Reference electrode of an electrochemical sensor

Abstract

The reference electrode of the electrochemical sensor includes a substrate having a well, a channel, and a conductive element. The well extends from a first surface of the substrate toward a second surface of the substrate. The channel is within the substrate. A longitudinal axis of the channel can be substantially perpendicular to a longitudinal axis of the well. The channel has a first end connected to the well and a second end in contact with the conductive element. The channel and the well form a flow path for the conductive medium. The flow path can be coupled to an operating element, such as a stirring element or a heating element, that facilitates flow of the membrane.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims priority to U.S. Provisional Application No. 63 / 322,415, filed on March 22, 2022, entitled "REFERENCE ELECTRODES OF ELECTROCHEMICAL SENSORS", which is assigned to the assignee of this specification and incorporated herein by reference.

[0002] This disclosure generally relates to electrochemical sensors, and more specifically, to reference electrodes of electrochemical sensors.

Background Art

[0003] An electrochemical sensor is a type of chemical sensor in which an electrode is used as a transducer element in the presence of an analyte. An electrochemical sensor can convert information associated with an electrochemical reaction (e.g., a reaction between an electrode and an analyte) into an applicable qualitative or quantitative signal. An electrochemical sensor can generate an electronic output in a digital signal for further analysis.

Summary of the Invention

[0004] The following presents a simplified summary of one or more aspects in order to provide a basic understanding of such aspects. This summary is not an extensive overview of all contemplated aspects, nor is it intended to identify key or critical elements of all aspects or to delineate the scope of any or all aspects. Its sole purpose is to present some concepts of one or more aspects in a simplified form as a prelude to the more detailed description that is presented later.

[0005] In some embodiments, the techniques described herein relate to a reference electrode in an electrochemical sensor, the reference electrode comprising a substrate having an inner wall defining a flow channel of a conductive medium, the flow channel being disposed within the substrate; a conductive element within the substrate in contact with the flow channel; and a stirring element coupled to the flow channel and configured to change the size or shape of at least a portion of the flow channel.

[0006] In some embodiments, the techniques described herein relate to a reference electrode, and the stirring element is configured to change the size or shape of at least a portion of the flow path by applying mechanical stress to the inner wall of the flow path in response to an electric field applied to the stirring element.

[0007] In some embodiments, the techniques described herein relate to a reference electrode, and the stirring element is configured to change the size or shape of at least a portion of the flow path by applying mechanical stress to the inner wall of the flow path in response to a radio frequency signal applied to the stirring element.

[0008] In some embodiments, the techniques described herein relate to a reference electrode, and the stirring element includes an electroactive polymer or piezoelectric material.

[0009] In some embodiments, the techniques described herein relate to a reference electrode, and the flow path includes a well in a substrate, extending from a first surface of the substrate toward a second surface of the substrate, with the first surface being opposite to the second surface; and a channel located in the substrate and between the first surface and the second surface, with a first end of the channel connected to the well, a second end of the channel in contact with a conductive element, and the first end being opposite to the second end.

[0010] In some embodiments, the techniques described herein relate to a reference electrode, and a stirring element is positioned in the wall of the channel and configured to change the size or shape of the channel.

[0011] In some embodiments, the techniques described herein relate to a reference electrode, which further includes an additional stirring element positioned in the wall of a well and configured to change the size or shape of the well.

[0012] In some embodiments, the techniques described herein relate to a reference electrode, the channel further comprising a second well in a substrate, the second well extending from a first surface to a second surface, the second end of the channel being connected to the second well, and the first end being on the opposite side of the second end.

[0013] In some embodiments, the techniques described herein relate to an electrochemical sensor comprising one or more working electrodes in a substrate and a reference electrode electrically coupled to the one or more working electrodes, wherein the reference electrode comprises a channel of a conductive medium defined by the inner wall of the substrate and disposed within the substrate, a conductive element in the substrate in contact with the channel, and a stirring element coupled to the channel and configured to change the size or shape of at least a portion of the channel.

[0014] In some embodiments, the techniques described herein relate to an electrochemical sensor, wherein the stirring element is configured to change the size or shape of at least a portion of the flow path by applying mechanical stress to the inner wall of the flow path in response to an electric field applied to the stirring element.

[0015] In some embodiments, the techniques described herein relate to an electrochemical sensor, wherein the stirring element is configured to change the size or shape of at least a portion of the flow path by applying mechanical stress to the inner wall of the flow path in response to a radio frequency signal applied to the stirring element.

[0016] In some embodiments, the techniques described herein relate to electrochemical sensors, wherein the stirring element includes an electroactive polymer or piezoelectric material.

[0017] In some embodiments, the techniques described herein relate to an electrochemical sensor, the channel comprising: a well in a substrate extending from a first surface of the substrate toward a second surface of the substrate, the first surface being opposite to the second surface; and a channel located in the substrate and between the first surface and the second surface, the first end of the channel being connected to the well, the second end of the channel being in contact with a conductive element, the first end being opposite to the second end.

[0018] In some embodiments, the techniques described herein relate to electrochemical sensors, in which the stirring element is located in the wall of a channel or a well.

[0019] In some embodiments, the techniques described herein relate to an electrochemical sensor, wherein the channel further includes a second well in a substrate, the second well extending from a first surface to a second surface, and the second end of the channel is connected to the second well.

[0020] In some embodiments, the techniques described herein relate to an electrochemical sensor, wherein one or more working electrodes include a working well configured such that the working electrode is at least partially filled with an ion-selective membrane different from the conductive medium.

[0021] In some embodiments, the techniques described herein relate to an electrochemical sensor, wherein one or more working electrodes include a first working electrode and a second working electrode, and a portion of the flow path is located between the working electrode and the second working electrode.

[0022] In some embodiments, the techniques described herein relate to a reference electrode in an electrochemical sensor, the reference electrode comprising a substrate including an inner wall defining a fluid flow path, a conductive element within the substrate in contact with the flow path, and a heating element coupled to the flow path and configured to raise the temperature of at least a portion of the flow path.

[0023] In some embodiments, the techniques described herein relate to a reference electrode, and the heating element is configured to increase the temperature of at least a portion of the flow path in response to a current applied to the heating element.

[0024] In some embodiments, the techniques described herein relate to a reference electrode, and the heating element includes metal.

[0025] In some embodiments, the techniques described herein relate to a reference electrode, and the flow path includes a first well in a substrate that extends from a first surface of the substrate toward a second surface of the substrate, with the first surface on the opposite side of the second surface, and a channel disposed within the substrate and between the first surface and the second surface, where a first end of the channel is connected to the first well, a second end of the channel touches a conductive element, and the first end is on the opposite side of the second end.

[0026] In some embodiments, the techniques described herein relate to a reference electrode, and the heating element is disposed on a wall of the channel or a wall of the well.

[0027] In some embodiments, the techniques described herein relate to a reference electrode, and the flow path further includes a second well in the substrate that extends from the first surface toward the second surface, and the second end of the channel is connected to the second well.

[0028] In some embodiments, the techniques described herein relate to a reference electrode, and the reference electrode further includes one or more other heating elements, and each of the heating element and the one or more other heating elements is disposed at a different location along the flow path.

[0029] In some embodiments, the techniques described herein relate to an electrochemical sensor, the electrochemical sensor including one or more working electrodes within a substrate and a reference electrode electrically coupled to the one or more working electrodes, the reference electrode including a flow path of a conductive medium disposed within the substrate, a conductive element within the substrate that touches the flow path, and a heating element coupled to the flow path and configured to raise the temperature of at least a portion of the flow path.

[0030] In some embodiments, the techniques described herein relate to an electrochemical sensor, the heating element being configured to raise the temperature of at least a portion of the flow path in response to a current applied to the heating element.

[0031] In some embodiments, the techniques described herein relate to an electrochemical sensor, the heating element including a metal.

[0032] In some embodiments, the techniques described herein relate to an electrochemical sensor, the flow path including a first well within the substrate that extends from a first surface of the substrate toward a second surface of the substrate, the first surface being on an opposite side of the second surface, and a channel within the substrate and disposed between the first surface and the second surface, a first end of the channel being connected to the well, a conductive element touching a second end of the channel, the first end being on an opposite side of the second end.

[0033] In some embodiments, the techniques described herein relate to an electrochemical sensor, the heating element being disposed on a wall of the channel or a wall of the well.

[0034] In some embodiments, the techniques described herein relate to an electrochemical sensor, the flow path further including a second well within the substrate that extends from the first surface toward the second surface, and a second end of the channel being connected to the second well.

[0035] In some embodiments, the techniques described herein relate to an electrochemical sensor, which further comprises one or more other heating elements, each of which is located at a different location along the flow path.

[0036] In some embodiments, the techniques described herein relate to electrochemical sensors, which further include one or more counter electrodes.

[0037] To achieve the aforementioned and related objectives, one or more embodiments shall have features that are fully described below and specifically indicated in the claims. The following description and accompanying drawings shall illustrate in detail certain exemplary features of one or more embodiments. However, these features represent only a small number of the various ways in which the principles of various embodiments may be employed, and this description is intended to include all such embodiments and their equivalents. [Brief explanation of the drawing]

[0038] To provide a more complete understanding of this disclosure and its features and advantages, the following description may be referred to in conjunction with the accompanying drawings, where similar reference numbers represent similar parts.

[0039] [Figure 1] This disclosure shows exemplary electrochemical sensors according to some embodiments. [Figure 2A] Exemplary working electrodes according to some embodiments of the present disclosure are shown. [Figure 2B] Exemplary working electrodes according to some embodiments of the present disclosure are shown. [Figure 3A] Exemplary reference electrodes according to some embodiments of the present disclosure are shown. [Figure 3B] Exemplary reference electrodes according to some embodiments of the present disclosure are shown. [Figure 4A] Another exemplary reference electrode is shown according to some embodiments of the present disclosure. [Figure 4B]Another exemplary reference electrode is shown according to some embodiments of the present disclosure. [Figure 4C] Another exemplary reference electrode is shown according to some embodiments of the present disclosure. [Figure 4D] Another exemplary reference electrode is shown according to some embodiments of the present disclosure. [Figure 5] This disclosure shows exemplary electrochemical sensors, including multiple reference electrodes, according to some embodiments of this disclosure. [Figure 6] This disclosure shows another exemplary electrochemical sensor according to several embodiments. [Figure 7] Further exemplary electrochemical sensors according to some embodiments of the present disclosure are shown. [Figure 8A] The present disclosure illustrates an exemplary system, including an electrochemical sensor with a cap, according to some embodiments of this disclosure. [Figure 8B] The present disclosure illustrates an exemplary system, including an electrochemical sensor with a cap, according to some embodiments of this disclosure. [Figure 9] This disclosure illustrates another exemplary system, including an electrochemical sensor with a cap, according to some embodiments of this disclosure. [Figure 10] The present disclosure describes exemplary electrochemical sensors, including a reference electrode having multiple channels, according to several embodiments of this disclosure. [Figure 11] This disclosure presents another exemplary electrochemical sensor, including a reference electrode having multiple channels, according to some embodiments of this disclosure. [Figure 12] The following are exemplary reference electrodes having a channel coupled to a valve, according to some embodiments of the present disclosure. [Figure 13] The following are exemplary reference electrodes, including a stirring element, according to some embodiments of the present disclosure. [Figure 14] The following are exemplary interfaces between a stirring element and a wall according to several embodiments of the present disclosure. [Figure 15] The following are exemplary stirring elements having different shapes according to several embodiments of the present disclosure. [Figure 16]The following are exemplary reference electrodes, including a heating element, according to some embodiments of the present disclosure. [Figure 17] The following are exemplary interfaces between a heating element and a wall according to some embodiments of the present disclosure. [Figure 18] The following are exemplary heating elements having different shapes according to several embodiments of the present disclosure. [Figure 19] The present disclosure shows exemplary electrochemical sensors, including a reference electrode on a sample channel, according to several embodiments of this disclosure. [Figure 20A] The following are some embodiments of the present disclosure that show a reference electrode comprising multiple layers. [Figure 20B] The following are some embodiments of the present disclosure that show a reference electrode comprising multiple layers. [Figure 21] The present disclosure shows valves for controlling the inlet and outlet of a reference electrode, according to some embodiments of this disclosure. [Modes for carrying out the invention]

[0040] Electrochemical sensors offer advantages such as simple measurement procedures, short response times, and sufficient sensitivity and selectivity. Electrochemical sensors are widely used in many applications. Biosensors are an example of such sensors. A biosensor is an analytical device that converts biological responses into electrical signals.

[0041] Electrochemical sensors typically include multiple types of electrodes that come into contact with an electrolyte. These electrodes may include a working electrode (or sensing electrode), a reference electrode, and a counter electrode. The working electrode often includes two main components: a recognition element and a transducer. The recognition element selectively reacts with the analyte. This reaction is then converted into an electrical signal by the transducer. The recognition element and transducer form the sensing electrode of the electrochemical sensor. An electrochemical sensor may include multiple working electrodes. The reference electrode is usually held at a constant electrode potential relative to the working electrode. In potentiometric sensors, the sensor response is the potential (voltage) difference measured between the sensing electrode (the electrode where the chemical phenomenon of interest occurs) and the reference electrode, which has a stable reference potential unaffected by the analyte. The reference electrode acts as a stable reference voltage for measurement. In amperometric (or voltametric, etc.) sensors, the sensor response is the current measured between the sensing electrode and the counter electrode (where the counter reaction occurs). Typically, in amperometric sensors (or voltametric sensors, etc.), a bias voltage is applied to the sensing electrode to facilitate a chemical reaction or physical process. In the latter case, the bias voltage is applied to the reference electrode, so it is important that the reference electrode potential is stable. In addition, the presence of a substance interacting with the working electrode / electrolyte interface can cause a current flow between the working electrode and the counter electrode as a result of a reduction / oxidation (redox) reaction at the working electrode. In some cases, this can cause a change in electrode potential, which is a result of the interaction between the analyte and the working electrode. In some other cases, this is a change in impedance or resistance, which may be proportional to the concentration of the analyte.

[0042] High-quality reference electrodes are crucial in electrochemical sensors. Reference electrodes are the fundamental unit of many chemical sensors that rely on electrochemical measurements, including glucose sensors and fire alarms. The stability and robustness of the reference electrode affect the accuracy, sensitivity, and lifespan of the sensor.

[0043] This disclosure relates to an electrochemical sensor including a reference electrode. An exemplary electrochemical sensor includes one or more reference electrodes and one or more working electrodes in a substrate. The electrochemical sensor may also include one or more counter electrodes in the substrate. The reference electrode may include wells and channels defined by the inner wall of the substrate, as well as a conductive element. The wells extend from a first surface of the substrate toward a second surface of the substrate. The channels are located within the substrate. The longitudinal axis of the channels may be substantially perpendicular to the longitudinal axis of the wells. The channels have a first end connected to a well, and the conductive element is in contact with a second end of the channels. The reference electrode may include additional wells extending from the first surface toward a second surface. The additional wells may be connected to a second end of a channel and may be in contact with a conductive element. The channels and wells form a flow channel for a conductive medium. For example, the conductive medium may be a solid or semi-solid film, or a liquid. The flow channel may be coupled to a stirring element or a heating element that facilitates the flow of the conductive medium. A stirring or heating element may help reduce blockage or precipitate buildup, or, in certain applications, may improve the efficiency of the analytical process.

[0044] Compared to conventional reference electrodes, the reference electrode in this disclosure has lower drift and is more stable. The use of one or more channels restricts ion diffusion between the test solution (or sample) and the reference electrode, thus providing a more stable reference electrode potential and, consequently, more accurate sensor measurements. Miniaturized reference electrodes can be produced. Miniaturized reference electrodes can be used in microfluidic applications or other applications requiring miniaturization.

[0045] The following detailed description presents various descriptions of specific embodiments. However, the technological innovations described herein can be embodied in many different ways, for example, as defined and covered by the claims or selected embodiments. In the following description, similar reference numerals may refer to drawings in which identical or functionally similar elements may be shown. It will be understood that the elements illustrated in the drawings are not necessarily drawn to scale. Furthermore, it will be understood that a particular embodiment may include more elements than illustrated in the drawings, or a subset of the elements illustrated in the drawings. Moreover, some embodiments may incorporate any preferred combination of features from two or more drawings.

[0046] Other features and advantages of this disclosure will be apparent from the following description and claims.

[0047] As described herein, one aspect of the technology may involve collecting and using data available from various sources to improve quality and experience. This disclosure intends that, in some examples, such collected data may include personal information. This disclosure intends that entities involved with such personal information respect and uphold privacy policies and practices.

[0048] The following disclosure describes various exemplary embodiments and examples for implementing the features and functions of this disclosure. Certain components, configurations, or features are described below in relation to various exemplary embodiments, but these are merely examples used to simplify this disclosure and are not intended to limit it. Of course, it will be understood that in developing any actual embodiment, many implementation-specific decisions must be made to achieve the developer's specific goals, including compliance with different systems, businesses, or legal constraints depending on the embodiment. Furthermore, it will be understood that while such development efforts can be complex and time-consuming, they are still routine work for those skilled in the art who are interested in this disclosure.

[0049] This specification may refer to spatial relationships between various components and spatial orientations of various aspects of components, as depicted in the accompanying drawings. However, as will be recognized by those skilled in the art after a full reading of this disclosure, the devices, components, members, apparatus, etc. described herein can be arranged in any desired orientation. Therefore, the use of terms such as “above,” “below,” “upper,” “lower,” “top,” “bottom,” or other similar terms to describe spatial relationships between various components or spatial orientations of aspects of such components should be understood to describe the relative relationships between components or the spatial orientations of aspects of such components, respectively, so that the components described herein can be oriented in any desired direction. When used to describe a range of dimensions or other properties (e.g., time, pressure, temperature, length, width, etc.) of an element, operation, or condition, the phrase “between X and Y” represents a range that may include X and Y.

[0050] In addition, the terms “comprise,” “comprising,” “include,” “including,” “have,” and “having,” or any other variations thereof, are intended to encompass non-exclusive inclusion. For example, a method, process, device, or system that includes a list of elements is not necessarily limited to those elements alone, but may include other elements that are not explicitly listed or that are specific to such a method, process, device, or system. Also, the term “or” refers to an inclusive “or” rather than an exclusive “or.”

[0051] Each of the systems, methods, and devices disclosed herein has several innovative aspects, and no single one is solely responsible for all of the desirable attributes disclosed herein. Details of one or more embodiments of the subject matter described herein are given in the following description and accompanying drawings.

[0052] Figure 1 shows an exemplary electrochemical sensor 100 according to several embodiments of the present disclosure. The electrochemical sensor 100 includes a substrate 110, a working electrode 120 (referred to separately as “working electrode 120”), and a reference electrode 130. In some embodiments, the electrochemical sensor 100 may include fewer, more, or different components. For example, the electrochemical sensor 100 may include one or more counter electrodes, for example, in embodiments where the electrochemical sensor 100 is a voltametric or amperometric sensor.

[0053] The substrate 110 may include plastic materials, semiconductor materials (e.g., silicon, glass, etc.), ceramic materials, other types of materials, or some combination thereof. The substrate 110 may be manufactured using injection molding, lamination, flexible / lamination or additive manufacturing techniques, or other suitable techniques, for example, depending on the specific requirements of the electrochemical sensor application.

[0054] The working electrode 120 includes electrically conductive contacts (also called "conductive contacts"). The conductive contacts include a conductive material, which may be a metal, such as gold (Au). In some embodiments, the conductive contacts are recessed into the substrate. For example, the working electrode 120 includes a working well and conductive contacts on (e.g., below) the end of the working well. In other embodiments, the conductive contacts may be recessed or protruding. In the embodiment of Figure 1, the working electrodes 120 are aligned along a line 105 parallel to the Y-axis. The center of each working electrode 120 lies on line 105. In other embodiments, the centers of the working electrodes may be slightly off-center from line 105. The working electrodes 120 may be substantially aligned or unaligned.

[0055] Line 105 may indicate the location of the sample channel (not shown in Figure 1) of the electrochemical sensor 100. For example, line 105 may indicate the location of a portion of the sample channel, such as the centerline or edge of the sample channel. The sample channel can supply the test sample (or analyte) to the working well of the working electrode 120. The test sample may be a fluid, such as blood or other types of biological fluids. The sample channel may also be used to provide a working film to the working electrode 120.

[0056] The reference electrode 130 includes two wells 133 and 135 defined in the substrate 110, as well as a conductive contact touching well 135. In some embodiments, well 133 may be inside the sample channel, while well 135 may be outside the sample channel. Well 133 may be in direct contact with the test sample. For example, the test sample may be supplied through the sample channel, may be in a container surrounding the electrochemical sensor 100, or may be distributed or placed in well 133. However, well 135 may not be in direct contact with the test sample. In the embodiment of Figure 1, well 133 is aligned with the working electrode 120 along the Y-axis, while well 135 is not aligned with the working electrode 120. In other embodiments, well 133 may be substantially aligned with the working electrode 120, aligned with a subset of the working electrode 120, or not aligned with the working electrode 120. The distance 137 from well 133 to well 135, for example, the distance 137 from the center of well 133 to the center of well 135 in the XY plane, is in the range of 1 mm to 2.5 mm. In some embodiments, the working electrode 120 is coupled to a sample channel (or other source) that provides a test sample to the working well of the working electrode 120. The sample channel may also be used to provide a working film to the working electrode 120.

[0057] There are no conductive contacts touching well 133. The conductive contacts of the reference electrode 130 include a conductive material, which may be a metal (e.g., silver (Ag), gold (Au), platinum (Pt)), or another conductive material (e.g., carbon). In some embodiments, the conductive contacts may include other materials. In one embodiment, the conductive contacts include a mixture of a metal (e.g., Ag) and a nonmetal (e.g., silver chloride (AgCl)). For example, the metal can be formed on the nonmetal through plating, e.g., selective plating. The two wells 133 and 135 may be connected through a channel between them. The channel may be separated from the sample channel. For example, the channel may be located between a first surface and a second surface of the substrate 110 and may extend laterally between well 133 and well 135. Wells 133 and 135, as well as the channel, may be at least partially filled with a reference medium, e.g., a reference electrode electrolyte. The reference medium may be a solid, semi-solid, or liquid and may be called a reference film. The conductive junction of the reference electrode 130 can be coupled to the test solution / sample or the working electrode 120 through wells 133 or 135. In this approach, the diffusion of ions (e.g., Cl) to and from the conductive junction of the reference electrode 130 is limited by lateral ion diffusion across wells 133 and 135 and the channel, instead of conventional bulk diffusion. Lateral ion diffusion takes longer than conventional bulk diffusion.

[0058] Figures 2A and 2B show exemplary working electrodes 220 according to several embodiments of the present disclosure. The working electrode 220 may be an embodiment of the working electrode 120 of Figure 1. As shown in Figure 2A, the working electrode 220 is disposed within a substrate 210. The substrate 210 may be an embodiment of the substrate 110 of Figure 1. The substrate 210 has surfaces 213 and 215 that face each other along the Z-axis.

[0059] The working electrode 220 includes a working well 223 and a conductive element 225. The working well 223 extends from surface 213 toward surface 215. The working well 223 has an opening 227 and an end 229. The opening 227 is on surface 213. The end 229 is between surface 213 and surface 215. The conductive element 225 is below the end 229.

[0060] As shown in Figure 2B, the working well 223 is partially filled with a working film 240. The working film 240 is in contact with the conductive element 225. In some embodiments, the working film 240 is an ion-selective film (ISE) or other layer to impart selectivity.

[0061] Figures 3A and 3B show exemplary reference electrodes 330 according to several embodiments of the present disclosure. The reference electrode 330 may be an embodiment of the reference electrode 130 of Figure 1. The reference electrode 330 is located within a substrate 310. The substrate 310 may be an embodiment of the substrate 110 of Figure 1. As shown in Figure 3A, the substrate 310 has surfaces 313 and 315 facing each other along the Z-axis.

[0062] The reference electrode 330 includes a well 333, a channel 335, and a conductive element 337. The well 333 extends from surface 313 toward surface 315. The well 333 has an opening 332 and an end 334. The opening 332 is on surface 313. The end 334 is between surface 313 and surface 315. The channel 335 extends along the X-axis. One end of the channel 335 is connected to the end 334 of the well 333. The other end of the channel 335 is connected to the conductive element 337. In some embodiments, the longitudinal axis of the channel 335 may be perpendicular to or substantially perpendicular to the longitudinal axis of the well 333. For example, the well 333 has a longitudinal axis along the Z-axis, and the channel 335 has a longitudinal axis along the X-axis. The well 333 and the channel 335 constitute a flow path. The flow path can, for example, allow the flow of the reference film toward the conductive element 337. Additionally, the wells 333 and channel 335 constitute a "serpentine" path for ion diffusion, which takes longer than bulk diffusion.

[0063] As shown in Figure 3B, portions of well 333 and channel 335 are filled with the reference film 340. In some embodiments, well 333 may be completely filled with the reference film 340. In the embodiment of Figure 3B, channel 335 is completely filled with the reference film 340. In other embodiments, well 335 may not be completely filled with the reference film 340. The reference film 340 is in contact with the conductive element 337. In some embodiments, the reference film 340 is a viscous material (e.g., an electrolyte). After solvent evaporation, the reference film 340 may become a solid material (e.g., a solid organic material). Although not shown in Figures 3A and 3B, the reference electrode 330 may include or be bonded to a hole in the substrate 310 through which the reference film 340 is permeated from the substrate 310 when introduced to the reference electrode 330.

[0064] Figures 4A to 4D show another exemplary reference electrode 430 according to several embodiments of the present disclosure. The reference electrode 430 may be an embodiment of the reference electrode 130 in Figure 1. The reference electrode 430 is located within a substrate 410. The substrate 410 may be an embodiment of the substrate 110 in Figure 1. As shown in Figure 4A, the substrate 410 has surfaces 413 and 415 that face each other along the Z-axis.

[0065] The reference electrode 430 includes wells 433 and 439, a channel 435, and a conductive element 437. Well 433 extends along the Z-axis from surface 413 to surface 415. Well 433 has an opening 432 and an end 434. The opening 432 is on surface 413. The end 434 is between surface 413 and surface 415. Well 439 also extends along the Z-axis from surface 413 to surface 415. Well 439 has an opening 436 and an end 438. The opening 436 is on surface 413. The end 438 is between surface 413 and surface 415.

[0066] Channel 435 extends along the X-axis. One end of channel 435 is connected to the end 434 of well 433. The other end of channel 435 is connected to the end 438 of well 439. In some embodiments, end 434 may at least partially overlap with the end of channel 435. Similarly, end 438 may at least partially overlap with the other end of channel 435. In some embodiments, the longitudinal axis of channel 435 may be perpendicular or substantially perpendicular to the longitudinal axis of well 433 or 439. For example, well 433 or 439 has a longitudinal axis along the Z-axis, and channel 435 has a longitudinal axis along the X-axis. Wells 433 and 439 and channel 435 constitute a flow path. The flow path may allow, for example, the flow of a reference film toward a conductive element 437. Furthermore, the wells 433 and 439 containing the film 440, as well as the channel 435, constitute a “serpentine” path for ion diffusion, which takes longer than bulk diffusion. For example, well 439 may have more reference film 450 on the conductive element 437 (compared to, for example, Figure 3B), which may provide a longer lifetime. Although not shown in Figures 4A and 4B, the reference electrode 430 may include or be bonded to a hole in the substrate 410 through which the reference film 440 is introduced from the substrate 410 when it is introduced onto the reference electrode 430.

[0067] As shown in Figure 4B, a portion of well 433, channel 435, and a portion of well 439 are filled with the reference film 440. In some embodiments, well 433 or 439 may be completely filled with the reference film 440. In the embodiment of Figure 4B, channel 435 is completely filled with the reference film 440. In other embodiments, well 435 may not be completely filled with the reference film 440. The reference film 440 is in contact with the conductive element 437. In some embodiments, the reference film 440 is a viscous material (e.g., an electrolyte). The reference film 440 may become a solid material (e.g., a solid organic material) after solvent evaporation.

[0068] As shown in Figure 4C, a portion of well 433 and a portion of channel 435 are filled with a reference film 450, while the other portion of channel 435 and a portion of well 439 are filled with a different reference film 460. In some embodiments, well 433 or 439 may be completely filled with reference film 450 or 460. In the embodiment of Figure 4D, channel 435 is completely filled with reference films 450 and 460. In other embodiments, channel 435 may not be completely filled with reference films 450 and 460. The reference film 460 is in contact with the conductive element 437. In some embodiments, the reference film 450 may be a solid film (e.g., a solid organic material), and the reference film 460 may be an aqueous film. The reference film 460 may include a gel such as sodium chloride (NaCl) gel or potassium chloride (KCl) gel. The reference film 460 may include a polymer, such as Nafion (a trademark name for a sulfonated tetrafluoroethylene-based fluorine copolymer). The reference film 460 may contain other materials. An example of the reference film 460 is 1% agarose containing NaCl.

[0069] In Figures 3A, 3B, 4A, and 4B, channels 335 and 435 have a rectangular shape in the XZ plane. In other embodiments, channels 335 or 435 may have different shapes depending on the specific requirements of the application. For example, the dimensions of channel 335 along the Z axis may increase or decrease in the direction from the well 333 along the X axis to the conductive element 337. Similarly, the dimensions of channel 435 along the Z axis may increase or decrease in the direction from the well 434 along the X axis to the end 438. Also, the longitudinal axis of channel 335 or 435 may not be perpendicular to the longitudinal axis of the well.

[0070] Figure 5 shows an exemplary electrochemical sensor 500 including a plurality of reference electrodes 530 and 540 according to several embodiments of the present disclosure. The reference electrodes 530 and 540 may have similar or different components. One embodiment of a reference electrode 530 or 540 may be a reference electrode 130, 330, or 430. The reference electrode 530 includes two wells 533 and 535. Well 535 is associated with a conductive material that may be located below the end of well 535. The reference electrode 530 may also include a channel (not shown in Figure 5) that connects well 533 to well 535 to form a flow path. The reference electrode 540 includes three wells 543, 545, and 547. Well 543 is located between well 545 and well 547. Each of the wells 545 and 547 is associated with a conductive material that may be located below the end of the well. The reference electrode 540 may also include a first channel (not shown in Figure 5) connecting well 543 to well 545 to form a first flow path, and a second channel (not shown in Figure 5) connecting well 543 to well 547 to form a second flow path. In some embodiments, the benefit of having two wells 545 and 547 having conductive elements is that it has redundancy so that if one of the conductive elements fails, the reference electrode 540 still has a conductive element that can function.

[0071] In other embodiments, the electrochemical sensor 500 may include more reference electrodes 530 or 540. The electrochemical sensor 500 also includes a substrate 510 and a working electrode 520 (referred to separately as “working electrode 520”). In some embodiments, the reference electrodes 530 and 540 are electrically coupled to different working electrodes 520. In other embodiments, the reference electrodes 530 and 540 may be electrically coupled to the same working electrode 520. Also, the reference electrodes 530 and 540 may operate simultaneously or at different times. In some embodiments, each reference electrode includes a valve, for example, located in a channel between two wells of the reference electrode. The valve may control the flow of a reference membrane within the reference electrode and thus control the operation of the reference electrode. For example, the reference electrode can be turned off by closing the valve, which prevents the flow of the reference membrane, and can be turned on by opening the valve. The valve may be used to select the reference electrode to use.

[0072] Figure 6 shows another exemplary electrochemical sensor 600 according to some embodiments of the present disclosure. The electrochemical sensor 600 includes a substrate 610, a working electrode 620 (referred to separately as “working electrode 620”), a reference electrode 630, and a sample channel 640. The reference electrode 630 has a single well located outside the sample channel 640. The well may not be in direct contact with the sample.

[0073] Figure 7 shows yet another exemplary electrochemical sensor 700 according to some embodiments of the present disclosure. The electrochemical sensor 700 includes a substrate 710, a working electrode 720 (referred to separately as “working electrode 720”), and a reference electrode 730. In some embodiments, the electrochemical sensor 700 may include fewer, more, or different components. For example, the electrochemical sensor 700 may include one or more counter electrodes, for example, in embodiments where the electrochemical sensor 700 is a voltametric or amperometric sensor. The reference electrode 730 includes an elongated well filled with a reference film. The elongated well 733 has one end located between the working electrodes 720 and the other end coupled to a conductive element 735. For example, the conductive element 735 may be located on the other end of the elongated well 733. In the embodiment of Figure 7, the elongated well 733 extends along the X-axis and has a longitudinal axis along the X-axis. In some embodiments, the reference electrode 730 includes a polymer (e.g., PVC (polyvinyl chloride), LCP (liquid crystal polymer), etc.) that covers part or all of the elongated well 733 in order to define the elongated well 733. In other cases, for example, using Flex technology, a solder resist may be placed beneath the polymer to ensure that the copper is not exposed.

[0074] In the embodiment shown in Figure 7, the elongated well 733 has a racetrack shape in the XY plane. In other embodiments, the elongated well 733 may have different shapes in the XY plane, such as rectangle, ellipse, trapezoid, etc. Also, the length of the elongated well 733 along the X axis may vary. Different shapes, depths, and locations of the wells, channels, and relative openings can be optimized according to the specific requirements of the application.

[0075] Figures 8A and 8B show exemplary electrochemical sensors 800 including a cap 810 according to some embodiments of the present disclosure. Figure 8B is a cross-sectional view of Figure 8A along line BB. The electrochemical sensor 800 also includes a working electrode 820 (referred to separately as “working electrode 820”), a reference electrode, and a sample channel 840. The interface between the electrode and the sample is inside the sample channel 840. The cap 810 defines the sample channel 840 on which the working electrode 820 is located. In some embodiments, the cap 810 is on a substrate on which the working electrode 820 and the reference electrode are located, such as substrate 110, 210, 310, 410, 510, 610, or 710. The cap 810 may partially cover the surface of the substrate, for example, covering the entire surface except for the sample channel 840. The reference electrode may include a first well 833 having an opening inside the sample channel 840, and a second well 839 having an opening outside the sample channel 840. The cap 810 does not cover or seal the first well 833 of the reference electrode. The first well can receive, for example, a test sample provided to the electrochemical sensor 800 through the sample channel 840. In the embodiments of Figures 8A and 8B, the film 830 is located inside the first well 833. The film 830 may be one of the films described above. The cap 810 may cover (or seal) the opening of the second well 839 of the reference electrode. The second well 839 may not be in direct contact with the test sample. The conductive element 835 may be in contact with the second well 839. The cap 810 may include a rubber gasket, adhesive tape, epoxy resin, a lid, a cartridge, a microfluidic substrate, etc. Cap 810 may be used in conjunction with other electrochemical sensors, such as any of the other electrochemical sensors described herein.

[0076] Figure 9 shows another exemplary electrochemical sensor 900, including a cap 910, according to some embodiments of the present disclosure. The electrochemical sensor 900 also includes a working electrode 920 (referred to separately as “working electrode 920”), a reference electrode, and a sample channel 940. The cap 910 may be the same as or similar to the cap 810 in Figures 8A and 8B. The cap 910 defines the sample channel 940 in which the working electrode 920 is located. The reference electrode may include a well having an opening inside the sample channel 940. The opening is not covered by the cap 910. The reference electrode may also include another well outside the sample channel 940, which may be covered or sealed by the cap 910. The membrane 930 is located in the well of the reference electrode inside the sample channel 940.

[0077] Figure 10 shows an exemplary electrochemical sensor 1000, according to several embodiments of the present disclosure, which includes a reference electrode 1030 having multiple channels 1035a-c. The electrochemical sensor 1000 also includes a substrate 1010 and a working electrode 1020 (referred to separately as “working electrode 1020”). In some embodiments, the electrochemical sensor 1000 may include fewer, more, or different components. For example, the electrochemical sensor 1000 may include one or more counter electrodes.

[0078] The reference electrode 1030 includes a well 1033, channels 1035a-c (collectively referred to as "channel 1035"), and wells 1037a-c (collectively referred to as "well 1037"). In some embodiments, well 1033 is inside the sample channel and may be in direct contact with the sample provided through the sample channel. Well 1037 may be outside the sample channel and may not be in direct contact with the sample. Each channel 1035 provides a flow path from well 1033 to well 1037. For example, channel 1035 has a first end connected to the end of well 1033 and a second end connected to well 1037, the second end opposite the first end. Conductive elements are coupled to each well 1037. For example, a conductive element may be located above the end of well 1037 connected to the corresponding channel 1035. The conductive elements may operate independently. In some embodiments, the conductive elements are insulated from each other, and different potentials can be applied to the conductive elements simultaneously.

[0079] Wells 1033, 1035, and 1037 form channels for a reference film that a conductive element can contact. Since there are three channels 1035 and three wells 1037, the reference electrode 1030 can provide three channels, all of which begin in well 1033. In other embodiments, the reference electrode 1030 may include a different number of channels 1035 and a different number of wells 1037.

[0080] Figure 11 shows another exemplary electrochemical sensor 1100, according to some embodiments of the present disclosure, which includes a reference electrode 1130 having multiple channels 1135a-c. The electrochemical sensor 1100 also includes a substrate 1110 and a working electrode 1120 (referred to separately as “working electrode 1120”). In some embodiments, the electrochemical sensor 1100 may include fewer, more, or different components. For example, the electrochemical sensor 1100 may include one or more counter electrodes.

[0081] The reference electrode 1130 includes a well 1133, channels 1135a-c (collectively referred to as "channels 1135"), and another well 1137. Each channel 1135 provides a flow path from well 1133 to well 1137. For example, a channel 1135 has a first end connected to the end of well 1133 and a second end connected to well 1137, the second end opposite the first end. A conductive element is coupled to well 1137 and may be in contact with the reference film flowing into well 1137 through some or all of the three channels 1135. For example, the conductive element may be above the end of well 1137 connected to channel 1135. Wells 1133, 1135, and 1137 form a flow path for the reference film. The flow of the reference film can begin in well 1133, then branch into the three channels 1135, and merge in well 1137. In some embodiments, channel 1135 can be shut off, for example, by closing a valve.

[0082] Figure 12 shows an exemplary reference electrode 1230 having channels 1235a and 1235b coupled to valves 1239a and 1239b, according to some embodiments of the present disclosure. The reference electrode 1230 also includes wells 1233 and wells 1237a and 1237b. Channel 1235a is connected to wells 1233 and 1237a. Channel 1235b is connected to wells 1233 and 1237b. Thus, the reference electrode 1230 forms two flow paths. Valve 1239a is positioned along channel 1235a and can control the flow of the conductive medium in channel 1235a. For example, when valve 1239a is open, the reference film can flow from well 1233 to well 1237a through channel 1235a, so that the reference film can reach the conductive element below well 1237a. When valve 1239a is closed, valve 1239a acts as a barrier preventing the reference membrane from flowing through channel 1235a and reaching the conductive element. Similarly, valve 1239b is positioned along channel 1235b and can control the flow within channel 1235b. Valves 1239a and 1239b can be used to select which conductive elements to enable / make available. In some embodiments, the conductive element (below well 1237a) is configured for use for wireless communication, and an additional conductive element (below well 1237b) is configured for use for wired communication. In some embodiments, valve 1239a or 1239b may be a hydrophobic valve. In other embodiments, valve 1239a or 1239b may be a barrier that can be destroyed by applying energy, such as voltage, pulsed current, or mechanical vibration. In some embodiments, the valve may include an electroactive polymer located on the wall of channel 1235a or 1235b. When a stimulus (e.g., an electric field) is applied to the valve, the electroactive polymer can obstruct the flow of fluid within the channel. For example, the electroactive polymer may expand to close the valve. The material, shape, and structure of the valve can be optimized according to the specific requirements of the application.

[0083] Figure 13 shows an exemplary reference electrode 1330 including stirring elements 1340a-d according to some embodiments of the present disclosure. The reference electrode 1330 includes wells 1333 and 1339, a channel 1335 between wells 1333 and 1339, and a conductive element 1337 located beneath well 1339 in a substrate 1310. The wells 1333 and 1339 and the channel 1335 form a flow path for the conductive medium. The stirring elements 1340a-d (collectively referred to as “stirring elements 1340”) are located on the walls of wells 1333 and channel 1335. As shown in Figure 13, stirring elements 1340a and 1340b are located on the outer surface of channel 1335, stirring element 1340c is located on the outer surface of channel 1335, and stirring element 1340d is located on another outer surface of channel 1335. In other embodiments, the reference electrode 1330 may include a different number of stirring elements 1340, stirring elements 1340 positioned in different locations (e.g., on the walls of the well 1339), or stirring elements 1340 of different sizes or shapes. The reference electrode 1330 may also include different flow paths, for example, a flow path including one well and one channel.

[0084] The agitator element 1340 is configured to agitate the flow of a reference membrane in the flow path. For example, the agitator element 1340 can create movement to manipulate or move the fluid present in the flow path. The agitator element 1340 can exert mechanical stress on the flow path in response to, for example, an electrical signal (e.g., an electric field), a radio frequency signal, or other type of signal. The mechanical stress can change the size or shape of the flow path (e.g., the size or shape of the well 1333 or channel 1335), which can accelerate or recede the flow of the reference membrane in the flow path. The agitator element 1340 may include an electroactive polymer or a piezoelectric material.

[0085] Figure 14 shows exemplary interfaces between a stirring element 1420 and a wall 1410 in a flow channel within a reference electrode, according to several embodiments of the present disclosure. The stirring element 1420 may be an embodiment of the stirring element 1340 in Figure 13. The wall 1410 may be the wall of the well 1333 or the wall of the channel 1335. As shown in Figure 14, the interface is not smooth. Rather, the stirring element 1420 has protrusions corresponding to recesses in the wall 1410. Such a “rough” interface can improve adhesion between the stirring element 1420 and the wall 1410. It can also affect how the structure moves, refracts, or bends to interact with the fluid in the flow channel.

[0086] Figures 15A to 15C show exemplary stirring elements 1520a to c having different shapes according to several embodiments of the present disclosure. Stirring elements 1520a to c are positioned in walls 1510a to c, respectively. Figure 15A shows stirring element 1520a having a circular shape in the XY plane. Figure 15B shows stirring element 1520b having a ring shape in the XY plane. Figure 15C shows stirring element 1520c having a rectangular shape in the XY plane. In other embodiments, stirring elements may have shapes different from those shown in Figures 15A to 15C. Stirring elements may be patterned based on the needs of the application. The pattern or shape will affect the movement of the channel structure and how the fluid is manipulated. The relative shape and interface between the stirring element and the wall can be modified to optimize the required movement and, therefore, the level of stirring imparted to the membrane or material in the channel. The stirring element may be a piezoelectric material (e.g., lead zirconate titanate (PZT), barium titanate, gallium nitride, zinc oxide, polyvinylidene fluoride, ferroelectric materials having a perovskite structure, etc.), or an electroactive polymer (e.g., polyvinylidene fluoride, ionic polymer-metal composites, stimulus-responsive gels, electrostrictive graft polymers, etc., ferroelectric polymers), or a conductive material such that the application of a stimulus (e.g., voltage, current, etc.) deforms, expands, contracts, or distorts the structure, and then imparts a stirring force to the medium / fluid / membrane in the channel. In certain embodiments, the stimulus may be applied in a pulsed or intermittent manner to impart a pulsed or intermittent stirring force to manipulate the fluid / medium / membrane in the channel. The shape / size / thickness / interface between the stirring element and the adjacent layer can be modified and optimized according to the specific requirements of the application.

[0087] Figure 16 shows an exemplary reference electrode 1630, including heating elements 1640A-D, according to several embodiments of the present disclosure. The reference electrode 1630 includes wells 1633 and 1639, a channel 1635 between wells 1633 and 1639, and a conductive element 1637 located beneath well 1639 in a substrate 1610. The wells 1633 and 1639 and the channel 1635 form a channel for the reference film. The heating elements 1640a-d (collectively referred to as “heating elements 1640”) are located on the walls of well 1633 and channel 1635. As shown in Figure 16, heating elements 1640a and 1640b are located on the outer surface of channel 1635, heating element 1640c is located on the outer surface of channel 1635, and heating element 1640d is located on another outer surface of channel 1635. In other embodiments, the reference electrode 1630 may include a different number of heating elements 1640, heating elements 1640 located in different places (e.g., on the wall of the well 1639), or heating elements 1640 of different sizes or shapes. The heating elements can be located on the inner surface of the flow path. The reference electrode 1630 may also include different flow paths, for example, a flow path containing one well and one channel. The heating elements 1640 are configured to raise the temperature of the fluid in the flow path. The heating elements 1640 can supply heat to the fluid in response to, for example, an electrical signal (e.g., an electric field) or other types of signals. The shape, location, and number of heating elements can be optimized according to the required applied and convective current or heat treatment stage. This also applies to piezoelectric elements that can be incorporated into the structure to manipulate or agitate the fluid.

[0088] Figure 17 shows exemplary interfaces between a heating element 1720 and a wall 1710 of a flow path according to several embodiments of the present disclosure. The heating element 1720 may be an embodiment of the heating element 1640 in Figure 16. The wall 1710 may be the wall of the well 1333 or the wall of the channel 1335. As shown in Figure 17, the interface is not smooth. Rather, the heating element 1720 has protrusions corresponding to recesses in the wall 1710. Such a “rough” interface can improve adhesion between the heating element 1720 and the wall 1710. This principle can also be applied to the addition of an electroactive or piezoelectric layer. In a manner in which layers of bimetallic plates are bent or flexed, defined sections of the channel can also be constructed to refract / move and manipulate fluid in a desired manner, which can be optimized according to specific applications of the electrochemical sensor.

[0089] Figures 18A to 18C show exemplary heating elements 1820a to c having different shapes according to several embodiments of the present disclosure. Heating elements 1820a to c are arranged in walls 1810a to c, respectively. Figure 18A shows heating element 1820a having a circular shape in the XY plane. Figure 18B shows heating element 1820b having a ring shape in the XY plane. Figure 18C shows heating element 1820c having a rectangular shape in the XY plane. In other embodiments, heating elements may have shapes different from those shown in Figures 18A to 18C. Heating elements may be patterned based on the needs of the application. The shape, location, and number of elements may be optimized according to the specific heat flow or heat treatment step required by the particular application of the electrochemical sensor.

[0090] Figure 19 shows an exemplary electrochemical sensor 1900, including a reference electrode on a sample channel 1940, according to several embodiments of the present disclosure. In this example, the sample channel 1940 may represent any source of sample, including a container into which the electrochemical sensor 1900 is immersed, or an aliquot of sample to be distributed or coated onto the electrochemical sensor 1900. The electrochemical sensor 1900 also includes a substrate 1010 and a working electrode (not shown in Figure 19). The substrate 1910 may be one embodiment of the substrate 110 in Figure 1. In some embodiments, the electrochemical sensor 1900 may include fewer, more, or different components. For example, the electrochemical sensor 1900 may include one or more counter electrodes. The shape, location, and position of the stirring element can be modified and optimized according to the requirements of a particular application.

[0091] A sample channel 1940 may be used to provide a test sample and a working film to the working electrode. As shown in Figure 19, the reference electrode is located below the sample channel 1940. The reference electrode includes a well 1933, a channel 1935, a conductive element 1937, another well 1939, and a stirring element 1950 (referred to separately as “stirring element 1950”). In some embodiments, the conductive element 1937 is located on the surface of the channel 1935. The channel 1935 is connected to the well 1933 and constitutes a fluid passage to the conductive element 1937. The channel 1935 is also connected to the well 1939. The well 1939 may function as a vent from which air (e.g., air from the solidification of the fluid) can be discharged from the reference electrode.

[0092] As shown in Figure 19, the stirring element 1950 is coupled to wells 1933 and 1939 and channel 1935. The stirring element 1950 can alter the flow of fluid in the channel. In some embodiments, the stirring element 1950 is a coil that can be energized by a radio frequency signal. The coil can stir, for example, pulsate, the fluid in a defined vicinity in response to the radio frequency signal. The characteristics of the coil (e.g., shape, structure, material, stacking of adjacent materials, etc.) can be determined based on the specific application of the electrochemical sensor 1900. The stirring element 1950 can be integrated with the sample channel 1940 so that an external radio frequency signal can be applied to the stirring element 1950. Although not shown in Figure 19, the electrochemical sensor 1900 may include a heating element for heating the fluid in the channel. The stirring element 1950 may include an electroactive polymer or piezoelectric material. The size or shape of the stirring element 1950 can be optimized according to the specific application of the electrochemical sensor and the required operation of the fluid.

[0093] Figures 20A and 20B show a reference electrode 2000 comprising multiple layers 2010, 2020, and 2030 according to several embodiments of the present disclosure. Figure 20A is a perspective view of the reference electrode 2000. Figure 20B is a side view of the reference electrode 2000. Layers 2010, 2020, and 2030 are stacked together along the Z-axis. Layer 2020 is located between layers 2010 and 2030.

[0094] As shown in Figure 20A, layer 2010 includes wells 2040a, 2050a, and 2060a, channels 2055a and 2065a, and valves 2070a and 2080a. Channel 2055a is connected to wells 2040a and 2050a and forms a first flow path. Channel 2065a is connected to wells 2040a and 2060a and forms a second flow path. Valve 2070a is coupled to channel 2055a and controls the first flow path. Valve 2080a is coupled to channel 2065a and controls the second flow path.

[0095] Similarly, the third layer 2030 includes wells 2040b, 2050, and 2060b, channels 2055b and 2065b, and valves 2070 and 2080b. As shown in the figure, well 2050 and valve 2070 of the third layer 2030, corresponding to well 2050a and valve 2070a, are covered by the second layer 2020 and are not shown. Channel 2055b is connected to wells 2040b and 2050a and forms a first flow path. Channel 2065b is connected to wells 2040b and 2060b and forms a second flow path. Valve 2070 of the third layer, corresponding to valve 2070a, is coupled to channel 2055b and controls the first flow path. Valve 2080b is coupled to channel 2065b and controls the second flow path. In some embodiments, each well of the third layer 2030 may be aligned with a well of layer 2010. For example, well 2040b may be aligned with well 2040a, well 2050 (not shown) may be aligned with its corresponding well 2050a, and well 2060b may be aligned with well 2060b. Alternatively, channel 2055b may be aligned with channel 2055b, and channel 2065b may be aligned with channel 2065b, or each layer may operate independently in different / non-aligned paths or networks. Fluid may also flow from a well or channel in layer 2010 through layer 2020 to the corresponding well or channel in layer 2030. In other embodiments, some or all of layers 2010, 2020, and 2030 may operate independently. The flow channels or flow networks of a layer may differ from those of another layer. A layer may have flow channels or flow networks (e.g., wells, channels, or both) that are not aligned with another layer. The system can be constructed so that fluids can flow laterally within layers and between layers. This can enable systems in which materials can be processed / analyzed in parallel on multiple layers.

[0096] The second layer 2020 includes a valve 2090 (referred to separately as “valve 2090”). The valve 2090 controls the flow of fluid from layer 2010 to layer 2030. In other embodiments, the second layer 2020 may include a different number of valves 2090, and the valves 2090 may have different locations in the second layer 2020. In some embodiments, the number or location of valves in layer 2020 may be determined to allow fluid to flow between layers 2010, 2020, and 2030. The number or location of valves in layer 2020 may be determined based on the location of other components of layer 2010 or 2030, such as channels 2055a, 2055b, 2065a, and 2065b, wells 2040a, 2040b, 2050a, 2050 (not shown), 2060a, and 2060b, or ports. The number or position of valves in layer 2020 may also be determined based on the requirements or processing steps of the system in which the reference electrode 2000 is used. The reference electrode 2000 may be used in a vertically integrated system in which the fluid can flow not only laterally within a single layer but also vertically between layers. Valves can be used to control both lateral and vertical flow. The valves used in the system may be constructed using the materials / structures described above. The shape / size / materials / structure of the valves can be modified and optimized according to the specific requirements of the application.

[0097] Figure 21 shows valves 2110 and 2120 controlling the inlet 2130 and outlet 2140 of a reference electrode 2100 according to some embodiments of the present disclosure. Valve 2110 is coupled to the inlet 2130. When valve 2110 is open, fluid can flow into the reference electrode 2100 through the inlet 2130. Valve 2120 is coupled to the outlet 2140. When valve 2120 is open, fluid can flow out of the reference electrode 2100 through the outlet 2140. A dashed line represents an exemplary flow path 2150 within the reference electrode 2100.

[0098] Many other embodiments become apparent from the detailed description and accompanying drawings mentioned above. These embodiments are described in the following clauses.

[0099] Clause 1. A reference electrode in an electrochemical sensor, the reference electrode comprising: a substrate having an inner wall defining a flow path of a conductive medium, wherein the flow path is disposed within the substrate; a conductive element within the substrate in contact with the flow path; and a stirring element coupled to the flow path and configured to change the size or shape of at least a portion of the flow path.

[0100] Clause 2. The reference electrode according to Clause 1, wherein the stirring element is configured to change the size or shape of at least a portion of the flow path by applying mechanical stress to the inner wall of the flow path in response to an electric field applied to the stirring element.

[0101] Clause 3. The reference electrode according to Clause 1, wherein the stirring element is configured to change the size or shape of at least a portion of the flow path by applying mechanical stress to the inner wall of the flow path in response to a radio frequency signal applied to the stirring element.

[0102] Clause 4. The reference electrode according to Clause 1, wherein the stirring element comprises an electroactive polymer or piezoelectric material.

[0103] Clause 5. A reference electrode according to any one of Clauses 1 to 4, comprising: a well in a substrate, which extends from a first surface of the substrate toward a second surface of the substrate, with the first surface being opposite to the second surface; and a channel located in the substrate and between the first surface and the second surface, with a first end of the channel connected to the well, a second end of the channel in contact with a conductive element, and the first end being opposite to the second end.

[0104] Clause 6. The reference electrode as described in Clause 5, wherein a stirring element is positioned in the wall of the channel and configured to change the size or shape of the channel.

[0105] Clause 7. The reference electrode as described in Clause 6, further comprising an additional stirring element positioned in the wall of the well and configured to change the size or shape of the well.

[0106] Clause 8. The reference electrode according to Clause 5 or 6, wherein the channel further includes a second well in the substrate, the second well extending from the first surface to the second surface, the second end of the channel being connected to the second well, and the first end being on the opposite side of the second end.

[0107] Clause 9. An electrochemical sensor comprising one or more working electrodes in a substrate, and a reference electrode electrically coupled to one or more working electrodes, wherein the reference electrode comprises a channel of a conductive medium defined by the inner wall of the substrate and disposed in the substrate, a conductive element in the substrate in contact with the channel, and a stirring element coupled to the channel and configured to change the size or shape of at least a portion of the channel.

[0108] Clause 10. The electrochemical sensor according to Clause 9, wherein the stirring element is configured to change the size or shape of at least a portion of the flow path by applying mechanical stress to the inner wall of the flow path in response to an electric field applied to the stirring element.

[0109] Clause 11. The electrochemical sensor according to Clause 9, wherein the stirring element is configured to change the size or shape of at least a portion of the flow path by applying mechanical stress to the inner wall of the flow path in response to a radio frequency signal applied to the stirring element.

[0110] Clause 12. An electrochemical sensor according to Clause 9, wherein the stirring element comprises an electroactive polymer or piezoelectric material.

[0111] Clause 13. An electrochemical sensor according to any one of Clauses 9 to 12, comprising: a well in a substrate, which extends from a first surface of the substrate toward a second surface of the substrate, with the first surface being opposite to the second surface; and a channel located in the substrate and between the first surface and the second surface, with a first end of the channel connected to the well, a second end of the channel in contact with a conductive element, and the first end being opposite to the second end.

[0112] Clause 14. An electrochemical sensor as described in Clause 13, wherein the stirring element is located on the wall of the channel or the wall of the well.

[0113] Clause 15. The electrochemical sensor according to Clause 13, wherein the channel further includes a second well in the substrate, the second well extending from a first surface to a second surface, and the second end of the channel is connected to the second well.

[0114] 16. An electrochemical sensor according to any one of the clauses 9 to 15, comprising a working well configured such that one or more working electrodes are at least partially filled with an ion-selective membrane different from the conductive medium.

[0115] Clause 17. An electrochemical sensor according to any one of Clauses 9 to 16, wherein one or more working electrodes include a first working electrode and a second working electrode, and a portion of the flow path is located between the working electrode and the second working electrode.

[0116] Clause 18. A reference electrode in an electrochemical sensor, the reference electrode comprising: a substrate including an inner wall defining a fluid flow path; a conductive element within the substrate in contact with the flow path; and a heating element coupled to the flow path and configured to raise the temperature of at least a portion of the flow path.

[0117] Clause 19. The reference electrode according to Clause 18, wherein the heating element is configured to raise the temperature of at least a portion of the flow path in response to a current applied to the heating element.

[0118] Clause 20. The heating element is a reference electrode as described in Clause 18, which includes a metal.

[0119] Clause 21. A reference electrode according to any one of Clauses 18 to 20, comprising: a first well in a substrate, the first well extending from a first surface of the substrate toward a second surface of the substrate, the first surface being opposite to the second surface; and a channel located in the substrate and between the first surface and the second surface, the first end of the channel being connected to the first well, the second end of the channel being in contact with a conductive element, and the first end being opposite to the second end.

[0120] Clause 22. The reference electrode as described in Clause 21, wherein the heating element is located in the wall of the channel or the wall of the well.

[0121] Clause 23. The reference electrode according to Clause 21, wherein the channel further includes a second well in the substrate, the second well extending from the first surface to the second surface, and the second end of the channel is connected to the second well.

[0122] A reference electrode according to any one of the clauses 18 to 23, further comprising one or more other heating elements, each of which is located at a different location along the flow path.

[0123] Clause 25. An electrochemical sensor comprising one or more working electrodes in a substrate and a reference electrode electrically coupled to one or more working electrodes, wherein the reference electrode comprises a flow channel of a conductive medium disposed in the substrate, a conductive element in the substrate in contact with the flow channel, and a heating element coupled to the flow channel and configured to raise the temperature of at least a portion of the flow channel.

[0124] Clause 26. The electrochemical sensor according to Clause 25, wherein the heating element is configured to raise the temperature of at least a portion of the flow path in response to an electric current applied to the heating element.

[0125] Clause 27. An electrochemical sensor as described in Clause 25, wherein the heating element includes a metal.

[0126] Clause 28. An electrochemical sensor according to any one of Clauses 25 to 27, comprising: a first well in a substrate, the first well extending from a first surface of the substrate toward a second surface of the substrate, the first surface being opposite to the second surface; and a channel located in the substrate and between the first surface and the second surface, the first end of the channel being connected to the well, and a conductive element touching the second end of the channel, the first end of the channel being opposite to the second end.

[0127] Clause 29. An electrochemical sensor as described in Clause 28, wherein the heating element is located in the wall of the channel or the wall of the well.

[0128] Clause 30. The electrochemical sensor according to Clause 28 or 29, wherein the channel further includes a second well in the substrate, the second well extending from a first surface to a second surface, and the second end of the channel is connected to the second well.

[0129] Clause 31. An electrochemical sensor according to any one of Clauses 25 to 30, further comprising one or more other heating elements, each of which is located at different locations along the flow path.

[0130] 32. An electrochemical sensor according to any one of the clauses 25 to 31, further comprising one or more counter electrodes.

[0131] It can be understood that not all objectives or benefits can necessarily be achieved by any particular embodiment described herein. Therefore, for example, a person skilled in the art will recognize that a particular embodiment may be configured to operate in a manner that achieves or optimizes one benefit or group of benefits as taught herein, without necessarily achieving other objectives or benefits that can be taught or suggested herein.

[0132] It may also be essential to note that all specifications, dimensions, and relationships outlined herein (e.g., the number of reference electrodes, channels, wells, etc.) are presented for illustrative and teaching purposes only. Such information may be substantially altered without departing from the spirit of this disclosure or the scope of the appended claims. This specification applies to only one non-limiting example, and therefore they should be interpreted as such. The above description has described exemplary embodiments with reference to specific arrangements of components. Various modifications and changes may be made to such embodiments without departing from the scope of the appended claims. Accordingly, the description and drawings should be considered illustrative, not limiting.

[0133] It should be noted that, with respect to the numerous embodiments provided herein, interactions may be described with respect to two, three, four, or more components. However, this is done for clarification and illustrative purposes only. It should be understood that the system can be arranged in any preferred manner. According to design options for similar designs, any of the components, modules, and elements illustrated in the drawings can be combined into various possible configurations, all of which are clearly within the broad scope of this specification.

[0134] Please note that any references in this specification to various features (e.g., elements, structures, modules, components, steps, operations, characteristics, etc.) included in “one embodiment,” “exemplary embodiment,” “one embodiment,” “another embodiment,” “several embodiments,” “various embodiments,” “other embodiments,” or “alternative embodiments” are intended to mean that any such features are included in one or more embodiments of this disclosure, but may be combined in the same embodiment or not necessarily.

[0135] Numerous other changes, substitutions, modifications, alterations, and modifications may be apparent to those skilled in the art, and this disclosure may be intended to encompass all such changes, substitutions, modifications, alterations, and modifications as they fall within the scope of the appended claims. All optional features of the systems and methods described above may also be implemented in relation to the methods or systems described herein, and it should be noted that the details in the examples may be used in any one or more embodiments.

[0136] In interpreting the claims attached to this specification, the applicant wishes to note, in order to assist the United States Patent and Trademark Office (USPTO) and any reader of any patent issued in this application, that (a) since none of the attached claims exist as of the filing date of this specification, the applicant does not intend to exercise Section 112(f) of the United States Patent Act unless the terms “means for” or “steps for” are used specifically in a particular claim, and (b) nothing in this specification intends to limit this disclosure in any way that may not be otherwise reflected in the attached claims. [Explanation of symbols]

[0137] 100 Electrochemical Sensors 110 circuit boards 130 Reference electrode 225 Conductive elements 1340 Stirring element 2150 flow path

Claims

1. A reference electrode in an electrochemical sensor, wherein the reference electrode is A substrate including an inner wall that defines a flow path for a conductive medium, wherein the flow path is disposed within the substrate. circuit board and A conductive element within the substrate, which is in contact with the flow path, The system comprises a stirring element connected to the flow path and configured to change the size or shape of at least a portion of the flow path, A reference electrode wherein the stirring element is configured to change the size or shape of at least a portion of the flow path by applying mechanical stress to the inner wall of the flow path in response to an electric field or radio frequency signal applied to the stirring element.

2. The reference electrode according to claim 1, wherein the stirring element comprises an electroactive polymer or a piezoelectric material.

3. The aforementioned flow path A well within the substrate, extending from the first surface of the substrate to the second surface of the substrate. The first surface extends, and the well on the opposite side of the second surface, A reference electrode according to claim 1 or 2, comprising: a channel located within the substrate and between the first surface and the second surface, wherein a first end of the channel is connected to the well, a second end of the channel is in contact with the conductive element, and the first end is on the opposite side of the second end.

4. The reference electrode according to claim 3, wherein the stirring element is positioned in the wall of the channel and is configured to change the size or shape of the channel.

5. The reference electrode according to claim 4, further comprising an additional stirring element positioned in the wall of the well and configured to change the size or shape of the well.

6. The aforementioned flow path The reference electrode according to any one of claims 3 to 5, further comprising a second well in the substrate, the second well extending from the first surface toward the second surface, the second end of the channel connected to the second well, and the first end on the opposite side of the second end.

7. It is an electrochemical sensor, One or more working electrodes in the substrate, The system comprises a reference electrode electrically coupled to one or more working electrodes, wherein the reference electrode is A flow channel for a conductive medium defined by the inner wall of the substrate, wherein the flow channel is disposed within the substrate. A conductive element within the substrate, which is in contact with the flow path, The system comprises a stirring element connected to the flow path and configured to change the size or shape of at least a portion of the flow path, An electrochemical sensor wherein the stirring element is configured to change the size or shape of at least a portion of the flow path by applying mechanical stress to the inner wall of the flow path in response to an electric field or radio frequency signal applied to the stirring element.

8. The electrochemical sensor according to claim 7, wherein the stirring element includes an electroactive polymer or a piezoelectric material.

9. The aforementioned flow path A well in the substrate, which extends from a first surface of the substrate toward a second surface of the substrate, wherein the first surface is on the opposite side of the second surface, The electrochemical sensor according to claim 7 or 8, comprising: a channel located within the substrate and between the first surface and the second surface, wherein a first end of the channel is connected to the well, a second end of the channel is in contact with the conductive element, and the first end is on the opposite side of the second end.

10. The electrochemical sensor according to claim 9, wherein the stirring element is located in the wall of the channel or the wall of the well.

11. The aforementioned flow path The electrochemical sensor according to claim 9 or 10, further comprising a second well in the substrate, wherein the second well extends from the first surface toward the second surface, and the second end of the channel is connected to the second well.

12. The electrochemical sensor according to any one of claims 7 to 11, comprising a working well configured such that one or more working electrodes is at least partially filled with an ion-selective film different from the conductive medium.

13. The electrochemical sensor according to any one of claims 7 to 12, wherein the one or more working electrodes include a first working electrode and a second working electrode, and a portion of the flow path is located between the first working electrode and the second working electrode.

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