim 7 or the device of claim 8; Optionally, the electrochemical detection is neurotransmitter detection; Optionally, the neurotransmitter includes central neurotransmitters or peripheral neurotransmitters; Optionally, the peripheral neurotransmitters are derived from the heart, blood vessels, kidneys, lungs, spleen, liver, fat, intestines, pancreas, skeletal muscle, bladder, and skin; Optionally, the central neurotransmitter originates from the brain; Optionally, the neurotransmitter detection includes in vitro neurotransmitter detection or in vivo neurotransmitter detection; Optionally, the electrochemical detection is an in vitro neurotransmitter detection, and the sample for detection is selected from biological samples; Optionally, the biological sample is selected from slice samples or cell samples; Optionally, the neurotransmitter is selected from monoamine neurotransmitters or their metabolites, or vitamin C; Optionally, the monoamine neurotransmitters include at least one of dopamine, adrenaline, noradrenaline, histamine, and serotonin; Optionally, the metabolite of the monoamine neurotransmitter is selected from at least one of 3,4-dihydroxyphenylglycol, 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindoleacetic acid.

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