Reference electrode for electrochemical sensors

Abstract

The reference electrode of the electrochemical sensor includes a substrate having a well, a channel, and an internal 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 the conductive element is in contact with a second end of the channel. The channel and the well form a flow path for a conductive medium. A valve is coupled to the flow path and configured to control flow of the conductive medium through the flow path to the conductive element.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 322,400, filed March 22, 2022, entitled "REFERENCE ELECTRODES OF ELECTROCHEMICAL SENSORS," which is assigned to the assignee hereof and incorporated herein by reference.

[0002] The present disclosure relates generally to electrochemical sensors, and more particularly to reference electrodes for electrochemical sensors. [Background technology]

[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. Electrochemical sensors 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. Electrochemical sensors 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, and is not 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 aspects, techniques described herein relate to a reference electrode in an electrochemical sensor, the reference electrode including a substrate including an interior wall defining a flow path for a conductive medium, a conductive element in the substrate that touches an end of the flow path, and a valve coupled to the flow path and configured to control the flow of the conductive medium to the conductive element through the flow path.

[0006] In some aspects, the techniques described herein relate to a reference electrode that further includes an additional flow path for the conductive medium, the additional flow path being disposed within the substrate, an additional conductive element within the substrate that touches an end of the additional flow path, and an additional valve coupled to the additional flow path and configured to control additional flow of the conductive medium through the additional flow path to the additional conductive element.

[0007] In some aspects, the techniques described herein involve a reference electrode, where a portion of the flow path overlaps a portion of the additional flow path.

[0008] In some aspects, the techniques described herein involve a reference electrode, and the valve comprises an electroactive polymer configured to impede the flow of a conductive medium in response to an electrical stimulus.

[0009] In some aspects, the techniques described herein relate to a reference electrode, wherein the flow path includes a well in a substrate, the well extending from a first surface of the substrate toward a second surface of the substrate, the first surface being opposite the second surface, and a channel disposed in the substrate and between the first and second surfaces, the first end of the channel being connected to the well, the conductive element touching the second end of the channel, the first end being opposite the second end.

[0010] In some aspects, the techniques described herein involve a reference electrode and a valve positioned along the wall of the channel.

[0011] In some aspects, the techniques described herein relate to a reference electrode that further includes an additional conductive element in the substrate, an additional channel in the substrate connected to the well, where the additional conductive element contacts the additional channel, and an additional valve disposed along the additional channel.

[0012] In some aspects, the techniques described herein relate to a reference electrode, and the flow path includes 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 the second surface; a second well in the substrate, the second well extending from the first surface toward the second surface, the conductive element contacting the second well; and a channel disposed between the first surface and the second surface, the first end of the channel connected to the first well and the second end of the channel connected to the second well, the first end being opposite the second end.

[0013] In some aspects, the techniques described herein involve a reference electrode and a valve disposed within the channel.

[0014] In some aspects, the techniques described herein relate to a reference electrode that further includes a third well in the substrate, an additional conductive element in the substrate that contacts the third well, an additional channel connected to the first well and the third well, and an additional valve disposed along the additional channel.

[0015] In some aspects, the techniques described herein relate to an electrochemical sensor including one or more working electrodes in a substrate and a reference electrode electrically coupled to the one or more working electrodes, the reference electrode including a substrate including an interior wall defining a flow path for a conductive medium, a conductive element in the substrate that touches an end of the flow path, and a valve coupled to the flow path and configured to control the flow of the conductive medium to the conductive element through the flow path.

[0016] In some aspects, the techniques described herein relate to an electrochemical sensor, the electrochemical sensor further including an additional flow path for a conductive medium, the additional flow path being disposed within the substrate, an additional conductive element within the substrate, the additional conductive element contacting an end of the additional flow path, and an additional valve coupled to the additional flow path and configured to control an additional flow of the conductive medium through the additional flow path to the additional conductive element.

[0017] In some aspects, the techniques described herein relate to an electrochemical sensor, wherein the valve includes an electroactive polymer configured to impede the flow of a conductive medium in response to an electrical stimulus.

[0018] In some aspects, the techniques described herein relate to electrochemical sensors, where the electrochemical sensors further include one or more counter electrodes.

[0019] In some aspects, the techniques described herein relate to an electrochemical sensor, wherein the flow path includes: 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 the second surface; a second well in the substrate, the second well extending from the first surface toward the second surface, the conductive element contacting the second well; and a channel disposed between the first surface and the second surface, the first end of the channel connected to the first well and the second end of the channel connected to the second well, the first end being opposite the second end, and the valve disposed on the channel.

[0020] In some aspects, the techniques described herein relate to a reference electrode in an electrochemical sensor, the reference electrode including: a first layer including a first flow path configured to facilitate a first flow of a conductive medium within the first layer, a first conductive element in contact with the first flow path, and a first valve coupled to the first flow path and configured to control the first flow of the conductive medium within the first layer; and a second layer above the second layer including a second flow path configured to facilitate a second flow of the conductive medium within the second layer, a second conductive element in contact with the second flow path, and a second valve coupled to the second flow path and configured to control the second flow of the conductive medium within the second layer.

[0021] In some aspects, the techniques described herein relate to a reference electrode, wherein the reference electrode further includes a third layer between the first layer and the second layer, the third layer including a third flow path connected to the first flow path and the second flow path.

[0022] In some aspects, the techniques described herein relate to a reference electrode, wherein the third layer further includes one or more third valves coupled to the third flow path, the one or more third valves configured to control the flow of the conductive medium from the first layer to the third layer.

[0023] In some aspects, the techniques described herein relate to a reference electrode, wherein the first flow path includes a well extending from a first surface of the first layer toward a second surface of the first layer, the first surface being opposite the second surface, and a channel disposed between the first surface and the second surface, the first end of the channel being connected to the well, the first conductive element contacting the second end of the channel, the first end being opposite the second end, and the first valve being disposed along the channel.

[0024] In some aspects, the techniques described herein relate to a reference electrode, wherein the first flow path includes: a first well extending from a first surface of the first layer toward a second surface of the first layer, the first surface being opposite the second surface; a second well extending from the first surface toward the second surface, the second well having a first conductive element contacting the second well; and a channel disposed between the first and second surfaces, the first end of the channel being connected to the first well and the second end of the channel being connected to the second well, the first end being opposite the second end, and the first valve being disposed along the channel.

[0025] To the accomplishment of the foregoing and related ends, the one or more aspects comprise the features hereinafter fully described and particularly pointed out in the claims. The following description and the annexed drawings set forth in detail certain illustrative features of the one or more aspects. These features are indicative, however, of but a few of the various ways in which the principles of the various aspects may be employed, and this description is intended to include all such aspects and their equivalents. [Brief explanation of the drawings]

[0026] To provide a more complete understanding of the present disclosure and its features and advantages, reference may be made to the following description taken in conjunction with the accompanying drawings, in which like reference numerals represent like parts and in which:

[0027] [Figure 1] 1 illustrates an exemplary electrochemical sensor according to some embodiments of the present disclosure. [Figure 2A] 1 illustrates an exemplary working electrode according to some embodiments of the present disclosure. [Figure 2B] 1 illustrates an exemplary working electrode according to some embodiments of the present disclosure. [Figure 3A] 1 illustrates an exemplary reference electrode according to some embodiments of the present disclosure. [Figure 3B] 1 illustrates an exemplary reference electrode according to some embodiments of the present disclosure. [Figure 4A] 1 illustrates another exemplary reference electrode according to some embodiments of the present disclosure. [Figure 4B] 1 illustrates another exemplary reference electrode according to some embodiments of the present disclosure. [Figure 4C] 1 illustrates another exemplary reference electrode according to some embodiments of the present disclosure. [Figure 4D] 1 illustrates another exemplary reference electrode according to some embodiments of the present disclosure. [Figure 5] 1 illustrates an exemplary electrochemical sensor including multiple reference electrodes, according to some embodiments of the present disclosure. [Figure 6] 1 illustrates another exemplary electrochemical sensor according to some embodiments of the present disclosure. [Figure 7] 1 illustrates yet another exemplary electrochemical sensor according to some embodiments of the present disclosure. [Figure 8A] 1 illustrates an exemplary system including an electrochemical sensor including a cap, according to some embodiments of the present disclosure. [Figure 8B] 1 illustrates an exemplary system including an electrochemical sensor including a cap, according to some embodiments of the present disclosure. [Figure 9]1 illustrates another exemplary system including an electrochemical sensor including a cap, according to some embodiments of the present disclosure. [Figure 10] 1 illustrates an exemplary electrochemical sensor including a reference electrode having multiple channels, according to some embodiments of the present disclosure. [Figure 11] 1 illustrates another exemplary electrochemical sensor including a reference electrode having multiple channels, according to some embodiments of the present disclosure. [Figure 12] 1 illustrates an exemplary reference electrode having a channel coupled to a valve, according to some embodiments of the present disclosure. [Figure 13] 1 illustrates an exemplary reference electrode including a stirring element, according to some embodiments of the present disclosure. [Figure 14] 1 illustrates an exemplary interface between a stirring element and a wall according to some embodiments of the present disclosure. [Figure 15] 10A-10C illustrate exemplary stirring elements having different shapes according to some embodiments of the present disclosure. [Figure 16] 1 illustrates an exemplary reference electrode including a heating element, according to some embodiments of the present disclosure. [Figure 17] 1 illustrates an exemplary interface between a heating element and a wall according to some embodiments of the present disclosure. [Figure 18] 10A-10C illustrate exemplary heating elements having different shapes according to some embodiments of the present disclosure. [Figure 19] 1 illustrates an exemplary electrochemical sensor including a reference electrode on a sample channel, according to some embodiments of the present disclosure. [Figure 20A] 1 illustrates a reference electrode including multiple layers, according to some embodiments of the present disclosure. [Figure 20B] 1 illustrates a reference electrode including multiple layers, according to some embodiments of the present disclosure. [Figure 21] 10 illustrates a valve controlling the inlet and outlet of a reference electrode according to some embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0028] Electrochemical sensors have 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. Biosensors are analytical devices that convert biological responses into electrical signals.

[0029] Electrochemical sensors typically contain multiple types of electrodes in 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 contains 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 the transducer form the sensing electrode of the electrochemical sensor. Electrochemical sensors may contain multiple working electrodes. The reference electrode is typically 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 a reference electrode with a stable reference potential that is unaffected by the analyte. The reference electrode serves as a stable reference voltage for the measurement. In amperometric (or voltammetric, 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 (or voltametric, etc.) sensors, 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 relative to a reference electrode, so again, it is important that the reference electrode potential be stable. In addition, the presence of a substance that interacts with the working electrode / electrolyte interface can cause current flow between the working and counter electrodes as a result of reduction / oxidation (redox) reactions at the working electrode. In some cases, this can cause a change in electrode potential, which is the result of an interaction between the analyte and the working electrode. In some other cases, it is a change in impedance or resistance, which can be proportional to the concentration of the analyte.

[0030] High-quality reference electrodes are important in electrochemical sensors. They are the basic unit of many chemical sensors that rely on such electrochemical measurements, including glucose sensors and fire alarms. The stability and robustness of the reference electrode affect the accuracy, sensitivity, and lifetime of the sensor.

[0031] The present 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 within a substrate. The electrochemical sensor may also include one or more counter electrodes within the substrate. The reference electrode may include a well and a channel defined by an interior wall of the substrate, 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. The longitudinal axis of the channel may be approximately perpendicular to the longitudinal axis of the well. The channel has a first end connected to the well, and the conductive element contacts the second end of the channel. The reference electrode may include an additional well extending from the first surface toward the second surface. The additional well may be connected to the second end of the channel and may contact the conductive element. The channel and well form a flow path for a conductive medium. For example, the conductive medium may be a solid or semi-solid film, or a liquid. The flow path may be coupled to a stirring element or a heating element that promotes the flow of the conductive medium. Agitation or heating elements may help alleviate blockages or sediment buildup, or may improve the efficiency of the analytical process in certain applications.

[0032] Compared to conventional reference electrodes, the reference electrode in the present disclosure has lower drift and is more stable. The use of one or more channels limits ion diffusion between the test solution (or sample) and the reference electrode, thus providing a more stable reference electrode potential and therefore more accurate sensor measurements. Miniaturized reference electrodes can be produced. Miniaturized reference electrodes can be used in microfluidic applications or other applications requiring miniaturization.

[0033] The following detailed description presents various descriptions of specific specific embodiments. However, the innovations described herein can be embodied in many different ways, as defined and covered, for example, by the claims or selected examples. The following description may refer to the drawings in which like reference numbers may indicate identical or functionally similar elements. It will be understood that the elements illustrated in the drawings are not necessarily drawn to scale. It will also be understood that certain embodiments may include more elements than are illustrated in the drawings, or a subset of the elements illustrated in the drawings. Furthermore, some embodiments may incorporate any suitable combination of features from two or more drawings.

[0034] Other features and advantages of the present disclosure will be apparent from the following description and from the claims.

[0035] As described herein, one aspect of the present technology may be to collect and use data available from various sources to improve quality and experience. The present disclosure contemplates that, in some instances, such collected data may include personal information. The present disclosure contemplates that entities involved with such personal information respect and honor privacy policies and practices.

[0036] The following disclosure describes various exemplary embodiments and examples for implementing the features and functionality of the present disclosure. While specific components, configurations, or features are described below in connection with various exemplary embodiments, these are merely examples used to simplify the disclosure and are not intended to be limiting. Of course, it will be understood that the development of any actual embodiment will require many implementation-specific decisions to be made to achieve the developer's particular goals, including compliance with system, business, or legal constraints that may vary from implementation to implementation. Moreover, it will be appreciated that such a development effort may be complex and time-consuming, but would nevertheless be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

[0037] Reference may be made herein to spatial relationships between various components and the spatial orientation of various aspects of components, as depicted in the accompanying drawings. However, as will be recognized by those skilled in the art after fully reading this disclosure, devices, components, members, apparatuses, etc. described herein may be disposed in any desired orientation. Accordingly, the use of terms such as "above," "below," "upper," "lower," "top," "bottom," or other similar terms to describe spatial relationships between various components or to describe the spatial orientation of aspects of such components should be understood to respectively describe the relative relationships between the components or the spatial orientation of aspects of such components, such that the components described herein may be oriented in any desired direction. When used to describe a range of a dimension or other characteristic (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.

[0038] Additionally, the terms "comprise," "comprising," "include," "including," "have," "having," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a method, process, device, or system that includes a list of elements is not necessarily limited to only those elements and may include other elements not expressly listed or inherent to such method, process, device, or system. Also, the term "or" refers to an inclusive "or," not an exclusive "or."

[0039] The systems, methods, and devices of the present disclosure each have several innovative aspects, no single one of which is solely responsible for all of the desirable attributes disclosed herein. The details of one or more implementations of the subject matter described herein are set forth in the following description and accompanying drawings.

[0040] 1 shows an exemplary electrochemical sensor 100 according to some embodiments of the present disclosure. Electrochemical sensor 100 includes a substrate 110, a working electrode 120 (individually referred to as "working electrode 120"), and a reference electrode 130. In some embodiments, electrochemical sensor 100 may include fewer, more, or different components. For example, electrochemical sensor 100 may include one or more counter electrodes, e.g., in embodiments where electrochemical sensor 100 is a voltammetric or amperometric sensor.

[0041] The substrate 110 may comprise a plastic material, a semiconductor material (e.g., silicon, glass, etc.), a ceramic material, other types of materials, or some combination thereof. The substrate 110 may be manufactured using, for example, injection molding, lamination, flexible / laminate or additive fabrication techniques, or other suitable techniques, depending on the particular requirements of the electrochemical sensor application.

[0042] The working electrodes 120 include electrically conductive contacts (also referred to as "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 electrodes 120 include a working well and a conductive contact on (e.g., below) the edge of the working well. In other embodiments, the conductive contacts may be recessed or protruding. In the embodiment of FIG. 1, the working electrodes 120 are aligned along a line 105 that is parallel to the Y-axis. The center of each working electrode 120 is on line 105. In other embodiments, the centers of the working electrodes may be slightly offset from line 105. The working electrodes 120 may be substantially aligned or non-aligned.

[0043] Line 105 may indicate the location of a sample channel (not shown in FIG. 1 ) of 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 may provide a test sample (or analyte) to a working well of working electrode 120. The test sample may be a fluid, such as blood, other types of biological fluids, etc. The sample channel may also be used to provide a working membrane for working electrode 120.

[0044] Reference electrode 130 includes two wells 133 and 135 defined in substrate 110 and a conductive contact touching well 135. In some embodiments, well 133 can be inside the sample channel, while well 135 can be outside the sample channel. Well 133 can be in direct contact with the test sample. For example, the test sample can be provided through the sample channel, the test sample can be in a container surrounding electrochemical sensor 100, or the test sample can be dispensed or placed in well 133. However, well 135 may not be in direct contact with the test sample. In the embodiment of FIG. 1 , well 133 is aligned with working electrode 120 along the Y-axis, but well 135 is not aligned with working electrode 120. In other embodiments, well 133 can be substantially aligned with working electrode 120, aligned with a subset of working electrode 120, or not aligned with working electrode 120. Distance 137 from well 133 to well 135, for example, 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, working electrode 120 is coupled with a sample channel (or other source) that provides a test sample to the working well of working electrode 120. The sample channel may also be used to provide a working membrane to working electrode 120.

[0045] No conductive contact touches the well 133. The conductive contact of the reference electrode 130 comprises a conductive material, which may be a metal (e.g., silver (Ag), gold (Au), platinum (Pt)), other conductive materials (e.g., carbon), etc. In some embodiments, the conductive contact may comprise other materials. In one example, the conductive contact comprises a mixture of a metal (e.g., Ag) and a non-metal (e.g., silver chloride (AgCl)). For example, the metal can be formed on the non-metal 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 the first and second surfaces of the substrate 110 and may extend laterally between the wells 133 and 135. The wells 133 and 135 and the channel may be at least partially filled with a reference medium, e.g., a reference electrode electrolyte. The reference medium may be solid, semi-solid, or liquid and may be referred to as a reference membrane. The conductive contact of the reference electrode 130 can be coupled to the test solution / sample or working electrode 120 through well 133 or 135. With such an approach, the diffusion of ions (e.g., Cl) to / from the conductive contact of the reference electrode 130 is limited by lateral ion diffusion across the wells 133 and 135 and the channel, instead of traditional bulk diffusion. Compared to traditional bulk diffusion, lateral ion diffusion is slower.

[0046] 2A and 2B show an exemplary working electrode 220 according to some embodiments of the present disclosure. Working electrode 220 may be an embodiment of working electrode 120 in FIG. 1. As shown in FIG. 2A, working electrode 220 is disposed within substrate 210. Substrate 210 may be an embodiment of substrate 110 in FIG. 1. Substrate 210 has surfaces 213 and 215 that face each other along the Z-axis.

[0047] Working electrode 220 includes a working well 223 and a conductive element 225. Working well 223 extends from surface 213 toward surface 215. Working well 223 has an opening 227 and an edge 229. Opening 227 is on surface 213. Edge 229 is between surfaces 213 and 215. Conductive element 225 is below edge 229.

[0048] 2B, the working well 223 is partially filled with a working membrane 240. The working membrane 240 is in contact with the conductive element 225. In some embodiments, the working membrane 240 is an ion-selective membrane (ISE) or other layer to impart selectivity.

[0049] 3A and 3B show an exemplary reference electrode 330 according to some embodiments of the present disclosure. Reference electrode 330 may be one embodiment of reference electrode 130 in FIG. 1. Reference electrode 330 is disposed within substrate 310. Substrate 310 may be one embodiment of substrate 110 in FIG. 1. As shown in FIG. 3A, substrate 310 has surfaces 313 and 315 that face each other along the Z-axis.

[0050] The reference electrode 330 includes a well 333, a channel 335, and a conductive element 337. The well 333 extends from the surface 313 toward the surface 315. The well 333 has an opening 332 and an end 334. The opening 332 is on the surface 313. The end 334 is between the surfaces 313 and 315. The channel 335 extends along the X-axis. An 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 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 form a flow path. The flow path may, for example, allow the flow of the reference membrane towards the conductive element 337. The well 333 and channel 335 also provide a "tortuous" path for ion diffusion, which takes longer than bulk diffusion.

[0051] As shown in FIG. 3B , the well 333 and a portion of the channel 335 are filled with a reference membrane 340. In some embodiments, the well 333 can be completely filled with the reference membrane 340. In the embodiment of FIG. 3B , the channel 335 is completely filled with the reference membrane 340. In other embodiments, the well 335 may not be completely filled with the reference membrane 340. The reference membrane 340 is in contact with the conductive element 337. In some embodiments, the reference membrane 340 is a viscous material (e.g., an electrolyte). The reference membrane 340 can become a solid material (e.g., a solid organic material) after solvent evaporation. Although not shown in FIGS. 3A and 3B , the reference electrode 330 can include or be bonded to a hole in the substrate 310 through which the reference membrane 340 is vented from the substrate 310 when the reference electrode 330 is introduced to the reference electrode 330.

[0052] 4A-4D show another exemplary reference electrode 430 according to some embodiments of the present disclosure. Reference electrode 430 may be an embodiment of reference electrode 130 in FIG. 1. Reference electrode 430 is disposed within substrate 410. Substrate 410 may be an embodiment of substrate 110 in FIG. 1. As shown in FIG. 4A, substrate 410 has surfaces 413 and 415 that face each other along the Z-axis.

[0053] 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 toward surface 415. Well 433 has an opening 432 and an edge 434. Opening 432 is on surface 413. Edge 434 is between surfaces 413 and 415. Well 439 also extends along the Z axis from surface 413 toward surface 415. Well 439 has an opening 436 and an edge 438. Opening 436 is on surface 413. Edge 438 is between surfaces 413 and 415.

[0054] The channel 435 extends along the X-axis. One end of the channel 435 is connected to the end 434 of the well 433. The other end of the channel 435 is connected to the end 438 of the well 439. In some embodiments, the end 434 may at least partially overlap the end of the channel 435. Similarly, the end 438 may at least partially overlap the other end of the channel 435. In some embodiments, the longitudinal axis of the channel 435 may be perpendicular or substantially perpendicular to the longitudinal axis of the well 433 or 439. For example, the well 433 or 439 has a longitudinal axis along the Z-axis, and the channel 435 has a longitudinal axis along the X-axis. The wells 433 and 439 and the channel 435 form a flow path. The flow path may, for example, allow the flow of a reference membrane toward the conductive element 437. Also, wells 433 and 439 containing membrane 440 and channel 435 provide a "tortuous" path for ion diffusion, which takes longer than bulk diffusion. For example, well 439 may have more reference membrane 450 on conductive element 437 (e.g., compared to FIG. 3B), which may provide a longer lifetime. Although not shown in FIGS. 4A and 4B, reference electrode 430 may include or be coupled to a hole in substrate 410 through which reference membrane 440 is vented from substrate 410 when introduced to reference electrode 430.

[0055] As shown in FIG. 4B , a portion of well 433, channel 435, and a portion of well 439 are filled with reference membrane 440. In some embodiments, well 433 or 439 can be completely filled with reference membrane 440. In the embodiment of FIG. 4B , channel 435 is completely filled with reference membrane 440. In other embodiments, well 435 may not be completely filled with reference membrane 440. Reference membrane 440 is in contact with conductive element 437. In some embodiments, reference membrane 440 is a viscous material (e.g., an electrolyte). Reference membrane 440 can become a solid material (e.g., a solid organic material) after solvent evaporation.

[0056] As shown in FIG. 4C , a portion of well 433 and a portion of channel 435 are filled with reference membrane 450, while another portion of channel 435 and a portion of well 439 are filled with a different reference membrane 460. In some embodiments, well 433 or 439 can be completely filled with reference membrane 450 or 460. In the embodiment of FIG. 4D , channel 435 is completely filled with reference membranes 450 and 460. In other embodiments, channel 435 may not be completely filled with reference membranes 450 and 460. Reference membrane 460 is in contact with conductive element 437. In some embodiments, reference membrane 450 can be a solid membrane (e.g., a solid organic material) and reference membrane 460 can be an aqueous membrane. Reference membrane 460 can include a gel, such as a sodium chloride (NaCl) gel or a potassium chloride (KCl) gel. Reference membrane 460 can include a polymer, such as Nafion (a trademarked name for a sulfonated tetrafluoroethylene-based fluorine-copolymer). Reference membrane 460 may comprise other materials. An example of reference membrane 460 is 1% agarose with NaCl.

[0057] Channels 335 and 435 in Figures 3A, 3B, 4A, and 4B have a rectangular shape in the XZ plane. In other embodiments, channel 335 or 435 may have a different shape depending on the specific requirements of the application. For example, the dimension of channel 335 along the Z axis may increase or decrease in the direction from well 333 to conductive element 337 along the X axis. Similarly, the dimension of channel 435 along the Z axis may increase or decrease in the direction from well 434 to end 438 along the X axis. Also, the longitudinal axis of channel 335 or 435 may not be perpendicular to the longitudinal axis of the well.

[0058] FIG. 5 illustrates an exemplary electrochemical sensor 500 including multiple reference electrodes 530 and 540 according to some embodiments of the present disclosure. Reference electrodes 530 and 540 may have similar or different components. One embodiment of reference electrode 530 or 540 may be reference electrode 130, 330, or 430. Reference electrode 530 includes two wells 533 and 535. Well 535 is associated with a conductive conductor that may be below the edge of well 535. Reference electrode 530 may also include a channel (not shown in FIG. 5 ) connecting well 533 to well 535 to form a flow path. Reference electrode 540 includes three wells 543, 545, and 547. Well 543 is located between well 545 and well 547. Each of wells 545 and 547 is associated with a conductive conductor that may be below the edge of the well. Reference electrode 540 may also include a first channel (not shown in FIG. 5) connecting well 543 to well 545 to form a first flow path, and a second channel (not shown in FIG. 5) connecting well 543 to well 547 to form a second flow path. In some embodiments, a benefit of having two wells 545 and 547 with conductive elements is redundancy, such that if one of the conductive elements fails, reference electrode 540 still has a conductive element that can function.

[0059] 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 (individually referred to as a "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, disposed in a channel between the two wells of the reference electrode. The valve may control the flow of a reference membrane within the reference electrode and thus 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 turned on by opening the valve. The valve can be used to select the reference electrode to use.

[0060] 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 (individually referred to as "working electrode 620"), a reference electrode 630, and a sample channel 640. The reference electrode 630 has a single well that is outside of the sample channel 640. The well may not be in direct contact with the sample.

[0061] FIG. 7 illustrates 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 (individually referred to as a “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, e.g., in embodiments where the electrochemical sensor 700 is a voltammetric or amperometric sensor. The reference electrode 730 includes an elongated well filled with a reference membrane. 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 FIG. 7, the elongated well 733 extends along the X-axis and has a longitudinal axis aligned with 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 to define the elongated well 733. In other cases, for example, using Flex technology, a solder resist may be placed under the polymer to ensure that no copper is exposed.

[0062] 7, the elongated wells 733 have a racetrack shape in the XY plane. In other embodiments, the elongated wells 733 may have different shapes in the XY plane, such as rectangular, oval, trapezoidal, etc. Also, the length of the elongated wells 733 along the X axis may vary. Different shapes, depths, and locations of the wells, channels, and relative openings may be optimized depending on the specific requirements of the application.

[0063] 8A and 8B show an exemplary electrochemical sensor 800 including a cap 810 according to some embodiments of the present disclosure. FIG. 8B is a cross-sectional view of FIG. 8A taken along line BB. The electrochemical sensor 800 also includes a working electrode 820 (individually referred to as the "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 in which the working electrode 820 is disposed. In some embodiments, the cap 810 is disposed on a substrate, such as substrate 110, 210, 310, 410, 510, 610, or 710, on which the working electrode 820 and the reference electrode are disposed. The cap 810 may partially cover the surface of the substrate, or may cover the entire surface except for the sample channel 840, for example. 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 a test sample provided to the electrochemical sensor 800, for example, through the sample channel 840. In the embodiment of FIGS. 8A and 8B , a membrane 830 is present in the first well 833. The membrane 830 may be one of the membranes 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 touch the second well 839. The cap 810 may include a rubber gasket, adhesive tape, epoxy, a lid, a cartridge, a microfluidic substrate, or the like. Cap 810 may be used with other electrochemical sensors, such as any of the other electrochemical sensors described herein.

[0064] FIG. 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 (individually referred to as the "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 of FIGS. 8A and 8B. The cap 910 defines a sample channel 940 in which the working electrode 920 is disposed. 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 a separate well outside the sample channel 940, which may be covered or sealed by the cap 910. A membrane 930 resides in the reference electrode's well, which is inside the sample channel 940.

[0065] 10 shows an exemplary electrochemical sensor 1000 including a reference electrode 1030 having multiple channels 1035a-c, according to some embodiments of the present disclosure. The electrochemical sensor 1000 also includes a substrate 1010 and a working electrode 1020 (individually referred to as a "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.

[0066] Reference electrode 1030 includes well 1033, channels 1035a-c (collectively referred to as "channels 1035"), and wells 1037a-c (collectively referred to as "wells 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 not 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 is opposite the first end. A conductive element is coupled to each well 1037. For example, the conductive element may be on 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 one another, and different potentials can be applied to the conductive elements simultaneously.

[0067] Well 1033, channel 1035, and well 1037 form a flow path for a reference membrane that a conductive element can contact. Because there are three channels 1035 and three wells 1037, reference electrode 1030 can provide three flow paths, all of which begin at well 1033. In other embodiments, reference electrode 1030 may include a different number of channels 1035 and a different number of wells 1037.

[0068] 11 shows another exemplary electrochemical sensor 1100 including a reference electrode 1130 having multiple channels 1135a-c, according to some embodiments of the present disclosure. The electrochemical sensor 1100 also includes a substrate 1110 and a working electrode 1120 (individually referred to as a "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.

[0069] Reference electrode 1130 includes 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, channel 1135 has a first end connected to the end of well 1133 and a second end connected to well 1137. The second end is opposite the first end. A conductive element is coupled to well 1137 and can contact a reference membrane that flows through some or all of the three channels 1135 to well 1137. For example, the conductive element can be on the end of well 1137 that is connected to channel 1135. Well 1133, channel 1135, and well 1137 form a flow path for the reference membrane. Reference membrane flow can begin at well 1133 and then branch into three channels 1135, which can meet at well 1137. In some embodiments, channels 1135 can be blocked, for example, by closing a valve.

[0070] 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. Reference electrode 1230 also includes well 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, reference electrode 1230 forms two flow paths. Valve 1239a is disposed along channel 1235a and can control the flow of conductive media within channel 1235a. For example, when valve 1239a is open, a reference membrane can flow from well 1233 to well 1237a through channel 1235a, allowing the reference membrane to reach the conductive element below well 1237a. When valve 1239a is closed, it becomes a barrier that prevents reference membrane from flowing through channel 1235a and reaching the conductive elements. Similarly, valve 1239b is positioned along channel 1235b and can control flow within channel 1235b. Valves 1239a and 1239b can be used to select which conductive elements are enabled / available. In some embodiments, the conductive elements (below well 1237a) are configured to be used for wireless communication, and the additional conductive elements (below well 1237b) are configured to be used for wired communication. In some embodiments, valve 1239a or 1239b can be a hydrophobic valve. In other embodiments, valve 1239a or 1239b can be a barrier that can be broken by applying energy, e.g., a voltage, a pulse of current, or mechanical vibration. In some embodiments, the valves can include an electroactive polymer located on the walls 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 can expand to close the valve. The material, shape, and structure of the valve can be optimized depending on the specific requirements of the application.

[0071] FIG. 13 shows an exemplary reference electrode 1330 including stirring elements 1340a-d, according to some embodiments of the present disclosure. Reference electrode 1330 includes wells 1333 and 1339, a channel 1335 between wells 1333 and 1339, and a conductive element 1337 below well 1339 in substrate 1310. Wells 1333 and 1339 and channel 1335 form a flow path for a conductive medium. Stirring elements 1340a-d (collectively referred to as "stirring elements 1340") are disposed on the walls of well 1333 and channel 1335. As shown in FIG. 13, stirring elements 1340a and 1340b are disposed on the outer surface of channel 1335, stirring element 1340c is disposed on the outer surface of channel 1335, and stirring element 1340d is disposed on another outer surface of channel 1335. In other embodiments, reference electrode 1330 may include a different number of stirring elements 1340, stirring elements 1340 located in different locations (e.g., on the walls of well 1339), or stirring elements 1340 of different sizes or shapes. Reference electrode 1330 may also include different flow paths, for example, a flow path including one well and one channel.

[0072] The agitating element 1340 is configured to agitate the flow of the reference membrane within the flow path. For example, the agitating element 1340 can create motion to manipulate or move fluid present within the flow path. The agitating element 1340 can exert mechanical stress on the flow path, for example, in response to an electrical signal (e.g., an electric field), a radio frequency signal, or another 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 the channel 1335), which can promote or retract the flow of the reference membrane within the flow path. The agitating element 1340 can include an electroactive polymer or a piezoelectric material.

[0073] FIG. 14 shows an exemplary interface between a stirring element 1420 and a wall 1410 of a flow path in a reference electrode, according to some embodiments of the present disclosure. The stirring element 1420 may be one embodiment of the stirring element 1340 of FIG. 13. The wall 1410 may be a wall of a well 1333 or a wall of a channel 1335. As shown in FIG. 14, the interface is not smooth. Rather, the stirring element 1420 has protrusions that correspond to recesses in the wall 1410. Such a "rough" interface can improve adhesion between the stirring element 1420 and the wall 1410 and can affect how the structure moves, flexes, or bends to interact with the fluid in the flow path.

[0074] 15A-15C show exemplary stirring elements 1520a-c having different shapes according to some embodiments of the present disclosure. Stirring elements 1520a-c are disposed on walls 1510a-c, respectively. FIG. 15A shows stirring element 1520a having a circular shape in the XY plane. FIG. 15B shows stirring element 1520b having a ring shape in the XY plane. FIG. 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 FIGS. 15A-15C. Stirring elements may be patterned based on application needs. The pattern or shape will affect the behavior of the channel structure and how fluid is manipulated. The relative shapes and interfaces between the stirring elements and the walls can be modified to optimize the required behavior and, therefore, the level of agitation imparted to the membrane or material within the channel. The stirring element can be a piezoelectric material (e.g., lead zirconate titanate (PZT), barium titanate, gallium nitride, zinc oxide, polyvinylidene fluoride, ferroelectric materials with perovskite structures, etc.), or an electroactive polymer (e.g., a ferroelectric polymer such as polyvinylidene fluoride, an ionic polymer-metal composite, a stimuli-responsive gel, or an electrostrictive grafted polymer), or a conductive material such that application of a stimulus (e.g., voltage, current, etc.) deforms or expands or contracts or distorts the structure, which in turn imparts a stirring force to the medium / fluid / membrane within the channel. In certain embodiments, the stimulus can be applied in a pulsed or intermittent manner to impart a pulsed or intermittent stirring force to manipulate the fluid / medium / membrane within the channel. The shape / size / thickness / interface between the stirring element and the adjacent layer can be modified and optimized depending on the specific requirements of the application.

[0075] FIG. 16 shows an exemplary reference electrode 1630 including heating elements 1640A-D, according to some embodiments of the present disclosure. Reference electrode 1630 includes wells 1633 and 1639, a channel 1635 between wells 1633 and 1639, and a conductive element 1637 below well 1639 in substrate 1610. Wells 1633 and 1639 and channel 1635 form the flow path of the reference membrane. Heating elements 1640a-d (collectively referred to as "heating elements 1640") are disposed on the walls of well 1633 and channel 1635. As shown in FIG. 16, heating elements 1640a and 1640b are disposed on the outer surface of channel 1635, heating element 1640c is disposed on the outer surface of channel 1635, and heating element 1640d is disposed 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 locations (e.g., on the walls of the well 1639), or heating elements 1640 of different sizes or shapes. The heating elements may be located on the inner surface of the flow path. The reference electrode 1630 may also include different flow paths, such as a flow path including one well and one channel. The heating elements 1640 are configured to increase the temperature of the fluid within the flow path. The heating elements 1640 may provide heat to the fluid, for example, in response to an electrical signal (e.g., an electric field) or other type of signal. The shape, location, and number of heating elements may be optimized depending on the required applied and convective current or heat treatment stage. This also applies to piezoelectric elements that may be incorporated into the structure to manipulate or agitate the fluid.

[0076] FIG. 17 shows an exemplary interface between a heating element 1720 and a wall 1710 of a flow channel, according to some embodiments of the present disclosure. The heating element 1720 may be one embodiment of the heating element 1640 of FIG. 16 . The wall 1710 may be a wall of the well 1333 or a wall of the channel 1335. As shown in FIG. 17 , the interface is not smooth. Rather, the heating element 1720 has protrusions that correspond 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 electroactive or piezoelectric layers. In the manner in which the bimetallic plate layer bends or flexes, the defined section of the channel can also be constructed to refract / move and manipulate fluid in a desired manner, which can be optimized depending on the particular application of the electrochemical sensor.

[0077] 18A-18C show exemplary heating elements 1820a-c having different shapes according to some embodiments of the present disclosure. The heating elements 1820a-c are disposed on the walls 1810a-c, respectively. FIG. 18A shows a heating element 1820a having a circular shape in the XY plane. FIG. 18B shows a heating element 1820b having a ring shape in the XY plane. FIG. 18C shows a heating element 1820c having a rectangular shape in the XY plane. In other embodiments, the heating elements may have shapes different from those shown in FIGS. 18A-18C. The heating elements may be patterned based on application needs. The shape, location, and number of elements may be optimized depending on the particular hot air flow or heat treatment step required by a particular application of the electrochemical sensor.

[0078] FIG. 19 shows an exemplary electrochemical sensor 1900 including a reference electrode on a sample channel 1940, according to some embodiments of the present disclosure. In this example, the sample channel 1940 may represent any source of sample, including a container in which the electrochemical sensor 1900 is immersed, or an aliquot of sample that is dispensed or applied to the electrochemical sensor 1900. The electrochemical sensor 1900 also includes a substrate 1010 and a working electrode (not shown in FIG. 19). The substrate 1910 may be an embodiment of the substrate 110 of FIG. 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 may be modified and optimized depending on the requirements of a particular application.

[0079] Sample channel 1940 can be used to provide a test sample and working membrane to the working electrode. As shown in FIG. 19 , a reference electrode is located below sample channel 1940. The reference electrode includes well 1933, channel 1935, conductive element 1937, another well 1939, and a stirring element 1950 (individually referred to as "stirring element 1950"). In some embodiments, conductive element 1937 is disposed on the surface of channel 1935. Channel 1935 is connected to well 1933 and provides a fluid flow path to conductive element 1937. Channel 1935 is also connected to well 1939. Well 1939 can function as a vent through which air (e.g., air from solidifying fluid) can be vented from the reference electrode.

[0080] As shown in FIG. 19 , a stirring element 1950 is coupled to wells 1933 and 1939 and channel 1935. The stirring element 1950 can alter the flow of fluid within the flow path. In some embodiments, the stirring element 1950 is a coil that can be energized by a radio frequency signal. The coil can agitate, e.g., pulsate, fluid within a defined proximity 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 particular application of the electrochemical sensor 1900. The stirring element 1950 can be integrated with the sample channel 1940 such that an external radio frequency signal can be applied to the stirring element 1950. Although not shown in FIG. 19 , the electrochemical sensor 1900 can include a heating element for heating the fluid within the flow path. The stirring element 1950 can include an electroactive polymer or a piezoelectric material. The size or shape of the stirring element 1950 can be optimized depending on the particular application of the electrochemical sensor and the desired manipulation of the fluid.

[0081] 20A and 20B show a reference electrode 2000 including multiple layers 2010, 2020, and 2030, according to some embodiments of the present disclosure. FIG. 20A is a perspective view of reference electrode 2000. FIG. 20B is a side view of reference electrode 2000. Layers 2010, 2020, and 2030 are stacked together along the Z-axis. Layer 2020 is between layers 2010 and 2030.

[0082] 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 to form a first flow path. Channel 2065a is connected to wells 2040a and 2060a to form 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.

[0083] Similarly, third layer 2030 includes wells 2040b, 2050, and 2060b, channels 2055b and 2065b, and valves 2070 and 2080b. As shown, well 2050 and valve 2070 of third layer 2030 corresponding to well 2050a and valve 2070a are covered by second layer 2020 and are not shown. Channel 2055b is connected to wells 2040b and 2050a to form a first flow path. Channel 2065b is connected to wells 2040b and 2060b to form 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 in third layer 2030 may be aligned with a well in layer 2010. For example, well 2040b may be aligned with well 2040a, well 2050 (not shown) may be aligned with corresponding well 2050a, and well 2060b may be aligned with well 2060b. Also, 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 / disjointed pathways or networks. Fluid may also flow from a well or channel in layer 2010, through layer 2020, and to a 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 network of a layer can be different from the flow channels or flow network of another layer. A layer can have flow channels or flow networks (e.g., wells, channels, or both) that are not aligned with another layer. Systems can be constructed to allow fluids to flow laterally within and between layers. This can enable systems in which materials can be processed / analyzed in parallel on multiple layers.

[0084] Second layer 2020 includes valves 2090 (individually referred to as "valves 2090"). Valves 2090 control the flow of fluid from layer 2010 to layer 2030. In other embodiments, second layer 2020 may include a different number of valves 2090, and valves 2090 may have different locations in 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 location of valves in layer 2020 can also be determined based on the requirements of the system or process step in which reference electrode 2000 is used. Reference electrode 2000 can be used in a vertically integrated system where fluid can flow laterally within a single layer as well as vertically between layers. Valves can be used to control lateral and vertical flow. Valves used in the system can be constructed using the materials / structures described above. The shape / size / materials / structure of the valves can be modified and optimized depending on the specific requirements of the application.

[0085] 21 shows valves 2110 and 2120 controlling an inlet 2130 and an outlet 2140 of a reference electrode 2100, according to some embodiments of the present disclosure. Valve 2110 is coupled to inlet 2130. When valve 2110 is open, fluid can enter the reference electrode 2100 through inlet 2130. Valve 2120 is coupled to outlet 2140. When valve 2120 is open, fluid can exit the reference electrode 2100 through outlet 2140. Dashed lines represent an exemplary flow path 2150 within the reference electrode 2100.

[0086] Many other aspects will become apparent from the foregoing detailed description and accompanying drawings, and these aspects are represented by the following clauses:

[0087] Clause 1. A reference electrode in an electrochemical sensor, the reference electrode comprising: a substrate including an interior wall defining a flow path for a conductive medium; a conductive element in the substrate, the conductive element touching an end of the flow path; and a valve coupled to the flow path and configured to control the flow of the conductive medium to the conductive element through the flow path.

[0088] Clause 2. The reference electrode of clause 1, further comprising: an additional flow path for the conductive medium, the additional flow path being disposed within the substrate; an additional conductive element within the substrate, the additional conductive element touching an end of the additional flow path; and an additional valve coupled to the additional flow path and configured to control additional flow of the conductive medium through the additional flow path to the additional conductive element.

[0089] Clause 3. The reference electrode of clause 2, wherein a portion of the flow path overlaps a portion of the additional flow path.

[0090] Clause 4. The reference electrode of any one of clauses 1-3, wherein the valve comprises an electroactive polymer configured to impede the flow of the conductive medium in response to an electrical stimulus.

[0091] Clause 5. A reference electrode described in any one of clauses 1 to 4, wherein the flow path includes a well in a substrate, the well extending from a first surface of the substrate toward a second surface of the substrate, the first surface being opposite the second surface, and a channel disposed in the substrate and between the first and second surfaces, the first end of the channel being connected to the well, the conductive element touching the second end of the channel, the first end being opposite the second end.

[0092] Clause 6. The reference electrode of clause 5, wherein the valve is disposed along a wall of the channel.

[0093] Clause 7. The reference electrode of clause 6, further comprising an additional conductive element in the substrate, an additional channel in the substrate connected to the well, the additional conductive element touching the additional channel, and an additional valve positioned along the additional channel.

[0094] Clause 8. A reference electrode described in any one of clauses 1 to 7, wherein the flow path includes: 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 the second surface; a second well in the substrate, the second well extending from the first surface toward the second surface, the conductive element contacting the second well; and a channel disposed between the first surface and the second surface, the first end of the channel being connected to the first well and the second end of the channel being connected to the second well, the first end being opposite the second end.

[0095] Clause 9. The reference electrode of clause 8, wherein the valve is disposed within the channel.

[0096] Clause 10. The reference electrode of clause 9, further comprising a third well in the substrate, an additional conductive element in the substrate that contacts the third well, an additional channel connected to the first well and the third well, and an additional valve positioned along the additional channel.

[0097] Clause 11. 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, the reference electrode comprising: a substrate including an interior wall defining a flow path for a conductive medium; a conductive element in the substrate, the conductive element touching an end of the flow path; and a valve coupled to the flow path and configured to control flow of the conductive medium to the conductive element through the flow path.

[0098] Clause 12. An electrochemical sensor as described in clause 11, further comprising an additional flow path for the conductive medium, the additional flow path being disposed within the substrate, an additional conductive element within the substrate, the additional conductive element touching an end of the additional flow path, and an additional valve coupled to the additional flow path and configured to control additional flow of the conductive medium through the additional flow path to the additional conductive element.

[0099] Clause 13. The electrochemical sensor of clause 11 or 12, wherein the valve comprises an electroactive polymer configured to impede the flow of the conductive medium in response to an electrical stimulus.

[0100] Clause 14. An electrochemical sensor according to any one of clauses 11 to 13, further comprising one or more counter electrodes.

[0101] Clause 15. An electrochemical sensor described in any one of clauses 11 to 14, wherein the flow path includes: 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 the second surface; a second well in the substrate, the second well extending from the first surface toward the second surface, the conductive element contacting the second well; and a channel disposed between the first surface and the second surface, the first end of the channel being connected to the first well and the second end of the channel being connected to the second well, the first end being opposite the second end, and the valve being disposed on the channel.

[0102] Clause 16. A reference electrode in an electrochemical sensor, the reference electrode comprising a first layer comprising a first flow path configured to facilitate a first flow of a conductive medium in the first layer, a first conductive element in contact with the first flow path, and a first valve coupled to the first flow path and configured to control the first flow of the conductive medium in the first layer; and a second layer over the second layer, the second layer comprising a second flow path configured to facilitate a second flow of the conductive medium in the second layer, a second conductive element in contact with the second flow path, and a second valve coupled to the second flow path and configured to control the second flow of the conductive medium in the second layer.

[0103] Clause 17. The reference electrode of clause 16, further comprising a third layer between the first layer and the second layer, the third layer comprising a third flow path connected to the first flow path and the second flow path.

[0104] Clause 18. The reference electrode of clause 17, wherein the third layer further comprises one or more third valves coupled to the third flow path, the one or more third valves configured to control the flow of the conductive medium from the first layer to the third layer.

[0105] Clause 19. A reference electrode described in any one of clauses 16 to 18, wherein the first flow path includes a well extending from a first surface of the first layer toward a second surface of the first layer, the first surface being opposite the second surface, and a channel disposed between the first surface and the second surface, the first end of the channel being connected to the well, the first conductive element contacting the second end of the channel, the first end being opposite the second end, and the first valve being disposed along the channel.

[0106] Clause 20. A reference electrode described in any one of clauses 16 to 19, wherein the first flow path includes: a first well extending from a first surface of the first layer toward a second surface of the first layer, the first surface being opposite the second surface; a second well extending from the first surface toward the second surface, the first conductive element contacting the second well; and a channel disposed between the first surface and the second surface, the first end of the channel being connected to the first well and the second end of the channel being connected to the second well, the first end being opposite the second end, and the first valve being disposed along the channel.

[0107] It may be understood that not necessarily all objectives or advantages may be achieved by any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that a particular embodiment may be configured to operate in a manner that achieves or optimizes one advantage or group of advantages as taught herein, without necessarily achieving other objectives or advantages that may be taught or suggested herein.

[0108] It may also be essential to note that all specifications, dimensions, and relationships (e.g., number of reference electrodes, channels, wells, etc.) outlined herein are presented for illustrative and teaching purposes only. Such information may be significantly changed without departing from the spirit of the present disclosure or the scope of the appended claims. This specification applies only to one non-limiting example, and therefore, they should be interpreted as such. In the foregoing description, exemplary embodiments have been described with reference to particular arrangements of components. Various modifications and changes can be made to such embodiments without departing from the scope of the appended claims. Accordingly, the description and drawings should be considered in an illustrative sense, and not a restrictive sense.

[0109] It should be noted that for many of the examples provided herein, interactions may be described in terms of two, three, four, or more components. However, this is done for purposes of clarity and illustration only. It should be understood that the system may be arranged in any suitable manner. Pursuant to similar design choices, any of the illustrated components, modules, and elements of the drawings may be combined into a variety of possible configurations, all of which are clearly within the broad scope of this specification.

[0110] It should be noted that, herein, references to various features (e.g., elements, structures, modules, components, steps, operations, characteristics, etc.) included in "one embodiment," "an exemplary embodiment," "another embodiment," "some embodiments," "various embodiments," "other embodiments," "alternative embodiments," etc., are intended to mean that any such features are included in one or more embodiments of the present disclosure, but may or may not necessarily be combined in the same embodiment.

[0111] Numerous other changes, substitutions, variations, alterations, and modifications may be ascertained by those skilled in the art, and the present disclosure may be intended to encompass all such changes, substitutions, variations, alterations, and modifications as fall within the scope of the appended claims. It should be noted that all optional features of the systems and methods described above may also be implemented with respect to the methods or systems described herein, and details in the examples may be used anywhere in one or more embodiments.

[0112] In order to assist the United States Patent and Trademark Office (USPTO) and, additionally, any reader of any patent issued on this application, in interpreting the claims appended hereto, Applicant wishes to note that (a) Applicant does not intend to invoke 35 U.S.C. §112(f) in any of the appended claims as they exist on the filing date hereof, unless the terms "means for" or "step for" are specifically used in a particular claim, and (b) nothing herein intends to limit the present disclosure in any manner that may not otherwise be reflected in the appended claims. [Explanation of symbols]

[0113] 100 Electrochemical Sensors 130 Reference electrode 110 Substrate 225 Conductive Elements 1239 Valve

Claims

1. A reference electrode in an electrochemical sensor, the reference electrode comprising: a substrate including an interior wall defining a flow path for a conductive medium; a conductive element in the substrate, the conductive element touching an end of the channel; a valve coupled to the flow path and configured to control the flow of the conductive medium through the flow path to the conductive element; The flow path is a well in the substrate, the well being formed with an opening on a first side of the substrate; a channel formed in the substrate to connect the well and the conductive element, the channel contacting the conductive element provided on a second side or contacting the conductive element formed inside the substrate at an intermediate portion of the channel; The conductive element is provided at a position different from the well in a plan view of the substrate.

2. an additional flow path for the conductive medium, the additional flow path being disposed within the substrate; an additional conductive element in the substrate, the additional conductive element touching an end of the additional flow channel; 10. The reference electrode of claim 1, further comprising: an additional valve coupled to the additional flow path and configured to control additional flow of the conductive medium through the additional flow path to the additional conductive element.

3. The reference electrode of claim 2 , wherein a portion of the flow path overlaps a portion of the additional flow path.

4. A reference electrode in an electrochemical sensor, the reference electrode comprising: a substrate including an interior wall defining a flow path for a conductive medium; a conductive element in the substrate, the conductive element touching an end of the channel; a valve coupled to the flow path and configured to control the flow of the conductive medium through the flow path to the conductive element; A reference electrode, wherein the valve comprises an electroactive polymer configured to impede the flow of the conductive medium in response to an electrical stimulus.

5. A reference electrode in an electrochemical sensor, the reference electrode comprising: a substrate including an interior wall defining a flow path for a conductive medium; a conductive element in the substrate, the conductive element touching an end of the channel; a valve coupled to the flow path and configured to control the flow of the conductive medium through the flow path to the conductive element; The flow path is a well in the substrate, the well extending from a first surface of the substrate to a second surface of the substrate, the first surface being opposite the second surface; a channel disposed within the substrate and between the first surface and the second surface, a first end of the channel connected to the well, the conductive element contacting a second end of the channel, the first end opposite the second end.

6. The reference electrode of claim 5 , wherein the valve is disposed along a wall of the channel.

7. additional conductive elements within the substrate; an additional channel in the substrate connected to the well, the additional conductive element contacting the additional channel; The reference electrode of claim 6 , further comprising: an additional valve disposed along the additional channel.

8. A reference electrode in an electrochemical sensor, comprising: a substrate including an interior wall defining a flow path for a conductive medium; a conductive element in the substrate, the conductive element touching an end of the channel; a valve coupled to the flow path and configured to control the flow of the conductive medium through the flow path to the conductive element; The flow path is a first well in the substrate, the first well extending from a first surface of the substrate toward a second surface of the substrate, the first surface being opposite the second surface; a second well in the substrate, the second well extending from the first surface toward the second surface, the conductive element contacting the second well; a channel disposed between the first surface and the second surface, a first end of the channel connected to the first well, a second end of the channel connected to the second well, and the first end opposite the second end.

9. The reference electrode of claim 8 , wherein the valve is disposed within the channel.

10. a third well in the substrate; an additional conductive element in the substrate, the additional conductive element contacting the third well; an additional channel connected to the first well and the third well; 10. The reference electrode of claim 9, further comprising: an additional valve disposed along the additional channel.

11. 1. An electrochemical sensor comprising: one or more working electrodes in the substrate; a reference electrode electrically coupled to the one or more working electrodes, the reference electrode comprising: a substrate including an interior wall defining a flow path for a conductive medium; a conductive element in the substrate, the conductive element touching an end of the channel; a reference electrode comprising: a valve coupled to the flow path and configured to control the flow of the conductive medium through the flow path to the conductive element; Equipped with The flow path is a well in the substrate, the well being formed with an opening on a first side of the substrate; a channel formed in the substrate to connect the well and the conductive element, the channel contacting the conductive element provided on the second side or contacting the conductive element formed inside the substrate at an intermediate portion of the channel; The electrochemical sensor, wherein the conductive element is provided at a position different from the well in a plan view of the substrate.

12. an additional flow path for the conductive medium, the additional flow path being disposed within the substrate; an additional conductive element in the substrate, the additional conductive element touching an end of the additional flow channel; 12. The electrochemical sensor of claim 11, further comprising: an additional valve coupled to the additional flow path and configured to control additional flow of the conductive medium through the additional flow path to the additional conductive element.

13. An electrochemical sensor comprising: one or more working electrodes in the substrate; a reference electrode electrically coupled to the one or more working electrodes, the reference electrode comprising: a substrate including an interior wall defining a flow path for a conductive medium; a conductive element in the substrate, the conductive element touching an end of the channel; a reference electrode comprising: a valve coupled to the flow path and configured to control the flow of the conductive medium through the flow path to the conductive element; An electrochemical sensor comprising: An electrochemical sensor, wherein the valve comprises an electroactive polymer configured to impede the flow of the conductive medium in response to an electrical stimulus.

14. The electrochemical sensor of any one of claims 11 to 13, further comprising one or more counter electrodes.

15. An electrochemical sensor comprising: one or more working electrodes in the substrate; a reference electrode electrically coupled to the one or more working electrodes, the reference electrode comprising: a substrate including an interior wall defining a flow path for a conductive medium; a conductive element in the substrate, the conductive element touching an end of the channel; a reference electrode comprising: a valve coupled to the flow path and configured to control the flow of the conductive medium through the flow path to the conductive element; An electrochemical sensor comprising: The flow path is a first well in the substrate, the first well extending from a first surface of the substrate toward a second surface of the substrate, the first surface being opposite the second surface; a second well in the substrate, the second well extending from the first surface toward the second surface, the conductive element contacting the second well; a channel disposed between the first surface and the second surface, a first end of the channel connected to the first well and a second end of the channel connected to the second well, the first end opposite the second end, and the valve disposed on the channel.

16. A reference electrode in an electrochemical sensor, the reference electrode comprising: A first layer, a first flow path configured to facilitate a first flow of a conductive medium within the first layer; a first conductive element contacting the first flow path; a first layer comprising: a first valve coupled to the first flow path and configured to control the first flow of the conductive medium within the first layer; a second layer over the first layer, said second layer comprising: a second flow path configured to facilitate a second flow of the conductive medium within the second layer; a second conductive element contacting the second flow path; a second layer comprising: a second valve coupled to the second flow path and configured to control the second flow of the conductive medium within the second layer; a reference electrode.

17. 17. The reference electrode of claim 16, further comprising a third layer between the first layer and the second layer, the third layer comprising a third flow path connected to the first flow path and the second flow path.

18. 18. The reference electrode of claim 17, wherein the third layer further comprises one or more third valves coupled to the third flow path, the one or more third valves configured to control the flow of the conductive medium from the first layer to the third layer.

19. The first flow path is a well extending from a first surface of the first layer toward a second surface of the first layer, the first surface being opposite the second surface; 19. The reference electrode of claim 16, comprising: a channel disposed between the first surface and the second surface, a first end of the channel connected to the well, the first conductive element touching a second end of the channel, the first end opposite the second end, and the first valve disposed along the channel.

20. The first flow path is a first well extending from a first surface of the first layer toward a second surface of the first layer, the first surface being opposite the second surface; a second well extending from the first surface toward the second surface, the first conductive element contacting the second well; 20. The reference electrode of claim 16, comprising: a channel disposed between the first surface and the second surface, a first end of the channel connected to the first well and a second end of the channel connected to the second well, the first end opposite the second end, and the first valve disposed along the channel.

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