Reference electrode for electrochemical sensors

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 March 22, 2022, entitled “REFERENCE ELECTRODES OF ELECTROCHEMICAL SENSORS,” which is assigned to the assignee hereof and incorporated by reference herein.

[0002] The present disclosure relates generally to electrochemical sensors, and more specifically 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. 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, 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, the flow path being disposed within the substrate, a conductive element within the substrate, the conductive element in contact with the flow path, and a stirring element coupled to the flow path and configured to change a size or shape of at least a portion of the flow path.

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

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

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

[0009] In some aspects, the techniques described herein relate to a reference electrode, where 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 and the second end of the channel being in contact with the conductive element, the first end being opposite the second end.

[0010] In some aspects, the techniques described herein relate to a reference electrode, and agitating elements are disposed on walls of a channel and configured to alter the size or shape of the channel.

[0011] In some aspects, the techniques described herein relate to a reference electrode, which is disposed on a wall of the well and further includes an additional stirring element configured to alter the size or shape of the well.

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

[0013] 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 flow path of a conductive medium defined by an inner wall of the substrate, the flow path being disposed in the substrate, a conductive element in the substrate, the conductive element in contact with the flow path, and a stirring element coupled to the flow path and configured to change a size or shape of at least a portion of the flow path.

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

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

[0016] In some aspects, the techniques described herein relate to electrochemical sensors, where the stirring element comprises an electroactive polymer or a piezoelectric material.

[0017] In some aspects, the techniques described herein relate to an electrochemical sensor, where 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 and the second end of the channel being in contact with the conductive element, the first end being opposite the second end.

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

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

[0020] In some aspects, the techniques described herein relate to an electrochemical sensor, where a working electrode of one or more working electrodes includes a working well configured to be at least partially filled with an ion-selective membrane distinct from a conductive medium.

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

[0022] 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 fluid flow path, a conductive element in the substrate, the conductive element in contact with the flow path, and a heating element coupled to the flow path and configured to increase a temperature of at least a portion of the flow path.

[0023] In some aspects, 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 aspects, the techniques described herein relate to a reference electrode and the heating element comprises a metal.

[0025] In some aspects, the techniques described herein relate to a reference electrode, where 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, and a channel disposed in the substrate and 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 in contact with the conductive element, the first end being opposite the second end.

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

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

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

[0029] 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 flow path for a conductive medium, the flow path being disposed in the substrate, a conductive element in the substrate and in contact with the flow path, and a heating element coupled to the flow path and configured to increase a temperature of at least a portion of the flow path.

[0030] In some aspects, the techniques described herein relate to an electrochemical sensor, where a heating element is configured to increase a temperature of at least a portion of a flow path in response to an electric current applied to the heating element.

[0031] In some aspects, the techniques described herein relate to electrochemical sensors, where the heating element comprises a metal.

[0032] In some aspects, the techniques described herein relate to an electrochemical sensor, where a 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, 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 contacting the second end of the channel, the first end being opposite the second end.

[0033] In some aspects, the techniques described herein relate to electrochemical sensors, where the heating element is disposed on a wall of a channel or a wall of a well.

[0034] In some aspects, the techniques described herein relate to an electrochemical sensor, wherein the flow path further comprises a second well in the substrate, the second well extending from the first surface to the second surface, and a second end of the channel connected to the second well.

[0035] In some aspects, the techniques described herein relate to an electrochemical sensor, the electrochemical sensor further comprising one or more other heating elements, the heating element and the one or more other heating elements each being disposed at a different location along the flow path.

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

[0037] 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 the description is intended to include all such aspects and their equivalents. [Brief description of the drawings]

[0038] 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:

[0039] [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. [Diagram 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] 1A-1D 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] 1 illustrates 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] 1 illustrates a valve controlling the inlet and outlet of a reference electrode according to some embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] 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.

[0041] Electrochemical sensors usually include several 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 includes two main components: a recognition element and a transducer. The recognition element selectively reacts with the analyte. This reaction is then converted to an electrical signal by the transducer. The recognition element and the transducer form the sensing electrode of the electrochemical sensor. An electrochemical sensor may include several 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 a reference electrode that has a stable reference potential that is not affected by the analyte. The reference electrode serves as a stable reference voltage for the measurement. In amperometric (or voltametric, etc.), 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, 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 relative to a reference electrode, so again, it is important that the reference electrode potential is stable. In addition, the presence of a substance that interacts with the working electrode / electrolyte interface can cause a current to flow between the working electrode and the counter electrode as a result of reduction / oxidation (redox) reactions at the working electrode. In some cases, it can cause a change in the electrode potential, which is the result of the 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.

[0042] High quality reference electrodes are important in electrochemical sensors. Reference electrodes 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.

[0043] 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 in a substrate. The electrochemical sensor may also include one or more counter electrodes in the substrate. The reference electrode may include a well and a channel defined by an inner 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 in 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 is in contact with a 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 be in contact with 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 membrane, 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.

[0044] Compared with conventional reference electrodes, the reference electrodes in the present disclosure have lower drift and are more stable. The use of one or more channels limits ion diffusion between the test liquid (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.

[0045] The following detailed description presents various descriptions of specific specific embodiments. However, the innovations described herein can be embodied in many different ways, for example, as defined and covered by the claims or selected examples. In the following description, reference may be made 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 be further 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.

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

[0047] As described herein, one aspect of the technology may be to collect and use data available from various sources to improve the 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 will respect and honor privacy policies and practices.

[0048] The following disclosure describes various exemplary embodiments and examples for implementing the features and functionality of the present disclosure. Although 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 vary from implementation to implementation. Moreover, it will be understood that such development efforts may be complex and time-consuming, but will nevertheless be routine for those of ordinary skill in the art having the benefit of this disclosure.

[0049] In this specification, reference may be made 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, devices, components, members, apparatuses, etc. described herein may be disposed in any desired orientation. Thus, 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 spatial orientations of aspects of such components should be understood to respectively describe the relative relationships between the components or the spatial orientations 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 dimensions or other characteristics (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," "having," or any other variations 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 that are inherent to such method, process, device, or system. Also, the term "or" refers to an inclusive "or" and not an exclusive "or."

[0051] 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 the accompanying drawings.

[0052] 1 illustrates 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.

[0053] The substrate 110 may include 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 injection molding, lamination, flexible / lamination or additive fabrication techniques, or other suitable techniques, depending on the particular requirements of the electrochemical sensor application, for example.

[0054] 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 the line 105. In other embodiments, the center of the working electrodes may be slightly offset from the line 105. The working electrodes 120 may be substantially aligned or non-aligned.

[0055] 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 a centerline or edge of the sample channel. The sample channel may provide a test sample (or analyte) to a working well of the 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 to the working electrode 120.

[0056] The reference electrode 130 includes two wells 133 and 135 defined in the substrate 110 and a conductive contact touching the well 135. In some embodiments, the well 133 can be inside the sample channel, while the well 135 can be outside the sample channel. The 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 the electrochemical sensor 100, or the test sample can be dispensed or placed in the well 133. However, the well 135 may not be in direct contact with the test sample. In the embodiment of FIG. 1, the well 133 is aligned with the working electrode 120 along the Y-axis, but the well 135 is not aligned with the working electrode 120. In other embodiments, the well 133 can 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. Distance 137 from well 133 to well 135, e.g., 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.

[0057] 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), and the like. 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 surface and the second surface 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 the well 133 or 135. With such an approach, the diffusion of ions (such as 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 slow.

[0058] 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 of FIG. 1. As shown in FIG. 2A, working electrode 220 is disposed within substrate 210. Substrate 210 may be an embodiment of substrate 110 of FIG. 1. Substrate 210 has surfaces 213 and 215 that face each other along the Z-axis.

[0059] 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 end 229. Opening 227 is on surface 213. End 229 is between surfaces 213 and 215. Conductive element 225 is below end 229.

[0060] 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.

[0061] 3A and 3B show an exemplary reference electrode 330, according to some embodiments of the present disclosure. Reference electrode 330 may be an embodiment of reference electrode 130 of FIG. 1. Reference electrode 330 is disposed within substrate 310. Substrate 310 may be an embodiment of substrate 110 of FIG. 1. As shown in FIG. 3A, substrate 310 has surfaces 313 and 315 that face each other along a Z-axis.

[0062] 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 surface 313 and the surface 315. The channel 335 extends along the X-axis. The 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 flow of the reference membrane towards the conductive element 337. The well 333 and the channel 335 also provide a "tortuous" path for ion diffusion, which takes longer than bulk diffusion.

[0063] As shown in FIG. 3B, the well 333 and a portion of the channel 335 are filled with the reference membrane 340. In some embodiments, the well 333 may 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 may 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 may include or be bonded to a hole in the substrate 310 through which air is vented from the substrate 310 when the reference membrane 340 is introduced to the reference electrode 330.

[0064] 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 of FIG. 1. Reference electrode 430 is disposed within a substrate 410. Substrate 410 may be an embodiment of substrate 110 of FIG. 1. As shown in FIG. 4A, substrate 410 has surfaces 413 and 415 that face each other along the Z-axis.

[0065] 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 end 434. Opening 432 is on surface 413. End 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 end 438. Opening 436 is on surface 413. End 438 is between surfaces 413 and 415.

[0066] The channel 435 extends along the X-axis. An end of the channel 435 is connected to an end 434 of the well 433. The other end of the channel 435 is connected to an 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 approximately 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 constitute a flow path. The flow path may, for example, allow the flow of the reference membrane towards the conductive element 437. Also, wells 433 and 439 containing membrane 440 and channel 435 provide a "tortuous" path for ion diffusion that 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), providing a longer lifespan. Although not shown in FIGS. 4A and 4B, reference electrode 430 may include or be bonded to a hole in substrate 410 through which air is vented from substrate 410 when reference membrane 440 is introduced to reference electrode 430.

[0067] 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 may 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 may become a solid material (e.g., a solid organic material) after solvent evaporation.

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

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

[0070] FIG. 5 illustrates an exemplary electrochemical sensor 500 including multiple reference electrodes 530 and 540 according to some embodiments of the present disclosure. The reference electrodes 530 and 540 may have similar or different components. An embodiment of the reference electrodes 530 or 540 may be the reference electrodes 130, 330, or 430. The reference electrode 530 includes two wells 533 and 535. The well 535 is associated with a conductive conductor that may be below the edge of the well 535. The reference electrode 530 may also include a channel (not shown in FIG. 5 ) that connects the well 533 to the well 535 to form a flow path. The reference electrode 540 includes three wells 543, 545, and 547. The well 543 is between the well 545 and the well 547. Each of the 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, the benefit of having two wells 545 and 547 with conductive elements is that there is redundancy, such that if one of the conductive elements fails, 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 (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 at the same time 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 in the reference electrode and may 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 turned on by opening the valve. The valve may be used to select the reference electrode to be used.

[0072] 6 illustrates 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.

[0073] 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, for example, in embodiments where the electrochemical sensor 700 is a voltammetric or amperometric sensor. The reference electrode 730 includes an elongated well that is filled with a reference membrane. The elongated well 733 has one end that is between the working electrodes 720 and the other end that is coupled to a conductive element 735. For example, the conductive element 735 may be 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 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 to define the elongated well 733. In other cases, for example, using Flex technology, a solder mask may be placed under the polymer to ensure that no copper is exposed.

[0074] In the embodiment of Figure 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.

[0075] 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 along line BB. The electrochemical sensor 800 also includes a working electrode 820 (individually referred to 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 in which the working electrode 820 is disposed. In some embodiments, the cap 810 is on a substrate in which the working electrode 820 and the reference electrode are disposed, such as substrate 110, 210, 310, 410, 510, 610, or 710. The cap 810 may partially cover the surface of the substrate, for example, may cover the entire surface except for the sample channel 840. The reference electrode may include a first well 833 with an opening inside the sample channel 840 and a second well 839 with 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 may receive a test sample provided to the electrochemical sensor 800, for example, through the sample channel 840. In the embodiment of FIG. 8A and FIG. 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, an adhesive tape, an epoxy resin, a lid, a cartridge, a microfluidic substrate, or the like. The cap 810 may be used with other electrochemical sensors, such as any of the other electrochemical sensors described herein.

[0076] FIG. 9 illustrates 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 a "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 another well outside the sample channel 940, which may be covered or sealed by the cap 910. The membrane 930 resides in the well of the reference electrode, which is inside the sample channel 940.

[0077] 10 illustrates 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 "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 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 may be inside the sample channel and 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 an end of well 1033 and a second end connected to well 1037. The second end 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 separately. In some embodiments, the conductive elements are insulated from one another such that different electrical potentials can be applied to the conductive elements simultaneously.

[0079] Well 1033, channel 1035, and well 1037 form a flow path for a reference membrane that a conductive element may 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.

[0080] 11 illustrates 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.

[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 the well 1133 to the well 1137. For example, the channel 1135 has a first end connected to an end of the well 1133 and a second end connected to the well 1137. The second end is opposite the first end. A conductive element is coupled to the well 1137 and may contact the reference membrane that flows through some or all of the three channels 1135 to the well 1137. For example, the conductive element may be on the end of the well 1137 that is connected to the channel 1135. The well 1133, the channel 1135, and the well 1137 form a flow path for the reference membrane. The flow of the reference membrane may start at the well 1133 and then branch into the three channels 1135 and may merge at the well 1137. In some embodiments, the channel 1135 can be blocked, for example, by closing a valve.

[0082] FIG. 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 a well 1233 and wells 1237a and 1237b. The channel 1235a is connected to the wells 1233 and 1237a. The channel 1235b is connected to the wells 1233 and 1237b. Thus, the reference electrode 1230 forms two flow paths. The valve 1239a is disposed along the channel 1235a and can control the flow of the conductive medium in the channel 1235a. For example, when the valve 1239a is open, the reference membrane can flow from the well 1233 to the well 1237a through the channel 1235a, so that the reference membrane can reach the conductive element below the well 1237a. When valve 1239a is closed, it becomes a barrier that prevents the reference membrane from flowing through channel 1235a to reach the conductive elements. Similarly, valve 1239b is disposed along channel 1235b and can control the flow in channel 1235b. Valves 1239a and 1239b can be used to select which conductive elements are enabled / available. In some embodiments, the conductive elements (under well 1237a) are configured to be used for wireless communication and the additional conductive elements (under 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., voltage, pulse of current, mechanical vibration. In some embodiments, the valve can 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 impede the flow of fluid in the channel. For example, the electroactive polymer can expand to close the valve. The materials, shape and structure of the valve can be optimized depending on the specific requirements of the application.

[0083] FIG. 13 illustrates 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 an outer surface of channel 1335, stirring element 1340c is disposed on an 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 a well and a channel.

[0084] The agitating element 1340 is configured to agitate the flow of the reference membrane in the flow path. For example, the agitating element 1340 can create a motion to manipulate or move a fluid present in the flow path. The agitating element 1340 can exert a 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 the channel 1335), which can promote or retract the flow of the reference membrane in the flow path. The agitating element 1340 can include an electroactive polymer or a piezoelectric material.

[0085] FIG. 14 illustrates 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 can be one embodiment of the stirring element 1340 of FIG. 13. The wall 1410 can 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. Also, this structure can affect how it moves, flexes, or bends to interact with the fluid in the flow path.

[0086] 15A-15C show exemplary stirring elements 1520a-c having different shapes according to some embodiments of the present disclosure. The stirring elements 1520a-c are disposed on the 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, the stirring elements may have shapes different from those shown in FIG. 15A-15C. The 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 shapes and interfaces between the stirring elements and the walls 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 with perovskite structure, etc.), or an electroactive polymer (e.g., ferroelectric polymers such as polyvinylidene fluoride, ionic polymer metal composites, stimuli-responsive gels, electrostrictive grafted polymers, etc.), or an electrically 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 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 layers may be modified and optimized depending on the specific requirements of the application.

[0087] FIG. 16 illustrates an exemplary reference electrode 1630 including heating elements 1640A-D, according to some embodiments of the present disclosure. The reference electrode 1630 includes wells 1633 and 1639, a channel 1635 between the wells 1633 and 1639, and a conductive element 1637 below the well 1639 in the substrate 1610. The wells 1633 and 1639 and the channel 1635 form a flow path of the reference membrane. The heating elements 1640a-d (collectively referred to as "heating elements 1640") are disposed on the walls of the well 1633 and the channel 1635. As shown in FIG. 16, the heating elements 1640a and 1640b are disposed on an outer surface of the channel 1635, the heating element 1640c is disposed on an outer surface of the channel 1635, and the heating element 1640d is disposed on another outer surface of the 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, for example a flow path including one well and one channel. The heating elements 1640 are configured to increase the temperature of the fluid in 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 currents or heat treatment stages. This also applies to piezoelectric elements that may be incorporated into the structure to manipulate or agitate the fluid.

[0088] FIG. 17 shows an exemplary interface between a heating element 1720 and a wall 1710 of a flow path according to some embodiments of the present disclosure. The heating element 1720 can be an embodiment of the heating element 1640 of FIG. 16. The wall 1710 can 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 way that the bimetallic plate layer bends or flexes, the defined section of the channel can also be constructed to refract / move and manipulate the fluid in a desired manner, which can be optimized depending on the particular application of the electrochemical sensor.

[0089] 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 the needs of the application. The shape, location, and number of elements may be optimized depending on the particular hot air flow or heat treatment step required by the particular application of the electrochemical sensor.

[0090] FIG. 19 illustrates 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.

[0091] The sample channel 1940 may be used to provide a test sample and a working membrane to the working electrode. As shown in FIG. 19, the reference electrode is below the sample channel 1940. The reference electrode includes a well 1933, a channel 1935, a conductive element 1937, another well 1939, and an agitation element 1950 (individually referred to as "agitation element 1950"). In some embodiments, the conductive element 1937 is disposed on the surface of the channel 1935. The channel 1935 is connected to the well 1933 and configures a fluid flow path to the conductive element 1937. The channel 1935 is also connected to the well 1939. The well 1939 may function as a vent through which air (e.g., air from solidification of the fluid) may be vented from the reference electrode.

[0092] As shown in FIG. 19 , the stirring element 1950 is coupled to the wells 1933 and 1939 and the channel 1935. The stirring element 1950 can modify the flow of fluid in 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, the fluid in 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 in 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 required manipulation of the fluid.

[0093] 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 the reference electrode 2000. FIG. 20B is a side view of the reference electrode 2000. The layers 2010, 2020, and 2030 are stacked together along the Z-axis. The layer 2020 is between the 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 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 to control the first flow path. Valve 2080a is coupled to channel 2065a to control 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, wells 2050 and valves 2070 of the third layer 2030 corresponding to wells 2050a and valves 2070a are covered by the 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 to control the first flow path. Valve 2080b is coupled to channel 2065b to control 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 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 with different / non-aligned pathways or networks. Fluid may also flow from a well or channel of layer 2010, through layer 2020, and into a corresponding well or channel of 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 be different from the flow channels or flow networks 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. Systems can be constructed that allow fluids to flow laterally within a layer and between layers. This may enable a system in which materials can be processed / analyzed in parallel on multiple layers.

[0096] The second layer 2020 includes valves 2090 (individually referred to as "valves 2090"). The valves 2090 control 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 the valves in the layer 2020 may be determined to allow fluid to flow between the layers 2010, 2020, and 2030. The number or location of the valves in the layer 2020 may be determined based on the location of other components of the layers 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 may also be determined based on the requirements of the system or process step in which reference electrode 2000 is used. Reference electrode 2000 may be used in a vertically integrated system where fluid may flow laterally within a single layer as well as vertically between layers. Valves may be used to control lateral and vertical flow. Valves used in the system may be constructed using the materials / structures described above. The shape / size / materials / structure of the valves may be modified and optimized depending on the specific requirements of the application.

[0097] 21 illustrates 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. The dashed lines represent an exemplary flow path 2150 within the reference electrode 2100.

[0098] Many other aspects will become apparent from the foregoing detailed description and the accompanying drawings, and these aspects are represented by the following clauses.

[0099] 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, the flow path being disposed within the substrate; a conductive element within the substrate, the conductive element in contact with the flow path; and a stirring element coupled to the flow path and configured to change a size or shape of at least a portion of the flow path.

[0100] Clause 2. The reference electrode of 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 a mechanical stress to an inner wall of the flow path in response to an electric field applied to the stirring element.

[0101] Clause 3. The reference electrode of 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 a mechanical stress to an inner wall of the flow path in response to a radio frequency signal applied to the stirring element.

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

[0103] Clause 5. A reference electrode as 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 and the second end of the channel being in contact with the conductive element, the first end being opposite the second end.

[0104] Clause 6. The reference electrode of clause 5, wherein the stirring element is disposed on a wall of the channel and configured to alter the size or shape of the channel.

[0105] Clause 7. The reference electrode of clause 6, further comprising an additional stirring element disposed in a wall of the well and configured to alter the size or shape of the well.

[0106] Clause 8. A reference electrode as described in clause 5 or 6, wherein the flow path further comprises a second well in the substrate, the second well extending from the first surface toward the second surface, the second end of the channel being connected to the second well, and the first end being opposite 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 the one or more working electrodes, the reference electrode comprising: a flow path of a conductive medium defined by an inner wall of the substrate, the flow path being disposed in the substrate; a conductive element in the substrate, the conductive element in contact with the flow path; and a stirring element coupled to the flow path and configured to change a size or shape of at least a portion of the flow path.

[0108] Clause 10. An electrochemical sensor as described in 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 a mechanical stress to an inner wall of the flow path in response to an electric field applied to the stirring element.

[0109] Clause 11. An electrochemical sensor as described in 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 a mechanical stress to an inner wall of the flow path in response to a radio frequency signal applied to the stirring element.

[0110] Clause 12. The electrochemical sensor of clause 9, wherein the stirring element comprises an electroactive polymer or a piezoelectric material.

[0111] Clause 13. An electrochemical sensor according to any one of clauses 9 to 12, wherein the flow path comprises: a well in a substrate, the well extending from a first surface of the substrate towards 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 and the second end of the channel being in contact with the conductive element, the first end being opposite the second end.

[0112] Clause 14. An electrochemical sensor according to clause 13, wherein the stirring element is disposed on a wall of the channel or on a wall of the well.

[0113] Clause 15. An electrochemical sensor as described in Clause 13, wherein the flow path further includes a second well in the substrate, the second well extending from the first surface toward the second surface, and a second end of the channel connected to the second well.

[0114] Clause 16. An electrochemical sensor as described in any one of clauses 9 to 15, wherein a working electrode of one or more working electrodes includes a working well configured to be at least partially filled with an ion selective membrane different from the conductive medium.

[0115] Clause 17. An electrochemical sensor described in any one of clauses 9 to 16, 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 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 interior wall defining a fluid flow path; a conductive element in the substrate, the conductive element in contact with the flow path; and a heating element coupled to the flow path and configured to increase a temperature of at least a portion of the flow path.

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

[0118] Clause 20. The reference electrode of clause 18, wherein the heating element comprises a metal.

[0119] Clause 21. A reference electrode described in any one of clauses 18 to 20, 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, and a channel disposed in the substrate and 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 in contact with the conductive element, the first end being opposite the second end.

[0120] Clause 22. The reference electrode according to clause 21, wherein the heating element is disposed on a wall of the channel or on a wall of the well.

[0121] Clause 23. A reference electrode as described in clause 21, wherein the flow path further includes a second well in the substrate, the second well extending from the first surface toward the second surface, and the second end of the channel connected to the second well.

[0122] Clause 24. A reference electrode as described in any one of clauses 18 to 23, further comprising one or more other heating elements, each of the heating element and the one or more other heating elements being positioned 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 the one or more working electrodes, the reference electrode comprising: a flow path for a conductive medium, the flow path being disposed in the substrate; a conductive element in the substrate, the conductive element contacting the flow path; and a heating element coupled to the flow path and configured to increase a temperature of at least a portion of the flow path.

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

[0125] Clause 27. The electrochemical sensor of clause 25, wherein the heating element comprises a metal.

[0126] Clause 28. An electrochemical sensor according to any one of clauses 25 to 27, wherein the flow path comprises: a first well in a substrate, the first well extending from a first surface of the substrate towards 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 contacting the second end of the channel, the first end being opposite the second end.

[0127] Clause 29. An electrochemical sensor according to clause 28, wherein the heating element is disposed on a wall of the channel or on a wall of the well.

[0128] Clause 30. An electrochemical sensor as described in clause 28 or 29, wherein the flow path further comprises 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 being connected to the second well.

[0129] Clause 31. An electrochemical sensor as described in any one of clauses 25 to 30, further comprising one or more other heating elements, each of the heating element and the one or more other heating elements being positioned at a different location along the flow path.

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

[0131] 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.

[0132] 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 altered 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 preceding description, exemplary embodiments have been described 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. Thus, the description and drawings should be considered in an illustrative sense, and not in a restrictive sense.

[0133] It should be noted that for many of the examples provided herein, the 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. Subject 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.

[0134] It should be noted that, in this specification, 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 be combined in the same embodiment.

[0135] Numerous other changes, substitutions, variations, modifications, and alterations may be ascertained by those skilled in the art, and the present disclosure may be intended to encompass all such changes, substitutions, variations, modifications, and alterations 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.

[0136] In order to assist the United States Patent and Trademark Office (USPTO) in interpreting the claims appended hereto, and in addition, any reader of any patents issued on this application, 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 of this specification, unless the words "means for" or "step for" are specifically used in a particular claim, and (b) nothing herein is intended to limit the present disclosure in any manner that may not otherwise be reflected in the appended claims. [Explanation of symbols]

[0137] 100 Electrochemical Sensors 110 Substrate 130 Reference electrode 225 Conductive Elements 1340 Stirring element 2150 Channel

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, the flow path being disposed within the substrate; a conductive element in the substrate, the conductive element being in contact with the flow path; a stirring element coupled to the flow path and configured to change a size or shape of at least a portion of the flow path.

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

3. 3. The reference electrode of claim 1 or 2, wherein the stirring element is configured to change the size or shape of at least a portion of the flow channel by applying a mechanical stress to the inner wall of the flow channel in response to a radio frequency signal applied to the stirring element.

4. The reference electrode of any one of claims 1 to 3, wherein the stirring element comprises an electroactive polymer or a piezoelectric material.

5. 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; 5. The reference electrode of claim 1, further comprising: a channel disposed within the substrate and between the first surface and the second surface, a first end of the channel being connected to the well and a second end of the channel being in contact with the conductive element, the first end being opposite the second end.

6. The reference electrode of claim 5 , wherein the stirring element is disposed in a wall of the channel and configured to modify a size or shape of the channel.

7. The reference electrode of claim 6 , further comprising an additional stirring element disposed in a wall of the well and configured to change the size or shape of the well.

8. The flow path is 8. The reference electrode of claim 5, further comprising a second well in the substrate, the second well extending from the first surface towards the second surface, a second end of the channel connected to the second well, and the first end opposite the second end.

9. 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 flow path for a conductive medium defined by an inner wall of the substrate, the flow path being disposed within the substrate; a conductive element in the substrate, the conductive element being in contact with the flow path; a stirring element coupled to the flow path and configured to change a size or shape of at least a portion of the flow path.

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

11. 11. The electrochemical sensor of claim 9 or 10, wherein the stirring element is configured to change the size or shape of at least a portion of the flow path by applying a mechanical stress to the inner wall of the flow path in response to a radio frequency signal applied to the stirring element.

12. The electrochemical sensor of any one of claims 9 to 11, wherein the stirring element comprises an electroactive polymer or a piezoelectric material.

13. 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; 13. The electrochemical sensor of claim 9, further comprising: 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 and a second end of the channel in contact with the conductive element, the first end being opposite the second end.

14. The electrochemical sensor of claim 13 , wherein the stirring element is disposed in a wall of the channel or in a wall of the well.

15. The flow path is 15. The electrochemical sensor of claim 13 or 14, further comprising a second well in the substrate, the second well extending from the first surface toward the second surface, and a second end of the channel connected to the second well.

16. 16. The electrochemical sensor of claim 9, wherein a working electrode of the one or more working electrodes comprises a working well configured to be at least partially filled with an ion selective membrane distinct from the conductive medium.

17. 17. The electrochemical sensor of claim 9, 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 between the first working electrode and the second working electrode.

18. A reference electrode in an electrochemical sensor, the reference electrode comprising: a substrate including an interior wall defining a fluid flow path; a conductive element in the substrate, the conductive element being in contact with the flow path; a heating element coupled to the flow path and configured to increase a temperature of at least a portion of the flow path.

19. The reference electrode of claim 18 , wherein the heating element is configured to increase the temperature of at least a portion of the flow path in response to an electrical current applied to the heating element.

20. 20. The reference electrode of claim 18 or 19, wherein the heating element comprises a metal.

21. 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; 21. The reference electrode of claim 18, comprising: a channel disposed in the substrate and 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 touching the conductive element, the first end being opposite the second end.

22. 22. The reference electrode of claim 21, wherein the heating element is disposed on a wall of the channel or a wall of the first well.

23. The flow path is 23. The reference electrode of claim 21 or 22, further comprising a second well in the substrate, the second well extending from the first surface toward the second surface, and a second end of the channel connected to the second well.

24. A reference electrode according to any one of claims 18 to 23, further comprising one or more other heating elements, the heating element and each of the one or more other heating elements being positioned at different locations along the flow path.

25. 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 flow path for a conductive medium, the flow path being disposed within the substrate; a conductive element in the substrate, the conductive element contacting the flow path; a heating element coupled to the flow path and configured to increase a temperature of at least a portion of the flow path.

26. 26. The electrochemical sensor of claim 25, wherein the heating element is configured to increase the temperature of at least a portion of the flow path in response to an electrical current applied to the heating element.

27. 27. The electrochemical sensor of claim 25 or 26, wherein the heating element comprises a metal.

28. 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; 28. The electrochemical sensor of claim 25, comprising: a channel disposed within the substrate and between the first surface and the second surface, a first end of the channel connected to the first well, the conductive element touching a second end of the channel, the first end being opposite the second end.

29. 29. The electrochemical sensor of claim 28, wherein the heating element is disposed on a wall of the channel or a wall of the first well.

30. The flow path is 30. The electrochemical sensor of claim 28 or 29, further comprising a second well in the substrate, the second well extending from the first surface toward the second surface, and the second end of the channel connected to the second well.

31. 31. The electrochemical sensor of claim 25, further comprising one or more other heating elements, the heating element and each of the one or more other heating elements being disposed at different locations along the flow path.

32. The electrochemical sensor of any one of claims 25 to 31, further comprising one or more counter electrodes.

Images