Electrochemical cell device and manufacturing method

Abstract

Electrochemical cells and methods for their production are provided. In particular, multi-well assay plates are provided that include multi-electrode wells. The multi-electrode wells contain multiple electrodes that are electrically isolated from one another, allowing different electrodes in different wells to be addressed in any suitable combination.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 485,187, filed February 15, 2023, entitled "ELECTROCHEMICAL CELL DEVICES AND METHODS OF MANUFACTURING," the entire contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION Embodiments of the present invention relate to systems, devices, and methods that use electrochemical cells in the performance of chemical, biochemical, and biological assays and analyses, and methods for their manufacture. [Background technology]

[0003] An assay is an investigative (analytical) procedure in chemistry, laboratory medicine, pharmacology, environmental biology, molecular biology, etc., to qualitatively assess or quantitatively measure the presence, amount, or functional activity of a target entity (e.g., an analyte). Assay systems may use electrochemical properties and procedures to qualitatively and quantitatively assess target entities. For example, assay systems may assess target entities by measuring the potential, current, and / or brightness in a sample region containing the target entity caused by an electrochemical process and by performing various analytical procedures (e.g., potentiometry, coulometry, voltammetry, optical analysis, etc.) on the measured data.

[0004] Assay systems utilizing electrochemical properties and procedures can include a sample region (e.g., a well, a well in a multiwell plate, etc.) with one or more electrodes (e.g., a working electrode, a counter electrode, and a reference electrode) for initiating and controlling the electrochemical process and measuring the resulting data. Depending on the design and configuration of the electrodes, assay systems can be classified as referenced and non-referenced systems. For example, the working electrode is the electrode in the assay system where the reaction of interest occurs. The working electrode is used in conjunction with a counter electrode to establish a potential difference, current, and / or electric field within the sample region. The potential difference can be divided between the interfacial potentials at the working and counter electrodes. In a non-referenced system, the interfacial potential (the force driving the reaction at the electrode) applied to the working electrode is not controlled or known. In a referenced system, the sample region includes a reference electrode that is separate from the working and counter electrodes. The reference electrode has a known potential (e.g., a reduction potential) that can be referenced during the reaction occurring in the sample region.

[0005] One example of these assay systems is the electrochemiluminescence (ECL) immunoassay. ECL immunoassays involve the use of ECL labels designed to emit light when electrochemically stimulated. Light is generated when a voltage is applied to electrodes located in the sample region holding the material under test. The voltage causes cyclic oxidation and reduction reactions, resulting in the generation and emission of light. In ECL, the electrochemical reaction responsible for ECL is driven by applying a potential difference between the working and counter electrodes.

[0006] Currently, both reference and non-reference assay systems have drawbacks in measuring and analyzing target entities. For non-reference assay systems, the unknown nature of the interfacial potential results in a lack of control in the electrochemical process, which can be further influenced by the design of the assay system. For example, in the case of an ECL immunoassay, the interfacial potential applied to the working electrode can be affected by the electrode area (working electrode and / or counter electrode), the composition of the solution, and any surface treatment of the electrode (e.g., plasma treatment). This lack of control has previously been addressed by choosing to ramp the potential difference from before the onset of ECL generation to after the end of ECL generation.

[0007] In a reference system, the potential may be known and controllable, but the addition of a reference electrode may increase the cost, complexity, size, etc. of the assay system. Furthermore, the addition of a reference electrode may limit the design and placement of the working and / or counter electrodes in the sample region due to the need to accommodate the extra electrode. Furthermore, both reference and non-reference assay systems may have slower read times due to the voltage signal required to operate the system. Reference systems may have higher costs due to the manufacturing of both the counter and reference electrodes.

[0008] A further difficulty with existing systems is the lack of flexibility regarding electrode addressability. Current systems lack the ability to address electrodes and electrode zones individually and independently of each other. This lack limits the operator's ability to design assays and experiments. Summary of the Invention [Problem to be solved by the invention]

[0009] Conventional assay systems, devices, and instruments suffer from these and other drawbacks. Therefore, there is a need for systems, devices, and methods that provide the operator with a choice between unreferenced and referenced electrode systems that mitigate the potential drawbacks of each system and highlight or optimize the potential advantages of each system. Furthermore, systems, devices, and methods that provide greater flexibility in electrode addressability are desirable. These drawbacks are addressed by the embodiments described herein. [Means for solving the problem]

[0010] Embodiments of the present disclosure include systems, devices, and methods for electrochemical cells that include auxiliary electrode designs, as well as electrochemical analyzers and devices that include electrochemical cells.

[0011] In some aspects, the technology described herein relates to a multi-well assay plate including: a top plate having top plate openings defining wells of the multi-well assay plate arranged in a well pattern, each well defined by a well area; a base plate including a substrate having a top surface and a bottom surface, the top surface mated to the top plate; and a plurality of well electrode structures, each including an electrode group patterned on the top surface and including an auxiliary electrode, an additional electrode, and a plurality of working electrodes electrically isolated from the remainder of the auxiliary electrode, the additional electrode, and the plurality of working electrodes; and an electrode contact group patterned on the bottom surface corresponding to the electrode group and including a plurality of electrode contacts, the electrode contact group including a plurality of working electrode contacts electrically connected to the corresponding working electrode, the auxiliary electrode contact electrically connected to the auxiliary electrode, and the additional electrode contact electrically connected to the additional electrode.

[0012] In some aspects, the technology described herein relates to a method of using a multi-well assay plate, the multi-well assay plate including a plurality of wells arranged in a well pattern, a plurality of well electrode structures each corresponding to a well of the plurality of wells, each including an electrode group patterned on the bottom of the well and having an auxiliary electrode, an additional electrode, and a plurality of working electrodes electrically isolated from the auxiliary electrode, the additional electrode, and the remainder of the plurality of working electrodes, the method including generating an electric potential between a selected working electrode and at least one of the selected auxiliary electrode and the selected additional electrode associated with the selected well electrode structure, maintaining substantial electrical isolation of the un-energized working electrode of the selected well electrode structure, and measuring a response to the electric potential.

[0013] In some aspects, the technology described herein relates to a method of making a multi-well assay plate, the method comprising: forming a plurality of holes in a substrate; applying a first conductive layer of material to a first side of the substrate, the first conductive layer filling the plurality of holes to form a plurality of vias; applying a second conductive layer of material to the first side of the substrate, the second conductive layer covering the first conductive layer to form a plurality of electrode contacts; applying a third conductive layer of material to a second side of the substrate, the third conductive layer forming a plurality of electrical traces, the plurality of electrical traces connecting first portions of the plurality of vias to a plurality of working electrodes, forming a plurality of additional electrode contact spots and a plurality of auxiliary electrode contact spots; and applying a fourth conductive layer of material to the second side of the substrate. applying a fifth conductive layer of material to the second side of the substrate covering the third conductive layer, applying a sixth conductive layer of material to the second side of the substrate, the sixth conductive layer forming the plurality of working electrodes, applying an insulating layer of material to the second side of the substrate, the insulating layer exposing the plurality of auxiliary electrodes, the plurality of additional electrodes, and the plurality of working electrodes and insulating the remainder of the second side of the substrate, and adhering the substrate to a top plate having top plate openings defining wells of a multi-well assay plate arranged in a well pattern, each well defined by a well area.

[0014] In some aspects, the technology described herein relates to a multi-well assay plate including: a top plate having top plate openings defining wells of the multi-well assay plate arranged in a well pattern, each well defined by a well area; a base plate including a substrate having a top surface and a bottom surface, the top surface mated to the top plate; and a plurality of well electrode structures, each including an electrode group patterned on the top surface and having an auxiliary electrode and a plurality of working electrodes electrically isolated from the auxiliary electrode and the remainder of the plurality of working electrodes; and an electrode contact group patterned on the bottom surface corresponding to the electrode group and including a plurality of working electrode contacts electrically connected to the corresponding working electrode and the auxiliary electrode contacts electrically connected to the auxiliary electrode by auxiliary electrical traces, wherein an insulating layer is provided between the auxiliary electrical traces and the plurality of working electrodes.

[0015] In some aspects, the technology described herein relates to an electrochemical cell for performing electrochemical analyses, the electrochemical cell including a plurality of working electrode zones disposed on a surface of the cell and defining a pattern, at least one auxiliary electrode disposed on the surface, and at least one additional electrode disposed on the surface, wherein each of the plurality of working electrode zones is electrically isolated from each other and from the auxiliary electrode and the additional electrode.

[0016] In some aspects, the technology described herein relates to a multi-well assay plate comprising: a top plate having top plate openings defining wells of the multi-well assay plate arranged in a well pattern, each well defined by a well area; a base plate comprising a substrate having a top surface and a bottom surface, the top surface mated to the top plate; and a plurality of well electrode structures, each well electrode structure including an electrode group patterned on the top surface and having at least one auxiliary electrode and at least one working electrode, and a plurality of working electrode contacts electrically connected to the working electrode and auxiliary electrode contacts electrically connected to the auxiliary electrode.

[0017] In some aspects, the technology described herein includes a multi-well assay plate, the top plate having top plate openings defining wells of the multi-well assay plate arranged in a well pattern, each well defined by a well area; a base plate including a substrate having a top surface and a bottom surface, the top surface mated to the top plate; a plurality of well electrode structures arranged on the top surface; and a plurality of zigzag auxiliary electrode contacts arranged in an auxiliary electrode contact array on the bottom surface, each zigzag auxiliary electrode contact array. a plurality of zigzag auxiliary electrode contacts, each having a top horizontal portion extending from a first end of the central longitudinal portion in a first horizontal direction and a bottom horizontal portion extending from a second end of the central longitudinal portion in a second horizontal direction different from the first direction, each zigzag auxiliary electrode contact corresponding to a well electrode structure; and a plurality of working electrode contacts arranged in an interrupted working electrode array on the top surface, the interrupted working electrode array having top horizontal portions of the zigzag auxiliary electrode contacts in a first auxiliary electrode contact array spaced apart from the bottom horizontal portions of the zigzag auxiliary electrode contacts in a second auxiliary electrode contact array. a plurality of auxiliary electrode bases arranged in a row on the top surface at positions corresponding to the bottom surface positions of the top horizontal portion and the bottom horizontal portion; a plurality of working electrode bases arranged in a row on the top surface at a position corresponding to the bottom surface position of the central vertical portion, the plurality of working electrode bases arranged in a row on the top surface at a position corresponding to the bottom surface position of the central vertical portion, each of the plurality of working electrode bases having a central portion configured to form one or more electrodes, a top extension portion extending away from the central portion in a second horizontal direction, and a bottom extension portion extending away from the central portion in a first horizontal direction; a plurality of vias providing conductive through-holes from the bottom surface to the top surface, the plurality of vias being arranged in rows corresponding to the interrupted working electrode rows, each via connecting the top horizontal portion to a corresponding auxiliary electrode base, the bottom horizontal portion to a corresponding auxiliary electrode base, the working electrode contact to the corresponding top extension portion, and the working electrode contact to the corresponding bottom extension portion; and an insulating layer disposed on the top surface and configured to define at least one auxiliary electrode and at least one working electrode for each well electrode structure.

[0018] In some aspects, the technology described herein includes a multi-well assay plate, the top plate having top plate openings defining wells of the multi-well assay plate arranged in a well pattern, each well defined by a well area; a base plate including a substrate having a top surface and a bottom surface, the top surface mated to the top plate; a plurality of well electrode structures arranged on the top surface; and a plurality of zigzag auxiliary electrode contacts arranged in an auxiliary electrode contact array on the bottom surface, each zigzag auxiliary electrode contact array. a plurality of zigzag auxiliary electrode contacts, each having a top horizontal portion extending from a first end of the central longitudinal portion in a first horizontal direction and a bottom horizontal portion extending from a second end of the central longitudinal portion in a second horizontal direction different from the first direction, each zigzag auxiliary electrode contact corresponding to a well electrode structure; and a plurality of working electrode contacts arranged in an interrupted working electrode array on the top surface, the interrupted working electrode array having top horizontal portions of the zigzag auxiliary electrode contacts in a first auxiliary electrode contact array spaced apart from the bottom horizontal portions of the zigzag auxiliary electrode contacts in a second auxiliary electrode contact array. a plurality of auxiliary electrode bases arranged in a row on the top surface at positions corresponding to the bottom surface positions of the top horizontal portion and the bottom horizontal portion; a plurality of working electrode bases arranged in a row on the top surface at a position corresponding to the bottom surface position of the central vertical portion, the plurality of working electrode bases arranged in a row on the top surface at a position corresponding to the bottom surface position of the central vertical portion, each of the plurality of working electrode bases having a central portion configured to form one or more electrodes, a top extension portion extending away from the central portion in a second horizontal direction, and a bottom extension portion extending away from the central portion in a first horizontal direction; a plurality of vias providing conductive through-holes from the bottom surface to the top surface, the plurality of vias being arranged in rows corresponding to the interrupted working electrode rows, each via connecting the top horizontal portion to a corresponding auxiliary electrode base, the bottom horizontal portion to a corresponding auxiliary electrode base, the working electrode contact to the corresponding top extension portion, and the working electrode contact to the corresponding bottom extension portion; and an insulating layer disposed on the top surface and configured to define at least one auxiliary electrode and at least one working electrode for each well electrode structure.

[0019] The foregoing and other features and advantages of the present invention will be apparent from the following description of embodiments herein, as illustrated in the accompanying drawings. The accompanying drawings, which are incorporated in and form a part of this specification, further serve to explain the principles of the various embodiments described herein and to enable those skilled in the art to make and use the various embodiments described herein. The drawings are not necessarily drawn to scale. [Brief explanation of the drawings]

[0020] [Figures 1A-1C] 1A-1D show several views of an electrochemical cell according to embodiments disclosed herein. [Figure 2A] 1 shows a top view of a multi-well plate including multiple sample regions according to embodiments disclosed herein. [Figure 2B] 1 shows a multi-well plate for use in an assay device containing multiple sample regions according to embodiments disclosed herein. [Figure 2C] 1D shows a side view of the sample area of ​​the multi-well plate of FIG. 1C according to embodiments disclosed herein. [Figures 3A-3F] 1A-1C or the multiwell plate of FIGS. 2A-2C, according to embodiments disclosed herein. [Figures 4A-4F] 1A-1C or the multiwell plate of FIGS. 2A-2C, according to embodiments disclosed herein. [Figures 5A-5C] 1A-1C or the multiwell plate of FIGS. 2A-2C, according to embodiments disclosed herein. [Figures 6A-6F] 1A-1C or the multiwell plate of FIGS. 2A-2C, according to embodiments disclosed herein. [Figures 7A-7F]1A-1C or the multiwell plate of FIGS. 2A-2C, according to embodiments disclosed herein. [Figures 8A-8D] 1A-1C or the multiwell plate of FIGS. 2A-2C, according to embodiments disclosed herein. [Figure 9A-9B] 1 illustrates an example of an assay device according to embodiments disclosed herein. [Figures 10A-10B] 10 illustrates the decay time of an auxiliary electrode according to an embodiment. [Figure 11] 1 illustrates a process for conducting electrochemical analyses and procedures using pulse waveforms according to embodiments disclosed herein. [Figures 12A-12B] 1 illustrates an example of a pulse waveform according to embodiments disclosed herein. [Figure 13] 1 illustrates a process for performing ECL analysis and procedures using pulse waveforms according to embodiments disclosed herein. [Figures 14A-14C] 1 shows ECL test results performed using pulse waveforms according to embodiments disclosed herein. [Figures 15A-15L] 1 shows ECL test results performed using pulse waveforms according to embodiments disclosed herein. [Figure 16] 1 shows ECL test results performed using pulse waveforms according to embodiments disclosed herein. [Figure 17] 1 shows ECL test results performed using pulse waveforms according to embodiments disclosed herein. [Figure 18] 1 illustrates a process for performing an ECL analysis using a pulse waveform according to embodiments disclosed herein. [Figure 19] 1 illustrates a process for performing an ECL analysis using a pulse waveform according to embodiments disclosed herein. [Figure 20] 1 illustrates a process for fabricating a well according to an embodiment disclosed herein. [Figures 21A-21F]1 illustrates exemplary stages in a process for fabricating a well according to embodiments disclosed herein. [Figure 22A] 1 illustrates exemplary stages in a process for fabricating a well according to embodiments disclosed herein. [Figure 22B] 1 shows an embodiment of a well according to the present disclosure. [Figures 23A-23D] 10A-10C show some examples of electrode configurations that have been tested according to embodiments disclosed herein. [Figures 24A-24C] 1 shows test results performed on various multi-well plates according to embodiments disclosed herein. [Figures 25A-25C] 1 shows test results performed on various multi-well plates according to embodiments disclosed herein. [Figures 26A-26D] 1 shows test results performed on various multi-well plates according to embodiments disclosed herein. [Figures 27A-27C] 1 shows test results performed on various multi-well plates according to embodiments disclosed herein. [Figure 28] 1 shows test results performed on various multi-well plates according to embodiments disclosed herein. [Figure 29] 10 illustrates tests performed to optimize waveforms for coating plasma-treated versus standard electrodes according to embodiments disclosed herein. [Figure 30] 10 illustrates tests performed to optimize waveforms for coating plasma-treated versus standard electrodes according to embodiments disclosed herein. [Figure 31A-31B] 10 illustrates tests performed to optimize waveforms for coating plasma-treated versus standard electrodes according to embodiments disclosed herein. [Figure 32A-32B] 10 illustrates tests performed to optimize waveforms for coating plasma-treated versus standard electrodes according to embodiments disclosed herein. [Figure 33A-33B]10 illustrates tests performed to optimize waveforms for coating plasma-treated versus standard electrodes according to embodiments disclosed herein. [Figure 34A-34B] 10 illustrates tests performed to optimize waveforms for coating plasma-treated versus standard electrodes according to embodiments disclosed herein. [Figure 35] 10 illustrates tests performed to optimize waveforms for coating plasma-treated versus standard electrodes according to embodiments disclosed herein. [Figures 36A-36D] 10 illustrates tests performed to optimize waveforms for coating plasma-treated versus standard electrodes according to embodiments disclosed herein. [Figure 37] 1 illustrates an electrochemical cell having individually addressable electrodes according to embodiments disclosed herein. [Figures 38A-38C] 1 illustrates a portion of a multi-well plate having wells containing individually addressable electrode electrochemical cells according to embodiments disclosed herein. [Figures 39A-39L] 1A-1C illustrate aspects of the construction of an individually addressable electrode electrochemical cell according to embodiments disclosed herein. [Figures 40A-40N] 1 illustrates aspects of a substrate configuration including multiple individually addressable electrode electrochemical cells according to embodiments disclosed herein. [Figures 41A-41M] 1A-1C show different views of a substrate including multiple individually addressable electrode electrochemical cells according to embodiments disclosed herein. [Figures 42A-42I] 1 illustrates features of an orientation-neutral electrode contact pattern according to embodiments disclosed herein. [Figures 43A-43D] 1 illustrates features of an orientation-neutral electrode contact pattern according to embodiments disclosed herein. [Figures 44A-44C] 1 illustrates aspects of an electrical connector according to embodiments disclosed herein. [Figures 45A-45F] 1 illustrates aspects of an electrical connector according to embodiments disclosed herein. [Figures 46A-46E] 1 illustrates features of a flexible connector according to an embodiment of the present invention. [Figures 47A-47H] 1 shows an embodiment of a multi-well assay plate including an auxiliary electrode and a reference electrode. [Figures 48A-48F] 1 illustrates aspects of a substrate configuration consistent with a three-electrode well electrode structure according to embodiments herein. [Figure 49A-49B] 1 illustrates a well electrode structure employing a crossover insulating structure according to an embodiment herein. [Figures 50A-50J] 1 illustrates aspects of a substrate configuration including multiple working electrode structures employing a crossover insulating structure according to embodiments disclosed herein. [Figures 51A-51C] 10 illustrates an alternative electrode arrangement consistent with embodiments herein. [Figure 52A-52B] Each shows the top surface of a substrate configured to accommodate a non-isolated working electrode well structure. [Fig. 52D-52O] 1 illustrates aspects of a substrate configured to accommodate a non-isolated working electrode well structure, according to embodiments herein. [Figure 53] 52A-52L show contact connection patterns suitable for multi-well assay plates consistent with FIGS. 52A-52L. [Fig. 54A-54J] 1 illustrates aspects of the structure of a substrate consistent with a three-electrode well electrode structure with crossover insulating layers consistent with embodiments herein. [Figures 55A-55J] 1 illustrates aspects of the structure of a substrate consistent with a three-electrode well electrode structure with crossover insulating layers consistent with embodiments herein. [Figures 56A-56K] 1 shows a three-electrode well structure consistent with embodiments herein. DETAILED DESCRIPTION OF THE INVENTION

[0021] Specific embodiments of the present invention are described below with reference to the figures. The following detailed description is merely exemplary in nature and is not intended to limit the invention, its application, and uses. Furthermore, there is no intention to be bound by any expressed or implied theory presented in the preceding technical field, background, brief summary, or the following detailed description.

[0022] Embodiments of the present disclosure relate to electrochemical cells including auxiliary electrode designs, as well as electrochemical analytical apparatus and devices including electrochemical cells. In embodiments, the auxiliary electrode is designed to include a redox couple (e.g., Ag-AgCl) that provides a stable interfacial potential. While specific electrode materials and compositions may be mentioned in various places throughout this disclosure, the disclosure is not so limited, and any suitable electrode material or composition may be employed. In certain embodiments, the redox couple may be generated by doping materials, compounds, etc., although other methods of generating the redox couple are contemplated as well. Auxiliary electrodes with reduction-oxidation couples that define a stable interfacial potential allow the auxiliary electrodes to function as dual-function electrodes. That is, one or more auxiliary electrodes simultaneously operate as counter and reference electrodes. Because the auxiliary electrodes operate as dual-function electrodes, the space occupied by the auxiliary electrodes within the electrochemical cell is reduced, thereby enabling additional configurations and numbers of working electrode zones to be included within the electrochemical cell.

[0023] In embodiments, the use of one or more auxiliary electrodes also improves the read time of electrochemical analyzers and devices during electrochemical analytical processes, such as ECL processes. While conventional non-reference ECL systems typically use a slow voltage ramp through the voltage that provides maximum ECL to provide tolerance to fluctuations in the potential at the auxiliary electrode, the use of an auxiliary electrode of the present invention, such as an auxiliary electrode comprising a redox couple, provides improved control over this potential, allowing the use of more efficient and faster waveforms, such as short voltage pulses or fast voltage ramps.

[0024] FIG. 1A illustrates an example of an electrochemical cell 100 according to one embodiment of the present invention. As shown in FIG. 1A, the electrochemical cell 100 defines a working space 101 in which electrical energy is utilized to drive one or more chemical reactions. Within the working space (or sample region) 101, the electrochemical cell 100 may include one or more auxiliary electrodes 102 and one or more working electrode zones 104. The auxiliary electrodes 102 and the working electrode zones 104 may be in contact with an ionic medium 103. The electrochemical cell 100 may operate via a reduction-oxidation (redox) reaction triggered by the introduction of electrical energy through the auxiliary electrodes 102 and the working electrode zones 104. In some embodiments, the ionic medium 103 may include an electrolyte solution, such as water or another solvent, in which ions, such as salts, are dissolved. In some embodiments, the ionic medium 103 or the surface of the working electrode 102 may include luminescent species that generate and emit photons during the redox reaction, as described in more detail below. During operation of the electrochemical cell 100, an external voltage may be applied to one or more of the auxiliary electrode 102 and the working electrode zone 104 to cause a redox reaction to occur at these electrodes.

[0025] As described herein, in use, the auxiliary electrode has an electrode potential that can be defined by a redox reaction occurring at the electrode. The potential can be defined, according to certain non-limiting embodiments, by (i) a reduction-oxidation (redox) couple confined to the surface of the electrode or (ii) a reduction-oxidation (redox) couple in solution. As described herein, a redox couple includes a pair of elements, chemicals, or compounds that interconvert via a redox reaction, e.g., one element, chemical, or compound that is an electron donor and one element, chemical, or compound that is an electron acceptor. An auxiliary electrode with a reduction-oxidation couple that defines a stable interfacial potential can function as a dual-function electrode. That is, one or more auxiliary electrodes 102 can provide functionality associated with both a counter electrode and a reference electrode in a three-electrode electrochemical system by providing high current flow (the function of a counter electrode in a three-electrode system) while providing the ability to define and control the potential at the working electrode (the function of a reference electrode in a three-electrode system). The one or more auxiliary electrodes 102 can act as counter electrodes by providing a potential difference with one or more of the one or more working electrode zones 104 during redox reactions occurring within the electrochemical cell 100 in which the one or more auxiliary electrodes 102 are disposed. Depending on the chemical structure and composition of the one or more auxiliary electrodes 102, the one or more auxiliary electrodes 102 may also act as reference electrodes for determining a potential difference with one or more of the working electrode zones 104.

[0026] In embodiments, the auxiliary electrode 102 may be formed from a chemical mixture of elements and alloys having a chemical composition that allows the auxiliary electrode 102 to function as a reference electrode. The chemical mixture (e.g., the ratio of elements and alloys in the chemical composition of the auxiliary electrode) can provide a stable interfacial potential during the reduction or oxidation of the chemical mixture, generating a quantifiable amount of charge throughout the reduction-oxidation reactions occurring within the electrochemical cell 100. While certain reactions described herein may be referred to as reduction or oxidation reactions, it is understood that the electrodes described herein can support both reduction and oxidation reactions, depending on the applied voltage. The specific reference to reduction or oxidation reactions does not limit the functionality of the electrode to a particular type of reaction. In some embodiments, the chemical mixture of one or more auxiliary electrodes 102 may include an oxidizer that provides a stable interfacial potential during the reduction of the chemical mixture, and the amount of oxidizer in the chemical mixture may be equal to or greater than the amount of oxidizer required to provide the entire reduction-oxidation reaction within the electrochemical cell that occurs during the electrochemical reaction. In embodiments, the auxiliary electrode 102 is formed from a chemical mixture that provides an interfacial potential during reduction of the chemical mixture, resulting in the generation of a quantifiable amount of charge throughout the reduction-oxidation reactions occurring within the electrochemical cell 100. The chemical mixture of the auxiliary electrode 102 includes an oxidizing agent that supports redox reactions during operation of the electrochemical cell 100, for example, during biological, chemical, and / or biochemical assays and / or analyses, such as ECL generation and analysis.

[0027] In one embodiment, the amount of oxidant in the chemical mixture of the one or more auxiliary electrodes 102 is equal to or greater than the amount of oxidant required for all of the redox reactions that occur within the electrochemical cell 100 during one or more biological, chemical, and / or biochemical assays and / or analyses, such as, for example, ECL generation. For example, a sufficient amount of the chemical mixture in the one or more auxiliary electrodes 102 remains after the redox reactions have occurred for an initial biological, chemical, and / or biochemical assay and / or analysis, thereby allowing one or more additional redox reactions to occur throughout a subsequent biological, chemical, and / or biochemical assay and / or analysis.

[0028] In some embodiments, the amount of oxidant in the chemical mixture of the one or more auxiliary electrodes 102 is based, at least in part, on the ratio of the exposed surface area (also referred to as area surface area) of each of the one or more working electrode zones 104 to the exposed surface area of ​​the one or more auxiliary electrodes 102. As described herein, the exposed surface area (also referred to as area surface area) of the one or more auxiliary electrodes 102 refers to the two-dimensional (2D) cross-sectional area of ​​the one or more auxiliary electrodes 102 exposed to the ionic medium 103. That is, as shown in FIG. 1B , the auxiliary electrodes 102 may be formed in a three-dimensional (3D) shape extending in the Z direction from the bottom surface of the electrochemical cell 100. The exposed surface area of ​​the auxiliary electrode 102 may correspond to a 2D cross-sectional area taken in the XY plane. In embodiments, the 2D cross-sectional area may be taken at any point of the auxiliary electrode 102, for example, at the interface with the bottom surface 120. 1B depicts the auxiliary electrode 102 as a regularly shaped cylinder, the auxiliary electrode 102 can have any shape, whether regular or irregular. Similarly, the exposed surface area of ​​one or more working electrode zones 104 refers to the 2D cross-sectional area of ​​the one or more auxiliary electrode zones 104 exposed to the ionic medium 103, similar to the 2D cross-sectional area of ​​the auxiliary electrode 102 depicted in FIG. 1B, for example. In certain embodiments, the areal surface area (exposed surface area) can be distinguished from the true surface area, which includes the actual surface of the electrode and accounts for any height or depth in the z-dimension. Using these examples, the areal surface area is less than or equal to the true surface area.

[0029] In embodiments, one or more auxiliary electrodes 102 may be formed from a chemical mixture including a redox couple that provides an interfacial potential at or near the standard reduction potential of the redox couple. In some embodiments, one or more auxiliary electrodes 102 may include a mixture of silver (Ag) and silver chloride (AgCl), or other suitable metal / metal halide couple. In some embodiments, one or more auxiliary electrodes 102 formed from an Ag-AgCl mixture may provide an interfacial potential at or near the standard reduction potential of Ag-AgCl, approximately 0.22 V. Other examples of chemical mixtures may include metal oxides with multiple metal oxidation states, such as manganese oxide, or other metal / metal oxide couples, such as silver / silver oxide, nickel / nickel oxide, zinc / zinc oxide, gold / gold oxide, copper / copper oxide, platinum / platinum oxide, etc. In some embodiments, the chemical mixture may provide an interfacial potential in the range of approximately 0.1 V to approximately 3.0 V. Table 1 lists example reduction potentials of redox couples in chemical mixtures that may be included in one or more auxiliary electrodes 102. Those skilled in the art will understand that the example reduction potentials are approximations and may vary, for example, by + / - 5.0%, based on chemical composition, temperature, impurities in the chemical mixture, or other conditions.

[0030] [Table 1]

[0031] In embodiments, the chemical mixture of the redox couple at the one or more auxiliary electrodes can be based on a molar ratio of the redox couple that falls within a particular range. In some embodiments, the chemical mixture has a molar ratio of Ag to AgCl within the particular range, for example, greater than or equal to about 1. In some embodiments, the one or more auxiliary electrodes 102 may maintain a controlled interfacial potential until all of the one or more chemical moieties participating in the redox reaction are oxidized or reduced.

[0032] In some embodiments, the one or more auxiliary electrodes 102 are approximately 1.56×10 -5 ~5.30×10 -4 C / mm 2In some embodiments, the one or more auxiliary electrodes 102 may include a redox couple that passes a current of about 0.5 mA to 4.0 mA throughout the redox reaction of the redox couple to generate ECL in the range of about 1.4 V to 2.6 V. In some embodiments, the one or more auxiliary electrodes 102 may include a redox couple that passes an average current of about 2.39 mA throughout the redox reaction to generate ECL in the range of about 1.4 V to 2.6 V.

[0033] In embodiments, the one or more auxiliary electrodes 102 may be such that the amount of oxidant in the redox couple is equal to or greater than the amount of charge required to pass through the auxiliary electrode to complete the electrochemical analysis. In some embodiments, the one or more auxiliary electrodes 102 may be equal to or greater than about 3.07×10 -7 ~3.97×10 -7 In some embodiments, the one or more auxiliary electrodes 102 may comprise an exposed surface area of ​​mm 2 Approximately 1.80 x 10 -7 ~2.32×10 -7 moles of oxidant (1.16 x 10 -4 ~1.5×10 -4 moles / in 2 In some embodiments, the one or more auxiliary electrodes 102 may comprise less than mm of the total (or aggregate) exposed surface area of ​​the one or more working electrode zones 104. 2 At least about 3.7 x 10 per -9 moles of oxidant (2.39 x 10 -6 moles / in 2 In some embodiments, one or more auxiliary electrodes may comprise less than mm of the total (or aggregate) exposed surface area of ​​one or more working electrode zones 104. 2 At least about 5.7 x 10 per -9 moles (3.69 x 10 -6 moles / in 2 ) oxidizing agent.

[0034] In embodiments, one or more auxiliary electrodes 102 may include a redox couple, and when a voltage or potential is applied, the reaction of the species in the redox couple is the predominant redox reaction occurring at the one or more auxiliary electrodes 102. In some embodiments, the applied potential is less than the defined potential required to reduce water or perform water electrolysis. In some embodiments, less than 1 percent of the current is associated with water reduction. In some embodiments, less than 1 current per unit area (exposed surface area) of the one or more auxiliary electrodes 102 is associated with water reduction.

[0035] In embodiments, the one or more auxiliary electrodes 102 (and one or more working electrode zones 104) may be formed using any type of manufacturing process, e.g., printing, vapor deposition, lithography, etching, etc. In embodiments, the form of the metal / metal halide chemical mixture may depend on the manufacturing process. For example, if the one or more auxiliary electrodes 102 (and one or more working electrode zones 104) are printed, the chemical mixture may be in the form of an ink or paste. In some embodiments, one or more additional substances may be added to the one or more auxiliary electrodes 102 and / or one or more working electrode zones 104 using a doping process.

[0036] A working electrode zone 104 may be a location on an electrode where a reaction of interest can occur. The reaction of interest may be chemical, biological, biochemical, or electrical in nature (or any combination of two or more of these types of reactions). As described herein, an electrode (auxiliary electrode and / or working electrode) may be a continuous / adjacent area where a reaction can occur, and an electrode "zone" may be a portion (or the entirety) of an electrode where a particular reaction of interest occurs. In some embodiments, the working electrode zone 104 may include the entire electrode, while in other embodiments, two or more working electrode zones 104 may be formed within and / or on a single electrode. For example, the working electrode zone 104 may be formed by an individual working electrode. In this example, the working electrode zone 104 may be configured as a single electrode formed from one or more conductive materials. In another example, the working electrode zone 104 may be formed by isolating a portion of a single working electrode. In this example, the single working electrode may be formed from one or more conductive materials, and the working electrode zones may be formed by electrically isolating regions ("zones") of the single working electrode using insulating materials, such as dielectrics, to create electrically isolated working electrode zones. In any embodiment, the working electrode zones 104 may be formed from any type of conductive material, such as metals, metal alloys, carbon compounds, doped metals, etc., and combinations of conductive and insulating materials.

[0037] In embodiments, the working electrode zone 104 may be formed from a conductive material. For example, the working electrode zone 104 may include a metal such as gold, silver, platinum, nickel, steel, iridium, copper, aluminum, or a conductive alloy. In some embodiments, the working electrode zone 104 may include an oxide-coated metal (e.g., aluminum oxide-coated aluminum). In some embodiments, the working electrode zone 104 may be formed from a carbon-based material such as carbon, carbon black, graphitic carbon, carbon nanotubes, carbon fibrils, graphite, carbon fibers, and mixtures thereof. In some embodiments, the working electrode zone 104 may be formed from a conductive carbon-polymer composite, conductive particles dispersed in a matrix (e.g., carbon ink, carbon paste, metal ink), and / or a conductive polymer. In some embodiments, as disclosed in further detail below, the working electrode zone 104 may be formed from carbon and silver layers fabricated using screen printing of carbon and silver inks. In some embodiments, the working electrode zone 104 may be formed from a semiconductor material (eg, silicon, germanium) or a semiconductor film such as indium tin oxide (ITO), antimony tin oxide (ATO), or the like.

[0038] In embodiments, as described in further detail below, the one or more auxiliary electrodes 102 and one or more working electrode zones 104 may be formed with different electrode designs (e.g., different sizes and / or shapes, different numbers of auxiliary electrodes 102 and working electrode zones 104, different positioning and patterns within the electrochemical cell 100, etc.) to improve the electrochemical properties and analyses (e.g., ECL analyses) performed by apparatuses and devices including the electrochemical cell. FIG. 1C shows an example of an electrode design 150 for an electrochemical cell 100 including multiple working electrode zones. As shown in FIG. 1C, the electrochemical cell 100 can include ten working electrode zones 104 and a single auxiliary electrode 102. Various other examples of electrode designs are discussed below with reference to FIGS. 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, and 8A-8D.

[0039] In embodiments, the configuration and arrangement of working electrode zones 104 within the electrochemical cell 100 may be defined according to the adjacency between the working electrode zones 104 and / or the adjacency between the working electrode zones 104 and one or more auxiliary electrodes 102. In some embodiments, adjacency may be defined as the relative number of adjacent working electrode zones 104 and / or one or more auxiliary electrodes 102. In some embodiments, adjacency may be defined as the relative distance between adjacent working electrode zones 104 and / or one or more auxiliary electrodes 102. In some embodiments, adjacency may be defined as the relative distance from the working electrode zones 104 and / or one or more auxiliary electrodes 102 to other features of the electrochemical cell 100, such as the perimeter of the electrochemical cell.

[0040] In embodiments herein, for example, the one or more auxiliary electrodes 102 and one or more working electrode zones 104 of each electrochemical cell 100 may be formed to have respective sizes such that the ratio of the collective exposed surface area of ​​the one or more working electrode zones 104 to the exposed surface area of ​​the one or more auxiliary electrodes 102 is greater than 1, although other ratios are also contemplated for electrochemical cells 100 (e.g., ratios less than or equal to 1 or greater than 1). In some embodiments herein, for example, each of the one or more auxiliary electrodes 102 and / or one or more working electrode zones 104 may be formed in a circular shape having a surface area that substantially defines a circle, but may also be in other shapes (e.g., rectangular, square, oval, cloverleaf, or any other regular or irregular geometric shape).

[0041] In embodiments herein, for example, one or more auxiliary electrodes 102 and / or one or more working electrode zones 104 may be formed in a wedge shape having a wedge-shaped surface area, also referred to herein as a trefoil shape. That is, one or more auxiliary electrodes 102 and / or one or more working electrode zones 104 may be formed with two opposing boundaries having different dimensions and two side boundaries connecting the two opposing boundaries. For example, the two opposing boundaries may include a wide boundary and a narrow boundary, where the wide boundary has a longer length than the narrow boundary. In some embodiments, the wide boundary and / or the narrow boundary may have an obtuse angle, e.g., a rounded corner, at the connection to the side boundary. In some embodiments, the wide boundary and / or the narrow boundary may have an acute angle, e.g., a square corner, at the connection to the side boundary. In embodiments, the wedge shapes described herein may be generally trapezoidal with rounded or square corners. In embodiments, the wedge shapes described herein may be generally triangular with flattened or rounded apexes and rounded or angular corners. In embodiments, the wedge shape may be utilized to maximize the available area at the bottom surface 120 of the electrochemical cell. For example, if the working area 101 of the electrochemical cell is circular, one or more working electrode zones 104 having a wedge shape may be positioned such that a wide boundary is adjacent the periphery of the working area 101 and a narrow boundary is adjacent the center of the working area 101.

[0042] In embodiments, electrochemical cell 100 can be included in an apparatus or device for performing electrochemical analyses. In some embodiments, electrochemical cell 100 can form part of a well for an assay device that performs electrochemical analyses, such as ECL immunoassays, as described below. In some embodiments, electrochemical cell 100 can form a flow cell within a cartridge used in an analytical device or instrument, such as an ECL cartridge (e.g., such as those provided in U.S. Pat. Nos. 10,184,884 and 10,935,547), a flow cytometer, etc. Those skilled in the art will understand that electrochemical cell 100 can be utilized in any type of instrument or device in which controlled redox reactions occur.

[0043] 2A-2C show several views of a sample area ("well") 200 containing an electrochemical cell (e.g., electrochemical cell 100) including an auxiliary electrode design for use in an assay device for biological, chemical, and / or biochemical analysis according to one embodiment of the present disclosure. Those skilled in the art will appreciate that FIGS. 2A-2C show one example of a well in an assay device, and that existing components shown in FIGS. 2A-2C may be removed and / or additional components may be added without departing from the scope of the embodiments described herein.

[0044] As shown in the top view of FIG. 2A, a base plate 206 of a multiwell plate 208 (shown in FIG. 2B) can include a plurality of wells 200. The base plate 206 can include a surface that forms the bottom of each well 200 and can include one or more auxiliary electrodes 102 and one or more working electrode zones 104 disposed on and / or within the surface of the base plate 206 of the multiwell plate 208. As shown in the perspective view of FIG. 2B, the multiwell plate 208 can include a top plate 210 and a base plate 206. The top plate 210 can define wells 200 extending from an upper surface of the top plate 210 to the base plate 206, which forms the bottom surface 207 of each well 200. In operation, light is generated when a voltage is applied between one or more working electrode zones 104 and one or more auxiliary electrodes 102 disposed within the wells 200 that hold the material under test. The applied voltage causes cyclical oxidation and reduction reactions, resulting in the production and emission of photons (light). The emitted photons can then be measured to analyze the material under test.

[0045] Depending on whether the reaction occurring at the working electrode zone 104 accepts or supplies electrons, the reaction at the working electrode zone 104 is either reduction or oxidation, respectively. In embodiments, the working electrode zone 104 may be derivatized or modified to immobilize assay reagents, such as binding reagents, on the electrode. For example, the working electrode zone 104 may be modified to attach antibodies, antibody fragments, proteins, enzymes, enzyme substrates, inhibitors, cofactors, antigens, haptens, lipoproteins, liposaccharides, bacteria, cells, intracellular components, cellular receptors, viruses, nucleic acids, antigens, lipids, glycoproteins, carbohydrates, peptides, amino acids, hormones, protein-binding ligands, drugs, and / or combinations thereof. Similarly, for example, the working electrode zone 104 may be modified to attach non-biological entities, such as, but not limited to, polymers, elastomers, gels, coatings, ECL tags, redox-active species (e.g., tripropylamine, oxalate), inorganic materials, chemical functional groups, chelators, linkers, etc. Reagents may be immobilized on one or more working electrode zones 104 by a variety of methods, including passive adsorption, specific binding, and / or forming covalent bonds to functional groups present on the surface of the electrode.

[0046] For example, ECL species can be attached to the working electrode zone 104 and induced to emit ECL for analytical measurements to determine the presence of a substance of interest in the fluid in the well 200. For example, species that can be induced to emit ECL (ECL-active species) have been used as ECL labels. Examples of ECL labels include: i) organometallic compounds in which the metal is derived from a noble metal that is resistant to corrosion and oxidation, including Ru- and Os-containing organometallic compounds such as the tris-bipyridyl-ruthenium (RuBpy) moiety; and ii) luminol and related compounds. Species that participate with the ECL label in the ECL process are referred to herein as ECL coreactants. Commonly used coreactants include tertiary amines such as triisopropylamine (TPA), oxalate, and persulfate for RuBpy-derived ECL, and hydrogen peroxide for luminol-derived ECL. The light generated by the ECL label can be used as a reporter signal in diagnostic procedures. For example, an ECL label can be covalently attached to a binding agent such as an antibody or nucleic acid probe, and the participation of the binding agent in the binding interaction can be monitored by measuring the ECL emitted from the ECL label. Alternatively, the ECL signal from an ECL-active compound can be indicative of its chemical environment.

[0047] In embodiments, the working electrode zone 104 and / or auxiliary electrode 102 (or other components of the well 200) may also be treated (e.g., pretreated) with materials and / or processes that improve the adhesion (e.g., absorption) of materials used in the electrochemical process (e.g., reagents, ECL species, labels, etc.) to the surface of the working electrode zone 104 and / or auxiliary electrode. In some embodiments, the working electrode zone 104 and / or auxiliary electrode 102 (or other components of the well 200) may be treated using a process (e.g., plasma treatment) that causes the surface of the working electrode zone 104 and / or auxiliary electrode 102 (or other components of the well 200) to exhibit hydrophilic properties (also referred to herein as "high binding" or "HB"). In some embodiments, the working electrode zone 104 and / or auxiliary electrode 102 (or other components of the well 200) may or may not be treated using a process that causes the surface of the working electrode zone 104 and / or auxiliary electrode 102 (or other components of the well 200) to exhibit hydrophobic properties (also referred to herein as "standard" or "Std").

[0048] As shown in FIG. 2C, which is a side cross-sectional view of a portion of the multiwell plate 208 of FIG. 2B, multiple wells 200 may be included on the multiwell plate 208, three of which are shown in FIG. 2C. Each well 200 may be formed by a top plate 210 including one or more sidewalls 212 that form the boundaries of the electrochemical cell 100. The one or more sidewalls 212 extend from the bottom surface of the top plate 210 to the top surface of the top plate 210. The wells 200 may be adapted to hold one or more fluids 250, such as ionic media as described above. In certain embodiments, one or more wells 200 may be adapted to hold gases and / or solids instead of, or in addition to, the one or more fluids 250. In embodiments, the top plate 210 may be secured to the base plate 206 using an adhesive 214 or other connecting material or device.

[0049] The multiwell plate 208 may include any number of wells 200. For example, as shown in FIGS. 2A and 2B, the multiwell plate 208 may include 96 wells 200. Those skilled in the art will appreciate that the multiwell plate 208 may include any number of wells 200, such as 6 wells, 24 wells, 384 wells, 1536 wells, etc., formed in a regular or irregular pattern. In other embodiments, the multiwell plate 208 may be replaced by a single-well plate or any other device suitable for performing biological, chemical, and / or biochemical analyses and / or assays. While the wells 200 are shown in a circular configuration (thus forming a cylinder) in FIGS. 2A-2C, other shapes are contemplated as well, including ovals, squares, and / or other regular or irregular polygons. Furthermore, the shape and configuration of the multiwell plate 108 can take multiple forms and is not necessarily limited to the rectangular array shown in these figures.

[0050] In some embodiments, as described above, the working electrode zone 104 and / or auxiliary electrode 102 used in the multiwell plate 108 may be non-porous (hydrophobic). In some embodiments, the working electrode zone 104 and / or auxiliary electrode 102 may be porous electrodes (e.g., carbon fiber or fibril mats, sintered metals, and metal films deposited on filtration membranes, paper, or other porous substrates). When configured as porous electrodes, the working electrode zone 104 and / or auxiliary electrode 102 may employ filtration of solutions through the electrode to: i) increase mass transport to the electrode surface (e.g., increase the kinetics of binding of molecules in solution to molecules on the electrode surface), ii) capture particles on the electrode surface, and / or iii) remove liquid from the wells.

[0051] In the above-described embodiment, each of the auxiliary electrodes 102 in the wells 200 is formed from a chemical mixture that provides a defined potential during reduction of the chemical mixture such that a quantifiable amount of charge is generated throughout the reduction-oxidation reaction occurring in the wells 200. The chemical mixture of the auxiliary electrodes 102 includes an oxidizing agent that supports the reduction-oxidation reaction, which may be used during biological, chemical, and / or biochemical assays and / or analyses, such as, for example, ECL generation and analysis. In one embodiment, the amount of oxidizing agent in the chemical mixture of the auxiliary electrodes 102 is equal to or greater than the amount of oxidizing agent required for the amount of charge passing through the auxiliary electrode and / or the amount of charge required to drive an electrochemical reaction at a working electrode in at least one well 200 during one or more biological, chemical, and / or biochemical assays and / or analyses, such as ECL generation. In this regard, a sufficient amount of the chemical mixture in the auxiliary electrode 102 remains after the redox reaction for the initial biological, chemical, and / or biochemical assay and / or analysis has occurred, thereby allowing one or more additional redox reactions to occur throughout the subsequent biological, chemical, and / or biochemical assay and / or analysis. In another embodiment, the amount of oxidant in the chemical mixture of the auxiliary electrode 102 is based at least in part on the ratio of the exposed surface area of ​​each of the plurality of working electrode zones to the exposed surface area of ​​the auxiliary electrode.

[0052] In embodiments, one or more auxiliary electrodes 102 of well 200 may be formed from a chemical mixture including a redox couple, as described above. In some embodiments, one or more auxiliary electrodes 102 of well 200 may be formed from a chemical mixture including a mixture of silver (Ag) and silver chloride (AgCl), or other suitable metal / metal halide couples. Other examples of chemical mixtures may include metal oxides with multiple metal oxidation states, such as manganese oxide, or other metal / metal oxide couples, such as silver / silver oxide, nickel / nickel oxide, zinc / zinc oxide, gold / gold oxide, copper / copper oxide, platinum / platinum oxide, etc. In embodiments, the auxiliary electrodes 102 (and working electrode zone 104) may be formed using any type of manufacturing process, such as printing, vapor deposition, lithography, etching, etc. In embodiments, the form of the metal / metal halide chemical mixture may depend on the manufacturing process. For example, if the auxiliary electrodes are printed, the chemical mixture may be in the form of an ink or paste.

[0053] For certain applications, such as ECL generation, various embodiments of the auxiliary electrode 102 can be adapted to prevent electrode polarization throughout the ECL measurement by including a sufficiently high concentration of available redox species. The auxiliary electrode 102 can be formed by printing the auxiliary electrode 102 onto a multiwell plate 208 using an Ag / AgCl chemical mixture (e.g., ink, paste, etc.) having a defined ratio of Ag to AgCl. In one embodiment, the amount of oxidizer in the auxiliary electrode chemical mixture is based, at least in part, on the ratio of Ag to AgCl in the auxiliary electrode chemical mixture. In one embodiment, the auxiliary electrode chemical mixture having Ag and AgCl includes about 50 percent or less AgCl, e.g., 34 percent, 10 percent, etc.

[0054] In some embodiments, one or more auxiliary electrodes 102 in a well 200 may comprise less than or equal to mm of the total working electrode area in the well 200. 2 At least about 3.7 x 10 per -9In some embodiments, one or more auxiliary electrodes 102 in a well 200 may contain more than one mole of oxidant in the well 200 than the mm of total working electrode area in the well. 2 At least about 5.7 x 10 per -9 The catalyst may contain molar amounts of oxidizing agent.

[0055] In various embodiments, one or more auxiliary electrodes 102 and working electrode zones 104 may be formed with different electrode designs (e.g., different sizes and / or shapes, different numbers of auxiliary electrodes 102 and working electrode zones 104, different positioning and patterns within the well, etc.) to improve electrochemical analyses (e.g., ECL analyses) performed by assay devices including one or more of the wells 200, examples of which are discussed below with reference to FIGS. 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, and 8A-8D. In embodiments herein, for example, the one or more auxiliary electrodes 102 and one or more working electrode zones 104 of each well 200 may be formed with respective sizes such that the ratio of the collective exposed surface area of ​​the working electrode zones 104 to the exposed surface area of ​​the auxiliary electrodes 102 is greater than 1, although other ratios are also contemplated for the wells 200 (e.g., ratios less than or greater than 1). In embodiments herein, for example, the auxiliary electrode 102 and / or working electrode zone 104 may each be formed in a circular shape having a surface area that substantially defines a circle, but may also be other shapes (e.g., rectangular, square, oval, cloverleaf, or any other regular or irregular geometric shape). In embodiments herein, for example, the auxiliary electrode 102 and / or working electrode zone 104 may be formed in a wedge shape having a wedge-shaped surface area, with a first side or end of the wedge-shaped surface area adjacent to the sidewall of the well 200 being larger than a second side or end of the wedge-shaped surface area adjacent to the center of the well 200. In other embodiments, the second side or end of the wedge-shaped surface area is larger than the first side or end of the wedge-shaped surface area. For example, the auxiliary electrode 102 and working electrode zone 104 may be formed in a pattern that maximizes the space available for the auxiliary electrode 102 and working electrode zone 104.

[0056] In some embodiments, one or more auxiliary electrodes 102 and / or one or more working electrode zones 104 may be formed with a wedge shape, with two opposing boundaries having different dimensions, and two side boundaries connecting the two opposing boundaries. For example, the two opposing boundaries may include a wide boundary and a narrow boundary, with the wide boundary having a longer length than the narrow boundary. In some embodiments, the wide boundary and / or the narrow boundary may have an obtuse angle, e.g., a rounded corner, at the connection to the side boundary. In some embodiments, the wide boundary and / or the narrow boundary may have an acute angle, e.g., a square corner, at the connection to the side boundary. In embodiments, a wedge shape may be utilized to maximize the available area on the bottom surface 120 of the electrochemical cell. For example, if the working area 101 of the electrochemical cell is circular, one or more working electrode zones 104 having a wedge shape may be positioned such that the wide boundary is adjacent to the periphery of the working area 101 and the narrow boundary is adjacent to the center of the working area 101.

[0057] In embodiments according to the present disclosure, the auxiliary electrode 102 and one or more working electrode zones 104 of each well 200 can be formed on the bottom of the well 200 according to different positioning configurations or patterns. Different positioning configurations or patterns can improve electrochemical analyses (e.g., ECL analyses) performed by assay devices including one or more of the wells 200, examples of which are discussed below with reference to FIGS. 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, and 8A-8D. The auxiliary electrode 102 and working electrode zones 104 may be positioned within the well according to a desired geometric pattern. For example, the auxiliary electrode 102 and working electrode zones 104 may be formed in a pattern that minimizes the number of adjacent working electrode zones 104 for each of the working electrode zones 104 out of the total number of working electrode zones 104. This may allow more working electrode zones to be positioned adjacent to the auxiliary electrode 102. For example, as shown in FIGS. 3A-3F and described in detail below, the working electrode zones 104 may be formed in a circular or semicircular shape that minimizes the number of adjacent working electrode zones 104.

[0058] In another example, as shown in FIGS. 3A-3F , the auxiliary electrodes 102 and working electrode zones 104 of each well 200 may be formed in a pattern in which the number of adjacent working electrode zones 104 is two or less. For example, the working electrode zones 104 may be formed in a circular or semicircular pattern that is adjacent to a parameter of the well (e.g., the sidewall 212) such that a maximum of two working electrode zones 104 are adjacent. In this example, the working electrode zones 104 form an incomplete circle such that two working electrode zones 104 have only one adjacent or neighboring working electrode zone 104. In another example, the auxiliary electrodes 102 and working electrode zones 104 of each well 200 may be formed in a pattern in which at least one of the working electrode zones 104 is adjacent to three or more other working electrode zones 104 out of the total number of working electrode zones 104. For example, as shown in Figures 5A-5C, which are described in detail below, the auxiliary electrodes 102 and working electrode zones 104 may be formed in a star pattern, where the number of adjacent auxiliary electrodes 102 and / or working electrode zones 104 depends on the number of points in the star pattern.

[0059] In one embodiment according to the present disclosure, the auxiliary electrode 102 and one or more working electrode zones 104 of each well 200 can be formed in a pattern configured to improve mass transport of material to each of the working electrode zones 104. For example, during orbital or rotational vibration or mixing, mass transport of material to zones in the center of the well 200 may be relatively slow compared to zones further from the center, and the pattern may be configured to improve mass transport by minimizing or eliminating the number of working electrode zones 104 located in the center of the well 200. That is, during operation, the well 200 may undergo orbital motion or "vibration" to mix or combine the fluids contained within the well 200. The orbital motion may generate vortices within the well 200, resulting in, for example, more liquid and faster liquid movement near the sidewalls 212 (periphery) of the well 200. For example, as shown in Figures 2A-2F, 3A-3F, 5A-5F, 6A-6F, and 7A-7D, which are described in detail below, the working electrode zones 104 may be circular or semicircular and arranged around the periphery of the well 200. Furthermore, due to the orbital oscillatory motion, any variation in substance concentration within the well may depend on the radial distance from the center of the well. In a concentric arrangement, the working electrode zones 104 are each approximately the same distance from the center of the well and therefore may have similar substance concentrations, even if the substance concentration is not uniform throughout the well.

[0060] In one embodiment according to the present disclosure, the auxiliary electrode 102 and one or more working electrode zones 104 of each well 200 may be formed in a pattern configured to reduce the meniscus effect caused by introducing liquid into one or more of the wells 200 of the multiwell plate 108. For example, as shown in FIG. 2C , the fluid 250 in the well 200 may form a curved upper surface, or meniscus 152, within the well 200. The curved upper surface may be caused by several factors, such as surface tension, electrostatic effects, and fluid motion (e.g., due to orbital vibration). Due to the meniscus effect, photons (light) emitted during light emission experience different optical effects (e.g., refraction, diffusion, scattering, etc.) based on the photon's optical path through the liquid. That is, when light is emitted from a material within the well 200, different levels of liquid may cause different optical effects (e.g., refraction, diffusion, scattering, etc.) on the emitted light depending on where the light exits through the liquid. This pattern can mitigate the meniscus effect by positioning each of the working electrode zones 104 at approximately equal distances from each sidewall 212 of the well 200. Therefore, photons emitted from the working electrode zones 104 travel a similar optical path through the liquid. In other words, the pattern ensures that all working electrode zones 104 are equally affected by the meniscus effect, minimizing, for example, potential differential effects of the meniscus. Therefore, if the working electrode zones 104 are positioned at different locations relative to the level of the liquid in the well 200, the emitted light may experience different optical distortions. For example, as shown in FIGS. 3A-3F, 4A-4F, 6A-6F, 7A-7F, and 8A-8D, which are described in detail below, the working electrode zones 104 may be formed in a circular or semicircular shape and positioned around the perimeter of the well 200. Therefore, light emitted at the working electrode zones 104 may experience the same optical distortion and be treated equally.

[0061] In one embodiment according to the present disclosure, the auxiliary electrode 102 and one or more working electrode zones 104 in each well 200 can be formed in a pattern configured to minimize mass transport differences (e.g., provide more uniform mass transport) to the working electrode zones during mixing of the liquid in one or more of the wells 200 of the multiwell plate 208 (e.g., vortices formed in a cylindrical well using an orbital shaker). For example, the pattern can be configured to reduce vortex effects by minimizing or eliminating the number of working electrode zones 104 located at or near the center of each well 200. For example, as shown in Figures 2A-2F, 3A-3F, 5A-5F, 6A-6F, 7A-7D, and 8A, which are described in detail below, the working electrode zones 104 can be formed in a circular or semicircular shape and positioned near the periphery of the well 200.

[0062] In one embodiment according to the present disclosure, the auxiliary electrode 102 and one or more working electrode zones 104 of each well 200 may be formed in a geometric pattern. For example, the geometric pattern may include a circular or semicircular pattern of working electrode zones 104, each of which may be positioned at approximately equal distances from the sidewall of the well 200, and the auxiliary electrode 102 may be positioned within a perimeter (either the entire perimeter or only a portion thereof) defined by the circular or semicircular pattern of working electrode zones 104, although other shapes and / or patterns are also contemplated. For example, if the well 200 is embodied as a square well, the working electrode zones 104 may be arranged in a square or rectangular ring pattern around the entire perimeter or only a portion of the perimeter of the well 200.

[0063] In another embodiment, for example, the geometric pattern may include a pattern in which the working electrode zones 104 define a star pattern, and the auxiliary electrode 102 may be disposed between two adjacent working electrode zones 104 that define two adjacent points of the star pattern. For example, the star pattern may be formed with the auxiliary electrodes 102 forming the "points" of the star pattern and the working electrode zones 104 forming the interior structure of the star pattern. For example, in a five-point star pattern, the auxiliary electrodes 102 may form five "points" of the star pattern, and the working electrode zones 104 may form an interior "pentagonal" structure, as shown in FIGS. 5A-5C, which are described in more detail below. In some embodiments, the star pattern may also be defined as one or more concentric circles, and one or more working electrodes 104 and / or one or more auxiliary electrodes may be disposed in a circular pattern around one or more concentric circles, as illustrated in FIGS. 5A-5C, which are described in more detail below.

[0064] 3A and 3B illustrate an embodiment of an electrode design 301 for a well 200 having circular working electrode zones 104 arranged in an open ring pattern. According to the exemplary, non-limiting embodiment shown in FIG. 3A, the bottom 207 of the well 200 can include a single auxiliary electrode 102. In other embodiments, more than one auxiliary electrode 102 can be included in the well 200 (e.g., two, three, four, five, etc.). In embodiments, the auxiliary electrode 102 can be formed to have a generally circular shape. In other embodiments, the auxiliary electrode 102 can be formed to have other shapes (e.g., rectangular, square, oval, cloverleaf, or any other regular or irregular geometric shape).

[0065] In embodiments, the well 200 may include ten working electrode zones 104. In other embodiments, fewer or more than ten working electrode zones 104 may be included in the well 200 (e.g., one, two, three, four, etc.). In embodiments, the working electrode zones 104 may be formed to have a generally circular shape. In other embodiments, the working electrode zones 104 may be formed to have other shapes (e.g., a rectangle, a square, an oval, a cloverleaf, or any other regular or irregular geometric shape).

[0066] The working electrode zones 104 may be arranged relative to one another in a semicircular or substantially "C-shaped" pattern adjacent to the perimeter "P" of the well 200 at a distance "D1." In some embodiments, the distance D1 may be the minimum distance between the boundary of the working electrode zone 104 and the perimeter P. That is, each of the working electrode zones 104 may be positioned at an equal distance D1 from the perimeter P of the well 200, and each of the working electrode zones 104 is equally spaced from one another by a distance "D2" (also referred to as the working electrode (WE) pitch). In some embodiments, the distance D2 may be the minimum distance between the boundaries of two adjacent working electrode zones 104. In some embodiments, the two working electrode zones 104A, 104B may be spaced from one another by a sufficient distance to form a gap "G." The gap "G" may provide a pitch distance between the two working electrode zones that is greater than the remainder of the pitch distance between the remainder of the working electrode zones. In certain embodiments, gap G may allow electrical traces or contacts to be electrically coupled to the auxiliary electrode 102 without electrically interfering with the working electrode zone 104, thereby maintaining electrical isolation between the auxiliary electrode 102 and the working electrode zone 104. For example, gap G may be formed at a distance sufficient to allow electrical traces to be formed between adjacent working electrode zones 104 while remaining electrically isolated. Thus, the size of gap G may be determined at least in part by the choice of manufacturing method for constructing the electrochemical cell. Thus, in embodiments, the larger pitch distance of gap "G" may be at least 10%, at least 30%, at least 50%, or at least 100% greater than the pitch distance D2 between the remainder of the working electrode zones 104.

[0067] In certain embodiments, distance D1 may be unequal between one or more working electrode zones 104 and the perimeter P of the well 200. In further embodiments, distance D2 may be unequal between two or more of the working electrode zones 104. The auxiliary electrode 102 may be positioned at the center of the C-shaped pattern at an equal distance “D3” (also referred to as the WE-AUXILIARY pitch) from each of the working electrode zones 104, although in other embodiments, distance D3 may vary for one or more of the working electrode zones 104 as measured to the auxiliary electrode 102. In certain embodiments, as shown, distance D1, distance D2, distance D3, and distance G may be measured from the nearest relative point on the perimeter of the respective feature, e.g., the working electrode zone 104, the auxiliary electrode 102, or the perimeter P. In some embodiments, distance D3 may be the minimum distance between the boundary of the working electrode zone 104 and the boundary of the auxiliary electrode. One skilled in the art will understand that distances can be measured from any relative point on a feature to generate a repeatable pattern, e.g., a geometric pattern.

[0068] Although these figures show a single auxiliary electrode 102, two or more auxiliary electrodes may be included, as shown in Figure 3C. Furthermore, although the auxiliary electrode 102 is shown in these figures as being located approximately (or exactly) in the center of the well 200, the auxiliary electrode 102 may be located elsewhere in the well 200, as shown in Figure 3D. Additionally, although these figures illustrate ten working electrode zones 104, a greater or lesser number of working electrode zones 104 may be included, as shown in Figures 3E and 3F.

[0069] The electrochemical cells shown in Figures 3A-3F can include electrodes of Ag, Ag / AgCl, carbon, carbon composites, and / or other carbon-based materials, and / or any other electrode material described herein.

[0070] In embodiments, the size of the auxiliary electrode 102 and / or the working electrode zone 104 may be varied. For example, as shown in Table 2A, the size of each of the working electrode zones 104 may be equal, and the size of the auxiliary electrode 102 may be varied, such as by varying its diameter. Those skilled in the art will appreciate that the dimensions included in Table 2A are approximate and may vary by, for example, + / - 5.0%, based on conditions such as manufacturing tolerances.

[0071] [Table 2]

[0072] Table 2A above provides exemplary values ​​for well geometry. For example, as noted above, in paragraph

[0057] , an Ag / AgCl electrode according to an embodiment of the present invention has a well geometry of approximately 3.07×10 -7 Molar ~ 3.97 x 10 -7 In addition to the above mentioned geometry, both the working and auxiliary electrodes may be approximately 10 microns (3.937×10 -4 The thickness may be in inches. Table 2B provides approximate values ​​and ranges for the moles of oxidant in the auxiliary electrode per auxiliary electrode area and volume. Table 2C provides approximate values ​​and ranges for the moles of oxidant in the auxiliary electrode per working electrode area and volume. The values ​​and ranges shown in Tables 2B and 2C are provided using inches as units. One skilled in the art will recognize that these values ​​may also be converted to mm.

[0073] [Table 3]

[0074] [Table 4]

[0075] 4A and 4B illustrate non-limiting, exemplary embodiments of an electrode design 401 for a well 200 having non-circular working electrode zones 104 arranged within the well in an open ring pattern, similar to that described above with reference to FIGS. 3A and 3B. The non-circular working electrode zones 104 shown in FIGS. 4A and 4B (and FIGS. 4C-4F) may be wedge-shaped or trefoil-shaped. In embodiments, the non-circular shaped working electrode zones 104 may allow for improved use of the area within the well 200. The use of non-circular shaped working electrode zones 104 may allow larger working electrode zones 104 and / or more working electrode zones 104 to be formed within the well 200. By forming these non-circular shapes, the working electrode zones 104 may be more tightly packed within the well 200. Thus, the ratio of working electrode zones 104 to auxiliary electrodes 102 may be maximized. Additionally, because the working electrode zone 104 can be made larger, the working electrode zone 104 can be manufactured, eg, printed, more reliably.

[0076] 4A, well 200 may include a single auxiliary electrode 102. In other embodiments, two or more (1) auxiliary electrodes 102 may be included in well 200 (e.g., two, three, four, five, etc.). In embodiments, auxiliary electrode 102 may be formed to have a generally circular shape. In other embodiments, auxiliary electrode 102 may be formed to have other shapes (e.g., rectangular, square, oval, clover, or any other regular or irregular geometric shape).

[0077] In embodiments, the well 200 may include ten working electrode zones 104. In other embodiments, fewer or more than ten working electrode zones 104 may be included in the well 200 (e.g., one, two, three, four, etc.). Each of the working electrode zones 104 may be formed to have a non-circular shape, for example, a wedge or triangular shape with one or more rounded or arcuate corners, although in other embodiments, the corners are not rounded, thus forming a polygonal shape such as a triangle.

[0078] The working electrode zones 104 may be arranged relative to one another in a semicircular or substantially "C-shaped" pattern adjacent the perimeter "P" of the well 200 at a distance "D1." In some embodiments, the distance D1 may be the minimum distance between the boundary of the working electrode zone 104 and the perimeter P. That is, each of the working electrode zones 104 may be positioned an equal distance D1 from the perimeter P of the well 200, and each of the working electrode zones 104 is equally spaced apart from one another by a distance "D2." In some embodiments, the distance D2 may be the minimum distance between the boundaries of two adjacent working electrode zones 104. In some embodiments, the two working electrode zones 104A, 104B may be spaced apart from one another by a sufficient distance to form a gap "G." In certain embodiments, the distance D1 may be unequal between one or more working electrode zones 104 and the perimeter P of the well 200. In further embodiments, the distance D2 may be unequal between two or more of the working electrode zones 104. The auxiliary electrode 102 may be positioned at the center of the C-shaped pattern an equal distance "D3" from each of the working electrode zones 104, although in other embodiments, distance D3 may vary relative to one or more of the working electrode zones 104 as measured to the auxiliary electrode 102. In certain embodiments, as shown, distances D1, D2, D3, and G may be measured from the nearest point on the perimeter of the respective feature, e.g., the working electrode zone 104, the auxiliary electrode 102, or the perimeter P. In some embodiments, distance D3 may be the minimum distance between the boundary of the working electrode zone 104 and the boundary of the auxiliary electrode. One skilled in the art will understand that distances can be measured from any relative point on the feature to generate a repeatable pattern, e.g., a geometric pattern.

[0079] Although these figures show a single auxiliary electrode 102, two or more auxiliary electrodes may be included, as shown in Figures 4C and 4D. Furthermore, although the auxiliary electrode 102 is shown in these figures as being located approximately (or exactly) in the center of the well 200, the auxiliary electrode 102 may be located elsewhere in the well 200, as shown in Figure 4D. Additionally, although these figures illustrate ten working electrode zones 104, a greater or lesser number of working electrode zones 104 may be included, as shown in Figures 4E and 4F.

[0080] In certain embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may be equal. In other embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may be varied. In one example, the size of the auxiliary electrode 102 may be constant, and the size of the working electrode zone 104 may be varied, such as by varying the radius of the auxiliary electrode 102. Table 3A includes example dimensions of the working electrode zone 104 and the auxiliary electrode 102 for embodiments including a wedge-shaped or trefoil-shaped working electrode zone 104 shown in FIGS. 4A-4F. Those skilled in the art will understand that the dimensions included in Table 3 are approximate and may vary by, for example, + / - 5.0% based on conditions such as manufacturing tolerances.

[0081] The electrochemical cells shown in Figures 4A-4F can include electrodes of Ag, Ag / AgCl, carbon, carbon composites, and / or other carbon-based materials, and / or any other electrode material described herein.

[0082] [Table 5]

[0083] Table 3A above provides exemplary values ​​for trefoil electrode well geometry dimensions. For example, as noted above, in paragraph

[0057] , an Ag / AgCl electrode according to an embodiment of the present invention has a well geometry of approximately 3.07×10 -7 Molar ~ 3.97 x 10 -7In addition to the above mentioned geometry, both the working and auxiliary electrodes may be approximately 10 microns (3.937×10 -4 The thickness may be in inches. Table 3B provides approximate values ​​and ranges for the moles of oxidant in the auxiliary electrode per auxiliary electrode area and volume. Table 3C provides approximate values ​​and ranges for the moles of oxidant in the auxiliary electrode per working electrode area and volume. The values ​​and ranges shown in Tables 3B and 3C are provided using inches as units. One skilled in the art will recognize that these values ​​may also be converted to mm.

[0084] [Table 6]

[0085] [Table 7]

[0086] 5A and 5B show a non-limiting example embodiment of an electrode design 401 for a well 200 having working electrode zones 104 arranged in a star pattern (also referred to herein as a penta-pattern), where the working electrode zones 104 are circular. As illustrated in FIG. 5A , the well 200 may include five auxiliary electrodes 102, each of which may be formed in a generally circular shape (although other numbers of auxiliary electrodes, different shapes, etc. are also contemplated). In this example, the well 200 may also include ten working electrode zones 104, each of which may be formed in a generally circular shape. The star pattern may be created by arranging multiple working electrode zones 104 in one of an inner circle and an outer circle relative to each other, with each working electrode zone 100 located in the outer circle being positioned at the angular midpoint relative to two adjacent working electrode zones 104 located in the inner circle. Each of the working electrode zones 104 in the inner circle may be spaced a distance “R1” from the center of the well 200. Each of the working electrode zones 104 in the outer circle may be spaced a distance "R2" from the center of the well 200. In the star pattern, each auxiliary electrode 102 may be positioned an equal distance "D4" from two of the working electrode zones 104 located in the outer circle.

[0087] In one embodiment, as shown, distances R1, R2, and D4 may be measured from the nearest point on the perimeter of the respective feature, e.g., working electrode zone 104, auxiliary electrode 102, or perimeter P. Those skilled in the art will appreciate that the distances may be measured from any relative point on the feature to generate a repeatable geometric pattern.

[0088] These figures illustrate ten working electrode zones 104, although a greater or lesser number of working electrode zones 104 may be included, as illustrated in Figure 5C. Additionally, although Figures 5A-5C show circular working electrode zones 104, in other embodiments, the working electrode zones 104 may be formed having other shapes (e.g., rectangular, square, oval, cloverleaf, or any other regular or irregular geometric shape). Other embodiments may include hybrid designs of electrode configurations, such as, for example, a star-shaped pattern including wedge-shaped working electrode zones and / or auxiliary electrodes.

[0089] The electrochemical cells shown in Figures 5A-5F can include electrodes of Ag, Ag / AgCl, carbon, carbon composites, and / or other carbon-based materials, and / or any other electrode material described herein.

[0090] In certain embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may be equal. In other embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may vary. In one example, as shown in Table 4A, the size of the working electrode zone 104 may be constant, and the size of the auxiliary electrode 102 may vary, such as by changing its diameter. One skilled in the art will understand that the dimensions included in Table 4A are approximate and may vary, for example, by + / - 5.0%, based on conditions such as manufacturing tolerances.

[0091] [Table 8]

[0092] Table 4A above provides exemplary values ​​for the 10-spot pentaelectrode well geometry. For example, as noted above, in paragraph

[0057] , an Ag / AgCl electrode according to an embodiment of the present invention has a well size of approximately 3.07×10 -7 Molar ~ 3.97 x 10 -7 In addition to the above mentioned geometry, both the working and auxiliary electrodes may be approximately 10 microns (3.937×10 -4The thickness may be in inches. Table 4B provides approximate values ​​and ranges for the moles of oxidant in the auxiliary electrode per auxiliary electrode area and volume. Table 4C provides approximate values ​​and ranges for the moles of oxidant in the auxiliary electrode per working electrode area and volume. The values ​​and ranges shown in Tables 4B and 4C are provided using inches as units. One skilled in the art will recognize that these values ​​may also be converted to mm.

[0093] [Table 9]

[0094] [Table 10]

[0095] 6A and 6B show exemplary, non-limiting embodiments of electrode designs 601 for wells 200 having non-circular shaped (e.g., trefoil-shaped or wedge-shaped) working electrode zones 104 arranged in a closed ring pattern. As shown in FIG. 6A, the wells 200 may include a single auxiliary electrode 102. In other embodiments, two or more (1) auxiliary electrodes 102 may be included in the wells 200 (e.g., two, three, four, five, etc.). In embodiments, the auxiliary electrodes 102 may be formed to have a generally circular shape. In other embodiments, the auxiliary electrodes 102 may be formed to have other shapes (e.g., rectangular, square, oval, cloverleaf, or any other regular or irregular geometric shape).

[0096] In embodiments, the well 200 may also include more or less than 10 working electrode zones 104. For example, FIGS. 6A and 6B show an embodiment with 12 working electrode zones 104, FIGS. 6C and 6D show an embodiment with 11 working electrode zones 104, FIG. 6E shows an embodiment with 14 working electrode zones 104, and FIG. 6F shows an embodiment with 7 working electrode zones 104. The working electrode zones 104 may be formed to have a non-circular shape, such as a wedge shape or a triangular shape with one or more rounded or arcuate corners, also known as a trefoil shape. In a closed ring pattern, the working electrode zones 104 may be arranged in a circle around the perimeter of the well 200, each in a pattern adjacent a perimeter "P" of the well 200 by a distance "D1." In some embodiments, the distance D1 may be the minimum distance between the boundary of the working electrode zone 104 and the perimeter P. That is, each of the working electrode zones 104 may be positioned an equal distance D1 from the perimeter P of the well 200, and each of the working electrode zones 104 may be equally spaced from one another by a distance "D2." In some embodiments, distance D2 may be the minimum distance between the boundaries of two adjacent working electrode zones 104. In certain embodiments, distance D1 may be unequal between one or more working electrode zones 104 and the perimeter P of the well 200. The auxiliary electrode 102 may be positioned at the center of the C-shaped pattern an equal distance "D3" from each of the working electrode zones 104, although in other embodiments, distance D3 may vary for one or more of the working electrode zones 104, as measured to the auxiliary electrode 102. In some embodiments, distance D3 may be the minimum distance between the boundary of the working electrode zone 104 and the boundary of the auxiliary electrode. In one embodiment, as shown, distance D1, distance D2, and distance D3 may be measured from the nearest point on the perimeter of the respective feature, e.g., working electrode zone 104, auxiliary electrode 102, or perimeter P. Those skilled in the art will appreciate that the distances may be measured from any relative point on the feature to generate a repeatable pattern, e.g., a geometric pattern.

[0097] Although these figures show a single auxiliary electrode 102, two or more auxiliary electrodes may be included, as shown in Figure 6C. Furthermore, although the auxiliary electrode 102 is shown in these figures as being located approximately (or exactly) in the center of the well 200, the auxiliary electrode 102 may be located elsewhere in the well 200, as shown in Figure 6D. Additionally, although these figures illustrate ten working electrode zones 104, a greater or lesser number of working electrode zones 104 may be included, as shown in Figures 6E and 6F.

[0098] The electrochemical cells shown in Figures 6A-6F can include electrodes of Ag, Ag / AgCl, carbon, carbon composites, and / or other carbon-based materials, and / or any other electrode material described herein.

[0099] In certain embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may be equal. In other embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may be varied. In one example, the size of the auxiliary electrode 102 may be constant, and the size of the working electrode zone 104 may be varied, such as by varying the radius of the auxiliary electrode 102. Table 5A includes example dimensions of the working electrode zone 104 and the auxiliary electrode 102 for the embodiment shown in FIGS. 6A-6F. One skilled in the art will understand that the dimensions included in Table 5A are approximate and may vary by, for example, + / - 5.0% based on conditions such as manufacturing tolerances.

[0100] [Table 11]

[0101] Table 5A above provides exemplary values ​​for closed trilobe electrode well geometry dimensions. For example, as noted above, in paragraph

[0057] , an Ag / AgCl electrode according to an embodiment of the present invention has a well geometry of approximately 3.07×10 -7 Molar ~ 3.97 x 10 -7In addition to the above mentioned geometry, both the working and auxiliary electrodes may be approximately 10 microns (3.937×10 -4 The thickness may be in inches. Table 5B provides approximate values ​​and ranges for the moles of oxidant in the auxiliary electrode per auxiliary electrode area and volume. Table 5C provides approximate values ​​and ranges for the moles of oxidant in the auxiliary electrode per working electrode area and volume. The values ​​and ranges shown in Tables 5B and 5C are provided using inches as units. One skilled in the art will recognize that these values ​​may also be converted to mm.

[0102] [Table 12]

[0103] [Table 13]

[0104] In embodiments, it may be beneficial to eliminate sharp corners in a trefoil electrode design. For example, FIG. 6A shows a trefoil design with sharp corners, and FIG. 6B shows a trefoil design with rounded corners. The rounded corners may reduce the area of ​​the working electrode zone 104 by, for example, 1-5%, but may provide additional benefits. For example, sharp corners may interfere with uniform distribution of solution. Sharp corners may also present small features that are more difficult to accurately image. Therefore, reducing sharp corners may be beneficial, even though it results in a smaller working electrode zone 104.

[0105] 7A and 7B show exemplary, non-limiting embodiments of an electrode design 701 for a well 200 having a closed-ring design with circular electrodes. As shown in FIG. 7A, the well 200 may include a single auxiliary electrode 102. In other embodiments, more than one auxiliary electrode 102 may be included in the well 200 (e.g., two, three, four, five, etc.). In embodiments, the auxiliary electrode 102 may be formed to have a generally circular shape. In other embodiments, the auxiliary electrode 102 may be formed to have other shapes (e.g., rectangular, square, oval, cloverleaf, or any other regular or irregular geometric shape).

[0106] In embodiments, the well 200 may include ten working electrode zones 104. In other embodiments, fewer or more than ten working electrode zones 104 may be included in the well 200 (e.g., one, two, three, four, etc.). In embodiments, the working electrode zones 104 may be formed to have a generally circular shape. In other embodiments, the working electrode zones 104 may be formed to have other shapes (e.g., a rectangle, a square, an oval, a cloverleaf, or any other regular or irregular geometric shape).

[0107] In a closed ring pattern, the working electrode zones 104 may be arranged in a circle around the perimeter of the well 200 such that each is in a pattern adjacent to the perimeter "P" of the well 200 by a distance "D1." In some embodiments, the distance D1 may be the minimum distance between the boundary of the working electrode zone 104 and the perimeter P. That is, each of the working electrode zones 104 may be positioned an equal distance D1 from the perimeter P of the well 200, and each of the working electrode zones 104 are equally spaced apart from each other by a distance "D2" (also referred to as the working electrode (WE-WE) pitch). In some embodiments, the distance D2 may be the minimum distance between the boundaries of two adjacent working electrode zones 104. In certain embodiments, the distance D1 may be unequal between one or more working electrode zones 104 and the perimeter P of the well 200. In further embodiments, the distance D2 may be unequal between two or more of the working electrode zones 104.

[0108] The auxiliary electrode 102 may be positioned at the center of the ring pattern an equal distance "D3" (referred to as the WE-AUXILIARY pitch) from each of the working electrode zones 104, although in other embodiments, the distance D3 may vary for one or more of the working electrode zones 104 as measured to the auxiliary electrode 102. In some embodiments, the distance D3 may be the minimum distance between the boundary of the working electrode zone 104 and the boundary of the auxiliary electrode. In certain embodiments, as shown, the distances D1, D2, and D3 may be measured from the nearest relative point on the perimeter of the respective feature, e.g., the working electrode zone 104, the auxiliary electrode 102, or the perimeter P. One skilled in the art will understand that the distances may be measured from any relative point on the feature to generate a repeatable pattern, e.g., a geometric pattern.

[0109] In a further example, the distance from the working electrode zone to the auxiliary electrode (WE-Auxiliary distance) may be measured from the center of the working electrode zone 104 to the center of the auxiliary electrode 102. Examples of WE-Auxiliary distances include 0.088 inches for a 10-spot open concentric design, 0.083 inches for a 10-trefoil open concentric design with sharp corners, 0.087 inches for a 10-trefoil open concentric design with rounded corners, 0.080 inches for a 10-trefoil closed concentric design with sharp corners, 0.082 inches for a 10-trefoil closed concentric design with rounded corners, and 0.086 inches for a 10-spot closed concentric design. In a penta design, the WE-Auxiliary distance may be 0.062 inches between the inner working electrode zone 104 and the auxiliary electrode 102 and 0.064 inches between the outer working electrode zone 104 and the auxiliary electrode 102. The WE-Auxiliary distance values ​​provided herein may vary by 5%, 10%, 15%, 25% or more without departing from the scope of the present disclosure. In embodiments, the WE-Auxiliary distance values ​​may be varied according to the size and configuration of the working electrode zone 104 and the auxiliary zone 102.

[0110] Although these figures show a single auxiliary electrode 102, two or more auxiliary electrodes may be included, as shown in Figure 7C. Furthermore, although the auxiliary electrode 102 is shown in these figures as being located approximately (or exactly) in the center of the well 200, the auxiliary electrode 102 may be located elsewhere in the well 200, as shown in Figure 7D. Additionally, although these figures illustrate ten working electrode zones 104, a greater or lesser number of working electrode zones 104 may be included, as shown in Figures 7E and 7F.

[0111] The electrochemical cells shown in Figures 7A-7F can include electrodes of Ag, Ag / AgCl, carbon, carbon composites, and / or other carbon-based materials, and / or any other electrode material described herein.

[0112] In certain embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may be equal. In other embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may vary. In one example, as shown in Table 6A, the size of the working electrode zone 104 may be constant, and the size of the auxiliary electrode 102 may vary, such as by changing its diameter. Those skilled in the art will understand that the dimensions included in Table 6A are approximate and may vary, for example, by + / - 5.0%, based on conditions such as manufacturing tolerances.

[0113] [Table 14]

[0114] Table 6A above provides exemplary values ​​for closed spot electrode well geometry. For example, as noted above, in paragraph

[0057] , an Ag / AgCl electrode according to an embodiment of the present invention has a well geometry of approximately 3.07×10 -7 Molar ~ 3.97 x 10 -7 In addition to the above mentioned geometry, both the working and auxiliary electrodes may be approximately 10 microns (3.937×10 -4The thickness may be in inches. Table 6B provides approximate values ​​and ranges for the moles of oxidant in the auxiliary electrode per auxiliary electrode area and volume. Table 6C provides approximate values ​​and ranges for the moles of oxidant in the auxiliary electrode per working electrode area and volume. The values ​​and ranges shown in Tables 6B and 6C are provided using inches as units. One skilled in the art will recognize that these values ​​may also be converted to mm.

[0115] [Table 15]

[0116] [Table 16]

[0117] Tables 2A-6C provide example dimensions for the spot sizes of the working electrode zone 104 and the auxiliary electrode 102. The selection of the spot sizes of the working electrode zone 104 and the auxiliary electrode 102 can be important for optimizing the results of the ECL process. For example, maintaining the proper ratio between the working electrode zone 104 area and the auxiliary electrode 102 area can be important for ensuring that the auxiliary electrode 102 has sufficient reducing capacity to complete ECL generation for a selected voltage waveform without saturating. In another example, a larger working electrode zone 104 can provide greater binding capacity and increase the ECL signal. A larger working electrode zone 104 can also facilitate manufacturing by avoiding small features and because any manufacturing tolerances are a smaller percentage of the overall size. In embodiments, the area of ​​the working electrode zone 104 can be maximized to increase the ECL signal, binding capacity, and facilitate manufacturing, while being limited by the need to maintain an adequate insulating dielectric barrier between the working electrode zone 104 and the auxiliary electrode 102.

[0118] 8A-8D show exemplary, non-limiting embodiments of an electrode design 801 for a well 200 having a closed-ring design with a circular working electrode zone and a complex-shaped auxiliary electrode 102. As shown in FIG. 8A, the well 200 can include two complex-shaped auxiliary electrodes 102. In other embodiments, as shown in FIG. 8D, fewer (or more) auxiliary electrodes 102 than two can be included in the well 200. In embodiments, the auxiliary electrode 102 can be formed to have a complex shape, such as a "gear," "cog," "ring," "washer," "elliptical," "wedge," or other complex shape, as described above. For example, as shown in FIG. 8B, the interior of the auxiliary electrode 102 can be formed in a circular shape with an outer semicircular space 802 (e.g., a "gear" or "cog" shape) corresponding to the working electrode zone 104. Similarly, as shown in FIG. 8C, the exterior of the auxiliary electrode 102 can be formed in a hollow ring shape with an inner semicircular space 804 (e.g., a "washer" shape) corresponding to the working electrode zone 104.

[0119] In embodiments, the well 200 may include ten working electrode zones 104. In other embodiments, fewer or more than ten working electrode zones 104 may be included in the well 200 (e.g., one, two, three, four, etc.). In embodiments, the working electrode zones 104 may be formed to have a generally circular shape. In other embodiments, the working electrode zones 104 may be formed to have other shapes (e.g., a rectangle, a square, an oval, a cloverleaf, or any other regular or irregular geometric shape).

[0120] In embodiments, the working electrode zone 104 may be circularly disposed between two auxiliary electrodes 102. In this configuration, the outer semicircular space 802 and the inner semicircular space 704 allow the two auxiliary electrodes 102 to partially surround the working electrode zone. The outer one of the two auxiliary electrodes 102 may be spaced a distance “D1” from the working electrode zone 104, where D1 is measured from the midpoint of the inner semicircular space to the boundary of the working electrode zone 104. In some embodiments, the distance D1 may be the minimum distance between the outer one of the two auxiliary electrodes 102 and the working electrode zone 104. In certain embodiments, the distance D1 may be unequal between one or more working electrode zones 104 and the outer two auxiliary electrodes 102. Each of the working electrode zones 104 may be equally spaced apart from one another by a distance “D2.” In some embodiments, the distance D2 may be the minimum distance between the boundaries of two adjacent working electrode zones 104. In further embodiments, the distance D2 may be unequal between two or more of the working electrode zones 104. The inner one of the two auxiliary electrodes 102 may be spaced a distance "D3" from the working electrode zone 104, where D3 is measured from the midpoint of the outer semicircular space to the edge of the working electrode zone 104. In some embodiments, the distance D3 may be the minimum distance between the boundary of the working electrode zone 104 and the boundary of the auxiliary electrode. In certain embodiments, the distance D1 may be unequal between one or more working electrode zones 104 and the inner one of the two auxiliary electrodes 102.

[0121] In one embodiment, as shown, distance D1, distance D2, and distance D3 may be measured from the nearest relative point on the perimeter of the respective feature, for example, working electrode zone 104 or auxiliary electrode 102. Those skilled in the art will appreciate that the distances may be measured from any relative point on the feature to generate a repeatable geometric pattern.

[0122] The electrochemical cells shown in Figures 8A-8D can include auxiliary electrodes of Ag / AgCl, carbon, and / or any other auxiliary electrode material described herein.

[0123] As described above, the electrochemical cell 100 may be utilized in devices and apparatus for performing electrochemical analyses. For example, the multiwell plate 208 containing the wells 200 described above may be used in any type of apparatus that supports the performance of biological, chemical, and / or biochemical assays and / or analyses, such as an apparatus for performing ECL analyses. FIG. 9A illustrates a generalized assay apparatus 900 in which the multiwell plate 208 containing the wells 200 may be used for electrochemical analyses and procedures, according to one embodiment herein. Those skilled in the art will appreciate that FIG. 9A illustrates one example of an assay apparatus, and that existing components shown in FIG. 9A may be removed and / or additional components may be added to the assay apparatus 900 without departing from the scope of the embodiments described herein.

[0124] 9A , the multiwell plate 208 may be electrically coupled to a plate electrical connector 902. The plate electrical connector 902 may be coupled to a voltage / current source 904. The voltage / current source 904 may be configured to selectively supply a controlled voltage and / or current to the wells 200 of the multiwell plate 208 (e.g., the electrochemical cell 100) via the plate electrical connector 902. For example, the plate electrical connector 1502 may be configured to align and / or mate with electrical contacts of the multiwell plate 208 that are coupled to one or more auxiliary electrodes 102 and / or one or more working electrode zones 102 to enable voltage and / or current to be supplied to the wells 200 of the multiwell plate 208.

[0125] In some embodiments, the plate electrical connector 902 may be configured to allow one or more wells 200 to be activated simultaneously (including one or more of the working electrode zones and auxiliary electrodes), or two or more of the working electrode zones and / or auxiliary electrodes may be activated individually. In certain embodiments, a device, such as one used to perform scientific analyses, may be electrically coupled to one or more apparatuses (e.g., a plate, a flow cell, etc.). The coupling between the device and the one or more apparatuses may include the entire surface of the apparatus (e.g., the entire bottom of the plate) or a portion of the apparatus. In some embodiments, the plate electrical connector 902 may be configured to allow one or more of the wells 200 to be selectively addressable, e.g., to allow voltage and / or current to be selectively applied to some of the wells 200 and a signal to be read from the detector 910. For example, as shown in FIG. 9B , a multiwell plate 208 may include 96 wells 200 arranged in rows labeled “A” through “H” and columns labeled “1” through “12.” In some embodiments, plate electrical connector 902 may include a single electrical strip connecting all of the wells 200 in one of rows A-H or one of columns 1-12. Thus, all of the wells 200 in one of rows A-H or one of columns 1-12 may be simultaneously activated by, for example, a voltage and / or current provided by voltage / current source 904. Similarly, all of the wells 200 in one of rows A-H or one of columns 1-12 may simultaneously have a signal read by, for example, detector 910.

[0126] In some embodiments, the plate electrical connector 902 may include a matrix of individual electrical connections, vertical wires 952 and horizontal wires 950, connecting the individual wells 200 in rows A-H and columns 1-12. The plate electrical connector 902 (or voltage / current source 904) may include switches or other electrical connection devices that selectively establish electrical connections to the vertical wires 952 and horizontal wires 950. Thus, one or more wells 200 in one of rows A-H or one of columns 1-12 may be individually activated, e.g., with a voltage and / or current provided by the voltage / current source 904, as illustrated in FIG. 9B . Similarly, one or more wells 200 in one of rows A-H or one of columns 1-12 may be individually read simultaneously, e.g., with a signal read by the detector 910. In this example, the one or more wells 200 to be individually activated are selected based on the index of one or more wells 200, e.g., well A1, well A2, etc.

[0127] In some embodiments, the plate electrical connector 902 may be configured to allow one or more working electrode zones 104 and / or one or more auxiliary electrodes 102 to be activated simultaneously. In some embodiments, the plate electrical connector 902 may be configured to allow one or more of the auxiliary electrodes 102 and / or working electrode zones 104 in each well 200 to be selectively addressable, e.g., to allow voltage and / or current to be selectively applied to individual ones of the auxiliary electrodes 102 and / or working electrode zones 104 and a signal to be read from the detector 910. Similar to the wells 200 described above, for each well 200, the one or more working electrode zones 104 may include separate electrical contacts that allow the plate electrical connector 902 to be electrically connected to each of the one or more working electrode zones 104 of the well 200. Similarly, for each well 200, the one or more auxiliary electrodes 102 may include separate electrical contacts that allow the plate electrical connector 902 to be electrically connected to each of the one or more auxiliary electrodes 102 of the well 200.

[0128] Although not shown, the plate electrical connector 902 (or other component of the assay device 900) may include any number of electrical components, e.g., electrical wires, switches, multiplexers, transistors, etc., to allow particular wells 200, auxiliary electrodes 102, and / or working electrode zones 104 to be selectively electrically coupled to the voltage / current source 904 to allow voltage and / or current to be selectively applied. Similarly, although not shown, the plate electrical connector 902 (or other component of the assay device 900) may include any number of electrical components, e.g., electrical wires, switches, multiplexers, transistors, etc., to allow particular wells 200, auxiliary electrodes 102, and / or working electrode zones 104 to selectively read signals from the detector 910.

[0129] To control the voltage and / or current supplied, in certain embodiments, one or more computer systems 906 may be coupled to the voltage / current sources 904. In other embodiments, the voltage / current sources 904 may supply the potential and / or current without the assistance of a computer system, for example, manually. The computer system 906 may be configured to control the voltage and / or current supplied to the wells 200. Similarly, in embodiments, the computer system 906 may be utilized to store, analyze, display, transmit, etc., data measured during electrochemical processes and procedures.

[0130] The multiwell plate 208 may be housed within a housing 908. The housing 908 may be configured to support and house the components of the assay device 900. In some embodiments, the housing 908 may be configured to maintain experimental conditions (e.g., airtight, light-tight, etc.) to accommodate operation of the assay device 900.

[0131] In embodiments, the assay device 900 may include one or more detectors 910 that measure, capture, store, analyze, etc., data related to the electrochemical processes and procedures of the assay device 900. For example, the detectors 910 may include a photodetector 912 (e.g., a camera, photodiode, etc.), a voltmeter, an ammeter, a potentiometer, a temperature sensor, etc. In some embodiments, one or more of the detectors 910 may be integrated into other components of the assay device 900, such as the plate electrical connector 902, the voltage and current source 904, the computer system 906, the housing 908, etc. In some embodiments, one or more of the detectors 910 may be integrated into the multiwell plate 208. For example, one or more heaters, temperature controllers, and / or temperature sensors may be integrated into the electrode design of each of the wells 200, as described below.

[0132] In embodiments, the one or more photodetectors 912 may be, for example, film, photomultiplier tubes, photodiodes, avalanche photodiodes, charge-coupled devices ("CCDs"), or other photodetectors or cameras. The one or more photodetectors 912 may be a single detector for detecting sequential emissions, or may include multiple detectors and / or sensors for detecting and spatially resolving simultaneous emissions at single or multiple wavelengths of emitted light. The emitted and detected light may be visible light or may be emitted as invisible radiation, such as infrared or ultraviolet radiation. The one or more photodetectors 912 may be fixed or movable. The emitted light or other radiation may be steered or modified during passage to the one or more photodetectors 912 using, for example, lenses, mirrors, and fiber optic light guides or light conduits (single, multiple, fixed, or movable) positioned on or adjacent to any component of the multiwell plate 208. In some embodiments, the surfaces of the working electrode zone 104 and / or auxiliary electrode 102 may themselves be utilized to guide or enable the transmission of light.

[0133] As discussed above, in embodiments, multiple detectors can be used to detect and resolve the simultaneous emission of various optical signals. In addition to the examples already provided herein, the detectors can include one or more beam splitters, mirror lenses (e.g., 50% silvered mirrors), and / or other devices for transmitting optical signals to two or more different detectors (e.g., multiple cameras, etc.). These multiple-detector embodiments can include, for example, setting one detector (e.g., a camera) in a high-gain configuration to capture and quantify low-power signals, while setting the other in a low-gain configuration to capture and quantify high-power signals. In embodiments, the high-power signal can be 2x, 5x, 10x, 100x, 1000x, or more times more powerful than the low-power signal. Other examples are contemplated as well.

[0134] Referring to the beam splitter example described above, a beam splitter with a particular ratio (e.g., a 90:10 ratio for two sensors, although other ratios and / or numbers of sensors are also contemplated) can be used to detect and resolve the emitted light. In this 90:10 example, 90% of the incident light may be directed to a first sensor using a high-gain configuration for low light levels, and the remaining 10% may be directed to a second sensor using a low-gain configuration for high light levels. In embodiments, the loss of 10% of light to the first sensor can be compensated (at least in part) based on various factors, e.g., the selected sensor / sensor technology, binning techniques, etc., to reduce noise.

[0135] In some embodiments, each sensor may be the same type (e.g., CCD / CMOS), while in other embodiments, different types may be employed (e.g., the first sensor may be a high-sensitivity, high-performance CCD / CMOS sensor, while the second sensor may be a lower-cost CCD / CMOS sensor). In other examples (e.g., with larger sized sensors), the light may be split so that 90% of the signal is imaged onto one half of the sensor and the remaining 10% is imaged onto the other half (e.g., 90 / 10 as described above, but other ratios are also possible). The dynamic range can be further expanded by optimizing the optics of this technique, for example, by using multiple sensors to achieve a 99:1 ratio, with one sensor (e.g., a camera) being highly sensitive within the first dynamic range and the second sensor starting with its lowest sensitivity higher than the first. When properly optimized, the amount of light each receives can be maximized, thus improving overall sensitivity. In these examples, techniques for minimizing and / or eliminating crosstalk may be employed, for example, by energizing the working electrode zones in a sequential manner. Advantages provided by these examples include simultaneous detection of low and high light levels, which can eliminate the need for dual excitation (e.g., multi-pulse techniques) and therefore reduce and / or otherwise improve ECL read times.

[0136] In embodiments, the one or more photodetectors 912 may include one or more cameras (e.g., charge-coupled device (CCD), complementary metal-oxide semiconductor (CMOS) image sensor, etc.) that capture images of the wells 200 to capture photons emitted during operation of the assay device 900. In some embodiments, the one or more photodetectors 912 may include a single camera that captures images of all wells 200 in the multiwell plate 208, a single camera that captures images of a subset of the wells 200, multiple cameras that capture images of all of the wells 200, or multiple cameras that capture images of a subset of the wells 200. In some embodiments, each well 200 in the multiwell plate 200 may include a camera that captures images of the well 200. In some embodiments, each well 200 in the multiwell plate 200 may include multiple cameras that capture images of a single working electrode zone 104 or a subset of the working electrode zones 104 within each well 200. In any embodiment, the computer system 906 may include hardware, software, and combinations thereof, including logic for analyzing images captured by one or more photodetectors 912 and extracting luminance data for performing ECL analysis. In some embodiments, the computer system 906 may include hardware, software, and combinations thereof, including logic for segmenting and enhancing an image, for example, if the image includes data for multiple wells 200, multiple working electrode zones 104, etc., to focus on a portion of the image that includes one or more of the wells 200, one or more of the working electrode zones 104, etc. Thus, the assay device 900 can provide flexibility, as the photodetector 912 can capture all light from multiple working electrode zones 104, and the computer system 906 can use imaging processing to resolve the luminescence data for each working electrode zone 104.In this manner, the assay device 900 can operate in various modes, such as singleplex mode (e.g., one working electrode zone), 10plex mode (e.g., all working electrode zones 104 in a 10 working electrode zone well 200), or a general multiplex mode (e.g., a subset of all working electrode zones within a single well 200 or across multiple wells 200 simultaneously, e.g., including five working electrode zones 104 for multiple 10 working electrode zone wells simultaneously).

[0137] In some embodiments, the one or more photodetectors 912 may include one or more photodiodes for detecting and measuring photons emitted during chemiluminescence. In some embodiments, each well 200 of the multiwell plate 200 may include a photodiode for detecting and measuring photons emitted within the well 200. In some embodiments, each well 200 of the multiwell plate 200 may include multiple photodiodes for detecting and measuring photons emitted from a single working electrode zone 104 or a subset of working electrode zones 104 within each well 200. Thus, the assay device 900 can operate in various modes. For example, in a sequential or "time-resolved" mode, the assay device 900 may apply a voltage and / or current to the five working electrode zones 104 individually. The photodiodes may then sequentially detect / measure the light coming from each of the five working electrode zones 104. For example, a voltage and / or current may be applied to the first of the five working electrode zones 104, and the emitted photons may be detected and measured by the corresponding photodiode. This may be repeated sequentially for each of the five working electrode zones 104. Similarly, in this example, the sequential mode of operation may be performed for working electrode zones 104 located in the same well 200, for working electrode zones 104 located in different wells 200, for working electrode zones 104 located in subsets or "sectors" of multiple wells 200, or combinations thereof. Similarly, in some embodiments, the assay device 900 may operate in a multiplex mode in which one or more working electrode zones 104 are simultaneously activated by application of voltage and / or current, and the emitted photons are detected and measured by multiple photodiodes for multiplexing. The multiplex mode of operation may be performed for working electrode zones 104 located in the same well 200, for working electrode zones 104 located in different wells 200, for working electrode zones 104 located in subsets or "sectors" of wells 200 in a multiwell plate 208, or combinations thereof.

[0138] In the above-described embodiment, the working electrode zone 104 undergoes a natural decay in the intensity of emitted photons after the voltage applied to the working electrode zone 104 is removed. That is, when a voltage is applied to the working electrode zone 104, a redox reaction occurs and photons are emitted at an intensity determined by the applied voltage and the substance undergoing the redox reaction. When the voltage is removed, the substance undergoing the redox reaction continues to emit photons at a decaying intensity for a period based on the chemical properties of the substance. In this manner, when the working electrode zones 104 are activated sequentially, the assay device 900 (e.g., computer system 906) can be configured to implement a delay in activating successive working electrode zones 104. The assay device 900 (e.g., computer system 906) can determine and implement a delay in activating successive working electrode zones 104 to prevent photons from a previously fired working electrode zone 104 from interfering with photons emitted from a currently activated working electrode zone 104. For example, Figure 10A shows the ECL decay during various voltage pulses, and Figure 10B shows the ECL decay time using a 50 ms pulse. In the example of Figure 10B, intensity data was determined by taking multiple images during and after a 50 ms long, 1800 mV voltage pulse. To improve time resolution, an image frame (or photon detection) was taken every 17 ms. The 50 ms voltage pulse was imaged in three frames (e.g., images 1-3, 17 ms x 3 = 51 ms), as shown in Figure 10B. Any photon, e.g., ECL signal, emitted after Image 3 is due to a decay in photon (e.g., ECL) intensity after the working electrode zone 104 is turned off. In Figure 10B, Image 4 captures additional ECL signal after the working electrode zone 104 is turned off, indicating that there may be some small luminescence chemistry continuing after the driving force (e.g., applied voltage potential) for this chemistry is deactivated. That is, since the working electrode zone 104 switches to 0 mV 1 ms after the end of the 1800 mV voltage pulse, polarization effects are unlikely to affect the delay.In embodiments, the assay device 900 (e.g., computer system 906) may be configured to utilize such data regarding different voltage pulses and delay activation of successive working electrode zones 104. In this manner, implementing delays enables the assay device 900 to minimize crosstalk between working electrode zones 104 and / or wells 200, have high throughput when performing ECL operations, etc.

[0139] In any embodiment, the use of one or more auxiliary electrodes 102 improves the operation of the assay device 900. In some embodiments, the use of one or more auxiliary electrodes 102 improves the read time of the detector 910. For example, using Ag / AgCl for one or more auxiliary electrodes 102 improves ECL read time for several reasons. For example, the use of an electrode (e.g., auxiliary electrode 102) having a redox couple (Ag / AgCl in this particular embodiment) may provide a stable interfacial potential, allowing the electrochemical analysis process to use voltage pulses rather than voltage ramps. The use of voltage pulses improves read time because the entire pulse waveform can be applied at a voltage potential that generates ECL throughout the entire duration of the waveform. Tables 7 and 8 below include improved read times (in seconds) for various configurations of the assay device 900 using one or more auxiliary electrodes 102. The examples in these tables are the total read time for all wells of a 96-well plate (where each well contains a single working electrode (or single working electrode zone) or ten working electrodes (or ten working electrode zones)). For these read times, analysis was performed on all working electrodes (or working electrode zones) from all 96 wells (either one or ten, depending on the experiment). In Table 7 below, "spatial" refers to the mode of operation in which all working electrode zones 104 are activated simultaneously, and images are captured and processed for resolution. "Time-resolved" refers to the sequential mode, as described above. Time resolution has the added advantage of allowing adjustments to ECL image collection (e.g., adjusting binning to adjust the dynamic range). The "Current Plate RT" column contains the read time of the non-auxiliary electrode (e.g., carbon electrode). The last three columns of the table contain the difference between the read time of the non-auxiliary electrode and the read time of the auxiliary electrode (e.g., Ag / AgCl). For time-resolved measurements (using these examples with 10 working electrode zones per well in both Tables 7 and 8), the subplex read time falls between the 1 working electrode zone (WE) and 10 WE read times. For the "B" experiment, the read time improvement was not calculated because the non-auxiliary electrode plate cannot be operated in time-resolved mode.Table 8 contains similar data where the assay device 900 includes a photodiode, as described above. Those skilled in the art will understand that the values ​​contained in Tables 7 and 8 are approximate and may vary by + / - 5.0% based on conditions such as the operating conditions and parameters of the assay device.

[0140] [Table 17]

[0141] [Table 18]

[0142] For Tables 7 and 8, "WE" can refer to either the working electrode or the working electrode zone.

[0143] In contrast, with voltage ramps in ECL applications, there are periods during which voltage is applied but ECL is not generated (e.g., at a portion of the beginning of the ramp and / or at a portion of the end of the ramp). For example, as described in more detail below, FIGS. 29 and 30 (using carbon-based and Ag / AgCl-based electrodes, respectively) illustrate a 3-second ramp time (1.0 V / s) applied to the electrodes. With this waveform, there are periods during which ECL is not generated despite the applied potential. In other words, when applying a ramp waveform, there is a percentage (e.g., 5%, 10%, 15%, etc.) of the total waveform duration during which ECL is not generated while the potential is applied. These percentages can vary based on several factors, including the type of material used to form the electrodes, the relative and absolute size of the electrodes, etc. FIGS. 29 and 30 show non-limiting, representative examples of specific percentages during which ECL was not generated for this particular ramp waveform.

[0144] In any of the above-described embodiments, the use of working electrode zones 104 with different sizes and configurations provides various advantages to the assay device 900. For ECL applications, the optimal working electrode size and location may depend on the exact nature of the application and the type of photodetector used to detect ECL. In binding assays using binding reagents immobilized on the working electrode, binding capacity and binding efficiency and speed generally increase with increasing working electrode zone size. In ECL instruments using imaging detectors (e.g., CCD or CMOS devices), the benefits of a larger working electrode zone for binding capacity and efficiency can be balanced by the improved sensitivity of these devices in terms of total photon counts when light is generated in a smaller working electrode zone and imaged on fewer imaging device pixels. The location of the working electrode zone 104 can affect the performance of the assay device 900. In some embodiments, the spot location, size, and geometry affect the amount of photon reflection, scattering, or loss on the well sidewalls, which can affect both the amount of desired light detected and the amount of undesired light (e.g., stray light from adjacent working electrode zones or wells) detected as coming from the working electrode zone of interest. In some embodiments, the performance of the assay device 900 may be improved by having a design that does not have a working electrode zone 104 located at the center of the well 200, and by having working electrode zones 104 located at a uniform distance from the center of the well 200. In some embodiments, one or more working electrode zones 104 located at radially symmetric positions within the well 200 can improve the operation of the assay device 900 because, as described above, the optical focusing and meniscus interaction are the same for all of the one or more working electrode zones 104 within the well 200. One or more working electrode zones 104 located at a fixed distance (e.g., in a circular pattern) allows the assay device to utilize shortened pulse waveforms, e.g., reduced pulse widths. In embodiments, a design in which one or more working electrode zones 104 have their nearest neighbor as one or more auxiliary electrodes 102 (e.g., no intervening working electrode zone between them) improves the performance of the assay device 900.

[0145] In embodiments, as briefly described above, the assay device 900 (e.g., computer system 906) may be configured to control the voltage / current source 904 to provide a voltage and / or current with a pulsed waveform, e.g., direct current, alternating current, or direct current that mimics alternating current, although other waveforms of various periods, frequencies, and amplitudes are contemplated as well (e.g., negative ramp sawtooth waveforms, square waveforms, rectangular waveforms, etc.). These waveforms may also include various duty cycles, e.g., 10%, 20%, 50%, 65%, 90%, or any other percentage between 0 and 100. The computer system 906 may selectively control the magnitude and duration of the pulse waveform, as described in more detail below. In one embodiment, as described above, the computer system 906 may be configured to selectively provide a pulsed waveform to one or more of the wells 200. For example, the voltage and / or current may be provided to all of the wells 200. Similarly, for example, the pulse waveform may be provided to selected wells 200 (e.g., individually or in sectors, such as groupings of subsets of wells, e.g., 4, 16, etc.). For example, as described above, the wells 200 may be individually addressable or may be addressable in groups or subsets of two or more wells. In one embodiment, the computer system 906 may also be configured to selectively provide the pulse waveform to one or more of the working electrode zones 104 and / or auxiliary electrodes 102 in the manner described above (e.g., individually addressable or addressable in groups of two or more auxiliary electrodes). For example, the pulse waveform may be provided to all working electrode zones 104 in the well 200 and / or addressed to one or more selected working electrode zones 104 in the well 200. Similarly, for example, the pulse waveform may be provided to all auxiliary electrodes 102 and / or addressed to one or more selected auxiliary electrodes 102.

[0146] In embodiments, the pulse waveform provided by the voltage / current source 904 may be designed to improve the electrochemical analysis and procedure of the assay device 900. Figure 11 shows a flowchart illustrating a process 1100 for operating an assay device using a pulse waveform, according to one embodiment herein.

[0147] In operation 1102, the process 1100 includes applying a voltage pulse to one or more working electrode zones 104 or one or more auxiliary electrodes 102 in the well. For example, the computer system 906 can control the voltage / current source 904 to provide the voltage pulse to one or more working electrode zones 104 or one or more auxiliary electrodes 102.

[0148] In embodiments, the pulse waveform may include various waveform types, such as direct current, alternating current, or direct current simulating alternating current, although other waveforms of different durations, frequencies, and amplitudes are also contemplated (e.g., negative ramp sawtooth waveforms, square waveforms, rectangular waveforms, etc.). These waveforms may also include various duty cycles, such as 10%, 20%, 50%, 65%, 90%, or any other percentage between 0 and 100. Figures 12A and 12B show two example pulse waveforms. As shown in Figure 12A, the pulse waveform may be a square wave having a voltage V for a time T. Examples of voltage pulses are also described with reference to Figures 14A, 14B, 15A-15L, 16, and 17, e.g., 1800 mV in 500 ms, 2000 mV in 500 ms, 2200 mV in 500 ms, 2400 mV in 500 ms, 1800 mV in 100 ms, 2000 mV in 100 ms, 2200 mV in 100 ms, 2400 mV in 50 ms, 1800 mV in 50 ms, 2000 mV in 50 ms, 2200 mV in 50 ms, 2400 mV in 50 ms, etc. As shown in Figure 17, the pulse waveform may be a combination of two types of waveforms, e.g., a square wave modulated with a sine wave. The resulting ECL signal also modulates at the sinusoidal frequency; therefore, the assay device 900 may include a filter or lock-in circuit to focus on the ECL signal exhibiting the sinusoidal frequency and to filter out electronic noise or stray light that does not exhibit the sinusoidal frequency. While Figures 12A and 12B show example pulse waveforms, those skilled in the art will understand that the pulse waveform may have any structure in which the potential rises to a predetermined voltage (or voltage range) over a predetermined period of time. Those skilled in the art will understand that the voltage pulse and pulse waveform parameters described herein (e.g., duration, duty cycle, and pulse height in volts) are approximate and may vary, for example, by + / - 5.0%, based on conditions such as the operating parameters of the voltage / current source.

[0149] In operation 1104, the process 1100 includes measuring a potential difference between one or more working electrode zones 104 and one or more auxiliary electrodes 102. For example, the detector 910 can measure a potential difference between the working electrode zone 104 and the auxiliary electrode 102 in the well 200. In some embodiments, the detector 910 can provide the measurement data to the computer system 1506.

[0150] In operation 1106, process 1100 includes performing an analysis based on the measured potential difference and other data. For example, computer system 906 can perform an analysis on the potential difference and other data. The analysis can be any process or procedure, such as potentiometry, coulometry, voltammetry, optical analysis (described further below), etc. In embodiments, the use of pulse waveforms allows for specific types of analysis to be performed. For example, there may be a number of different redox reactions occurring in the sample that are activated when the applied potential exceeds a certain level. By using pulse waveforms of specified voltages, assay device 900 can selectively activate some of these redox reactions and not others.

[0151] In one embodiment, the disclosure provided herein can be applied to a method for performing an ECL assay.Specific examples of methods for performing an ECL assay are provided in U.S. Patent Nos. 5,591,581, 5,641,623, 5,643,713, 5,705,402, 6,066,448, 6,165,708, 6,207,369, 6214552, and 7842246, and published PCT applications Nos. 87 / 06706 and 98 / 12539, which are incorporated herein by reference.

[0152] In embodiments, the pulse waveform provided by voltage / current source 904 can be designed to improve the ECL emitted during ECL analysis. For example, the pulse waveform can improve the ECL emitted during ECL analysis by providing a stable, constant potential, thereby producing a stable, predictable ECL emission. Figure 13 shows a flowchart illustrating a process 1300 for operating an ECL device using a pulse waveform, according to one embodiment of the present disclosure.

[0153] In operation 1302, the process 1300 includes applying a voltage pulse to one or more working electrode zones 104 or auxiliary electrodes 102 in a well of the ECL device. For example, the computer system 906 can control the voltage / current source 904 to provide the voltage pulse to one or more working electrode zones 104 or one or more auxiliary electrodes 102. In embodiments, the one or more auxiliary electrodes 102 may include a redox couple, and when a voltage or potential is applied, the reaction of species in the redox couple is the predominant redox reaction occurring at the one or more auxiliary electrodes 102. In some embodiments, the applied potential is less than the defined potential required to reduce water or perform water electrolysis. In some embodiments, less than 1 percent of the current is associated with water reduction. In some embodiments, less than 1 current per unit area (exposed surface area) of the one or more auxiliary electrodes 102 is associated with water reduction.

[0154] In embodiments, the pulse waveform may include various waveform types, such as DC, AC, DC simulating AC, etc., although other waveforms of different durations, frequencies, and amplitudes are also contemplated (e.g., negative ramp sawtooth waveforms, square waveforms, rectangular waveforms, etc.). Figures 12A and 12B above show two examples of pulse waveforms. The pulse waveform may be a square wave having a voltage V for a time T. Examples of voltage pulses are also described with reference to Figures 14A, 14B, 15A-15L, 16, and 17, e.g., 1800 mV in 500 ms, 2000 mV in 500 ms, 2200 mV in 500 ms, 2400 mV in 500 ms, 1800 mV in 100 ms, 2000 mV in 100 ms, 2200 mV in 100 ms, 2400 mV in 100 ms, 1800 mV in 50 ms, 2000 mV in 50 ms, 2200 mV in 50 ms, 2400 mV in 50 ms, etc. These waveforms may include various duty cycles, e.g., 10%, 20%, 50%, 65%, 90%, or any other percentage between 0 and 100.

[0155] In operation 1304, the process 1300 includes capturing luminescence data from the electrochemical cell over a period of time. For example, one or more photodetectors 912 may capture luminescence data emitted from the wells 200 and transmit the luminescence data to the computer system 906. In one embodiment, the period of time may be selected to allow the photodetectors to collect ECL data. In some embodiments, the one or more photodetectors 912 may include a single camera that captures images of all wells 200 in the multiwell plate 208, or multiple cameras that capture images of a subset of the wells 200. In some embodiments, each well 200 in the multiwell plate 200 may include a camera that captures images of the well 200. In some embodiments, each well 200 in the multiwell plate 200 may include multiple cameras that capture images of a single working electrode zone 104 or a subset of the working electrode zones 104 within each well 200. Thus, the assay device 900 can provide flexibility because the camera can capture all light from multiple working electrode zones 104 and the computer system 906 can use imaging processing to resolve the luminescence data for each working electrode zone 104. In this manner, the assay device 900 can operate in a variety of modes, such as singleplex mode (e.g., one working electrode zone), 10plex mode (e.g., all working electrode zones 104 in a 10 working electrode zone well 200), or a general multiplex mode (e.g., a subset of all working electrode zones within a single well 200 or across multiple wells 200 simultaneously, e.g., including five working electrode zones 104 for multiple 10 working electrode zone wells simultaneously).

[0156] In some embodiments, the assay device 900 may include a photodiode corresponding to each well 200 of the multiwell plate 200 to detect and measure photons emitted within the well 200. In some embodiments, the assay device 900 may include multiple photodiodes corresponding to each well 200 of the multiwell plate 200 to detect and measure photons emitted from a single working electrode zone 104 or a subset of working electrode zones 104 within each well 200. Thus, the assay device 900 can operate in various modes. For example, the assay device 900 may apply voltage and / or current individually to one or more, e.g., five, working electrode zones 104 from the multiwell plate 208. The working electrode zones 104 may be located within a single well 200, different wells 200, or a combination thereof. The photodiodes may then sequentially detect / measure the light coming from each of the five working electrode zones 104. For example, a voltage and / or current may be applied to a first of five working electrode zones 104, and the emitted photons may be detected and measured by a corresponding photodiode. This may be repeated sequentially for each of the five working electrode zones 104. Similarly, in this example, the sequential mode of operation may be performed for working electrode zones 104 located in the same well 200, for working electrode zones 104 located in different wells 200, for working electrode zones 104 located in subsets or "sectors" of wells 200, or combinations thereof. Similarly, in some embodiments, the assay device 900 may operate in a multiplex mode, in which one or more working electrode zones 104 are activated simultaneously by application of a voltage and / or current, and the emitted photons may be detected and measured by multiple photodiodes for multiplexing.The multiplexed mode of operation may be performed for working electrode zones 104 within the same well 200, for working electrode zones 104 located in different wells 200, for working electrode zones 104 located in subsets or "sectors" of wells 200 in a multiwell plate 208, or any combination thereof. Figures 14A, 14B, 15A-15L, 16, and 17 below show tests of several waveforms used in ECL analysis.

[0157] In embodiments, by applying a pulse waveform to generate ECL, read times and / or exposure times may be improved, resulting in faster and more efficient ECL data generation, collection, observation, and analysis. Furthermore, as described above, the use of one or more auxiliary electrodes 102 improves the operation of the assay device 900. Also, various exposure approaches (e.g., single exposure, double exposure, triple exposure (or more)) that can use different (or equal) exposure times may be used to improve ECL collection, acquisition, observation, and analysis, for example, by improving dynamic range extension (DRE), binning, etc. In some embodiments, the use of one or more auxiliary electrodes 102 improves the read time of the detector 910. For example, the use of Ag / AgCl in one or more auxiliary electrodes 102 improves ECL read times for several reasons. For example, the use of an electrode (e.g., auxiliary electrode 102) with a redox couple (Ag / AgCl in this particular embodiment) may provide a stable interfacial potential, allowing the electrochemical analysis process to use voltage pulses rather than voltage ramps. The use of voltage pulses improves read times because the entire pulse waveform can be applied at a voltage potential that generates ECL throughout the entire waveform. The "time-resolved" or sequential mode also has the added benefit of allowing adjustments to ECL image collection (e.g., adjusting binning to adjust dynamic range). Furthermore, as described above, the assay device 900 (e.g., computer system 906) can be configured to delay activation of successive working electrode zones 104 using such data regarding different voltage pulses. In this manner, implementing delays allows the assay device 900 to minimize crosstalk between working electrode zones 104 and / or wells 200, have high throughput when performing ECL operations, and the like.

[0158] In operation 1306, the process 1300 includes performing an ECL analysis on the luminescence data. For example, the computer system 906 can perform the ECL analysis on the luminescence data. In some embodiments, the luminescence data, e.g., signal, resulting from a given target entity on the binding surface, e.g., binding domain, of the working electrode zone 104 and / or auxiliary electrode 102 may have a range of values. These values ​​can be correlated with a quantitative measurement (e.g., ECL intensity) to provide an analog signal. In other embodiments, a digital signal (signal present or absent) can be obtained from each working electrode zone 104 to indicate either the presence or absence of an analyte. Statistical analysis can be used for both techniques or to convert multiple digital signals to provide a quantitative result. Some analytes may require a digital presence / absence signal indicating a threshold concentration. Analog and / or digital formats can be utilized separately or in combination. Other statistical methods, such as techniques for determining concentration through statistical analysis of binding across a concentration gradient, can also be utilized. Multiple linear arrays of data with a concentration gradient can be generated using multiple different specific binding reagents used in different wells 200 and / or different working electrode zones 104. The concentration gradient can consist of distinct binding domains that present different concentrations of binding reagent.

[0159] In embodiments, a control assay solution or reagent, such as a read buffer, may be utilized on the working electrode zone of the well 200. The control assay solution or reagent may provide uniformity for each analysis to control for signal variations (e.g., variations due to degradation, perturbations, aging, thermal shifts, noise in the electronic circuitry, and noise in the photodetection device of the multiwell plate 208). For example, multiple overlapping working electrode zones 104 (containing the same binding reagent or different binding reagents specific to the same analyte) may be used for the same analyte. In another example, a known concentration of analyte may be utilized, or a control assay solution or reagent may be covalently bound to a known amount of ECL label, or a known amount of ECL label in solution may be used.

[0160] In embodiments, the data collected and generated in process 1300 can be utilized in a variety of applications. The collected and generated data can be stored, for example, in the form of a database consisting of a collection of clinical or research information. The collected and generated data can also be used for rapid forensic or personal identification. For example, the use of multiple nucleic acid probes when exposed to a human DNA sample can be used for a signature DNA fingerprint that can be easily used to identify clinical or research samples. The collected and generated data can be used to identify conditions (e.g., diseases, radiation levels, etc.), the presence of organisms (e.g., bacteria, viruses, etc.), etc.

[0161] The above describes an exemplary flow of exemplary process 1300. The process shown in FIG. 13 is exemplary only, and variations exist without departing from the scope of the embodiments disclosed herein. As noted above, steps may be performed in a different order than described, additional steps may be performed, and / or fewer steps may be performed. In embodiments, the use of a pulse waveform in combination with an auxiliary electrode provides various advantages to ECL assays. The auxiliary electrode allows luminescence to be generated more quickly without the use of a lamp.

[0162] Figures 14A-14C, 15A-15L, 16, and 17 are graphs showing the results of ECL analysis using various pulse waveforms. Figures 15A-15L show raw data plotted against BTI concentration for model binding assays using various pulse waveforms. Figures 15A-15L show a comparison between the use of pulse waveforms applied to wells using an Ag / AgCl auxiliary electrode (labeled according to the pulse parameters) and the use of a ramp waveform (1 s at 1.4 V / s) applied to wells using a carbon electrode as a control (labeled control lot). Figures 14A-14C summarize the performance of model binding assays with various pulse waveforms as shown in Figures 15A-15L. Figures 16 and 17 are described in more detail below. In these tests, the model binding assay was used to measure the effect of ECL generation conditions on the amount of ECL generated from a controlled amount of ECL-labeled binding reagent bound to the working electrode zone through a specific binding interaction. In this model system, the ECL-labeled binding reagent was an IgG antibody (SULFO-TAG, Meso Scale Diagnostics, LLC) labeled with both biotin and an ECL label. Various concentrations of this binding reagent (designated "BTI" or "BTI HC" for the BTI high control) were added to the wells of a 96-well plate containing an integrated screen-printed carbon ink working electrode with an immobilized layer of streptavidin in each well. Two types of plates were used: the control plate was an MSD Gold 96-well Streptavidin QuickPlex plate (Meso Scale Diagnostics, LLC) with a screen-printed carbon ink counter electrode, and the test plate was similar in design but with a screen-printed Ag / AgCl auxiliary electrode instead of the counter electrode. The plate was incubated to allow the BTI in the wells to bind to the working electrode via the biotin-streptavidin interaction.After incubation was complete, the plates were washed to remove free BTI, ECL read buffer (MSD Read Buffer Gold, Meso Scale Diagnostics, LLC) was added, and the plates were analyzed by applying a defined voltage waveform between the working and auxiliary electrodes and measuring the emitted ECL. The Ag:AgCl ratio in the auxiliary electrode ink of the test plates was approximately 50:50. Twelve waveforms were used, using four different potentials (1800 mV, 2000 mV, 2200 mV, and 2400 mV) at three different times or pulse widths (500 ms, 100 ms, and 50 ms). One test plate was tested for each waveform. A control plate was tested using a standard ramp waveform.

[0163] Assay performance data were determined and calculated for the plates tested with each waveform. The mean, standard deviation, and %CV were calculated for each sample and plotted as data points with error bars. The signals measured for BTI solutions ranging from 0 (blank sample to measure assay background) to 2 nM were linearly fitted (slope, Y-intercept, and R 2 (where σ is the average background voltage and σ is the standard deviation) The detection limit was calculated based on the average background voltage + / - 3*standard deviation ("stdev") and the linear fit of the titration curve (shown in Figure 14C). Signals were also measured for 4, 6, and 8 nM BTI solutions. These signals were divided by the extrapolated signal from the linear fit of the titration curve (this ratio can be used to estimate the binding capacity of the streptavidin layer on the working electrode; a ratio significantly less than 1 indicates that the amount of BTI added is close to or exceeds the binding capacity). The ratio of the slope from the production control lot to the slope from each test plate was calculated. Figure 14A shows the results of these calculations for each pulse waveform. Each of the graphs in Figures 15A-15L shows the average ECL data collected for a ramp voltage applied to a multiwell plate with a carbon counter electrode from the control lot and different voltage pulses applied to a multiwell plate using an Ag / AgCl auxiliary electrode. Figures 14A-14C provide a summary of the data shown in Figures 15A-15L.

[0164] Additionally, signal, slope, background, and dark analyses (e.g., the signal resulting in the absence of ECL) were performed. Plots of the 2 nM signal (with 1stdev error bars) and slope were generated. Bar graphs of background and dark (with 1stdev error bars) and slope were generated. These results are shown in Figure 14B. As shown in Figures 14A and 14B, a pulse voltage of 1800 mV for 500 ms produces the highest average ECL reading. As shown in Figures 14A and 14B, the magnitude and / or duration of the pulse waveform affects the measured ECL signal. The change in 2 nM signal with waveform reflects the change in slope. The change in background also reflects the change in slope. The signal, background, and slope decreased with decreasing pulse duration. The signal, background, and slope decreased with increasing pulse potential. The change in signal, background, and slope with decreasing time decreased with increasing pulse potential. Simultaneous changes in signal, background, and slope with varying pulse potential and duration resulted in little change in assay sensitivity. The signal, background, and slope decreased with decreasing pulse duration. The signal, background, and slope decreased with increasing pulse potential. The change in signal, background, and slope with decreasing time decreased with increasing pulse potential. There was little change in assay sensitivity with simultaneous changes in signal, background, and slope with varying pulse potential and duration.

[0165] Titration curves were also analyzed for each pulse waveform. Plots of the average ECL signal versus BTI concentration were generated. Error bars based on 1stdev are included. Titration curves from the test plate were plotted on the primary y-axis. Titration curves were plotted on the secondary y-axis. The scale of the secondary y-axis was 0 to 90,000 counts ("cts") of detected photons. The scale of the primary y-axis was set to 90,000 divided by the slope ratio. The slope ratio for each test plate was calculated. Figures 15A-15L show the results of these calculations for each pulse waveform.

[0166] Regarding background, dark, and dark noise, the dark (1 & 2 cts) and dark noise (2 cts) were essentially unchanged for all waveform times tested. The background decreased with decreasing pulse duration. The background decreased with increasing applied pulse potential. The change in background with decreasing time decreased with increasing pulse potential. The background from 1800 mV over 50 ms was 6 ± 2 cts, just above the dark + dark noise.

[0167] As shown in Figures 15A-15L, the %CVs were comparable across all test plates and reference signals for all signals (8 replicates) except for background. The CV for background increased as the background signal approached dark and dark noise. Backgrounds above 40 cts (16 replicates) had good CVs of 55 (3.9%), 64 (5.1%), and 44 (5.4%). Below 40 cts, the CV increased to over 7%. All titrations from background to 2 nM HC were linearly fitted with R values ​​≥ 0.999.

[0168] Decreasing the highest concentration in the fitting range resulted in a smaller slope and a larger y-intercept. This indicates nonlinearity at the lower end of the titration curve (likely caused by different dilutions in the test samples). The y-intercepts for the other assays were essentially between zero and the measured background. All assays produced sublinear signals for 6 nM and 8 nM HC, and these decreases in binding capacity were similar for all assays. All assays produced a 4 nM signal within 2 stdev of the extrapolated 4 nM signal. After correction for the ratio of the production control lot slope to the test plate slope, the assay signal was within 3 stdev of the assay signal from the production control lot for 1 nM to 4 nM HC. At HC below 1 nM, the corrected signal was higher than the signal from the production control lot. At 0.0125 to 0.5 nM HC, the corrected signals from the test plate were within 3 stdev of each other. Corrected signals for assays performed in the same BTI solution were within 3 steps of each other at 0.0125 nM to 4 nM HC. As shown in the plot, the performance of assays measured at different pulse potentials and durations was within this range of variation of the performance of the control assay measured with the lamp.

[0169] As can be seen by comparing Figures 15A-15L with Figures 14A and 14B, the signal and slope decreased with decreasing pulse duration (500 ms, 100 ms, and 50 ms). The signal and slope decreased with increasing pulse potential (1800 mV, 2000 mV, 2200 mV, and 2400 mV). The change in signal and slope with decreasing pulse duration also decreased with increasing pulse potential. A correction factor (ratio of slopes) can compensate for signal changes with waveform changes. The calculated detection limits were similar for 11 of these waveforms (0.005 nM to 0.009 nM). The calculated detection limit for the 1800 mV, 500 ms pulse waveform was lower (0.0004 nM), likely due to subtle differences in the fit and measured background (CV).

[0170] Example 1 - ECL measurement equipment Referring now in detail to Figures 14A-14C, ECL measurements were performed in a 96-well plate specially configured for ECL assay applications by including integrated screen-printed electrodes. While the basic structure of the plate is similar to that described in U.S. Patent No. 7,842,246 (see, e.g., the description of Plates B, C, D, and E in Example 6.1), this design has been modified to incorporate the novel elements of the present disclosure. As with the previous design, the bottom of the wells is defined by a Mylar sheet with screen-printed electrodes on its top surface, providing integrated working and counter electrode surfaces (or, in some embodiments of the present invention, novel working and auxiliary electrodes) within each well. A patterned, screen-printed dielectric ink layer printed on top of the working electrode defines one or more exposed working electrode zones within each well. Conductive through-holes extending through the Mylar to screen-printed electrical contacts on the bottom surface of the Mylar sheet provide the electrical contacts necessary to connect an external electrical energy source to the electrodes.

[0171] ECL measurements on specially constructed plates were performed using an ECL plate reader designed to accept the plate, contact electrical contacts on the plate, apply electrical energy to the contacts, and image the ECL generated in the wells. For some measurements, modified software was used to allow customization of the timing and shape of the applied voltage waveform.

[0172] Exemplary plate readers include the MESO SECTOR S600 (www.mesoscale.com / en / products_and_services / instrumentation / sector_s_600) and the MESO QUICKPLEX SQ120 (www.mesoscale.com / en / products_and_services / instrumentation / quickplex_sq_120), both available from Meso Scale Diagnostics, LLC. Plate readers are described in U.S. Patent No. 6,977,722, issued December 20, 2005, and International Patent Application No. PCT / US2020 / 042104, entitled "Assay Apparatuses, Methods and Reagents," filed July 15, 2020, by Krivoy et al., each of which is incorporated herein by reference in its entirety. Other example devices are described in U.S. patent application Ser. No. 16 / 513,526, entitled "Graphical User Interface System," by Wohlstadter et al., filed July 16, 2019, and U.S. patent application Ser. No. 16 / 929,757, entitled "Assay Apparatuses, Methods and Reagents," by Krivoy et al., filed July 15, 2020, each of which is incorporated herein by reference in its entirety.

[0173] Example 2 - Fast pulse ECL measurement A model binding assay was used to demonstrate the use of a fast-pulse voltage waveform in combination with an Ag / AgCl auxiliary electrode to generate an ECL signal and to compare it with the performance observed with the conventional combination of a slow voltage ramp and a carbon counter electrode. The model binding assay was performed in a 96-well plate, where each well contained an integrated screen-printed carbon ink working electrode region supporting an immobilized layer of streptavidin. These screen-printed plates either contained a screen-printed carbon ink counter electrode (MSD Gold 96-Well Streptavidin Plate, Meso Scale Diagnostics, LLC.) or a plate with a similar electrode design except for the use of a screen-printed Ag / AgCl ink auxiliary electrode. In this model system, the ECL-labeled binding reagent was an IgG antibody (SULFO-TAG, Meso Scale Diagnostics, LLC) labeled with both biotin and an ECL label. Various concentrations of this binding reagent (referred to as "BTI" or "BTI HC" for the BTI high control) in 50 μL aliquots were added to the wells of the 96-well plate. The binding reagent was incubated in the wells with shaking for a time sufficient to deplete it from the assay solution by binding to the immobilized streptavidin on the working electrode. The plate was washed to remove the assay solution and then filled with ECL read buffer (MSD Read Buffer T2X, Meso Scale Diagnostics, LLC.). A standard waveform (100 ms ramp from 3200 mV to 4600 mV) was applied to the plate with the counter electrode. Twelve constant-voltage pulse waveforms at four different potentials (1800 mV, 2000 mV, 2200 mV, 2400 mV) and three different times or pulse widths (500 ms, 100 ms, and 50 ms) were evaluated on the plate with the Ag / AgCl auxiliary electrode. One plate was tested for each waveform. Figures 14A, 14B, and 15A-15L are graphs showing the results of the ECL analysis from this study.

[0174] Assay performance data were determined and calculated for the plates tested with each waveform. The mean, standard deviation, and %CV were calculated for each sample. Figures 15A-15L show plots of mean signal versus concentration of binding reagent, with the signal from the standard waveform plotted on a different y-axis than the signal from the potential pulse. Data points in the lower linear region of the plot, i.e., BTI concentrations ranging from 0 (blank sample to measure assay background) to 0.1 nM, were fitted with a line to determine the slope, standard error of the slope, y-intercept, standard error of the y-intercept, and R 2 All linear fits were performed using the R 2 Values ​​≥ 0.999 were obtained. Figures 14A and 14B show the 2 nM mean signal, 0 nM (assay background), and mean dark signal (empty wells) for each test condition, along with 1stdev error bars. Both figures also show the calculated slope for each condition. The detection limit, provided in terms of BTI concentration, was calculated based on the mean Y-intercept of the background + 3*standard deviation ("stdev") and a linear fit of the titration curve. The standard errors in the slope and Y-intercept and the background standard deviation were propagated to the error in the detection limit. Based on the volume of BTI per well and the number of ECL labels per BTI molecule (~0.071), the detection limit could be expressed in terms of the number of moles of ECL label required to generate a detectable signal (plotted in Figure 14E).

[0175] Figures 14C and 14D show that the ECL signal from a BTI on an electrode generated by a 500 ms pulse waveform at a potential of 1800 mV is comparable in half the time to the signal generated by a conventional 1000 ms ramp waveform. Figure 14C shows that for a particular pulse potential, ECL decreases as the pulse time decreases below 500 ms, but comparison with Figure 14D shows that the corresponding decrease in assay background signal remains significantly above the camera signal for a dark image of an empty well (i.e., an image without ECL excitation). This result demonstrates that very short pulses can be used to substantially reduce the time required to perform ECL measurements while maintaining overall sensitivity.

[0176] The calculated detection limit for a standard waveform (1000 ms ramp) using a carbon counter electrode was 2.4 ± 2.6 attomoles (10 -18 Figure 14E shows that the estimated detection limits for different excitation conditions tended to increase with decreasing pulse time, but significantly less than expected from a linear relationship. For example, the estimated detection limit for a 2000 mV, 100 ms pulse was slightly less than two times higher than the detection limit for a 1000 ms ramp, but was ten times slower. In addition, the increase in detection limit with decreasing pulse time was not always statistically significant. The detection limits for the 1800 mV, 500 ms, 2000 mV, 500 ms, 2000 mV, 100 ms, and 2200 mV, 500 ms pulses using an Ag / AgCl auxiliary electrode were within the error range of the detection limit for the standard waveform (1000 ms ramp) using a carbon counter electrode.

[0177] Figure 16 shows a graph illustrating the results of an ECL assay using a pulse waveform for a read buffer solution, e.g., read buffer T. For testing, an Ag / AgCl Std 96-1 IND plate printed with 50:50 ink was used. For testing, aliquots of MSD T4x (Y0140365) were diluted with molecular-grade water to create T3x, T2x, and T1x. The Ag / AgCl Std 96-1 IND plate was filled with 150 μL aliquots of these solutions, e.g., as shown in Figure 9B, with T4x in two adjacent rows of wells 200, T3x in two adjacent rows of wells 200, T2x in two adjacent rows of wells 200, and T1x in two adjacent rows of wells 200. These solutions were allowed to soak, covered, on the bench for 15 minutes ± 0.5 minutes. One plate was measured for each of the following waveforms: 1800 mV for 100 ms, 1800 mV for 300 ms, 1800 mV for 1000 ms, and 1800 mV for 3000 ms. The mean ECL signal and mean integrated current were calculated for 24 replicates per condition, and plots of mean vs. MSD T concentration (4, 3, 2, & 1) were generated.

[0178] As shown in Figure 16, the ECL signal and integrated current increased with increasing concentration of read buffer T. The ECL signal and integrated current increased with increasing pulse duration. The read buffer ECL signal increased linearly between T1x and T3x, but not between T3x and T4x. The integrated current increased linearly between T1x and T4x.

[0179] Figure 17 shows a graph illustrating the results of another ECL assay using pulse waveforms. Testing used Ag / AgCl Std 96-1 IND plates printed with 50:50 ink. The test method described above with respect to Figures 14A and 14B was used with different, longer pulse waveforms. One plate was measured with each of the following waveforms: 1800 mV for 3000 ms, 2200 mV for 3000 ms, 2600 mV for 3000 ms, and 3000 mV for 3000 ms. The average ECL signal and average integrated current were calculated for 24 replicates per condition, and plots of the average versus read buffer T concentration (4, 3, 2, & 1) were generated.

[0180] As shown in Figure 17, the ECL signal increased with increasing concentrations of read buffer T for pulse potentials of 1800 mV, 2200 mV, and 2600 mV. For the 3000 mV pulse, the ECL signal decreased between T1x and T2x, and then ECL increased until T4x. The integrated current increased with increasing concentrations of T for all pulse potentials. The integrated current for the 2600 mV and 3000 mV pulses was somewhat linear between T1x and T3x, but at T4x, the increase in current was less than linear with the concentration of read buffer T.

[0181] Example 3 - Reduction ability of Ag / AgCl auxiliary electrode To determine the reducing capacity of the auxiliary electrode, i.e., the amount of reduced charge that can pass through the electrode while maintaining a controlled potential, an assay plate with an integrated screen-printed carbon ink working electrode (as described in Example 2) and a screen-printed Ag / AgCl auxiliary electrode was used. To assess capacity in relation to the requirements of ECL experiments using pulsed ECL measurements, a pulsed voltage waveform was applied between the working and auxiliary electrodes while the total charge passing through the auxiliary electrode in the presence of an ECL read buffer containing TPA was measured. Two types of experiments were performed. In the first experiment (shown in Figure 16), a voltage pulse (1800 mV) near the optimal potential for ECL generation was applied and held for different times (100-3000 ms). In the second experiment (Figure 17), different pulse potentials (2200-3000 mV) were held for a fixed time (3000 ms). In both experiments, tolerance to changes in concentration or co-reactants and electrolytes in the read buffer composition was assessed by testing each voltage and time condition in the presence of components of MSD read buffer T at 1x to 4x the nominal working concentration of TPA. Each point in the graph represents the average of 24 replicate measurements.

[0182] The Ag / AgCl auxiliary electrode supports the oxidation of TPA at the working electrode under the potential applied in the experiment until the charge passing through the auxiliary electrode consumes all of the accessible oxidant (AgCl) in the auxiliary electrode. Figure 16 shows that the charge passing through the auxiliary electrode using an 1800 mV pulse increases approximately linearly with pulse duration and TPA concentration, demonstrating that the electrode capacity is sufficient to support pulses as long as 3000 ms at 1800 mV, even in the presence of higher-than-typical concentrations of TPA. Figure 17 shows an experiment designed to determine the capacity of the auxiliary electrode by increasing the potential using the longest pulse (3000 ms) from Figure 16 until the charge passing through the electrode reaches its maximum value. Data points collected using a 3000 mV potential show that the charge increases linearly with the concentration of ECL read buffer up to approximately 30 mC of total charge. Around 45 mC, the total charge plateaus, indicating depletion of the oxidant in the Ag / AgCl auxiliary electrode. A charge of 30 mC is equivalent to 3.1 x 10-7 moles of oxidant in the Ag / AgCl auxiliary electrode, and a charge of 45 mC is equivalent to 4.7 x 10-7 moles of oxidant in the Ag / AgCl auxiliary electrode.

[0183] Reduction capacity tests were also conducted to determine differences in reduction capacity due to spot pattern and auxiliary electrode size. Four different spot patterns were tested using a 2600 mV, 4000 ms reduction capacity waveform and standardized test solutions. Four spot patterns were tested: a 10-spot penta pattern (Figure 5A), a 10-spot open pattern (Figure 1C), a 10-spot closed pattern (Figure 7A), and a 10-spot open trefoil pattern (Figure 4A). The results for the penta, open, closed, and open trefoil patterns are reproduced in Tables A, B, C, and D below. As shown in Tables A-C, increasing the auxiliary electrode (labeled CE) area in the three different patterns increases the total measured charge (e.g., reduction capacity). As shown in Table D, multiple tests with the same auxiliary electrode area resulted in similar measured charges. Therefore, maximizing the auxiliary electrode area can be useful for increasing the total reduction capacity of Ag / AgCl auxiliary electrodes with multiple different spot patterns.

[0184] [Table 19]

[0185] [Table 20]

[0186] [Table 21]

[0187] [Table 22]

[0188] Further experiments were conducted to determine the amount of AgCl accessible to the redox reaction under various experimental conditions. Electrodes printed with an approximately 10-micron-thick Ag / AgCl ink film were used. Different electrode portions, ranging from 0% to 100%, were exposed to the solution, and the amount of charge passed was measured. The experimental results show that the amount of charge passed increases roughly linearly with increasing percentage of the electrode exposed to the solution. This indicates that reduction does not occur strongly or at all in the electrode portions not directly exposed to the test solution. Furthermore, the total amount of charge passed through the experimental electrode (2.03E+18e-) roughly corresponds to the total amount of electrons available within the experimental electrode, based on the total amount of Ag / AgCl in the printed electrode. This indicates that at a thickness of 10 microns and 100% solution contact, all or nearly all of the available AgCl may be accessible to the redox reaction. Therefore, for films less than 10 microns thick, all or nearly all of the available AgCl may be accessible during the reduction reaction.

[0189] In embodiments, the pulse waveform provided by voltage / current source 904 can be designed to enable the ECL device to capture different luminescence data over time to improve ECL analysis. Figure 18 shows a flowchart illustrating another process 1800 for operating an ECL device with a pulse waveform, according to one embodiment of the present disclosure.

[0190] In operation 1802, process 1800 includes applying a voltage pulse to one or more working electrode zones 104 or auxiliary electrodes 102 in a well of an ECL device, the voltage pulse causing a reduction-oxidation reaction in the well. For example, computer system 906 can control voltage / current source 904 to provide one or more voltage pulses to one or more working electrode zones 104 or auxiliary electrodes 102.

[0191] In embodiments, the voltage pulses can be configured to induce a reduction-oxidation reaction between one or more working electrode zones 104 and one or more auxiliary electrodes 102. As described above, based on a predetermined chemical composition of one or more auxiliary electrodes 102 (e.g., a mixture of Ag:AgCl), one or more auxiliary electrodes 102 can act as a reference electrode for determining a potential difference with one or more working electrode zones 104 and as a counter electrode for the working electrode zones 104. For example, a predetermined chemical mixture (e.g., the ratio of elements and alloys in the chemical composition) can provide an interfacial potential during reduction of the chemical mixture such that a quantifiable amount of charge is generated through the reduction-oxidation reaction occurring within the well 200. That is, the amount of charge passed during the redox reaction can be quantified, for example, by measuring the current at the working electrode zone 104. In some embodiments, the one or more auxiliary electrodes 102 can define the total amount of charge that can pass with an applied potential difference because, as AgCl is consumed, the interfacial potential at the auxiliary electrode 102 shifts more negatively relative to the reduction potential of water. This shifts the potential of the working electrode zone 104 to a lower potential (maintaining the applied potential difference) and turns off the oxidation reaction that occurred during the reduction of AgCl.

[0192] In embodiments, the pulse waveform may include various waveform types, such as DC, AC, DC simulating AC, etc., although other waveforms of different durations, frequencies, and amplitudes are also contemplated (e.g., negative ramp sawtooth waveforms, square waveforms, rectangular waveforms, etc.). Figures 12A and 12B above show two examples of pulse waveforms. The pulse waveform may be a square wave having a voltage V for a time T. Examples of voltage pulses are also described with reference to Figures 14A, 14B, 15A-15L, 16, and 17, e.g., 1800 mV in 500 ms, 2000 mV in 500 ms, 2200 mV in 500 ms, 2400 mV in 500 ms, 1800 mV in 100 ms, 2000 mV in 100 ms, 2200 mV in 100 ms, 2400 mV in 100 ms, 1800 mV in 50 ms, 2000 mV in 50 ms, 2200 mV in 50 ms, 2400 mV in 50 ms, etc. These waveforms may include various duty cycles, e.g., 10%, 20%, 50%, 65%, 90%, or any other percentage between 0 and 100.

[0193] In operation 1804, process 1800 includes capturing first luminescence data from the first reduction-oxidation reaction over a first time period. In operation 1806, process 1800 includes capturing second luminescence data from the second reduction-oxidation reaction over a second time period, where the first time period is not equal in duration to the second time period. For example, one or more photodetectors 910 may capture first and second luminescence data emitted from well 200 and transmit the first and second luminescence data to computer system 906. For example, in one embodiment, well 200 may contain a substance of interest that requires different time periods for photodetector 912 to capture luminescence data. Thus, photodetector 912 may capture ECL data over two different time periods. For example, one of the time periods may be a short time period (e.g., a short camera exposure time of the light generated from ECL) and one of the time periods may be a longer time period. These time periods may be affected by, for example, light saturation during ECL generation. From there, depending on the captured photons, the assay device 900 may use either a long exposure, a short exposure, or a combination of the two. In some embodiments, the assay device 900 may use a long exposure, or a combination of a long and short exposure. In some embodiments, if the captured photons exceed the dynamic range of the photodetector 912, the assay device 900 may use a short exposure. By adjusting / optimizing these, the dynamic range may be increased by one or two orders of magnitude. In certain embodiments, the dynamic range may be improved by implementing various multi-pulse and / or multi-exposure schemes. For example, a short exposure may be followed by a long exposure (e.g., exposure of a single working electrode, a single working electrode zone, two or more single working electrodes or working electrode zones (within a single well or across multiple wells), exposure of a single well, two or more wells or sectors, or two or more sectors, etc.). In these instances, it may be advantageous to use a longer exposure, unless the exposure is saturated. In that case, for example, a shorter exposure may be used.By making these adjustments (e.g., manually or with the aid of hardware, firmware, software, algorithms, computer-readable media, computing devices, etc.), the dynamic range can be improved. In another example, a first short pulse (e.g., 50 ms, although other durations are contemplated) may be applied to an electrode or a set of two or more electrodes, followed by a second, longer pulse (e.g., 200 ms, although other durations are contemplated) to each electrode or set of electrodes. Another approach may include reading the entire plate (e.g., 96 wells) with one or more first short pulses (e.g., 50 ms, although other durations are contemplated), and then reading the entire plate a second time with a second, longer pulse (e.g., 200 ms, although other durations are contemplated). In another example, a long pulse may be applied first, followed by application and / or alternating application of a short pulse, multiple short pulses, and / or long pulses, etc. In addition to one or more discrete pulses, composite or hybrid functions may be used using these or other durations, for example, to determine and / or model the response in the transition region (e.g., during the transition between pulses). Further, in the above example, a longer pulse may be used first, before a shorter pulse. The waveform and / or acquisition window may also be adjusted to improve dynamic range.

[0194] Furthermore, if additional information is known about one or more individual working electrodes and / or working electrode zones (e.g., a particular working electrode zone is known to contain a high amount of analyte), this information can be used to optimize exposure times to prevent camera saturation before taking a read and / or sample. Using the example of high analyte described above, shorter exposure times can be used because the signal is expected to be high in the dynamic range (and vice versa for electrodes where a low signal is expected). Accordingly, exposure times, pulse durations, and / or pulse intensities can be customized and / or optimized for individual wells, electrodes, etc., to improve overall read times, for example. Also, pixels from one or more ROIs can be continuously sampled to obtain ECL curves over time, which can be further utilized to determine how to truncate exposure times and extrapolate ECL generation curves beyond saturation. In another example, the camera can initially be set to perform short exposures, and then the signal intensity from the short exposures can be examined. This information can then be used to adjust the binning of the final exposure. In other examples, rather than adjusting binning, other parameters, such as waveform, capture window, or other current-based techniques, can be adjusted as well.

[0195] Additional techniques in which waveform and / or exposure are held constant can also be used. For example, if the intensity of pixels within one or more ROIs is measured and pixel saturation is observed, other aspects of ECL generation and / or measurement (e.g., current-ECL correlation, dark mask techniques in which a dark mask region around the ROI is observed, which can be used to update the estimated ECL for saturated electrodes and / or portions of electrodes, etc.) can be used to optimize the readout and / or readout time. These solutions avoid the need for fast analysis and / or reaction times to adjust waveform and / or exposure durations over relatively short periods of time (e.g., milliseconds). This is because, for example, ECL generation and / or capture can be performed in the same and / or similar manner, and analysis can be performed last.

[0196] Other techniques can also be used to improve dynamic range. For example, when applied to electrochemiluminescence (ECL) applications, because ECL labels fluoresce, pre-illumination and / or pre-exposure can be performed to obtain information about how much label is present in one or more wells, working electrodes, working electrode zones, etc. Information obtained from pre-illumination and / or pre-exposure can be used to optimize exposure and / or pulse duration to achieve further improvements in dynamic range and / or read time. In other embodiments, particularly for ECL, a correlation can exist between the current and one or more of the electrodes and the ECL signal, so the signal signature can inform the camera exposure time and / or applied waveform (e.g., stop waveform, decrease waveform, increase waveform, etc.). This can be further optimized by improving the accuracy and update rate of the current measurement and optimizing the current path to provide a better correlation between the current and the ECL signal.

[0197] Additional improvements in dynamic range may be realized with respect to particular imaging devices according to certain embodiments. By using a CMOS-based imaging device for ECL applications, for example, a particular region of interest (ROI) may be sampled and read at different times within one or more exposure ranges to optimize exposure time. For example, an ROI (e.g., a portion or all of a working electrode and / or working electrode zone) may comprise a fixed or variable number of pixels or a particular sample percentage of the electrode area (e.g., 1%, 5%, 10%, etc., although other percentages are contemplated). In this example, the pixels and / or sample percentage may be read early during exposure. Depending on the signal read from the ROI, the exposure time may be adjusted and / or optimized for a particular working electrode, working electrode zone, well, etc. In a non-limiting illustrative example, a subset of pixels may be sampled over a sample period. If the signal from that subset tends to be high, the exposure time may be reduced (e.g., from 3 seconds to 1 second, although longer or shorter durations are also contemplated). Similarly, if the signal tends to be low, a longer exposure time may be used (e.g., 3 seconds, although other durations are also contemplated). These adjustments may be made manually or with the aid of hardware, firmware, software, algorithms, computer-readable media, computing devices, etc. In other embodiments, the ROIs may be selected to be distributed in a manner that avoids any possible ring effect, which may occur, for example, due to non-uniformity in the light around the working electrode zone (e.g., a bright ring around the periphery of the working electrode zone with a darker spot in the center). To address this, an ROI may be selected that samples both the brighter and darker regions (e.g., a row of pixels from edge to edge, random sampling of pixels from both regions, etc.). Furthermore, pixels may be sampled continuously for one or more working electrode zones to determine an ECL generation curve over time. This sampled data may then be used to extrapolate the ECL generation curve for points beyond saturation.

[0198] In embodiments, different pulse waveforms may be used for the first and second periods. In embodiments, the pulse waveforms may differ in amplitude (e.g., voltage), duration (e.g., period), and / or waveform type (e.g., square, sawtooth, etc.). The use of different pulse waveforms may be advantageous when multiple types of electroactive species that require different activation potentials and can emit light at different wavelengths are used as ECL labels. For example, such ECL labels may be complexes based on ruthenium, osmium, hassium, iridium, etc.

[0199] In operation 1808, process 1800 includes performing ECL analysis on the first luminescence data and the second luminescence data. For example, the computer system 906 can perform ECL analysis on the luminescence data. These values ​​can be correlated with quantitative measurements (e.g., ECL intensity) to provide an analog signal. In other embodiments, a digital signal (signal present or absent) can be obtained from each working electrode zone 104 to indicate either the presence or absence of an analyte. Statistical analysis can be used for both techniques or to convert multiple digital signals to provide a quantitative result. Some analytes may require a digital presence / absence signal indicating a threshold concentration. Analog and / or digital formats can be utilized separately or in combination. Other statistical methods, such as techniques for determining concentration through statistical analysis of binding across a concentration gradient, can be utilized. Multiple different specific binding reagents can be used in different wells 200 and / or different working electrode zones 104 to generate multiple linear arrays of data with a concentration gradient. The concentration gradient can be composed of distinct binding domains presenting different concentrations of binding reagent.

[0200] In embodiments, a control assay solution or reagent, such as a read buffer, may be utilized on the working electrode zone of the well 200. The control assay solution or reagent may provide uniformity for each analysis to control for signal variations (e.g., variations due to degradation, perturbations, aging, thermal shifts, noise in the electronic circuitry, and noise in the photodetection device of the multiwell plate 208). For example, multiple overlapping working electrode zones 104 (containing the same binding reagent or different binding reagents specific to the same analyte) may be used for the same analyte. In another example, a known concentration of analyte may be utilized, or a control assay solution or reagent may be covalently bound to a known amount of ECL label, or a known amount of ECL label in solution may be used.

[0201] In embodiments, the data collected and generated in process 1800 can be utilized in a variety of applications. The collected and generated data can be stored, for example, in the form of a database consisting of a collection of clinical or research information. The collected and generated data can also be used for rapid forensic or personal identification. For example, the use of multiple nucleic acid probes when exposed to a human DNA sample can be used for a signature DNA fingerprint that can be easily used to identify clinical or research samples. The collected and generated data can be used to identify conditions (e.g., diseases, radiation levels, etc.), the presence of organisms (e.g., bacteria, viruses, etc.), etc.

[0202] In embodiments, although the process 1800 described above involves capturing luminescence data during two periods, the process 1800 may be used to capture luminescence data during any number of periods, e.g., three periods, four periods, five periods, etc. In this embodiment, different pulse waveforms may also be used for some or all of the periods. In embodiments, the pulse waveforms may differ in amplitude (e.g., voltage), duration (e.g., period), and / or waveform type (e.g., square, sawtooth, etc.).

[0203] The above describes an example flow of example process 1800. The process shown in Figure 18 is exemplary only, and variations exist without departing from the scope of the embodiments disclosed herein. Steps may be performed in a different order than described, additional steps may be performed, and / or fewer steps may be performed.

[0204] In embodiments, different configurations of pulse waveforms supplied by voltage / current source 904 can be utilized together to improve the ECL emitted during an ECL analysis. Figure 19 shows a flowchart illustrating another process 1900 for operating an ECL device with pulse waveforms, according to an embodiment herein.

[0205] In operation 1902, process 1900 includes applying a first voltage pulse to one or more working electrode zones 104 or auxiliary electrodes 102 in a well of an ECL device, the first voltage pulse causing a first reduction-oxidation reaction in the well. In operation 1904, process 1900 includes capturing first luminescence data from the first reduction-oxidation reaction over a first period of time.

[0206] In operation 1906, process 1900 includes applying a second voltage pulse to one or more working electrode zones or auxiliary electrodes in the well, the second voltage pulse causing a second reduction-oxidation reaction in the well. In operation 1908, process 1900 includes capturing second luminescence data from the second reduction-oxidation reaction over a second time period, the first time period not being of equal duration to the second time period.

[0207] In one embodiment, the voltage level (amplitude or magnitude) or pulse width (or duration) for the first voltage pulse and / or the second voltage pulse may be selected to cause a first reduction-oxidation reaction, and the first luminescence data corresponds to the first reduction-oxidation reaction that occurs. In one embodiment, the voltage level (amplitude or magnitude) or pulse width (or duration) for the first voltage pulse and / or the second voltage pulse may be selected to cause a second reduction-oxidation reaction, and the second luminescence data corresponds to the second reduction-oxidation reaction that occurs. In one embodiment, the magnitude of at least one of the first voltage pulse and the second voltage pulse may be selected based at least in part on the chemical composition of the counter electrode.

[0208] In operation 1910, the process 1900 includes performing an ECL analysis on the first luminescence data and the second luminescence data. For example, the computer system 906 can perform the ECL analysis on the luminescence data. In some embodiments, the luminescence data, e.g., signal, resulting from a given target entity on the binding surface, e.g., binding domain, of the working electrode zone 104 and / or auxiliary electrode 102 may have a range of values. These values ​​can be correlated with a quantitative measurement (e.g., ECL intensity) to provide an analog signal. In other embodiments, a digital signal (signal present or absent) can be obtained from each working electrode zone 104 to indicate either the presence or absence of an analyte. Statistical analysis can be used for both techniques or to convert multiple digital signals to provide a quantitative result. Some analytes may require a digital presence / absence signal indicating a threshold concentration. Analog and / or digital formats can be utilized separately or in combination. Other statistical methods, such as techniques for determining concentration through statistical analysis of binding across a concentration gradient, can be utilized. Multiple linear arrays of data with a concentration gradient can be generated using multiple different specific binding reagents used in different wells 200 and / or different working electrode zones 104. The concentration gradient can consist of distinct binding domains that present different concentrations of binding reagent.

[0209] In embodiments, a control assay solution or reagent, such as a read buffer, may be utilized on the working electrode zone of the well 200. The control assay solution or reagent may provide uniformity for each analysis to control for signal variations (e.g., variations due to degradation, perturbations, aging, thermal shifts, noise in the electronic circuitry, and noise in the photodetection device of the multiwell plate 208). For example, multiple overlapping working electrode zones 104 (containing the same binding reagent or different binding reagents specific to the same analyte) may be used for the same analyte. In another example, a known concentration of analyte may be utilized, or a control assay solution or reagent may be covalently bound to a known amount of ECL label, or a known amount of ECL label in solution may be used.

[0210] In embodiments, the data collected and generated in process 1900 can be utilized in a variety of applications. The collected and generated data can be stored, for example, in the form of a database consisting of a collection of clinical or research information. The collected and generated data can also be used for rapid forensic or personal identification. For example, the use of multiple nucleic acid probes when exposed to a human DNA sample can be used for a signature DNA fingerprint that can be easily used to identify clinical or research samples. The collected and generated data can be used to identify conditions (e.g., diseases, radiation levels, etc.), the presence of organisms (e.g., bacteria, viruses, etc.), etc.

[0211] The above describes an example flow of example process 1900. The process shown in Figure 19 is exemplary only, and variations exist without departing from the scope of the embodiments disclosed herein. Steps may be performed in a different order than described, additional steps may be performed, and / or fewer steps may be performed.

[0212] In any of the above-described processes 1300, 1800, and 1900, a voltage pulse may be selectively applied to one or more working electrode zones 104 and / or one or more auxiliary electrodes 102. For example, a voltage pulse may be supplied to all working electrode zones 104 and / or auxiliary electrodes 102 in one or more wells 106 of a multiwell plate 108. Similarly, for example, a voltage pulse may be supplied to a selected (or "addressable") set of working electrode zones 104 and / or auxiliary electrodes 102 in one or more wells 106 of a multiwell plate 208 (e.g., zone-by-zone, well-by-well, sector-by-sector (e.g., a group of two or more wells), etc.).

[0213] The systems, devices, and methods described herein may be applied in a variety of contexts. For example, the systems, devices, and methods may be applied to improve various aspects of ECL measurement and reader devices. Exemplary plate readers include those described above and throughout this application, for example, in paragraph

[0180] .

[0214] For example, by applying one or more voltage pulses to generate ECL as described herein, read and / or exposure times can be improved by more quickly and efficiently generating, collecting, observing, and analyzing ECL data. Furthermore, improved exposure times (e.g., single exposure, double (or more) exposures using different (or equal) exposure times) can help improve ECL generation, collection, observation, and analysis, for example, by improving dynamic range extension (DRE), binning, etc., for substances of interest that require different time periods to capture luminescence data, in one embodiment. Thus, emitted photons can be captured as ECL data over multiple different time periods, which can be affected, for example, by the level of light saturation during ECL generation. Dynamic range can be improved by implementing various multi-pulse and / or multi-exposure schemes. For example, a short exposure followed by a long exposure (e.g., exposure of a single working electrode, a single working electrode zone, two or more single working electrodes or working electrode zones (within a single well or across multiple wells), exposure of a single well, two or more wells, or sectors, or two or more sectors, etc.) can be used. In these instances, it may be advantageous to use a longer exposure, unless the exposure is saturated. For example, when performing a short exposure and a long exposure, if saturation occurs during the long exposure, that exposure may be discarded and the short exposure may be used. If neither saturates, the longer one may be used, providing better sensitivity. In that case, for example, a shorter exposure may be used. By making these adjustments (e.g., manually or with the aid of hardware, firmware, software, algorithms, computer-readable media, computing devices, etc.), the dynamic range may be improved, as described in detail above.

[0215] Additionally, the systems, devices, and methods described herein may be utilized in various ways to enable optimization of software, firmware, and / or control logic for hardware instruments such as the readers described above. For example, because the systems, devices, and methods described herein enable faster and more efficient generation, collection, observation, and / or analysis of ECL, the instruments may be optimized with improved software, firmware, and / or control logic, resulting in lower hardware costs required to perform ECL analysis (e.g., cheaper lenses, fewer and / or cheaper motors to drive the instruments, etc.). The examples provided herein are merely illustrative, and additional improvements to these instruments are contemplated.

[0216] In embodiments such as those described above, the wells 200 of the multiwell plate 208 may contain one or more fluids (e.g., reagents) for conducting an ECL assay. For example, the fluids may include an ECL coreactant (e.g., TPA), a read buffer, preservatives, additives, excipients, carbohydrates, proteins, detergents, polymers, salts, biomolecules, inorganic compounds, lipids, etc. In some embodiments, the chemical properties of the fluid within the wells 200 during the ECL process may alter the electrochemistry / ECL generation. For example, the relationship between the ion concentration of the fluid and the electrochemistry / ECL generation may depend on different liquid types, read buffers, etc. In embodiments, one or more auxiliary electrodes, as described above, may provide a constant interfacial potential regardless of the current passing therethrough. That is, a plot of current versus potential results in infinite current at a fixed potential.

[0217] In some embodiments, the fluid used (e.g., in the wells 200 of the multiwell plate 208) may contain an ionic compound, such as NaCl (e.g., a salt). In some embodiments, for example, a higher NaCl concentration in the fluid contained in the wells 200 may improve control of ECL generation through the ECL process. A plot of current versus potential for an auxiliary electrode 102 having a redox couple, such as Ag / AgCl, has a predetermined slope. In some embodiments, this slope depends on the composition and concentration of salt in the fluid contained in the wells 200. When Ag+ is reduced, the charge balance in the redox couple of the auxiliary electrode 102 must be balanced, which may require ions from the fluid to diffuse to the electrode surface. In some embodiments, the composition of the salt may change the slope of the current versus potential curve, which in turn affects, for example, the reference potential at the interface of the auxiliary electrode 102 containing Ag / AgCl relative to the current passing through it. Thus, in embodiments, the concentration of an ion, such as a salt, may be modified and controlled to maximize the current generated relative to the applied voltage.

[0218] In embodiments, the volume of fluid within the well 200 during the ECL process can alter the electrochemistry / ECL generation. In some embodiments, the relationship between the volumes of fluid within the well 200 can depend on the design of the electrochemical cell 100. For example, a working electrode zone 104 and an auxiliary electrode 102 separated by a relatively thick fluid layer can have more ideal electrochemical behavior, such as a spatially consistent interfacial potential. Conversely, a working electrode zone 104 and an auxiliary electrode 102 separated by a relatively thin fluid layer covering both can have non-ideal electrochemical behavior due to a spatial gradient in the interfacial potential across the electrodes. In some embodiments, the design and layout of one or more working electrode zones 104 and one or more auxiliary electrodes 102 can maximize the spatial distance between the working electrode zones 104 and the auxiliary electrode 102. For example, as shown in FIG. 3A, the working electrode zones 104 and the auxiliary electrode 102 can be positioned to maximize the spatial distance D1. The clearance may be maximized by reducing the number of working electrode zones 104, reducing the exposed surface area of ​​the working electrode zones 104, reducing the exposed surface area of ​​the auxiliary electrode 102, etc. Although not described, clearance maximization may be applied to the designs shown in Figures 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, and 8A-8D.

[0219] In embodiments, the multiwell plate 208 described above may form part of one or more kits for use in performing an assay, such as an ECL assay, in the assay device. The kit may include an assay module, such as the multiwell plate 208, and at least one assay component selected from the group consisting of binding reagents, enzymes, enzyme substrates, and other reagents useful in performing the assay. Examples include whole cells, cell surface antigens, intracellular particles (e.g., organelles or membrane fragments), viruses, prions, house dust mites or fragments thereof, viroids, antibodies, antigens, haptens, fatty acids, nucleic acids (and synthetic analogs), proteins (and synthetic analogs), lipoproteins, polysaccharides, lipopolysaccharides, glycoproteins, peptides, polypeptides, enzymes (e.g., phosphorylases, phosphatases, esterases, transglutaminases, transferases, oxidases, reductases, dehydrogenases, glycosidases), protein processing enzymes (e.g., proteases, kinases, protein phosphotases, ubiquitin-protein ligases, etc.), nucleic acid promoters, and the like. Assay reagents include, but are not limited to, cleaving enzymes (e.g., polymerases, nucleases, integrases, ligases, helicases, telomerases, etc.), enzyme substrates (e.g., substrates of the enzymes mentioned above), second messengers, cellular metabolites, hormones, pharmacological agents, tranquilizers, barbiturates, alkaloids, steroids, vitamins, amino acids, sugars, lectins, recombinant or derived proteins, biotin, avidin, streptavidin, luminescent labels (preferably electrochemiluminescent labels), electrochemiluminescent coreactants, pH buffers, blocking agents, preservatives, stabilizers, detergents, dessimilates, hygroscopic agents, read buffers, etc. Such assay reagents may be unlabeled or labeled (preferably with a luminescent label, most preferably an electrochemiluminescent label).In some embodiments, the kit may include an ECL assay module, e.g., a multiwell plate 208, and at least one assay component selected from the group consisting of: (a) at least one luminescent label (preferably an electrochemiluminescent label), (b) at least one electrochemiluminescent coreactant, (c) one or more binding reagents, (d) a pH buffer, (e) one or more blocking reagents, (f) a preservative, (g) a stabilizer, (h) an enzyme, (i) a detergent, (j) a desiccant, and (k) a moisture absorbent.

[0220] 20 shows a flowchart illustrating a process 2000 for fabricating wells containing working and auxiliary electrodes, according to one embodiment of the present disclosure. For example, the process 2000 may be used to fabricate one or more of the wells 200 of a multiwell plate 208 that contain one or more working electrode zones 104 and one or more auxiliary electrodes 102.

[0221] In operation 2002, process 2000 includes forming one or more working electrode zones 104 on a substrate. In embodiments, the one or more working electrodes may be formed using any type of fabrication process, such as screen printing, three-dimensional (3D) printing, vapor deposition, lithography, etching, and combinations thereof. In embodiments, the one or more working electrode zones 104 may be formed as a multi-layer structure that may be deposited and patterned.

[0222] In embodiments, one or more working electrodes may be contiguous / adjacent regions where a reaction may occur, and an electrode "zone" may be a portion (or entirety) of an electrode where a particular reaction of interest occurs. In some embodiments, a working electrode zone may include the entire working electrode, while in other embodiments, two or more working electrode zones may be formed within and / or on a single working electrode. For example, working electrode zones may be formed by individual working electrodes. In this example, the working electrode zones may be configured as a single working electrode formed from one or more conductive materials. In another example, working electrodes may be formed by isolating portions of a single working electrode. In this example, the single working electrode may be formed from one or more conductive materials, and the working electrode zones may be formed by electrically isolating regions ("zones") of the single working electrode using insulating materials such as dielectrics. In any embodiment, the working electrode may be formed from any type of conductive material, such as metals, metal alloys, carbon compounds, etc., and combinations of conductive and insulating materials.

[0223] In operation 2004, process 2000 includes forming one or more auxiliary electrodes 102 on the substrate. In embodiments, the one or more auxiliary electrodes can be formed using any type of fabrication process, such as screen printing, three-dimensional (3D) printing, vapor deposition, lithography, etching, and combinations thereof. In embodiments, the auxiliary electrode 102 can be formed as a multilayer structure that can be deposited and patterned. In embodiments, the one or more auxiliary electrodes can be formed from a chemical mixture that provides an interfacial potential during reduction of the chemical mixture, generating a quantifiable amount of charge throughout the reduction-oxidation reaction occurring in the well. The one or more auxiliary electrodes include an oxidizing agent that supports the reduction-oxidation reaction, which can be used during biological, chemical, and / or biochemical assays and / or analyses, such as, for example, ECL generation and analysis. In one embodiment, the amount of oxidizing agent in the chemical mixture of the one or more auxiliary electrodes is equal to or greater than the amount of oxidizing agent required for the entire reduction-oxidation reaction ("redox") occurring in at least one well during one or more biological, chemical, and / or biochemical assays and / or analyses, such as, for example, ECL generation. In this regard, a sufficient amount of the chemical mixture in the one or more auxiliary electrodes remains after the redox reaction for the initial biological, chemical, and / or biochemical assay and / or analysis has occurred, thereby allowing one or more additional redox reactions to occur throughout the subsequent biological, chemical, and / or biochemical assay and / or analysis. In another embodiment, the amount of oxidant in the chemical mixture of the one or more auxiliary electrodes is based at least in part on the ratio of the exposed surface area of ​​each of the plurality of working electrode zones to the exposed surface area of ​​the auxiliary electrode.

[0224] For example, one or more auxiliary electrodes may be formed from a chemical mixture including a mixture of silver (Ag) and silver chloride (AgCl), or other suitable metal / metal halide pairs. Other example chemical mixtures may include metal oxides having multiple metal oxidation states, such as manganese oxide, or other metal / metal oxide pairs, such as silver / silver oxide, nickel / nickel oxide, zinc / zinc oxide, gold / gold oxide, copper / copper oxide, platinum / platinum oxide, etc.

[0225] In operation 2006, the process includes forming an electrically insulating material to electrically isolate the one or more auxiliary electrodes from the one or more working electrodes. In embodiments, the electrically insulating material can be formed using any type of manufacturing process, such as screen printing, 3D printing, vapor deposition, lithography, etching, and combinations thereof. The electrically insulating material can include a dielectric.

[0226] In operation 2008, process 2000 includes forming additional electrical components on the substrate. In embodiments, the one or more auxiliary electrodes may be formed using any type of manufacturing process, such as screen printing, 3D printing, vapor deposition, lithography, etching, and combinations thereof. The additional electrical components may include through-holes, electrical traces, electrical contacts, etc. For example, through-holes may be formed in the layers or materials forming the working electrode zone 104, the auxiliary electrode 102, and the electrically insulating material so that electrical contact can be made between the working electrode zone 104 and the auxiliary electrode 102 without creating a short circuit with other electrical components. For example, one or more additional insulating layers may be formed on the substrate to support the electrical traces to which they are coupled while insulating the electrical traces.

[0227] In embodiments, additional electrical components may include an electric heater, a temperature controller, and / or a temperature sensor. The electric heater, the temperature controller, and / or the temperature sensor may support an electrochemical reaction, such as an ECL reaction, and electrode performance may be temperature-dependent. For example, a screen-printed resistive heater may be incorporated into the electrode design. The resistive heater, whether internal or external, may be powered and controlled by a temperature controller and / or a temperature sensor. These are self-regulating and are constructed to generate a specific temperature when a constant voltage is applied. Ink may assist in controlling the temperature during the assay or during plate reading. Ink (and / or heater) may also be useful when high temperatures are desired during the assay (e.g., in assays using PCR components). Temperature sensors may be printed on the electrodes (working and / or auxiliary electrodes) to provide actual temperature information.

[0228] 21A-21F illustrate a non-limiting example of a process for forming a working electrode zone 104 and an auxiliary electrode 102 in one or more wells 200, according to one embodiment of the present disclosure. While FIGS. 21A-21F illustrate the formation of two wells (as shown in FIG. 22A), one skilled in the art will understand that the process illustrated in FIGS. 21A-21F can be applied to any number of wells 200. Also, while FIGS. 21A-21F illustrate the formation of an auxiliary electrode 102 and a working electrode zone 104 in an electrode design similar to electrode design 701 shown in FIGS. 7A-7F, one skilled in the art will understand that the process illustrated in FIGS. 21A-21F can be used with any of the electrode designs described herein.

[0229] The process of fabricating the auxiliary electrode 102, working electrode zone 104, and other electrical components can be performed using a screen printing process, as described below, in which different materials are formed using inks or pastes. In embodiments, the auxiliary electrode 102 and working electrode zone 104 can be formed using any type of fabrication process, such as 3D printing, deposition, lithography, etching, and combinations thereof.

[0230] As shown in FIG. 21A , a first conductive layer 2102 may be printed on a substrate 2100. In embodiments, the substrate 2100 may be formed of any material (e.g., an insulating material) that provides support for the components of the well 200. In some embodiments, the first conductive layer 2102 may be formed of a metal, such as silver. Other examples of the first conductive layer 2102 may include metals such as gold, silver, platinum, nickel, steel, iridium, copper, aluminum, conductive alloys, etc. Other examples of the first conductive layer 2102 may include oxide-coated metals (e.g., aluminum coated with aluminum oxide). Other examples of the first conductive layer 2102 may include carbon-based materials such as carbon, carbon black, graphitic carbon, carbon nanotubes, carbon fibrils, graphite, carbon fiber, and mixtures thereof. Other examples of the first conductive layer 2102 may include conductive carbon-polymer composites.

[0231] The substrate 2100 may also include one or more through-holes or other types of electrical connections (e.g., traces, electrical contacts, etc.) for connecting components of the substrate 2100 and providing locations where electrical connections can be made to the components. For example, as shown, the substrate 2100 may include a first through-hole 2104 and a second through-hole 2106. The first through-hole 2104 may be electrically isolated from the first conductive layer 2102. The second through-hole 2106 may be electrically coupled to the first conductive layer 2102. Fewer or more holes are also contemplated. For example, the through-holes may be formed in the layers or materials that form the working electrode zone 104, the auxiliary electrode 102, and the electrically insulating material so that electrical contact can be made with the working electrode zone 104 and the auxiliary electrode 102 without creating a short circuit with other electrical components. For example, one or more additional insulating layers may be formed on the substrate to support the electrical traces to which they are coupled while insulating the electrical traces.

[0232] As shown in FIG. 21B , the second conductive layer 2108 can be printed on the first conductive layer 2102. In embodiments, the second conductive layer 2108 can be formed with a chemical mixture including a mixture of silver (Ag) and silver chloride (AgCl), or other suitable metal / metal halide pairs. Other examples of chemical mixtures may include metal oxides, as described above. In some embodiments, the second conductive layer 2108 can be formed to have approximately the same dimensions as the first conductive layer 2102. In some embodiments, the second conductive layer 2108 can be formed with dimensions larger or smaller than the first conductive layer 2102. The second conductive layer 2108 can be formed by printing the second conductive layer 2108 with an Ag / AgCl chemical mixture (e.g., ink, paste, etc.) having a predetermined Ag to AgCl ratio. In one embodiment, the amount of oxidizer in the auxiliary electrode chemical mixture is based, at least in part, on the ratio of Ag to AgCl in the auxiliary electrode chemical mixture. In one embodiment, the chemical mixture of the auxiliary electrode having Ag and AgCl comprises about 50 percent or less AgCl, e.g., 34 percent, 10 percent, etc. Although not shown, one or more additional intermediate layers (e.g., insulating layers, conductive layers, and combinations thereof) may be formed between the second conductive layer 2108 and the first conductive layer 2102.

[0233] As shown in FIG. 21C , a first insulating layer 2110 may be printed on the second conductive layer 2108. The first insulating layer 2110 may be formed of any type of insulating material, such as a dielectric, polymer, glass, or the like. The first insulating layer 2110 may be formed in a pattern that exposes two portions (“spots”) of the second conductive layer 2108 to form two auxiliary electrodes 102. The exposed portions may correspond to the desired shape and size of the auxiliary electrodes 102. In embodiments, the auxiliary electrodes 102 may be formed in any number, size, and shape, such as those described in the electrode designs described above with reference to, for example, FIGS. 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, 8A-8D, and 38A-39E.

[0234] As shown in FIGS. 21D and 21E, a third conductive layer 2112 may be printed on the insulating layer 2110, followed by printing a fourth conductive layer 2114 on the third conductive layer 2112. In an embodiment, the third conductive layer 2112 may be formed of a metal, such as Ag. In an embodiment, the fourth conductive layer 2114 may be formed of a composite material, such as a carbon composite. Other examples of the first conductive layer 2102 may include metals such as gold, silver, platinum, nickel, steel, iridium, copper, aluminum, conductive alloys, etc. Other examples of the first conductive layer 2102 may include oxide-coated metals (e.g., aluminum coated with aluminum oxide). Other examples of the first conductive layer 2102 may include other carbon-based materials such as carbon, carbon black, graphitic carbon, carbon nanotubes, carbon fibrils, graphite, carbon fiber, and mixtures thereof. Other examples of the first conductive layer 2102 may include conductive carbon-polymer composites. The third conductive layer 2112 and the fourth conductive layer 2114 may be formed in a pattern that forms the base of the working electrode zone and provides electrical coupling with the first through-holes 2104. In embodiments, the through-holes may be formed in any number, size, and shape as described in the electrode designs described above with reference to, for example, Figures 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, 8A-8D, and 38A-39E.

[0235] As shown in FIG. 21F , a second insulating layer 2116 may be printed on the fourth conductive layer 2114. The second insulating layer 2116 may be formed of any type of insulating material, such as a dielectric. The second insulating layer 2116 may be formed in a pattern that exposes 20 portions (“spots”) of the fourth conductive layer 2114, thereby forming 10 working electrode zones 104 for each well 200, as shown in FIG. 22A . The second insulating layer 2116 may be formed to expose the auxiliary electrode 102. Thus, printing or depositing the second insulating layer 2116 may control the size and / or area of ​​the working electrode zones 104 and the size and / or area of ​​the auxiliary electrode 102. The exposed portions may correspond to the desired shapes and sizes of the working electrode zones 104 and the auxiliary electrode 102. In embodiments, the working electrode zones 104 may be formed in any number, size, and shape, such as those described in the electrode designs discussed above with reference to Figures 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, 8A-8D, and 38A-39E. In certain embodiments, one or more of the described layers may be formed in a particular order to minimize contamination of layers (e.g., carbon-based layers, etc.).

[0236] In the above-described method, electrical conductivity between the auxiliary electrodes 102 is maintained by the conductive layer 2108, which is masked by the insulating layer 2110. This design allows the conductive connection between the auxiliary electrodes 102 to pass under the working electrode zones 104. FIG. 22B shows a further embodiment of a well 200 fabricated by a manufacturing method somewhat similar to that described above with respect to FIGS. 21A-F and 22A. As shown in FIG. 22B, the working electrode zones 104 may be arranged in a circular pattern with gaps, e.g., a C-shape. Each well 200 may have, for example, ten working electrode zones. In further embodiments, any suitable number of working electrode zones may be included. The gaps in the pattern of the working electrode zones 104 allow the conductive trace 2120 to pass between the auxiliary electrodes 102 of two wells 200. Because the conductive trace 2120 passes between, but does not cross, the auxiliary electrodes 102, working electrode zones 104, and conductive trace 2120 may be printed on the same layer during the manufacturing process. For example, in embodiments including individually addressable working electrode zones 104, the auxiliary electrode 102, working electrode zone 104, and conductive trace 2120 may each be printed as individual features on the same layer of the substrate. The C-shaped electrode design shown in FIG. 22B is not limited to use with a dual-well layout. Other layouts including different numbers of wells are also consistent with embodiments herein. For example, a single-well layout may include a C-shaped electrode layout. In another example, four or more wells 200 may be laid out in a C-shaped electrode layout with multiple conductive traces 2120 connecting the auxiliary electrodes 102 of each well 200 in the layout.

[0237] Figures 24A-24C, 25A-25C, 26A-26D, 27A-27C, 28, and 29 show test results performed on various multiwell plates according to embodiments herein. The tests included two different test lots. Each of the two different test lots included four different configurations of multiwell plates: a standard ("Std") 96-1 plate, a Std96ss plate (small spot plate), a Std96-10 plate, and a Std96ss "BAL" plate. The Std96-1 plate, as shown in Figure 23A, contains 96 wells 106 with one working electrode zone per well 106. The Std96ss plate, as shown in Figure 23B, contains 96 wells 106 with one working electrode zone per well 106. The Std96-10 plate contains 96 wells 106 with 10 working electrode zones per well 106, as shown in FIG. 23C. The Std96ss "BAL" plate has two auxiliary electrodes and a single working electrode zone, as shown in FIG. 23D. For each test lot, three sets of multiwell plates of each configuration were screen printed with different Ag / AgCl inks to generate different ratios of Ag / AgCl chemical mixtures, as shown in Table 8. Each of the above plates was configured with two auxiliary electrodes per well. The "BAL" configuration was configured with smaller auxiliary electrodes compared to the other configurations.

[0238] [Table 23]

[0239] The test also included a production control, which included a working electrode zone and counter electrode made of carbon, labeled Production Control in the figure.

[0240] Test solutions were tested using the electrode design described above to generate voltammetry, ECL traces (ECL intensity vs. applied potential difference), and integrated ECL signal measurements. The test solutions included three solutions: a 1 μM TAG (TAG refers to an ECL label or species that emits photons when electrically excited) solution in T1x, a 1 μM TAG solution in T2x, and MSD Free TAG 15,000 ECL (Y0260157). The 1 μM TAG solution in T1x contained 5.0 mM Tris(2,2'-bipyridine)ruthenium(II) chloride stock solution (Y0420016) and MSD T1x (Y0110066). The 1 μM TAG solution in T2x contained 5.0 mM Tris(2,2'bipyridine)ruthenium(II) chloride stock solution (Y0420016) and MSD T2x (Y0200024). The test solution also contained a read buffer solution containing MSD T1x (Y0110066). Measurements were performed on voltammetry, ECL traces, and the Free TAG 15,000 ECL test and MSD T1x ECL signals under the following conditions:

[0241] Voltammetry was measured using a standard three-electrode configuration (working electrode, reference electrode, and counter electrode) using one plate from each Ag / AgCl ink and one plate from each of the Std 96-1, Std96ss, and Std96-10 inventories. Reductive voltammetry was measured at the counter electrode. For reductive voltammetry, wells were filled with 150 μL of 1 μM TAG in T1x or 1 μM TAG in T2x and allowed to sit for at least 10 minutes. A waveform was applied to the Ag / AgCl plate at 100 mV / s, going from 0.1 V to -1.0 V and back to 0.1 V. A waveform was applied to the production control at 100 mV / s, going from 0 V to -3 V and back to 0 V. Three replicate wells of each solution were measured and averaged.

[0242] Oxidative voltammetry was measured at the working electrode. For oxidative voltammetry, wells were filled with 150 μL of 1 μM TAG in T1x or 1 μM TAG in T2x and allowed to sit for at least 10 minutes. For Ag / AgCl, a waveform was applied at 100 mV / s, going from 0 V to 2 V and back to 0 V. For the production control, a waveform was applied at 100 mV / s, going from 0 V to 2 V and back to 0 V. Three replicate wells of each solution were measured and averaged.

[0243] For ECL traces, one plate of each Ag / AgCl ink and one plate from each of the Std 96-1, Std 96ss, and Std 96-10 inventories were measured. Six wells were filled with 150 microliters (μL) of 1 micromolar (μM) TAG in T1x, and six wells were filled with 1 mM TAG in T2x. The plates were allowed to rest for at least 10 minutes. ECL was measured with a proprietary video system using the following parameters: Ag / AgCl: 0 V to 3000 mV in 3000 ms, imaged using 120 consecutive 25 ms frames (i.e., image exposure length); and production control: 2000 mV to 5000 mV in 3000 ms using 25 ms frames. Six replicate wells of each solution were averaged for ECL intensity vs. potential and current vs. potential.

[0244] Six plates of each AgCl ink, and six plates from the Std96-1, Std96ss, and Std96-10 inventories, were measured for integrated ECL signals: two plates were MSD T1x and four plates were "Free TAG 15,000 ECL." Plates were filled with 150 μL of "Free TAG 15,000 ECL" or MSD T1x and allowed to sit for at least 10 minutes. ECL was measured on a MESO QUICKPLEX SQ 120 instrument ("SQ120") using a 0V to 3000mV waveform for 3000ms for AgCl. ECL was measured on the SQ120 using a 2000mV to 5000mV waveform for 3000ms for the production control. Intra- and inter-plate values ​​were calculated. Test results are discussed below.

[0245] Figures 24A-24C show the results of ECL measurements performed on the Std96-1 plate. Figure 24A is a graph showing voltammetry measurements for the Std96-1 plate. In particular, Figure 24A shows the average voltammogram for the Std96-1 plate. As shown in Figure 24A, an increase in current occurred between the T1x and T2x solutions. The oxidation curves were similar for the three Ag / AgCl ink plates and the control plate. The onset of oxidation was approximately 0.8 V vs. Ag / AgCl. The peak potential was approximately 1.6 V vs. Ag / AgCl. When the CE was changed from carbon to Ag / AgCl, a shift in reduction occurred. The onset of water reduction on the carbon was ca. -1.8 V vs. Ag / AgCl. The onset of AgCl reduction was ca. 0 V vs. Ag / AgCl. An increase in the AgCl content of the Ag / AgCl ink resulted in an increase in the total amount of AgCl reduced. A small shoulder at -0.16 V occurred in the reduction voltammetry at Ag / AgCl, and the current increased between the T1x and T2x solutions. These results indicate that increasing the concentration of lead buffer from T1x to T2x increases the oxidation current. By incorporating AgCl into the auxiliary electrode, the onset of reduction shifted to the expected 0 V vs. the carbon reference electrode. Increasing AgCl in the ink increased the total amount of AgCl reduced without affecting the slope of the current vs. potential curve.

[0246] Figures 24B and 24C are graphs showing ECL measurements for the Std96-1 plate. In particular, Figures 24B and 24C show average ECL and current traces for the Std96-1 plate with either the T1x or T2x solution, as shown in Figure 24A. As shown, the three Ag / AgCl ink plates produced similar ECL traces. The onset of ECL occurred at ca. 1100 mV for the T1x and T2x solutions. The peak potential occurred at 1800 mV for the T1x solution and 1900 mV for the T2x solution. The ECL intensity returned to baseline at ca. 2250 mV. The three Ag / AgCl ink plates produced similar current traces, except for a drop in current for the ink ratio 1 (90 / 10 Ag:AgCl) with T2x at the end of the waveform. For the production plate, the onset of ECL shifted to ca. 3100 mV, and the peak potential shifted to ca. 4000 mV. The relative shift in ECL on the production plate corresponded to a shift in the onset of the reduction current measured in reference voltammetry. The full width at half maximum of the ECL trace on the production plate was wider than that on the Ag / AgCl ink plate, which correlated with the lower slope of the reduction current in reference voltammetry.

[0247] As shown in Figure 24C, the total current flowing during the waveform with a 90:10 ratio was smaller than with the other inks. This indicated that the 90:10 ratio may limit the amount of oxidation that can occur at the working electrode. For experiments where currents greater than the FT of T2x can flow using this waveform, a 50:50 ratio was chosen to ensure sufficient reduction capacity. As shown by testing, the Ag / AgCl ink provides a controlled potential for reduction at the auxiliary electrode 102. With Ag / AgCl, the auxiliary electrode 102 shifts the ECL response to the potential where TPA oxidation occurs when measured using a true Ag / AgCl reference electrode.

[0248] With respect to the auxiliary electrode 102, the amount of AgCl accessible within the auxiliary electrode 102 must be such that it is not completely consumed during the ECL measurement. For example, one mole of AgCl is required per mole of electrons passed during oxidation at the working electrode. Less than this amount of AgCl results in a loss of control of the interfacial potential at the working electrode zone 104. Loss of control refers to a situation in which the interfacial potential is not maintained within a specified range during the chemical reaction. One purpose of having a controlled interfacial potential is to ensure consistency and reproducibility of readings from well to well, plate to plate, screen lot to screen lot, etc.

[0249] Table 10 shows the intra- and inter-plate FT and T1x values ​​for the Std96-1 plates determined from ECL measurements. As shown in Table 10, the three Ag / AgCl ink plates yielded comparable values. The production plate yielded higher FT and T1x ECL signals. These higher signals can be attributed to a lower effective ramp rate due to the lower slope of the reductive voltammetry.

[0250] [Table 24]

[0251] Figures 25A-25C show the results of ECL measurements performed on a Std96ss plate. Figure 25A is a graph showing voltammetry measurements on the Std96ss plate. In particular, Figure 25A shows the average voltammogram for the Std96ss plate. As shown in Figure 25A, an increase in current occurred between the T1x and T2x solutions. The oxidation curves were similar for the three Ag / AgCl ink plates and the control plate. The onset of oxidation occurred at ca. 0.8 V vs. Ag / AgCl. The peak potential occurred at approximately 1.6 V vs. Ag / AgCl. When the auxiliary electrode was changed from carbon to Ag / AgCl, a shift in reduction occurred. The onset of water reduction at the carbon occurred at approximately -1.8 V vs. Ag / AgCl. The onset of AgCl reduction occurred at approximately 0 V vs. Ag / AgCl. The total amount of AgCl reduced increased with increasing AgCl content in the Ag / AgCl ink. A small shoulder at -0.16 V occurred in the reductive voltammetry in Ag / AgCl, and the current increased between the T1x and T2x solutions.

[0252] Figures 25B and 25C are graphs showing ECL measurements for Std96ss plates. In particular, Figures 25B and 25C show average ECL and current traces for Std96ss plates with either T1x or T2x solutions, as shown in Figure 10A. As shown, the three Ag / AgCl ink plates produced very similar ECL traces. The onset of ECL occurred at approximately 1100 mV for the T1x and T2x solutions. The peak potential occurred at 1675 mV for the T1x solution and 1700 mV for the T2x solution. The ECL intensity returned to baseline at approximately 2175 mV. The three Ag / AgCl ink plates produced similar current traces. For the production plate, the onset of ECL shifted to approximately 3000 mV and the peak potential shifted to approximately 3800 mV. The relative shift in ECL on the production plate corresponded to a shift in the onset of the reduction current measured in the reference voltammetry. The full width at half maximum of the ECL trace on the production plate was wider than that on the Ag / AgCl ink plate, which correlated with the lower slope of the reduction current in the reference voltammetry. The results shown in Figures 25A-25C are consistent with those in Figures 24A-24C, indicating that the changes resulting from the use of an Ag / AgCl electrode are robust across different electrode configurations.

[0253] Table 11 shows the intra- and inter-plate FT and T1x values ​​for the Std96ss plates determined from ECL measurements. As shown in Table 11, the three Ag / AgCl ink plates yielded comparable values. The production plate yielded higher FT and T1x ECL signals. These higher signals may be due to a lower effective ramp rate due to the lower slope of the reductive voltammetry. The higher background signal in the production plate may be due to a non-standard waveform in the reader used for the experiment.

[0254] [Table 25]

[0255] Figures 26A-26D show the results of ECL measurements performed on the Std96ss BAL plate. Figure 26A is a graph showing voltammetry measurements on the Std96ss BAL plate. In particular, Figure 26A shows the average voltammogram for the Std96ss BAL plate. As shown in Figure 26A, an increase in current occurred between the T1x and T2x solutions. The oxidation curves were similar for the three Ag / AgCl ink plates and the production control. The onset of oxidation occurred at approximately 0.8 V vs. Ag / AgCl. The peak potential occurred at ca. 1.6 V vs. Ag / AgCl. As the AgCl content of the Ag / AgCl ink increased, the total amount of AgCl reduced increased. A small shoulder at -0.16 V occurred in the reductive voltammetry on Ag / AgCl, where the current increased between the T1x and T2x solutions. The overall auxiliary electrode current was reduced compared to the Std96ss plate configuration due to the smaller electrode area. The slope of the current versus potential plot was smaller than that of the Std96ss plate configuration.

[0256] Figure 26B is a graph showing Std96ss vs. Std96ss BAL with T2x solution at an ink ratio of 3. As shown in Figure 26B, the oxidation peak current (approximately -0.3 mA) was similar for both of these formats. For most of the reduction current, Std96ss BAL was at a more negative potential than Std96ss.

[0257] Figures 26C and 26D are graphs showing ECL measurements for Std96ss BAL plates. In particular, Figures 26C and 26D show average ECL and current traces for Std96ss BAL plates with either T1x or T2x solutions. As shown, the three plates with Ag / AgCl counter electrodes produced similar ECL traces. ECL onset occurred at ca. 1100 mV for T1x and T2x solutions. Peak potentials occurred at 1750 mV for T1x solution and 1800 mV for T2x solution. ECL intensity returned to baseline at ca. 2300 mV. ECL onset was similar to that of Std96ss plates, but the peak potential and return to baseline were slower and shifted compared to Std96ss plates. The differences between the Std96ss plate and the Std96ss BAL plate can be attributed to a lower ramp rate resulting from the lower slope of reductive voltammetry on the smaller counter electrode. The three plates with Ag / AgCl counter electrodes produced similar current traces, except for a drop in current at 90 / 10 Ag:AgCl with T2x solution at the end of the waveform. The different behavior of the ink ratio 1 with T2x solution was also observed in the Std96-1 plate format. The results shown in Figures 26A-26D are consistent with those in Figures 24A-24C and 25A-25C, indicating that the changes resulting from the use of an Ag / AgCl electrode are robust across different electrode configurations.

[0258] Table 12 shows the intra- and inter-plate FT and T1x values ​​for the Std96ss BAL plate determined from ECL measurements. As shown in Table 12, the ECL signal is higher than the Std96ss plate configuration. The higher signal can be attributed to a lower effective ramp rate due to the lower slope of reductive voltammetry on the smaller counter electrode. As the AgCl content in the ink increased, the FT signal decreased.

[0259] [Table 26]

[0260] Figures 27A-27C show the results of ECL measurements performed on the Std96-10 plate. Figure 27A is a graph showing voltammetry measurements for the Std96-10 plate. In particular, Figure 27A shows the average voltammogram for the Std96-10 plate. As shown in Figure 27A, an increase in current occurred between the T1x and T2x solutions. The oxidation curves were similar for the three plates with the Ag / AgCl counter electrode and the production control. The onset of oxidation occurred at approximately 0.8 V vs. Ag / AgCl. The peak potential occurred at approximately 1.6 V vs. Ag / AgCl. A higher oxidation current was present in the production control. When the auxiliary counter electrode was changed from carbon to Ag / AgCl, a shift in reduction occurred. The onset of water reduction at the carbon occurred at approximately -1.8 V vs. Ag / AgCl. The onset of AgCl reduction occurred at approximately 0 V vs. Ag / AgCl. Increasing the AgCl content of the Ag / AgCl ink resulted in an increase in the total amount of reduced AgCl. A small shoulder at −0.16 V appeared in the reductive voltammetry on Ag / AgCl, where the current increased between the T1x and T2x solutions.

[0261] Figures 27B and 27C are graphs showing ECL measurements for the Std96-10 plate. In particular, Figures 27B and 27C show the average ECL and current traces for the Std96-10 plate with either T1x or T2x solution. As shown, the three plates with Ag / AgCl counter electrodes produced similar ECL traces. The onset of ECL occurred at approximately 1100 mV for the T1x and T2x solutions. The peak potential occurred at 1700 mV for the T1x solution and 1750 mV for the T2x solution. The ECL intensity returned to baseline at approximately 2250 mV. The three plates with Ag / AgCl counter electrodes produced similar current traces. For the production plate, the onset of ECL shifted to approximately 3000 mV, and the peak potential shifted to approximately 3800 mV. The relative shift in ECL on the production plate corresponded to the shift in the onset of the reduction current measured by reference voltammetry. The full width at half maximum of the ECL traces on the production plate was wider than that of the Ag / AgCl ink, which correlated with the lower slope of the reduction current in the reference voltammetry. The results shown in Figures 27A-27C are consistent with those in Figures 24A-24C, 25A-25C, and 26A-26D, indicating that the changes resulting from the use of an Ag / AgCl electrode are robust across different spot sizes.

[0262] Table 13 shows the intra- and inter-plate F T and T lx values ​​for the Std96-10 plates determined from ECL measurements. As shown in Table 13, the three plates with Ag / AgCl counter electrodes yielded comparable values. The production plate yielded lower F T and T lx ECL signals. The cause of the lower signals in the production plate is unknown but may be related to the higher oxidation currents measured in the reference voltammetry.

[0263] [Table 27]

[0264] As shown in the test results described above and in Figure 28, the auxiliary electrode with Ag / AgCl shifted the ECL in the non-reference system to a potential corresponding to oxidation measured in the reference system, i.e., a system including a separate reference electrode. For the auxiliary electrode composed of Ag / AgCl, the onset of ECL occurred at a potential difference of 1100 mV. The ECL peak occurred at potential differences (average across plate types) of -1833 mV for the Std96-1 plate, -1688 mV for the Std96ss plate, -1775 mV for the Std96ss BAL plate, and -1721 mV for the Std96-10 plate. The onset of oxidation current occurred at 0.8 V vs. Ag / AgCl. The peak oxidation current occurred at ca. 1.6 V vs. Ag / AgCl.

[0265] Additionally, as shown by the test results, the three ink formulations were tested with a range of Ag:AgCl ratios, and variations in the amount of AgCl were detectable in the referenced reductive voltammetry. All three formulations produced comparable ECL traces. There were some differences in the current vs. potential plots when ECL was measured in T2x solution. The current capacity appeared to be limited for Std96-1 and Std96ss BAL, which have a 90 / 10 Ag:AgCl ratio; these plate types have the largest working electrode to counter electrode area ratios. FT signals were comparable for the three formulations, except for the 96ssBAL plate type.

[0266] In the above example, the working electrode area of ​​the Std96-1 plate is 0.032171 in 2 The working electrode area of ​​the Std96ss plate is 0.007854 in 2 The auxiliary electrode area of ​​Std96-1 and Std96sspr is 0.002646 inches 2 The auxiliary electrode area of ​​the Std96ss BAL plate was estimated to be 0.0006459 in. 2The area ratios may be Std96-1:12.16, Std96ss:2.968, and Std96ss BAL:12.16. The ratio of peak reduction currents in the Std96ss plate and the Std96ss BAL plate is determined by the auxiliary electrode area of ​​the Std96ss BAL plate being 0.0007938 in 2 The ECL traces suggest that this reduction in counter electrode area is a necessary approach to unify the ECL traces from the Std96-1 and Std96ss BAL plates.

[0267] Example 4 - Effect of the area ratio of the working electrode to the auxiliary electrode on the performance of the Ag / AgCl auxiliary electrode Four different multiwell plate configurations were tested, each with a different ratio of working to auxiliary electrode area within each well, as indicated by the exposed working electrode area 104 and auxiliary electrode area 102 in the electrode patterns shown in Figures 23A-23D. The first, "Std96-1 plate" (Figure 23A), has wells with large working electrode areas (defined by dielectric ink patterned on the working electrode) bounded by two auxiliary electrode strips, and has the same electrode configuration as the plates used in Examples 2 and 3. The second, "Std96ss plate" (Figure 23B), is similar to the first, except that the dielectric ink on the working electrode area is patterned to expose only a smaller, circular, exposed working electrode area in the center of the well (providing a small spot or "ss" area). The third, "Std96-10" (Figure 23C), is similar to the first, except that the dielectric ink on the working electrode area is patterned to expose 10 small, circular exposed working electrode areas, providing a "10-spot" pattern of working electrode area within each well. The fourth, "Std96ss BAL" (Figure 23D), has the small exposed working electrode area of ​​the Std96ss pattern, but the area of ​​the exposed auxiliary electrode has been significantly reduced so that the ratio of working electrode area to counter electrode area remains similar to the Std96-1 configuration, maintaining a balance between these areas. For each configuration, the total exposed working electrode area, the total exposed auxiliary electrode area, and the area ratio of the working electrode to the counter electrode are provided in Table 14. To evaluate the effect of Ag / AgCl ink on auxiliary electrode performance, each of the electrode configurations was fabricated using auxiliary electrodes made with three different inks with different Ag-to-AgCl ratios, as described in Table 15. The Std96-1, Std96ss, and Std96-10 configurations were also compared to "control" or "production control" plates (MSD96 well, MSD96 Well Small Spot, and MSD 96 Well 10 Spot Plates, Meso Scale Diagnostics, LLC), which were similar plates with a conventional carbon ink counter electrode instead of an Ag / AgCl auxiliary electrode.

[0268] [Table 28]

[0269] [Table 29]

[0270] Different electrode configurations were evaluated by cyclic voltammetry in the presence of ECL lead buffer (1x and 2x MSD lead buffer T relative to the nominal working concentration) and by using tris(2,2'-bipyridine)ruthenium(II) chloride ("TAG") solutions in these lead buffers for ECL measurements. Voltammetry was measured using a standard three-electrode configuration (working electrode, reference electrode, and counter electrode) with a 3M KCl Ag / AgCl reference electrode. Oxidation of the ECL lead buffer at the working electrode 104 was measured by cycling from 0 V to 2 V at a scan rate of 100 mV / s, using the working electrode 104 and auxiliary electrode 102 as the working and counter electrodes, respectively. Reduction of the ECL lead buffer at the auxiliary electrode 102 was measured by cycling from -0.1 V to -1 V at a scan rate of 100 mV / s, using the auxiliary electrode 102 and working electrode 104 as the working and counter electrodes, respectively. To measure the reduction of ECL lead buffer at the carbon counter electrode of the "control" plate, a wider voltage range was required; the voltage was cycled from 0 V to -3 V at a scan rate of 100 mV / s. Wells were filled with 150 μL of ECL lead buffer and allowed to sit for at least 10 minutes before voltammetry. Each solution was measured in triplicate wells, and the voltammetry data were averaged.

[0271] The integrated ECL signal of the TAG solution was measured on a MESO QUICKPLEX SQ 120 instrument ("SQ120") using a 0V to 3000mV ramp over 3000ms (for test plates with an Ag / AgCl auxiliary electrode) and a 2000mV to 5000mV ramp over 3000ms (for control plates with a carbon ink counter electrode). All wells were filled with 150µL of MSD Free Tag ("FT", a TAG solution in MSD Read Buffer T 1X designed to provide approximately 15,000 signals within the ECL signal unit of the SQ120 instrument), and the plates were allowed to stand for at least 10 minutes. Two replicate plates (96 wells per plate) of T1X were run to measure background signal in the absence of TAG, and four replicate plates of FT were run to measure the ECL signal generated from TAG. After normalization for the area of ​​the exposed working electrode, the instrument reports a value proportional to the integrated ECL intensity over the duration of the applied waveform. Within- and between-plate means and standard deviations were calculated across the wells run for each solution and electrode configuration.

[0272] To measure ECL intensity as a function of time during ECL measurements, ECL measurements from TAG solutions were performed on a modified MSD plate reader with a proprietary video system. The same waveform and procedure as used for measuring integrated signals was used, except that ECL was imaged as a series of consecutive 120 × 25 ms frames captured over the course of a 3000 ms waveform, and a more concentrated solution of TAG (1 μM TAG in MSD read buffer T1X and 2X) was used. Each frame was background corrected using an image captured before the start of the waveform. The ECL intensity of each exposed working electrode area (or "spot") within the image was calculated by summing the intensities measured for each pixel within the area defined by the spot. For images with multiple spots within a well, the intensity values ​​for the spots within the well were averaged. The instrument also measured the current passing through the wells as a function of time during the ECL experiment. For each solution and electrode configuration, the mean and standard deviation of ECL intensity and current were calculated based on data from six replicate wells.

[0273] Voltammetric data for the Std96-1, Std96ss, Std96ss BAL, and Std96-10 plates are shown in Figures 24A, 25A, 26A, and 27A, respectively. The oxidation current at the working electrode 104 in this three-electrode configuration is largely independent of the nature of the auxiliary or counter electrode; in all instances, the onset of oxidation in the lead buffer occurs at approximately 0.8 V and the peak current occurs at approximately 1.6 V. The oxidation current increases with increasing concentrations of the tripropylamine ECL coreactant, from 1X lead buffer to 2X lead buffer, and the peak and integrated oxidation currents increase roughly proportionally to the exposed working electrode area (as shown in Table 14). The small differences observed between the currents of the test and control plates in some instances are likely related to differences in the carbon ink used to fabricate the working electrodes.

[0274] The reduction current measured at the auxiliary or counter electrode 102 showed an onset of reduction at approximately 0 V for the Ag / AgCl auxiliary electrode (related to the reduction of AgCl to Ag), compared to approximately 3100 mV for the carbon ink counter electrode (likely related to the reduction of water). An increase in the slope of the current onset and the overall integrated current was observed with 2X lead buffer T compared to 1X concentration, but this increase was small and may be related to the higher ion concentration at 2X. For a given combination of Ag / AgCl ink and lead buffer formulation, the reduction current measured at the auxiliary electrode for the Std96-1, Std96ss, and Std96-10 electrode configurations was largely independent of electrode configuration, since the geometry of the auxiliary electrode in these configurations was identical. When the percentage of AgCl in the Ag / AgCl ink was increased from 10% (Ratio 1) to 34% (Ratio 2) to 50% (Ratio 3), the reduction onset potential and the slope of the reduction onset current did not change significantly, indicating a relatively insensitive electrode potential to the percentage of AgCl. However, with increasing AgCl, the peak potential shifts negatively and the integrated current increases roughly proportional to the proportion of AgCl in the ink, indicating that increasing AgCl is associated with increased reduction ability. Comparing the reduction currents for the 96ss vs. 96ss BAL configurations (Figure 26B), the shape and peak potential are roughly the same, but the peak and integrated current for the 96ss BAL decrease roughly proportional to the reduction in auxiliary electrode area.

[0275] The ECL intensity from 1 μM TAG in MSD Read Buffer T 1X as a function of applied potential is provided in Figures 24B, 25B, 26C, and 27B for the Std96-1, Std96ss, Std96ss BAL, and Std96-10 electrode configurations, respectively. Similar plots for 1 μM TAG in MSD Read Buffer T2X are provided in Figures 24C, 25C, 26D, and 27C, respectively. All plots also provide plots of the associated current through the electrode as a function of potential. For each of the tested electrode configurations, the ECL traces generated using auxiliary electrodes with three different Ag / AgCl ink formulations were largely superimposable, indicating that even the Ag / AgCl formulation with the lowest percentage of AgCl (10%) had sufficient reducing capacity to complete ECL generation. For measurements of TAG in MSD Read Buffer T 1X with Ag / AgCl, the current traces were also largely superimposable. However, for measurements of TAG in MSD lead buffer T2X, particularly for the configurations with the lowest ratios of Ag / AgCl auxiliary electrode area to working electrode area (96-1 and 96ss BAL configurations), the current measured using the ink with the lowest AgCl percentage diverged at high potentials, showing a decrease in current with increasing potential. This divergence occurred at potentials near the end of the ECL peak and did not significantly affect the ECL trace, indicating that the 10% AgCl ink appears to be near the border of sufficient reduction capacity to complete ECL generation using the selected waveform, lead buffer, and electrode configuration.

[0276] Subtle changes in the shape of the peaks in the ECL traces were observed with changes in electrode configuration. For all configurations and both lead buffer concentrations, the onset of ECL generation occurred at approximately 3100 mV with the carbon ink counter electrode and 1100 mV with the Ag / AgCl auxiliary electrode. The onset potential with the Ag / AgCl auxiliary electrode is very close to the approximately 800 mV onset potential observed in the three-electrode system with an Ag / AgCl reference electrode. Although the onset potential is relatively independent of electrode configuration, slight differences in the potential at which peak ECL intensity occurs were observed. For the Std96-1 configuration, peak ECL with the Ag / AgCl auxiliary electrode occurs at approximately 1800 mV and 1900 mV for TAG in 1X and 2X lead buffer formulations, respectively. For the carbon counter electrode, the peaks are at 4000 and 4100 mV. The peak potential decreases as the ratio of working electrode area to auxiliary / counter electrode area decreases. This effect occurs because the current required at the working electrode to achieve peak ECL can be achieved with a lower current density, i.e., a lower potential drop, at the auxiliary / counter electrode. For the Std96-10 configuration, peak ECL using an Ag / AgCl auxiliary electrode occurs at approximately 1700 mV and 1750 mV for TAG in 1X and 2X lead buffer formulations, respectively. For the Std96ss configuration, which has the lowest electrode area ratio, peak ECL using an Ag / AgCl auxiliary electrode occurs at approximately 1675 mV and 1700 mV for TAG in 1X and 2X lead buffer formulations, respectively. The shape of the ECL curve can be made more consistent between configurations with varying working electrode area by balancing the auxiliary electrode area to maintain a constant ratio. The Std96ss BAL configuration has the working electrode area of ​​the Std96ss configuration, but the auxiliary electrode area has been reduced so that the electrode area ratio matches that of the Std96-1 configuration. For the Std96ss BAL configuration, peak ECL using an Ag / AgCl auxiliary electrode occurred at approximately 1750 mV and 1800 mV for TAG in 1X and 2X lead buffer formulations, respectively, which is higher than that observed in the Std966 configuration and approaches that observed in the Std96-1 configuration.The difference in peak potential between the Std96-1 and Std96ss BAL configurations may simply indicate that the actual area ratio achieved when printing the Std96ss plate may be lower than targeted in the screen printing design. ECL traces and currents for 1 μM TAG in MSD read buffer T2x for the three electrode configurations are compared in Figure 28.

[0277] The integrated ECL signal results for the Std96-1, Std96ss, Std96ss BAL, and Std96-10 electrode configurations are provided in Tables 16, 17, 18, and 19, respectively. Each table provides results for three different Ag / AgCl auxiliary electrode compositions and a control carbon counter electrode condition (Ag:AgCl = "n / a"). The table provides the starting potential (Vi), ending potential (Vf), and duration (T) of the ramp waveform used for that condition, as well as the average integrated ECL signal measured for the TAG solution (FT) and the background signal measured for the base buffer used in the TAG solution (T1X) in the absence of TAG. The coefficient of variation (CV) is also provided for within- and between-plate variations. Tables 16–19 demonstrate that the integrated signal was largely independent of electrode configuration and auxiliary / counter electrode ink composition. No clear trends in CV with electrode configuration or composition were observed, and the conditions with the highest CV were generally associated with a single outlier well or plate. Despite sharing the same working electrode geometry, a slightly higher signal was observed with the Std96ss BAL configuration than with the Std96ss configuration. The current required at the working electrode during ECL generation generated a higher current density with the smaller Std96ss BAL auxiliary electrode, placing the auxiliary electrode in a region of the current vs. voltage curve with a lower slope (Figure 26B). The net result was a slower effective voltage ramp rate at the working electrode, increasing the time for ECL generation.

[0278] [Table 30]

[0279] [Table 31]

[0280] [Table 32]

[0281] [Table 33]

[0282] Examples of voltage pulses are described above with reference to Figures 12A, 12B, 14A, 14B, 15A-15L, 16, and 17. In embodiments, the magnitude and duration of the pulse waveform can be tailored to the chemical mixture of the auxiliary electrode 102 and / or the configuration of the working electrode zone 104. Figures 14A, 14B, 15A-15L, 16, and 17 are graphs illustrating tests conducted to optimize the waveform for a high-binding standard plate. Tests were conducted with various configurations of a working electrode zone 104 formed of carbon, a counter electrode formed of carbon, and an auxiliary electrode 102 formed of various ratios of Ag / AgCl. In these tests, the voltage was ramped to determine the potential value that maximized ECL. The graphs show how the high-binding standard plate affects how ECL is generated with a change in potential and at what point in the curve. The results of the tests can be used to determine the optimal magnitude and / or duration of the pulse waveform.

[0283] Specifically, in the test, FT ECL traces were performed on uncoated standard ("Std") and high-binding ("HB") 96-1, 96ss, and 96-10 plates, as shown in Figures 8A-8D. 300k FT was measured on 12 different SI plate types: Std and HB 96-1, 96ss, and 96-10 production control plates, Std and HB 96-1, 96ss, and 96-10 Ag / AgCl plates with an ink ratio of 3 (50:50 Ag:AgCl ratio). Five waveforms were performed on each plate type (four replicates per well). The waveforms for the production plates were 2000 mV to 5000 mV for 3000 ms (1.0 V / s), 2000 ms (1.5 V / s), 1500 ms (2.0 V / s), 1200 ms (2.5 V / s), and 1000 ms (3.0 V / s). The waveforms for the Ag / AgCl plates were 0 mV to 3000 mV for 3000 ms (1.0 V / s), 2000 ms (1.5 V / s), 1500 ms (2.0 V / s), 1200 ms (2.5 V / s), and 1000 ms (3.0 V / s). The production and Ag / AgCl plates were measured with an ECL system using a video system to capture the luminescence data. To generate the graphs shown in Figures 14A, 14B, 15A-15L, 16, and 17, the ECL intensity at each potential was determined using a macro, and four replicates were averaged. Plots of the average ECL values ​​versus potential were generated.

[0284] Based on the tests performed, the ECL peak voltage was determined for each of the production plates and test plates, as shown in Table 20. The ECL peak voltage can be used to set the magnitude of the pulse waveform in the ECL process.

[0285] [Table 34]

[0286] As shown by Figures 26, 27, 28A, 28B, 29, 30, 31, 32A, and 32B, and as shown in Table 21, the ramp rate changed the measured ECL. As the ramp rate increased, the intensity increased and the signal decreased. As the ramp rate increased, the width of the ECL peak increased. The baseline intensity was defined as the average intensity over the first 10 frames. The onset potential was defined as the potential where the ECL intensity exceeded two times the average baseline. The return to baseline was defined as the potential where the ECL intensity was less than two times the baseline. The width was defined as the potential difference between the return potential and the onset potential.

[0287] For the Ag / AgCl auxiliary electrode 102, the amplitude increased from 175 mV to 525 mV between 1.0 V / s and 3.0 V / s for the carbon counter electrode. HB96-1 showed the largest change, while Std96ss showed the smallest change. For the Ag / AgCl counter electrode, the amplitude increased from 375 mV to 450 mV between 1.0 V / s and 3.0 V / s.

[0288] [Table 35]

[0289] For the Ag / AgCl auxiliary electrode 102, the amplitude increased from 175 mV to 525 mV between 1.0 V / s and 3.0 V / s for the carbon counter electrode. HB96-1 showed the largest change, while Std96ss showed the smallest change. For the Ag / AgCl counter electrode, the amplitude increased from 375 mV to 450 mV between 1.0 V / s and 3.0 V / s.

[0290] Example 5 - Effect of working electrode composition and ramp rate on ECL generation using an Ag / AgCl auxiliary electrode In this experiment, plates were prepared in the 96-1, 96ss, and 96-10 configurations as described in Example 4. Test plates with an Ag / AgCl auxiliary electrode ("Ag / AgCl") used the Ag / AgCl mixture of 50% AgCl described in Example 4 to provide more than sufficient reduction capacity for ECL generation with the selected electrode configuration. Control plates ("Carbon") were also prepared with a conventional carbon ink counter electrode instead of the Ag / AgCl auxiliary electrode. For each combination of electrode configuration and auxiliary / counter electrode composition, plates were made with working electrodes that had either a standard carbon ink electrode (denoted "Standard" or "Std") as used in the previous examples or a carbon electrode treated with oxygen plasma after printing (denoted "High Binding" or "HB").

[0291] These plates were used to generate ECL from TAGs (a solution designated "300k Free Tags" or "300k FT") dissolved in MSD Read Buffer T 1X at a concentration that provided an ECL signal of approximately 300,000 ECL numbers when analyzed in a Std96-1 plate on an MSD SECTOR Imager plate reader. For this example, analysis was performed using a video capture system (as described in Example 4) to measure the ECL time course during the ECL experiment. ECL was generated using a 3 V ramp waveform from 0 V to 3 V for the plate with the Ag / AgCl auxiliary electrode and from 2 V to 5 V for the plate with the carbon counter electrode. The effect of ramp rate was evaluated by testing each plate / electrode condition at five different ramp durations (ramp rates): 3.0 s (1.0 V / s), 2.0 s (1.5 V / s), 1.5 s (2.0 V / s), 1.2 s (2.5 V / s), and 1.0 s (3.0 V / s). Plots of ECL intensity versus applied potential for the control plate with a carbon counter electrode using the five different ramp rates are provided in Figures 29, 31A, 32A, 33A, and 34A, respectively. Similar plots for the test plate with an AgCl auxiliary electrode are provided in Figures 30, 31B, 32B, 33B, and 34B. Traces for the control and test plates are plotted together in Figure 35 for a ramp rate of 1.0 V / s.

[0292] For all ramp rates and electrode configurations, the onset of ECL was at a lower potential for the HB working electrode than for the Std working electrode, due to the lower potential for the onset of TPA oxidation (approximately 0.6 V for HB and approximately 0.8 V for Std vs. Ag / AgCl reference electrode). Relative to the control plate with a carbon counter electrode, the onset of ECL for the HB96-1 plate was at a higher potential than for the other HB electrode configurations, likely an effect of the higher reduction potential at the counter electrode required to support the high current required for the large-area working electrode of the 96-1 format. This large shift in onset potential was not observed when an Ag / AgCl auxiliary electrode was used, indicating that the potential at these electrodes was not sensitive to this change in current density. Figures 36A and 36B plot the integrated ECL intensity over the waveform as a function of ramp rate and show that the integrated ECL intensity decreases with ramp rate as less time is spent in the voltage region where ECL is generated. Figures 36D and 36E plot the ECL onset potential as a function of ramp rate and show that, compared to using a carbon counter electrode, the Ag / AgCl auxiliary electrode provides an ECL onset potential that is less sensitive to electrode configuration and ramp rate.

[0293] Figure 35 plots the ECL traces for the test plate (Ag / AgCl) and the control plate (carbon) at a 1.0 V / s ramp rate (colored curves). The plot also shows the cyclic voltammetry current vs. voltage traces for the oxidation of TPA in MSD Lead Buffer T 1X at the Std and HB carbon working electrodes (black curves). The plot shows that the higher ECL onset potential for Std vs. HB is related to the higher onset potential for TPA oxidation. The greater sensitivity of HB vs. Std to the effect of electrode configuration on ECL onset potential is likely due to the significantly higher TPA oxidation current observed with the HB electrode near the ECL onset potential. Table 22 shows the applied potentials that provided the maximum ECL intensity for each plate type measured with a 1.0 V / s waveform. With an Ag / AgCl auxiliary electrode, the ECL peak potential correlated with the area ratio of the working electrode to the counter electrode, with 96-1 > 96-10 > 96ss. The Ag / AgCl auxiliary electrode minimized the effect of the electrode area ratio on the ECL peak potential and shift on the HB plate, as well as the ECL onset potential on the HB plate.

[0294] [Table 36]

[0295] Various experiments were conducted using assay plates employing Ag / AgCl auxiliary and working electrode configurations in various configurations. Results from some of these experiments are described herein. Experiments were conducted to determine the difference in ECL signal intensity with varying working-to-auxiliary electrode ratios for different BTI concentrations and electrode configurations. For all configurations tested, including the concentric open-spot configuration (e.g., as shown in Figures 3A and 3B), the concentric closed-spot configuration (e.g., as shown in Figures 7A and 7B), the concentric open trefoil configuration (e.g., as shown in Figures 4A and 4B), and the concentric penta configuration (e.g., as shown in Figures 5A and 5B), an increase in ECL response intensity with increasing ratio was observed. This result was observed in situations where the increase in ratio was due to a change in auxiliary electrode size or a change in working electrode size.

[0296] In another experiment, differences in ECL signal intensity with incubation time were observed for different BTI concentrations and electrode configurations. For all configurations tested, including the concentric open spot configuration (e.g., as shown in Figures 3A and 3B), the concentric open trefoil configuration (e.g., as shown in Figures 4A and 4B), and the concentric penta configuration (e.g., as shown in Figures 5A and 5B), an increase in ECL signal was observed for 2 or 3 hours of incubation compared to 1 hour of incubation. An increase in ECL signal intensity was also observed for 3 hours of incubation compared to 2 hours of incubation. In a further experiment, differences in %CV with incubation time were observed between different electrode configurations with different BTI concentrations. The configurations tested were the concentric open spot configuration (e.g., as shown in Figures 3A and 3B), the concentric open trefoil configuration (e.g., as shown in Figures 4A and 4B), and the concentric penta configuration (e.g., as shown in Figures 5A and 5B). In the concentric open spot configuration, a decrease in %CV with increasing incubation time was observed. In the concentric open trefoil configuration, an increase in %CV with increasing incubation time from 1 hour to 2 hours was observed. In the concentric penta configuration, an increase in %CV with increasing incubation time from 1 hour to 2 hours and from 2 hours to 3 hours was observed.

[0297] In another experiment, gain differences were observed between different spots of an electrochemical cell in different electrode configurations with different working electrode zone to auxiliary electrode zone ratios. Test configurations included a non-concentric 10-spot arrangement, a concentric open spot arrangement (e.g., as shown in Figures 3A and 3B), and a concentric open trefoil arrangement (e.g., as shown in Figures 4A and 4B). The results, summarized in Table 23 below, show that the spread between minimum and maximum gain is reduced in the concentric open arrangement relative to the non-concentric layout. Thus, a concentric arrangement of working electrode zones may offer advantages in maintaining consistent gain across all spots or locations within a well.

[0298] [Table 37]

[0299] In embodiments, concentric, approximately equidistant electrode configurations may provide particular advantages for ECL procedures, as described above and throughout. Due to the symmetry of these designs (see, e.g., Figures 1C, 3A-3F, and 6A-7F), each spot or working electrode zone is similarly affected by the overall geometry of the well. For example, as described with respect to Figure 2C, the meniscus effect on the fluid filling the well is approximately equal for each of the concentrically arranged working electrode zones. This occurs because the meniscus is a radial effect, and the concentrically arranged working electrode zones are located approximately equidistant from the center of the well. In addition, as described above, mass transport effects may be equalized between different working electrode zones. During orbital or rotational vibration, the distribution of material within the well may depend on the distance from the center of the well due to mass transport effects over time. Thus, concentrically arranged working electrode zones help reduce or minimize variations that may result from uneven material distribution across the well. Also, because each of the working electrode zones is located approximately equidistant from the auxiliary electrode, any voltammetric effects that might otherwise occur due to unequal distances may be reduced or minimized.

[0300] The disclosures above and below provide electrochemical cells including a working electrode zone and an auxiliary electrode. Various designs are presented and described. In some examples, electrode configurations (e.g., concentric, separated, and equidistant) and the advantages they provide are described. In further examples, electrode compositions (e.g., Ag, Ag / AgCl, and / or any other material disclosed throughout (e.g., metal oxide, metal / metal oxide couple, etc.)) and the advantages they provide are described. It is understood that the scope of the embodiments described herein also includes examples of various electrode configurations and patterns (e.g., as shown in Figures 3A-8D and 37-43D) used with electrodes of other materials (e.g., carbon, carbon composites, and / or other carbon-based materials). The advantages offered by the electrochemical cell electrode configurations and geometries described herein can be realized in embodiments including electrodes of any of the materials described herein. Furthermore, the advantages provided by electrochemical cells employing Ag, Ag / AgCl, and / or any other material disclosed throughout (e.g., metal oxides, metal / metal oxide couples, etc.) to form electrodes as described herein may be realized in embodiments including other working electrode zone configurations (see, e.g., Figures 3A-4E of U.S. Pat. No. 7,842,246, issued Nov. 30, 2010, which is incorporated herein by reference in its entirety).

[0301] In embodiments, the electrochemical cells described herein may include individually addressable electrodes. As described throughout, electrochemical cells consistent with the present disclosure include working and auxiliary electrodes arranged according to a specific positioning and patterning. As described above with respect to FIG. 9, for example, in embodiments, the electrochemical cells of individual wells may be selectively addressable (e.g., electrically energizable). In further embodiments, as discussed below with respect to FIGS. 37, 38A-38C, 39A-39L, 40A-40N, 41A-41M, 42A-42I, and 43A-43D, individual electrodes within individual electrochemical cells (e.g., within individual wells) may be selectively addressable. This design allows any electrode (and any combination of electrodes) within the substrate of the electrochemical cell to be electrically addressed independently of each other electrode within the substrate.

[0302] FIG. 37 illustrates an electrochemical cell with individually addressable electrodes according to embodiments disclosed herein. The electrochemical cell 1001 includes multiple working electrode zones 1002 and at least one auxiliary electrode 1003. In the embodiment illustrated by this figure, the electrochemical cell 1001 may include 10 working electrode zones 1002 and one auxiliary electrode 1003. In other embodiments, fewer or more working electrode zones 1002 may alternatively be provided (e.g., 6, 7, 8, 12, etc.) and / or multiple auxiliary electrodes may be provided (e.g., 2, 4, 5, etc.). The following description of the individually addressable electrode electrochemical cell will refer to the 10 working electrode zone design (also referred to throughout as a 10-spot design) illustrated in FIG. 37. In a 10-spot design, the working electrodes may be referred to by their position within the well, e.g., 1-spot, 2-spot, 3-spot, etc. However, it will be understood that the devices, systems, and methods disclosed herein in connection with individually addressable electrode electrochemical cells are not limited to the particular 10-spot design, but may be applied to other patterns and arrangements of electrode zones, including at least those disclosed herein (e.g., those shown in Figures 3A-8C), as appropriate.

[0303] As described above, the working electrode zone may include an entire electrode, or in other embodiments, two or more working electrode zones may be formed within and / or on a single electrode. For example, as in the case of electrochemical cell 1001 formed by well electrode structure 3101 described below, the working electrode zones may be formed by individual working electrodes that are electrically isolated from one another. In another example, the working electrode zones may be configured as a single electrode formed from one or more conductive materials. In another example, as described above, for example, with respect to Figures 21A-21F, the working electrode zones may be formed by isolating portions of a single working electrode. In this example, the single working electrode may be formed from one or more conductive materials, and the working electrode zones may be formed by electrically isolating regions ("zones") of the single working electrode using an insulating material, such as a dielectric. The electrochemical cell 1001 is formed from a well electrode structure 3101 having individually electrically isolated working electrodes as discussed herein, although it is understood that the features, elements, and aspects of the well electrode structure 3101 may be modified or altered to achieve working electrode zones according to other aspects discussed herein, for example, working electrode zones formed by separate zones of a single electrode.

[0304] Figures 38A and 38B show a portion of a multiwell plate having wells containing individually addressable electrode electrochemical cells according to embodiments disclosed herein. Figure 38A is a perspective top view of a multiwell assay plate 2000. Figure 38A shows a top plate 3001 having top plate openings 3002 that define wells 3003 of the multiwell assay plate 2000 arranged in a well pattern, each well defined by a well area, as further described below. Figure 38A also shows a base plate 3010 including a substrate 3100, as shown in Figure 38B.

[0305] Figure 38B shows the substrate 3100 and its top surface 3190. In the exemplary multi-well assay plate 2000 shown in Figure 38A, the top surface 3190 is mated to the top plate 3001. Figure 38B shows various elements visible on the top surface 3190 of the substrate 3100 that help to form a plurality of well electrode structures 3101. Further elements and additional description of the well electrode structures 3101 are provided below. The well electrode structures 3101 help to define the electrochemical cell 1001 (Figure 37) comprising a plurality of working electrode zones 1002 and at least one auxiliary electrode 1003.

[0306] 38C shows the substrate 3100 and its bottom surface 3210. The bottom surface 3210 of the substrate 3100 features a plurality of electrode contacts 3201, which are arranged in electrode contact groups, as described below, and form part of a well electrode structure 3101.

[0307] 38A-38C show a multiwell assay plate 2000 having 96 wells 3003 in a 12x8 arrangement. Each well 3003 corresponds to a well electrode structure 3101. In further embodiments, any suitable number of wells 3003 and well electrode structures 3101 may be provided. Additionally, the multiwell assay plate 2000 shown in FIGS. 38A-38C is merely an example of one use of the well electrode structures 3101 described herein. The well electrode structures 3101 described herein may be used to form electrochemical cells 1001 for a variety of applications, including, for example, cartridge readers, plate-based analyzers, lateral flow-based testing devices, etc.

[0308] In embodiments, the well electrode structure 3101 may be formed on the substrate 3100 in a variety of ways, for example, via a sequential screen printing process, etching, deposition, lithography, and / or other methods for forming electrodes. In these examples, the well electrode structure 3101 may be printed layer by layer on the substrate 3100, although other methods are contemplated as well. In embodiments, the electrodes described throughout may be implemented on one or more circuits, such as, for example, a printed circuit board (PCB) and a thin flexible PCB, e.g., a flex circuit. Figures 39A-39L show aspects of the well electrode structure 3101 and the layering process.

[0309] 39A-42D illustrate the design and layout of a substrate 3100 for a multi-well assay plate 2000 having separate, individually addressable working electrodes 3102 and auxiliary electrodes 3103. Furthermore, the following description describes various manufacturing processes for achieving the described design and layout. Screen printing techniques provide one example of manufacturing a substrate 3100 having the layout and design disclosed herein. Alternative manufacturing methods, including various types of printing, deposition, lithography, etching, inkjet printing, flexography, gravure printing, and the like, can be used to manufacture the structures described herein without departing from the scope of the embodiments described herein. Additionally, the layouts and designs described herein can be applied to substrates 3100 of different materials, as may be appropriate for particular manufacturing techniques, e.g., printed circuit boards or flexible printed circuit boards (flex circuits). In embodiments, alternative manufacturing methods may include, use, or require alternative dimensions for manufacturing purposes.

[0310] In the following discussion of the layering process used to create the substrate 3100, various dimensions are discussed. As described below with respect to FIGS. 39A-39L, nominal dimensions are described. It is understood that these dimensional descriptions (whether the term nominal is used or not) include variations based on manufacturing tolerances and limitations. Additionally, the term approximate is also used to describe dimensions. As used below, approximate refers to variations in dimensions beyond those of manufacturing tolerances that do not interfere with the described functionality of the various structures.

[0311] The dimensions described below with respect to Figures 39A-39L are selected to allow for placement of all necessary features within the allowable space without interference between the features. Interference can refer to physical interference, e.g., two features intersecting in an unintended manner, and electrical interference, e.g., two features electrically affecting each other in an unintended manner. The dimensions described below are selected with consideration of manufacturing tolerances and limitations. Such concerns relate to both manufacturing tolerances within the production of a single layer, e.g., the tolerances associated with manufacturing various screens and templates for printing, and the tolerances associated with printing one or more features using a screen or template. The tolerances at issue also relate to manufacturing tolerances across multiple layers, e.g., print-to-print registration tolerances involved in aligning one layer with a subsequent layer. Due to these types of manufacturing errors, the potential for tolerance or error stackup must be considered. For example, to meet a requirement that two features remain a specific distance apart in the final product, the nominal distance between the two features may need to be greater than that specific distance to account for variations in the manufacturing process within a single layer. Furthermore, if these features are located on different layers on the substrate, the nominal distance must also be chosen to account for potential print-to-print registration errors.

[0312] FIG. 39A shows the electrode pattern of a portion of the top surface 3190 of the substrate 3100. The depicted portion shows features of four well electrode structures 3101 disposed on the top surface 3190 of the substrate 3100. Features belonging to one well electrode structure 3101 are shown outlined with a dashed border. As described below, the well electrode structure 3101 features are patterned on the top surface 3190 and bottom surface 3210 of the substrate 3100. The well electrode structures 3101 each include an electrode group 3104 that includes multiple working electrodes 3102 and auxiliary electrodes 3103. The working electrodes 3102 are electrode structures that form the working electrode zone 1002 of the electrochemical cell 1001. As described above, in the electrochemical cell 1001, the working electrode zone 1002 is formed by individually electrically isolated working electrodes, e.g., working electrode 3102. In further embodiments, as described above, the working electrode zone 1002 may be formed according to other principles and concepts described herein. The auxiliary electrode 3103 is an electrode structure that forms the auxiliary electrode 1003 of the electrochemical cell 1001. As will be described in more detail below with respect to the patterning process, each of the plurality of working electrodes 3102 is electrically isolated from the auxiliary electrode 3103 and the remainder of the plurality of working electrodes 3102.

[0313] Electrode group 3104 is disposed within well region 3106 defined by well perimeter 3105. Well region 3106 is configured to correspond to top plate opening 3002 to form the bottom of well 3003 when substrate 3100 is bonded to top plate 210.

[0314] 39B shows the electrode contact pattern for a portion of the bottom surface 3210 of the substrate 3100. Each electrode contact group 3204 (four shown) corresponds to an electrode contact group 3204 in a respective well electrode structure 3101. Features belonging to one well electrode contact group 3204 are shown outlined with a dashed boundary. Each electrode contact group 3204 includes multiple electrode contacts, including multiple working electrode contacts 3202 and auxiliary electrode contacts 3203. The electrode contact groups 3204 are disposed outside of the well region 3106.

[0315] A portion of the well electrode structure 3101 patterned on the top surface 3190 of the substrate 3100 is connected to a portion of the well electrode structure 3101 on the bottom surface 3210 of the substrate by a plurality of vias 3205 arranged in a plurality of via groups 3206, each via group 3206 corresponding to an electrode contact group 3204 and electrode group 3104 of a respective well electrode structure 3101. The via groups 3206 provide electrical connection between the electrode contact groups 3204 and the electrode groups 3104 as follows:

[0316] Each well electrode structure 3101 further includes an electrical trace group including a plurality of electrical traces 3107 patterned on the top surface 3190. Each electrical trace 3107 provides electrical connection between a via 3205 and either the working electrode 3102 or the auxiliary electrode 3103, as follows: Each electrical trace 3107 includes a via contact spot 3110 (shown in FIG. 39A ) electrically connected to a via 3205 (shown in FIG. 39B ) and positioned outside the well region 3106, an electrical bridge 3109 extending from the contact spot 3110 into the well region 3106, and an electrode contact spot 3108 electrically connected to the electrical bridge 3109 and to a corresponding electrode working electrode 3102 in the well region 3106. The electrical trace group also includes at least one auxiliary electrical trace 3112 including a via contact spot 3110 (shown in FIG. 39A ) electrically connected to a via 3205 (shown in FIG. 39B ) and positioned outside the well region 3106, an electrical bridge 3109 extending from the contact spot 3110 into the well region 3106, and an electrode contact spot 3108 electrically connected to the electrical bridge 3109 and the corresponding auxiliary electrode 3103. Thus, a continuous electrical path is established between each working electrode 3102 and the corresponding working electrode contact 3202 through the corresponding via 3205 and the corresponding electrical trace 3107 including the corresponding via contact spot 3110, the corresponding electrical bridge 3109, and the corresponding electrode contact spot 3108. Similarly, a continuous electrical path is established between each auxiliary electrode 3103 and a corresponding auxiliary electrode contact 3203 via a corresponding via 3205 and a corresponding auxiliary electrical trace 3112 including a corresponding via contact spot 3110, a corresponding electrical bridge 3109, and a corresponding electrode contact spot 3108.

[0317] As referred to herein, contact spots, e.g., via contact spots and electrode contact spots, can include layer structures configured to provide electrical contact with at least one other layer above or below, and optionally with at least one other electrical structure. Thus, via contact spots can include a material layer configured to provide contact with a via and an electrical bridge, thereby facilitating electrical connection between the two. Electrode contact spots can include a material layer configured to provide contact with an electrode and an electrical bridge, thereby facilitating electrical connection between the two. Contact spots can be any suitable shape, such as circular to support contact with a circular structure, like electrode contact spot 3108, curved to support contact with a curved structure, like electrode contact spot 4708 (FIGS. 47A / 47B), and / or any other suitable shape. In some embodiments, contact spots can have a shape similar to another material layer to which they are connected. In some embodiments, contact spots can have a shape different from another material layer to which they are connected.

[0318] In one example, the well electrode structure 3101 may be arranged as follows: The auxiliary electrode 3103 may be positioned approximately at the center of the well region 3106. The area of ​​the auxiliary electrode 3103 may encompass the center of the well region 3106. The auxiliary electrode 3103 may be approximately concentric with the well region 3106. The working electrode 3102 may be positioned in a circle approximately equidistant from the auxiliary electrode 3103. The working electrodes may be separated from each other within the circle by multiple working electrode spacings 3111. In an embodiment, at least one of the multiple working electrode spacings 3111 may be sized to allow placement therein or through an electrical bridge 3109 of an auxiliary electrical trace 3112 of multiple electrical traces 3112 connecting the auxiliary electrode 3103 to the auxiliary electrode contact 3203. Thus, the electrical bridge 3109 of the auxiliary electrical trace 3112 spans an adjacent working electrode 3102. Sizing at least one of the plurality of working electrode spacings 3111 may include, for example, positioning at least two adjacent working electrodes at a greater distance from each other relative to the remaining working electrodes 3102 (creating a gap between these two working electrodes). In this example, the working electrodes 3102 may form a C-shaped pattern. Thus, at least one of the plurality of working electrode spacings 3111 may be larger than the rest of the plurality of working electrode spacings 3111. In other embodiments, the distance between all adjacent working electrodes 3102 may be the same (or approximately the same), with a sufficient distance between each adjacent pair to allow for placement of one or more electrical bridges 3109 of one or more auxiliary electrical traces 3112 therein or therethrough. In this example, the working electrodes form a concentric circular shape. While these examples relate to circular wells, other well shapes (e.g., square, rectangular, oval, etc.) are contemplated as well.

[0319] 39A and 39B, each well electrode structure 3101 includes an electrode group 3104, an electrical trace group including a plurality of electrical traces 3107 and auxiliary traces 3112, an electrode contact group 3204, and a via group 3206. The well electrode structures 3101 can form an electrochemical cell 1001 as described herein.

[0320] 39C-39J show the individual layers involved in constructing the well electrode structure 3101 according to embodiments disclosed herein.

[0321] 39C shows a pattern of holes formed in the substrate. Holes 3115 are formed in the substrate 3100 outside of the well regions 3106. The holes 3115 may be formed by laser cutting, micro-drilling, or any other suitable method. The holes 3115 are formed in pairs, one for each via 3205, for redundancy purposes.

[0322] Holes 3115 may have a nominal diameter ranging from about 0.004 inches to 0.010 inches in diameter. Holes 3115 are spaced about 0.019 inches from the well area. The positioning of holes 3115 allows for a potential variation of about 0.019 inches in alignment between well area 3106 and top plate opening 3002 when substrate 3100 is attached to top plate 3001.

[0323] 39D shows a pattern of a layer applied to the bottom surface of the substrate to fill holes 3115 and form vias 3205. A conductive layer is applied to bottom surface 3210 of substrate 3100. The conductive layer flows through holes 3115, filling them and forming conductive vias 3205. The conductive layer is disposed on a plurality of via spots 3207, each via spot corresponding to one of a pair of holes 3115. In an embodiment, the conductive layer forming via spots 3207 may be silver or another conductive material, such as, for example, other metals (e.g., gold, platinum, nickel, steel, iridium, copper, aluminum), conductive inks, conductive alloys, etc.

[0324] The via spot 3207 is configured to be approximately circular, with a nominal dimension 3901 between the edge of the spot via 3207 and the edge of the hole 3115 being approximately 0.015 inches. Because the holes 3115 are arranged in pairs, the distance between the edge of the hole 3115 and the edge of the spot may vary and may be as large as approximately 0.018 inches in places. Selecting a nominal value of approximately 0.0015 inches for the nominal dimension 3901 accounts for potential alignment errors between the conductive layer forming the via spot 3207 and the hole 3115.

[0325] Figure 39E shows the pattern of layers applied to the bottom surface of the...

Claims

1. 1. A multi-well assay plate comprising: a top plate having top plate openings defining wells of the multi-well assay plate arranged in a well pattern, each well defined by a well area; a base plate comprising a substrate having a top surface and a bottom surface, the top surface being fitted to the top plate; and a plurality of well electrode structures, each comprising an electrode group patterned on the top surface and having an auxiliary electrode, an additional electrode, and a plurality of working electrodes electrically isolated from the remainder of the auxiliary electrode, the additional electrode, and the plurality of working electrodes; and an electrode contact group patterned on the bottom surface corresponding to the electrode group and including a plurality of working electrode contacts electrically connected to corresponding working electrodes, an auxiliary electrode contact electrically connected to the auxiliary electrode, and an additional electrode contact electrically connected to the additional electrode.

2. 10. The plate of claim 1, wherein each working electrode of a selected electrode group is configured to be energized independently from the energization of the remaining working electrodes of the plurality of working electrodes of the selected electrode group.

3. The plate of claim 1 , wherein the working electrodes of selected electrode groups are configured to be energized separately.

4. The plate of claim 1 , wherein each well electrode structure is electrically isolated from the remaining well electrode structures of the plurality of well electrode structures.

5. 10. The plate of claim 1, wherein the top surface further comprises an adhesive layer corresponding to the well pattern on the top surface, the well area being free of adhesive.

6. the electrode group is disposed within the well region, the electrode contact group is disposed outside the well region, and each of the plurality of well electrode structures is The plate of claim 1 further comprising a via group including a plurality of vias extending through the substrate and electrically connected to the plurality of electrode contacts.

7. 2. The plate of claim 1, wherein each of the plurality of well electrode structures further comprises an electrical trace group patterned on the top surface and including a plurality of electrical traces electrically connecting a plurality of vias to the electrode group.

8. 2. The plate of claim 1, wherein the additional electrode is electrically isolated from the auxiliary electrode.

9. The plate of claim 1 , wherein the additional electrode is configured to partially surround the auxiliary electrode.

10. 2. The plate of claim 1, wherein the additional electrode is configured with a border edge that is equidistant from a border edge of the auxiliary electrode.

11. 2. The plate of claim 1, wherein the plurality of electrical traces provide electrical connection between the group of electrode contacts arranged outside the well region and the group of electrodes arranged inside the well region.

12. 2. The plate of claim 1, wherein each of the plurality of electrical traces includes a via contact spot in electrical communication with a corresponding one of the plurality of vias outside the well region and extending at least about 0.015 inches from the corresponding via of the plurality of vias, an electrical bridge extending from the via contact spot into the well region, and an electrode contact spot connected to the electrical bridge inside the well region.

13. 2. The plate of claim 1, wherein the auxiliary electrode is positioned approximately at the center of the well region, the additional electrodes are positioned to at least partially surround the auxiliary electrode, and the working electrodes are arranged in a circle approximately equidistant from the auxiliary electrode.

14. 2. The plate of claim 1, wherein the working electrodes are separated from one another within the circle by a plurality of working electrode spacings, at least one of the plurality of working electrode spacings being sized to accommodate placement therein of an auxiliary electrical trace of the plurality of electrical traces that connects the auxiliary electrode to the auxiliary electrode contact.

15. 2. The plate of claim 1, wherein the working electrodes are separated from one another within the circle by a plurality of working electrode spacings, at least one of the plurality of working electrode spacings being sized to accommodate placement therein of an additional electrical trace of the plurality of electrical traces that connects the additional electrode to the additional electrode contact.

16. 2. The plate of claim 1, wherein the auxiliary electrode is formed from an Ag / AgCl layer, the additional electrode is formed from an Ag / AgCl layer, and the working electrode is formed from a silver layer, a first carbon conductive layer, and a second carbon conductive layer.

17. 2. The plate of claim 1, wherein the top surface of the substrate further comprises a first insulating layer arranged in a pattern that exposes the electrode groups of each of the plurality of electrode well structures and covers the remainder of the top surface of the substrate.

18. The plate of claim 1 , wherein the first insulating layer is configured to provide a single exposed portion that exposes the auxiliary electrode and the additional electrode.

19. 2. The plate of claim 1, wherein the first insulating layer is configured to provide a first exposed portion exposing the auxiliary electrode and a second exposed portion exposing the additional electrode, the first exposed portion and the second exposed portion not intersecting.

20. The plate of claim 1 , wherein the plurality of well electrode structures comprises 48 well electrode structures.

21. The plate of claim 1 , wherein the plurality of well electrode structures comprises 96 well electrode structures.

22. The plate of claim 1 , wherein the electrode contact groups are arranged in an orientation-neutral pattern.

23. 10. The plate of claim 1, wherein the additional electrode is electrically connected to a second additional electrode in an adjacent well electrode structure.

24. The plate of claim 1 , wherein the auxiliary electrode is electrically connected to a second auxiliary electrode in an adjacent well electrode structure.

25. 10. The plate of claim 1, wherein the multi-well assay plate is configured for use in an electrochemiluminescence (ECL) assay.

26. 1. A method of using a multi-well assay plate, the multi-well assay plate comprising: a plurality of wells arranged in a well pattern; and a plurality of well electrode structures, each well electrode structure corresponding to a well of the plurality of wells, each well electrode structure including an electrode group patterned on a bottom of the well and having an auxiliary electrode, an additional electrode, and a plurality of working electrodes electrically isolated from the auxiliary electrode, the additional electrode, and the remainder of the plurality of working electrodes, the method comprising: generating an electric potential between a selected working electrode and at least one of the selected auxiliary electrode and selected additional electrode associated with a selected well electrode structure; maintaining substantial electrical isolation of an unpowered working electrode of the selected well electrode structure; and measuring a response to the electric potential.

27. 27. The method of claim 26, further comprising generating a plurality of potentials between a selected working electrode and at least one of a corresponding auxiliary electrode and a corresponding additional electrode from a plurality of selected well electrode structures; maintaining substantial electrical isolation of unpowered working electrodes of each of the plurality of selected well electrode structures; and measuring a plurality of responses to the plurality of potentials.

28. 27. The method of claim 26, wherein generating the plurality of potentials and measuring the plurality of responses are performed substantially simultaneously.

29. 27. The method of claim 26, further comprising, after measuring the plurality of responses, sequentially generating a plurality of potentials for the un-powered working electrodes in each of the plurality of selected well electrode structures between each of the un-powered working electrode contacts and at least one of a corresponding auxiliary electrode and a corresponding additional electrode from each of the plurality of selected well electrode structures, maintaining substantial electrical isolation of the un-powered working electrodes in each of the plurality of selected well electrode structures, and measuring a plurality of responses to the sequential plurality of potentials.

30. 27. The method of claim 26, further comprising generating a second potential between a second selected working electrode and the at least one of a selected auxiliary electrode and an additional electrode associated with the selected well electrode structure, maintaining substantial electrical isolation of the unpowered working electrode of the selected well electrode structure, and measuring a second response to the second potential.

31. 27. The method of claim 26, wherein the multiwell assay plate further comprises an electrode contact group patterned on a bottom surface of the multiwell assay plate, the electrode contact group comprising a plurality of electrode contacts including a plurality of working electrode contacts electrically connected to corresponding working electrodes, an auxiliary electrode contact electrically connected to the auxiliary electrode, and an additional electrode contact electrically connected to the additional electrode, and wherein generating the potential comprises contacting the plurality of electrode contacts with a plate electrical connector including a plurality of pins arranged to correspond to the plurality of electrode contacts, and applying a voltage to a selected electrode contact from the plurality of electrode contacts, wherein the selected electrode contact is electrically connected to a selected working electrode from the plurality of working electrodes.

32. 27. The method of claim 26, further comprising generating the potential between only one selected working electrode, one selected auxiliary electrode, and optionally one selected additional electrode associated with the selected well electrode structure.

33. 27. The method of claim 26, further comprising generating the potential between a plurality of selected working electrodes that are less than all of the plurality of working electrodes of the electrode group and at least one of the selected auxiliary electrode and the selected additional electrode.

34. 27. The method of claim 26, further comprising depositing a biological sample in at least one well of the plurality of wells.

35. 27. The method of claim 26, further comprising loading the multi-well assay plate into an instrument configured to generate the electrical potential, wherein the multi-well assay plate is configured to be loaded orientation-neutral in a first orientation or a second orientation that is 180 degrees different from the first orientation.

36. 27. The method of claim 26, wherein the multiwell assay plate is a first multiwell assay plate, the method further comprising loading the first multiwell assay plate into an instrument configured to generate the electrical potential in a first orientation, and loading the second multiwell assay plate into the instrument in a second orientation that is 180° different from the first orientation, wherein the electrical potential generates valid assay electrical conditions in the first multiwell assay plate in the first orientation and in the second multiwell assay plate in the second orientation.

37. 27. The method of claim 26, wherein the selected working electrodes include all the working electrodes of a selected well electrode structure.

38. 27. The method of claim 26, wherein the selected working electrodes are selected from a set of electrode well structures, the set of working electrodes including an equal number of designated working electrodes from each well electrode structure of the set of well electrode structures, and the designated working electrodes of the set of working electrodes are positioned at the same respective locations within each well electrode structure of the set of well electrode structures.

39. 27. The method of claim 26, wherein the set of electrode well structures comprises 2x2, 4x4, or 8x8 sector electrode well structures.

40. 27. The method of claim 26, wherein the set of electrode well structures comprises 8 x 1 columns or 12 x 1 rows of electrode well structures.

41. 27. The method of claim 26, wherein generating the potential generates a potential in an electrochemiluminescence assay.

42. 1. A method of making a multi-well assay plate, comprising: forming a plurality of holes in a substrate; applying a first conductive layer of material to a first side of the substrate, the first conductive layer filling the plurality of holes to form a plurality of vias; applying a second conductive layer of material to the first side of the substrate, the second conductive layer overlying the first conductive layer to form a plurality of electrode contacts; applying a third conductive layer of material to a second side of the substrate, the third conductive layer forming a plurality of electrical traces, the plurality of electrical traces connecting first portions of the plurality of vias to a plurality of working electrodes, forming a plurality of additional electrode contact spots and a plurality of auxiliary electrode contact spots; and applying a fourth conductive layer of material to the second side of the substrate, the fourth conductive layer forming a plurality of auxiliary electrodes, forming a plurality of auxiliary electrode electrical traces, a plurality of additional electrodes, and a plurality of additional electrode electrical traces; applying a fifth conductive layer of material to the second side of the substrate, covering the third conductive layer; applying a sixth conductive layer of material to the second side of the substrate, the sixth conductive layer forming the plurality of working electrodes; applying an insulating layer of material to the second side of the substrate, the insulating layer exposing the plurality of auxiliary electrodes, the plurality of additional electrodes, and the plurality of working electrodes and insulating the remainder of the second side of the substrate; and adhering the substrate to a top plate having top plate openings defining wells of the multi-well assay plate arranged in a well pattern, each well defined by a well area.

43. 43. The method of claim 42, wherein the plurality of auxiliary electrodes, the plurality of additional electrodes, the plurality of working electrodes, the plurality of electrical traces, the plurality of vias, and the plurality of electrode contacts are arranged in a plurality of well electrode structures, each well electrode structure comprising: an electrode group patterned on the second surface and having an auxiliary electrode from the plurality of auxiliary electrodes electrically isolated from the plurality of auxiliary electrodes and the plurality of working electrodes, and an additional electrode electrically isolated from the plurality of additional electrodes and the plurality of working electrodes; an electrode contact group including a working electrode contact from the plurality of electrode contacts electrically connected to a corresponding working electrode, an auxiliary electrode contact electrically connected to the auxiliary electrode, and an additional electrode contact electrically connected to the additional electrode; a via group including a via from the plurality of vias connected to a corresponding one of the electrode contact groups; and an electrical trace group including an electrical trace from the plurality of electrical traces connecting a corresponding via in the via group to a corresponding electrode in the electrode group.

44. 43. The method of claim 42, wherein the auxiliary electrode is surrounded by a circularly arranged group of working electrodes from the plurality of working electrodes, and the additional electrode at least partially surrounds the auxiliary electrode.

45. 1. A multiwell assay plate comprising: a top plate having top plate openings defining wells of the multiwell assay plate arranged in a well pattern, each well defined by a well area; a base plate comprising a substrate having a top surface and a bottom surface, the top surface mated to the top plate; a plurality of well electrode structures, each comprising an electrode group patterned on the top surface and having an auxiliary electrode and a plurality of working electrodes electrically isolated from the auxiliary electrode and the remainder of the plurality of working electrodes; and an electrode contact group patterned on the bottom surface corresponding to the electrode group and including a plurality of electrode contacts electrically connected to corresponding working electrodes and auxiliary electrode contacts electrically connected to the auxiliary electrodes by auxiliary electrical traces, an insulating layer being provided between the auxiliary electrical traces and the plurality of working electrodes.

46. 46. ​​The plate of claim 45, wherein each of the plurality of well electrode structures further comprises an electrode trace group, the electrode trace group comprising a plurality of electrical traces patterned on the top surface and electrically connecting the plurality of vias to the electrode group.

47. 46. ​​The plate of claim 45, wherein each working electrode of a selected electrode group is configured to be electrically energized independently from the electrical energization of the remaining working electrodes of the plurality of working electrodes of the selected electrode group.

48. 46. ​​The plate of claim 45, wherein the electrode group is disposed within the well region and the electrode contact group is disposed outside the well region, and each of the plurality of well electrode structures further comprises a via group including a plurality of vias electrically connected to the plurality of electrode contacts and extending through the substrate.

49. 46. ​​The plate of claim 45, wherein each of the plurality of well electrode structures further comprises an electrode trace group, the electrode trace group including a plurality of electrical traces patterned on the top surface and electrically connecting the plurality of vias to the electrode group.

50. 46. ​​The plate of claim 45, wherein the top surface of the substrate includes a second insulating layer arranged in a pattern that exposes the electrode groupings and covers the remainder of the top surface of the substrate for each of the plurality of electrode well structures.

51. 46. ​​The plate of claim 45, wherein the plurality of well electrode structures comprises 48 or 96 well electrode structures.

52. 46. ​​The plate of claim 45, wherein the electrode contact groups are arranged in an orientation-neutral pattern.

53. 46. ​​The plate of claim 45, further comprising a bus bar patterned on the bottom surface of the substrate and configured to provide an electrical connection between the auxiliary electrode contact and an adjacent auxiliary electrode contact of an adjacent well electrode structure.

54. 46. ​​The plate of claim 45, further comprising a bus bar patterned on the bottom surface of the substrate and configured to provide electrical connection between the auxiliary electrode contacts and an additional central auxiliary electrode contact disposed on the bottom surface of the substrate opposite the auxiliary electrode disposed on the top surface of the substrate.

55. 46. ​​The plate of claim 45, wherein the at least one auxiliary electrode comprises Ag / AgCl.

56. 46. ​​The plate of claim 45, wherein the multi-well assay plate is configured for use in an electrochemiluminescence (ECL) assay.

57. 1. An electrochemical cell for performing electrochemical analyses, comprising: a plurality of working electrode zones disposed on a surface of the cell and defining a pattern; at least one auxiliary electrode disposed on the surface; and at least one additional electrode disposed on the surface, wherein each of the plurality of working electrode zones is electrically isolated from each other and from the auxiliary electrode and the additional electrode.

58. 58. The cell of claim 57, wherein the pattern is configured to provide sufficient distance between the working electrode zones to prevent electrical interference.

59. 59. The cell of claim 57 or 58, wherein individual working electrode zones of the plurality of working electrode zones are configured to be electrically energized while maintaining the remainder of the plurality of working electrode zones in an unenergized state.

60. 58. The cell of claim 57, wherein electrically energizing each of the plurality of working electrode zones comprises generating a potential between the each of the working electrode zones and one of the auxiliary electrode and the additional electrode.

61. 58. The cell of claim 57, wherein electrically energizing individual ones of the plurality of working electrode zones comprises generating multiple potentials between different ones of the individual working electrode zones and the auxiliary electrode.

62. 58. The cell of claim 57, wherein the electrochemical cell is part of a plate.

63. 58. The cell of claim 57, wherein the electrochemical cell is part of a cartridge.

64. 58. The cell of claim 57, wherein the electrochemical cell is part of a flow cell.

65. 58. The cell of claim 57, wherein the auxiliary electrode is disposed at a center of the electrochemical cell and the working electrode is disposed in a circle approximately equidistant from the auxiliary electrode, the circle including a gap configured to allow passage of an auxiliary electrical trace for connecting the auxiliary electrode to an auxiliary electrode contact.

66. 58. The cell of claim 57, wherein the electrochemical analysis comprises electrochemiluminescence (ECL) analysis.

67. 58. The cell of claim 57, wherein the at least one auxiliary electrode comprises Ag / AgCl.

68. 1. A multi-well assay plate comprising: a top plate having top plate openings that define wells of the multi-well assay plate arranged in a well pattern, each well being defined by a well area; a base plate including a substrate having a top surface and a bottom surface, the top surface being fitted to the top plate; and a plurality of well electrode structures, each well electrode structure including an electrode group patterned on the top surface and having at least one auxiliary electrode and at least one working electrode, and a plurality of working electrode contacts electrically connected to the working electrode and auxiliary electrode contacts electrically connected to the auxiliary electrode.

69. 69. The multi-well assay plate of claim 68, wherein the auxiliary electrode contact is in electrical contact with a second auxiliary electrode of an adjacent well electrode structure.

70. 69. The multi-well assay plate of claim 68, wherein said at least one working electrode comprises one working electrode.

71. 69. The multi-well assay plate of claim 68, wherein said at least one working electrode comprises ten electrically connected working electrodes.

72. 69. The multi-well assay plate of claim 68, wherein the at least one working electrode is formed from a first conductive layer, a second conductive layer, and a third conductive layer.

73. 73. The multi-well assay plate of claim 72, wherein said at least one auxiliary electrode is formed from said first conductive layer and a fourth conductive layer comprising Ag / AgCl.

74. 1. A multi-well assay plate comprising: a top plate having top plate openings defining wells of said multi-well assay plate arranged in a well pattern, each well defined by a well area; a base plate including a substrate having a top surface and a bottom surface, the top surface mated to the top plate; a plurality of well electrode structures arranged on the upper surface; a plurality of zigzag auxiliary electrode contacts arranged in a row of auxiliary electrode contacts on the bottom surface; a plurality of working electrode contacts arranged in an interrupted working electrode array on the top surface; a plurality of auxiliary electrode bases arranged in rows corresponding to the upper surface; a plurality of working electrode bases arranged in rows on the upper surface; a plurality of vias providing conductive through-holes from the bottom surface to the top surface, the plurality of vias being arranged in rows corresponding to the rows of interrupted working electrodes; an insulating layer disposed on the upper surface and configured to define at least one auxiliary electrode and at least one working electrode for each well electrode structure; 1. A multi-well assay plate comprising:

75. 75. The multi-well assay plate of claim 74, wherein said at least one auxiliary electrode comprises two auxiliary electrodes.

76. 75. The multi-well assay plate of claim 74, wherein said at least one working electrode comprises only one working electrode.

77. 75. The multi-well assay plate of claim 74, wherein said at least one working electrode comprises 10 working electrodes.

78. 75. The multi-well assay plate of claim 74, wherein said plurality of working electrode bases are formed in at least one additional conductive layer.

79. 75. The multi-well assay plate of claim 74, wherein said plurality of auxiliary electrode bases are formed in at least one additional conductive layer.

80. 75. The multi-well assay plate of claim 74, wherein said plurality of well electrode structures are configured for electrochemical analysis.

81. 81. The multi-well assay plate of claim 80, wherein said electrochemical analysis comprises electrochemiluminescence (ECL) analysis.

82. 75. The multi-well assay plate of claim 74, wherein said at least one auxiliary electrode comprises Ag / AgCl.

83. each zigzag auxiliary electrode contact includes a top lateral portion extending in a first lateral direction from a first end of a central longitudinal portion, and a bottom lateral portion extending in a second lateral direction from a second end of the central longitudinal portion, the second lateral direction being different from the first direction; Each zigzag auxiliary electrode contact corresponds to a well electrode structure; 75. The multi-well assay plate of claim 74.

84. 84. The multi-well assay plate of claim 83, wherein the plurality of auxiliary electrode bases are arranged at positions corresponding to bottom positions of the top lateral portion and the bottom lateral portion.

85. 84. The multi-well assay plate of claim 83, wherein the plurality of working electrode bases are arranged at positions corresponding to a bottom position of the central longitudinal portion, and each working electrode base has a central portion configured to form one or more electrodes, a top extension portion extending in the second horizontal direction away from the central portion, and a bottom extension portion extending in the first horizontal direction away from the central portion.

86. The vias in each row are: the top lateral portions are connected to corresponding auxiliary electrode bases, the bottom lateral portions are connected to corresponding auxiliary electrode bases, working electrode contacts are connected to corresponding top extension portions, and working electrode contacts are connected to corresponding bottom extension portions; 86. The multi-well assay plate of claim 85.

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