Electrochemical cell device and method for producing same

Abstract

A multi-well assay plate is provided. The multi-well assay plate includes at least a top plate defining a plurality of wells and a base plate having a substrate (3100) patterned with well electrode structures (3100). The well electrode structures are arranged in a plurality of sector electrode structures (3150), each including a working electrode busbar (3190) and a portion of an auxiliary electrode pattern (3191). The substrate further includes at least one working electrode contact (3204) patterned on a bottom surface (3210) and an auxiliary electrode contact pattern (3291) disposed on the bottom surface, the auxiliary electrode contact pattern including a cutout (3295).

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 296,287, filed January 4, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] SUMMARY OF THE DISCLOSURE Embodiments of the present invention relate to systems, devices, and methods that use electrochemical cells in conducting chemical, biochemical, and biological assays and analyses, and methods of manufacturing auxiliary electrodes. [Background technology]

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

[0004] Assay systems using electrochemical properties and procedures may include a sample area (e.g., a well, a well in a multi-well 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 for measuring the result data. Depending on the design and configuration of the electrodes, the assay system may be classified as a reference system and a non-reference system. For example, the working electrode is the electrode at which the reaction of interest occurs in the assay system. The working electrode is used in combination with the counter electrode to establish a potential difference, current flow, and / or electric field in the sample area. The potential difference may be divided between the interfacial potentials of the working and counter electrodes. In a non-reference system, the interfacial potential (the force that causes the reaction at the electrode) applied to the working electrode is not controlled or known. In a reference system, the sample area 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 while the reaction occurs in the sample area.

[0005] One example of these assay systems is the electrochemiluminescence (ECL) immunoassay. ECL immunoassays involve a process that uses ECL labels designed to emit light when electrochemically stimulated. Light generation occurs when a voltage is applied to electrodes located within the sample region that holds the material under test. The voltage causes cyclic oxidation and reduction reactions, which result in the generation and emission of light. In ECL, the electrochemical reaction responsible for ECL is brought about by applying a potential difference between working and counter electrodes. Summary of the Invention

[0006] According to an embodiment of the present specification, a multi-well assay plate is disclosed. The multi-well assay plate includes a top plate having top plate openings defining 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 mating with the top plate; a plurality of sector electrode structures forming a plurality of well electrode structures, each of the sector electrode structures including at least one working electrode bus bar deposited on the top surface and a portion of an auxiliary electrode pattern deposited on the top surface; at least one working electrode contact patterned on the bottom surface in electrical communication with the at least one working electrode bus bar; and an auxiliary electrode contact pattern disposed on the bottom surface in electrical communication with the auxiliary electrode pattern, the auxiliary electrode contact pattern including a first plurality of cutouts and a second plurality of cutouts. The first plurality of cutouts is configured to accommodate at least one working electrode contact, and the second plurality of cutouts is configured to generate a plurality of separation zones.

[0007] According to an embodiment of the present specification, a method of using a multi-well assay plate is disclosed, the multi-well assay plate includes a plurality of wells arranged in a well pattern, a plurality of sector electrode structures forming a plurality of well electrode structures corresponding to the plurality of wells, each including at least one working electrode bus bar deposited on a top surface of a substrate of the multi-well assay plate forming a plurality of working electrode zones, and a portion of the auxiliary electrode pattern deposited on the top surface forming a plurality of auxiliary electrode zones, an auxiliary electrode contact pattern in communication with the auxiliary electrode pattern and including a first plurality of cutouts and a second plurality of cutouts, a plurality of working electrode contacts deposited on a bottom surface of the multi-well assay plate within the first plurality of cutouts and electrically connected to the corresponding working electrode bus bar, and a plurality of separation zones disposed within the second plurality of cutouts. The method includes generating an electrical potential between a plurality of working electrode zones formed by at least one working electrode bus bar associated with a selected sector electrode structure and a plurality of auxiliary electrode zones associated with the selected sector electrode structure, maintaining substantial electrical isolation between the plurality of working electrode zones of the selected sector electrode structure and remaining working electrode zones of the remaining sector electrode structures, and measuring a response to the electrical potential.

[0008] According to an embodiment herein, a method of manufacturing a multi-well assay plate including a plurality of wells is disclosed, the method includes 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 and providing a plurality of working electrode contact bases and an auxiliary electrode contact pattern base, applying a second conductive layer of material to the first side of the substrate, the second conductive layer overlying the first conductive layer and forming a plurality of working electrode contacts and an auxiliary electrode contact pattern having a first plurality of cutouts and a second plurality of cutouts, the working electrode contacts being formed within the first plurality of cutouts and the separation zones being formed within the second plurality of cutouts, applying a third conductive layer of material to a second side of the substrate, the third conductive layer filling the plurality of working electrode busbar bases and the auxiliary electrode pattern base, and applying a second conductive layer of material to the second side of the substrate, the second conductive layer overlying the first conductive layer and forming a plurality of working electrode contacts and an auxiliary electrode contact pattern having a first plurality of cutouts and a second plurality of cutouts, the working electrode contacts being formed within the first plurality of cutouts and the separation zones being formed within the second plurality of cutouts. applying a fourth conductive layer of material to a second side of the substrate overlying the fourth conductive layer to form a plurality of working electrode busbars, the fourth conductive layer forming a carbon layer; applying a fifth conductive layer of material to a second side of the substrate overlying the fourth conductive layer to form a plurality of working electrode busbars, the sixth conductive layer forming an auxiliary electrode pattern; applying an insulating layer of material to the second side of the substrate overlying the auxiliary electrode pattern base, the sixth conductive layer forming an auxiliary electrode pattern; applying an insulating layer of material to the second side of the substrate, the insulating layer exposing the plurality of auxiliary electrode zones and the plurality of working electrode zones and insulating a remainder of the plurality of wells; and adhering the substrate to a top plate having top plate openings defining wells of a multi-well assay plate arranged in the well pattern, each well defined by a well area.

[0009] According to an embodiment herein, a substrate having a top surface and a bottom surface is disclosed, the substrate comprising: a plurality of electrochemical cells disposed on the top surface for performing electrochemical analysis, each including one or more working electrode zones disposed on a surface of the cell, each in electrical communication with one another, and at least one auxiliary electrode zone disposed on the surface of the cell; and an auxiliary electrode contact pattern disposed on the bottom surface and in electrical communication with the at least one auxiliary electrode, the auxiliary electrode contact pattern including a first plurality of cutouts and a second plurality of cutouts, the first plurality of cutouts configured to accommodate at least one working electrode contact in electrical communication with the one or more working electrode zones, and the second plurality of cutouts configured to generate a plurality of separation zones.

[0010] The details of one or more embodiments of this disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the technology described in this disclosure will become apparent from the specification and drawings, and from the claims.

[0011] These and other features and advantages of the present invention will become apparent from the following description of embodiments of the invention 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 description of the drawings]

[0012] [Figure 1A] 1A-1D show several views of an electrochemical cell according to embodiments disclosed herein. [Figure 1B] 1A-1D show several views of an electrochemical cell according to embodiments disclosed herein. [Figure 1C] 1A-1D show several views of an electrochemical cell according to embodiments disclosed herein. [Figure 2A]FIG. 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] FIG. 1D shows a side view of a sample area of ​​the multi-well plate of FIG. 1C according to embodiments disclosed herein. [Figure 3A] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 3B] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 3C] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 3D] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 3E] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 3F] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 4A] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 4B]1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 4C] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 4D] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 4E] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 4F] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 5A] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 5B] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 5C] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 6A] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 6B]1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 6C] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 6D] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 6E] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 6F] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 7A] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 7B] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 7C] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 7D] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 7E]1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 7F] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 8A] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 8B] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 8C] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 8D] 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. FIG. 1A shows an example of an electrode design for use in the electrochemical cell of FIGS. 1A-1C or the multi-well plate of FIGS. 2A-2C according to embodiments disclosed herein. [Figure 9A] 1 illustrates an example of an assay device according to embodiments disclosed herein. [Figure 9B] 1 illustrates an example of an assay device according to embodiments disclosed herein. [Figure 10A] 4 illustrates the decay time of an auxiliary electrode, according to an embodiment. [Figure 10B] 4 illustrates the decay time of an auxiliary electrode, according to an embodiment. [Figure 11] 1 illustrates a process for performing electrochemical analyses and procedures using pulse waveforms according to embodiments disclosed herein. [Figure 12A] 1 illustrates an example of a pulse waveform according to embodiments disclosed herein. [Figure 12B] 1 illustrates an example of a pulse waveform according to embodiments disclosed herein. [Figure 13] 1 illustrates a process for performing an ECL analysis and procedure using a pulse waveform according to embodiments disclosed herein. [Figure 14] 1 illustrates a process for performing an ECL analysis using a pulse waveform according to embodiments disclosed herein. [Figure 15] 1 illustrates a process for performing an ECL analysis using a pulse waveform according to embodiments disclosed herein. [Figure 16] 1 illustrates a process for fabricating a well according to embodiments disclosed herein. [Figure 17A] 1 illustrates exemplary stages in a process for fabricating a well according to embodiments disclosed herein. [Figure 17B] 1 illustrates exemplary stages in a process for fabricating a well according to embodiments disclosed herein. [Figure 17C] 1 illustrates exemplary stages in a process for fabricating a well according to embodiments disclosed herein. [Figure 17D] 1 illustrates exemplary stages in a process for fabricating a well according to embodiments disclosed herein. [Figure 17E] 1 illustrates exemplary stages in a process for fabricating a well according to embodiments disclosed herein. [Figure 17F] 1 illustrates exemplary stages in a process for fabricating a well according to embodiments disclosed herein. [Figure 18A] 1 illustrates exemplary stages in a process for fabricating a well according to embodiments disclosed herein. [Figure 18B] 1 shows an embodiment of a well according to the present disclosure. [Figure 19A] 1 shows some examples of electrode configurations that have been tested, according to embodiments disclosed herein. [Figure 19B] 1 shows some examples of electrode configurations that have been tested, according to embodiments disclosed herein. [Figure 19C]1 shows some examples of electrode configurations that have been tested, according to embodiments disclosed herein. [Figure 19D] 1 shows some examples of electrode configurations that have been tested, according to embodiments disclosed herein. [Figure 20A] 1 illustrates an electrochemical cell having electrodes according to embodiments disclosed herein. [Figure 20B] 1 illustrates an electrochemical cell having electrodes according to embodiments disclosed herein. [Figure 21A] 1 illustrates a portion of a multiwell plate having wells containing electrochemical cells according to embodiments disclosed herein. [Figure 21B] 1 illustrates a portion of a multiwell plate having wells containing electrochemical cells according to embodiments disclosed herein. [Figure 21C] 1 illustrates a portion of a multiwell plate having wells containing electrochemical cells according to embodiments disclosed herein. [Figure 21D] 1 illustrates a portion of a multiwell plate having wells containing electrochemical cells according to embodiments disclosed herein. [Figure 22A] 1 illustrates aspects of the structure of an electrochemical cell according to embodiments disclosed herein. [Figure 22B] 1 illustrates aspects of the structure of an electrochemical cell according to embodiments disclosed herein. [Figure 22C] 1 illustrates aspects of the structure of an electrochemical cell according to embodiments disclosed herein. [Figure 22D] 1 illustrates aspects of the structure of an electrochemical cell according to embodiments disclosed herein. [Figure 22E] 1 illustrates aspects of the structure of an electrochemical cell according to embodiments disclosed herein. [Figure 22F] 1 illustrates aspects of the structure of an electrochemical cell according to embodiments disclosed herein. [Figure 22G] 1 illustrates aspects of the structure of an electrochemical cell according to embodiments disclosed herein. [Fig. 22H] 1 illustrates aspects of the structure of an electrochemical cell according to embodiments disclosed herein. [Figure 22I] 1 illustrates aspects of the structure of an electrochemical cell according to embodiments disclosed herein. [Figure 22J] 1 illustrates aspects of the structure of an electrochemical cell according to embodiments disclosed herein. [Figure 22K] 1 illustrates aspects of the structure of an electrochemical cell according to embodiments disclosed herein. [Figure 22L] 1 illustrates aspects of the structure of an electrochemical cell according to embodiments disclosed herein. [Figure 22M] 1 illustrates aspects of the structure of an electrochemical cell according to embodiments disclosed herein. [Figure 22N] 1 illustrates aspects of the structure of an electrochemical cell according to embodiments disclosed herein. [Figure 22O] 1 illustrates aspects of the structure of an electrochemical cell according to embodiments disclosed herein. [Fig. 23A-23PP] 1 illustrates aspects of the structure of a substrate including multiple electrochemical cells according to embodiments disclosed herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Specific embodiments of the present invention will now be described with reference to the drawings. The following detailed description is merely exemplary in nature and is not intended to limit the present invention or its application and uses. Also, it is not intended to be limited by any of the express or implied theories presented in the preceding technical field, background, brief summary, or the following detailed description.

[0014] The embodiments of the present disclosure are directed to electrochemical cells including auxiliary electrode designs, and electrochemical analyzers 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. In certain embodiments, materials, compounds, etc. may be doped to generate a redox couple, although other methods of generating a redox couple are contemplated as well. The auxiliary electrode with a reduction-oxidation couple that defines a stable interfacial potential allows the auxiliary electrode to function as a dual-function electrode. That is, one or more auxiliary electrodes operate as counter electrodes and reference electrodes simultaneously. Because the auxiliary electrode operates as a dual-function electrode, the space occupied by the auxiliary electrode in the electrochemical cell is reduced, allowing additional configurations and numbers of working electrode zones to be included in the electrochemical cell.

[0015] In embodiments, the use of one or more auxiliary electrodes also improves the read time of electrochemical analyzers and devices during electrochemical analysis processes, such as ECL processes.In conventional non-reference ECL systems, it is common to use a slow voltage ramp that passes through the voltage that provides maximum ECL to provide tolerance to the potential fluctuations at the auxiliary electrode.The use of the auxiliary electrode of the present invention, such as the auxiliary electrode with 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.

[0016] FIG. 1A illustrates an example of an electrochemical cell 100 according to an 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 induce 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 by a reduction-oxidation (redox) reaction triggered by the introduction of electrical energy via the auxiliary electrodes 102 and the working electrode zones 104. In some embodiments, the ionic medium 103 may include an electrolyte solution, such as, for example, water, or other solvent in which ions, such as, for example, a salt, are dissolved. In some embodiments, as described in more detail below, the surface of the ionic medium 103 or the working electrode zones 104 may include luminescent species that generate and emit photons during a redox reaction. 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 at these electrodes.

[0017] As described herein, in use, the auxiliary electrode has an electrode potential that can be determined by a redox reaction occurring at the electrode. The potential can be determined, 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 by 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 determines a stable interfacial potential can function as a dual-function electrode. That is, one or more auxiliary electrodes 102 may provide the functions associated with both a counter electrode and a reference electrode in a three-electrode electrochemical system by providing a high current (function of a counter electrode in a three-electrode system) while providing the ability to determine and control the potential at the working electrode (function of a reference electrode in a three-electrode system). The auxiliary electrode(s) 102 may operate as a counter electrode by providing a potential difference with one or more of the working electrode zones 104 during redox reactions occurring within the electrochemical cell 100 in which the auxiliary electrode(s) 102 are located. Based on the chemical structure and composition of the auxiliary electrode(s) 102, the auxiliary electrode(s) 102 may operate as a reference electrode for determining a potential difference with one or more of the working electrode zones 104.

[0018] In embodiments, the auxiliary electrode 102 may be formed of 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) may provide a stable interfacial potential during the reduction of the chemical mixture such that a quantifiable amount of charge is generated through the reduction-oxidation reactions occurring in the electrochemical cell 100. In some embodiments, the chemical mixture of the one or more auxiliary electrodes 102 may include an oxidizer that provides a stable interfacial potential during the reaction of the chemical mixture, and the amount of oxidizer in the chemical mixture may be equal to or greater than the amount of oxidizer necessary to provide the entire reduction-oxidation reaction in the electrochemical cell occurring during the electrochemical reaction. In embodiments, the auxiliary electrode 102 is formed of a chemical mixture that provides an interfacial potential during the reaction of the chemical mixture such that a quantifiable amount of charge is generated through the reduction-oxidation reaction occurring in the electrochemical cell 100. The chemical mixture of the auxiliary electrode 102 includes an oxidizer that supports redox reactions during operation of the electrochemical cell 100, for example, during biological, chemical, and / or biochemical assays such as ECL generation and analysis.

[0019] In an embodiment, the amount of oxidant in the chemical mixture on the one or more auxiliary electrodes 102 is equal to or greater than the amount of oxidant required for the entirety of redox reactions to occur within the electrochemical cell 100 during a biological, chemical, and / or biochemical assay and / or analysis, such as, for example, ECL generation. For example, a sufficient amount of the chemical mixture on the one or more auxiliary electrodes 102 remains after a redox reaction has occurred for an initial biological, chemical, and / or biochemical assay and / or analysis, thereby allowing one or more additional redox reactions to occur throughout subsequent biological, chemical, and / or biochemical assays and / or analyses.

[0020] 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 the areal 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 the areal 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 that is in contact with the ionic medium 103. That is, as shown in FIG. 1B, the auxiliary electrodes 102 may be formed in a three-dimensional (3D) shape that extends 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 the 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 shows the auxiliary electrode 102 to be a regularly shaped cylinder, the auxiliary electrode 102 may 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 that is in contact with the ionizable medium 103, similar to the 2D cross-sectional area of ​​the auxiliary electrode 102 described in FIG. 1B, for example. In certain embodiments, the areal surface area (exposed surface area) may be distinguished from the true surface area, which includes the actual surface of the electrode, taking into account any height or depth in the z dimension. With these examples, the areal surface area is less than or equal to the true surface area.

[0021] In embodiments, the one or more auxiliary electrodes 102 may be formed with 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, the one or more auxiliary electrodes 102 may include silver (Ag) and silver chloride (AgCl), or other suitable metal / metal halide couples. In some embodiments, the one or more auxiliary electrodes 102 formed with a mixture of Ag-AgCl may provide an interfacial potential at or near the standard reduction potential of Ag-AgCl, which is about 0.22 V. Other examples of chemical mixtures may include metal oxides having 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, and the like. In some embodiments, the chemical mixture may provide an interfacial potential in the range of about 0.1 V to about 3.0 V. Table 1 provides examples of reduction potentials of redox couples of chemical mixtures that may be included in the one or more auxiliary electrodes 102. One of ordinary skill in the art will recognize that the example reduction potentials are approximations and can vary, for example, by + / - 5.0%, based on chemical composition, temperature, impurities in the chemical mixture, or other conditions.

[0022] [Table 1]

[0023] In embodiments, the chemical mixture of the redox couple at the one or more auxiliary electrodes may be based on a molar ratio of the redox couple that is within a specified range. In some embodiments, the chemical mixture has a molar ratio of Ag to AgCl that is within a specified range, for example, about 1 or greater. 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 involved in the redox reaction are oxidized or reduced.

[0024] In some embodiments, the auxiliary electrode(s) 102 comprise approximately 1.56×10 of the electrode surface area. -5 ~5.30×10 -4 C / mm2 In some embodiments, the one or more auxiliary electrodes 102 may include a redox couple that passes an average current of about 0.5 mA to 4.0 mA through the redox reaction of the redox couple and generates an 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 through the redox reaction and generates an ECL in the range of about 1.4 V to 2.6 V.

[0025] In embodiments, the auxiliary electrode(s) 102 may be equal to or greater than the amount of charge that the oxidant in the redox couple must pass through the auxiliary electrode to complete the electrochemical analysis. In some embodiments, the auxiliary electrode(s) 102 may be equal to or greater than about 3.07×10 -7 ~3.97×10 -7 In some embodiments, the auxiliary electrode 102 may comprise an exposed surface area of ​​1 mm 2 Approximately 1.80 x 10 -7 ~2.32×10 -7 In some embodiments, the auxiliary electrode(s) 102 may comprise at least one mole of oxidant within the total (or aggregate) exposed surface area of ​​the working electrode zone(s) 104 of 1 mm 2 At least about 3.7 x 10 -9 In some embodiments, the auxiliary electrode or electrodes may comprise at least 1 mm2 of the total (or aggregate) exposed surface area of ​​the working electrode zone or zones 104. 2 At least about 5.7 x 10 per -9 The mixture may contain moles of oxidizing agent.

[0026] In embodiments, the one or more auxiliary electrodes 102 may include a redox couple where the reaction of species in the redox couple is the primary redox reaction occurring at the one or more auxiliary electrodes 102 when a voltage or potential is applied. In some embodiments, the applied potential is less than a defined potential required to reduce water or to effect 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.

[0027] In embodiments, the auxiliary electrode(s) 102 (and working electrode zone(s) 104) may be formed using any type of manufacturing process, such as printing, 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 electrode(s) 102 (and working electrode zone(s) 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 auxiliary electrode(s) 102 and / or working electrode zone(s) 104 using a doping process.

[0028] A working electrode zone 104 may be a location on an electrode where a reaction of interest may occur. The reaction of interest may be chemical, biological, biochemical, 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 / unbroken area where a reaction may occur, and an electrode "zone" may be a portion (or the entirety) of an electrode where a particular reaction of interest occurs. In certain embodiments, the working electrode zone 104 may comprise the entire electrode, while in other embodiments, multiple 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 of one or more conductive materials. In other examples, 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 of one or more conductive materials, and the working electrode zones may be formed by electrically isolating regions ("zones") of the single working electrode with insulating materials, such as, for example, insulators. In any embodiment, the working electrode zones 104 may be formed of any type of conductive material, such as, for example, metals, metal alloys, carbon compounds, doped metals, and combinations of conductive and insulating materials.

[0029] In embodiments, the working electrode zone 104 may be formed of a conductive material. For example, the working electrode zone 104 may include a metal, such as, for example, gold, silver, platinum, nickel, steel, iridium, copper, aluminum, a conductive alloy, etc. In some embodiments, the working electrode zone 104 may include an oxide coating material (e.g., aluminum coated with aluminum oxide). In some embodiments, the working electrode zone 104 may be formed of a carbon-based material, such as, for example, 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 of 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, the working electrode zone 104 may be formed of a carbon layer and a silver layer fabricated using screen printing of carbon and silver inks, as described in more detail below. In some embodiments, the working electrode zone 104 may be formed of a semiconducting material (eg, silicon, germanium) or a semiconducting film, such as, for example, indium tin oxide (ITO), antimony tin oxide (ATO), and the like.

[0030] In embodiments, as described in more detail below, the one or more auxiliary electrodes 102 and the 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 arrangements and patterns within the electrochemical cell 100, etc.) to improve the electrochemical properties and analyses (e.g., ECL analyses) performed by the apparatus and devices including the electrochemical cell. FIG. 1C illustrates 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 may include ten working electrode zones 104 and a single auxiliary electrode 102. Various other examples of electrode designs are described below with reference to FIGS. 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, and 8A-8D.

[0031] In embodiments, the configuration and arrangement of the working electrode zones 104 within the electrochemical cell 100 may be defined according to the adjacency between the working electrode zones 104 and / or between the working electrode zones 104 and one or more auxiliary electrodes 102. In some embodiments, the 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, the adjacency may be defined as the relative distance between the working electrode zones 104 and / or one or more auxiliary electrodes 102. In some embodiments, the 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, for example, the perimeter of the electrochemical cell.

[0032] In embodiments herein, for example, the auxiliary electrode(s) 102 and the working electrode zone(s) 104 of each electrochemical cell 100 may be formed to have respective sizes such that the ratio of the aggregate exposed surface area of ​​the working electrode zone(s) 104 to the exposed surface area of ​​the auxiliary electrode(s) 102 is greater than 1, although other ratios (e.g., ratios equal to, less than, or greater than 1) are also contemplated for the electrochemical cell 100. In some embodiments herein, for example, each of the auxiliary electrode(s) 102 and / or the working electrode zone(s) 104 may be formed in a circular shape having a surface area that substantially defines a circle, although other shapes (e.g., rectangular, square, oval, quatrefoil, or any other regular or irregular geometric shape) are contemplated.

[0033] In embodiments according to the present disclosure, for example, the auxiliary electrode(s) 102 and / or the working electrode zone(s) 104 may be formed in a wedge shape having a wedge-shaped surface area. That is, the auxiliary electrode(s) 102 and / or the working electrode zone(s) 104 may be formed to have 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 be blunt, e.g., rounded corners at the connections with the side boundaries. In some embodiments, the wide boundary and / or the narrow boundary may be sharp, e.g., squared corners at the connections with the side boundaries. In embodiments, the wedge shape may be used 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 arranged such that a wide boundary is adjacent to the periphery of the working area 101 and a narrow boundary is adjacent to the center of the working area 101.

[0034] In embodiments, the electrochemical cell 100 may be included in an apparatus or device for performing electrochemical analyses. In some embodiments, as described below, the electrochemical cell 100 may form part of a well for an assay device performing electrochemical analyses, such as, for example, an ECL immunoassay. In some embodiments, the electrochemical cell 100 may form a cartridge used in an analytical device or apparatus, such as a flow cell in a flow cytometer. Those skilled in the art will appreciate that the electrochemical cell 100 may be used in any type of apparatus or device in which controlled redox reactions are performed.

[0035] 2A-2C show several views of a sample area ("well") 200 including 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 embodiments herein. 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. Additionally, while the well 200 shown in FIGS. 2A-2C is essentially circular, other well shapes may be included as well. For example, square wells, square wells with rounded corners, oval wells, rectangular wells, rectangular wells with rounded corners, triangular wells, hexagonal wells, etc.

[0036] As shown in FIG. 2A, a top view, a base plate 206 of a multiwell plate 208 (shown in FIG. 2B) may include a plurality of wells 200. The base plate 206 may form the bottom of each well 200 and may 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 FIG. 2B, a perspective view, the multiwell plate 208 may include a top plate 210 and a base plate 206. The top plate 210 may 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 emitted when a voltage is applied across the one or more working electrode zones 104 and one or more auxiliary electrodes 102 located within the wells 200 holding the material under test. The applied voltage causes cyclic oxidation and reduction reactions that result in the generation and emission of photons (light), which can then be measured to analyze the material under test.

[0037] Depending on whether the reaction occurring at the working electrode zone 104 accepts or donates 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, for example, 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, cell fractions, cell 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, and the like. Reagents may be immobilized on one or more of the working electrode zones 104 by a variety of methods including passive adsorption, specific binding, and / or through the formation of covalent bonds to functional groups present on the surface of the electrode.

[0038] For example, ECL species that can be induced to emit ECL may be attached to the working electrode zone 104 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 a noble metal that is resistant to corrosion and oxidation, including, for example, Ru-containing and Os-containing organometallic compounds such as tris-bipyridyl-ruthenium (RuBpy) moieties, and (ii) luminol and related compounds. Species involved in ECL labeling in the ECL process are referred to herein as ECL co-reactants. Commonly used co-reactants include, for example, tertiary amines such as triisopropylamine (TPA), oxalate, persulfate, for ECL from RuBpy, and hydrogen peroxide for ECL from luminol. The light generated by the ECL label may be used as a reporter signal in diagnostic procedures. For example, an ECL label may be covalently attached to a binding agent, such as an antibody or nucleic acid probe, and the participation of the binding agent in a binding interaction may be monitored by measuring the ECL emitted from the ECL label. Alternatively, an ECL signal from an ECL-active compound may be indicative of the chemical environment.

[0039] In embodiments, the working electrode zone 104 and / or auxiliary electrode 102 (or other components of the well 200) may be treated (e.g., pretreated) with materials and / or processes that improve attachment (e.g., adsorption) of materials used in the electrochemical process (e.g., reagents, ECL species, labels, etc.) to the surfaces 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 with a process (e.g., plasma treatment) that renders the surfaces of the working electrode zone 104 and / or auxiliary electrode 102 (or other components of the well 200) hydrophilic (also referred to herein as "high binding" or "HB"). In some embodiments, the working electrode zone 104 and / or the auxiliary electrode 102 (or other components of the well 200) may be untreated or may be treated with a process that renders the surface of the working electrode zone 104 and / or the auxiliary electrode 102 (or other components of the well 200) exhibiting hydrophobicity (also referred to herein as “standard” or “Std”).

[0040] As shown in FIG. 2C, which is a side cross-sectional view of a portion of the multiwell plate 208 of FIG. 2B, a plurality of 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 a bottom surface of the top plate 210 to a top surface of the top plate 210. The wells 200 may be adapted to hold one or more fluids 250, such as, for example, an ionic medium as described above. In certain embodiments, the one or more wells 200 may be adapted to hold gases and / or solids as an alternative or in addition to the one or more fluids 250. In embodiments, the top plate 210 may be secured to the base plate 206 by an adhesive 214 or other connecting material or device.

[0041] The multiwell plate 208 may include any number of wells 200. For example, as shown in Figures 2A and 2B, the multiwell plate 208 may include 96 wells 200. One skilled in the art will appreciate that the multiwell plate 208 may include any number of wells 200, such as, for example, 6, 24, 384, 1536 wells 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. Although the wells 200 are shown in a circular configuration in Figures 2A-2C (resulting in a cylinder), other shapes are contemplated as well, including ovals, squares, and / or other regular or irregular polygons. Additionally, the shape and configuration of the multiwell plate 108 may take many forms and is not necessarily limited to a rectangular array as shown in these figures.

[0042] 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 a porous electrode (e.g., a mat of carbon fibers or fibrils, sintered metal, and metal films deposited on a filtration membrane, paper, or other porous substrate). When configured as a porous electrode, the working electrode zone 104 and / or auxiliary electrode 102 may employ filtration of the solution through the electrode to: i) increase mass transport to the electrode surface (e.g., to increase the kinetics of binding of molecules in the solution to molecules on the electrode surface), ii) trap particles on the electrode surface, and / or iii) remove liquid from the wells.

[0043] In embodiments as described above, each of the auxiliary electrodes 102 in the wells 200 is formed with a chemical mixture that provides a defined potential during reduction of the chemical mixture such that a quantifiable amount of charge is generated through the reduction-oxidation reaction occurring in the wells 200. The chemical mixture of the auxiliary electrodes 102 includes an oxidizing agent that supports a reduction-oxidation reaction that may be used during a biological, chemical, and / or biochemical assay and / or analysis, such as, for example, ECL generation and analysis. In embodiments, 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 passed through the auxiliary electrode and / or the amount of charge required to effect an electrochemical reaction at the working electrode in at least one well 200 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 auxiliary electrodes 102 remains after a redox reaction has occurred for an initial biological, chemical, and / or biochemical assay and / or analysis to allow one or more additional redox reactions to occur throughout subsequent biological, chemical, and / or biochemical assays and / or analyses. 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 multiple working electrode zones to the exposed surface area of ​​the auxiliary electrode.

[0044] In embodiments, the one or more auxiliary electrodes 102 of the well 200 may be formed with a chemical mixture including a redox couple, as described above. In some embodiments, the one or more auxiliary electrodes 102 of the well 200 may be formed with 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, 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.

[0045] For certain applications, such as ECL generation, various embodiments of the auxiliary electrode 102 may be adapted to prevent polarization of the electrode throughout the ECL measurement by including a sufficiently high concentration of accessible redox species. The auxiliary electrode 102 may be formed by printing the auxiliary electrode 102 onto a multiwell plate 208 with an Ag-AgCl chemical mixture (e.g., ink, paste, etc.) having a defined Ag to AgCl ratio. In embodiments, the amount of oxidizer in the auxiliary electrode chemical mixture is based at least in part on the Ag to AgCl ratio in the auxiliary electrode chemical mixture. In embodiments, the auxiliary electrode chemical mixture having Ag and AgCl comprises about 50 percent or less of AgCl, e.g., 34 percent, 10 percent, etc.

[0046] In some embodiments, one or more auxiliary electrodes 102 in a well 200 may be arranged to cover a total working electrode area of ​​1 mm 2 At least about 3.7 x 10 -9In some embodiments, the one or more auxiliary electrodes 102 in the well 200 may comprise 1 mm 2 of total working electrode area in the well 200. 2 At least about 5.7 x 10 per -9 The mixture may contain moles of oxidizing agent.

[0047] In various embodiments, the 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 arrangements and patterns within a well, etc.) to improve an electrochemical analysis (e.g., ECL analysis) performed by an assay device including one or more of the wells 200, examples of which are described below with reference to Figures 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, and 8A-8D. In embodiments according to the present disclosure, for example, the one or more auxiliary electrodes 102 and the one or more working electrode zones 104 of each well 200 may be formed to have respective sizes such that the ratio of the total 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 (e.g., ratios equal to 1, or less than or greater than 1) are contemplated as well. In embodiments according to the present disclosure, for example, each of the auxiliary electrodes 102 and / or working electrode zones 104 may be formed in a circular shape having a surface area that substantially defines a circle, although other shapes (e.g., rectangular, square, oval, quatrefoil, or any other regular or irregular geometric shape) are contemplated. In embodiments according to the present disclosure, for example, the auxiliary electrodes 102 and / or working electrode zones 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 a sidewall of the well 200 being larger than a second side or end of the wedge-shaped surface area adjacent a 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. For example, the auxiliary electrodes 102 and working electrode zones 104 may be formed in a pattern that maximizes the space available to the auxiliary electrodes 102 and working electrode zones 104.

[0048] In some embodiments, the auxiliary electrode(s) 102 and / or the working electrode zone(s) 104 may be formed to have 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 be blunt, e.g., rounded corners at the connections with the side boundaries. In some embodiments, the wide boundary and / or the narrow boundary may be sharp, e.g., squared corners at the connections with the side boundaries. In embodiments, the wedge shape may be used 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, the working electrode zone(s) 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.

[0049] In embodiments according to the present disclosure, the auxiliary electrode 102 and one or more working electrode zones 104 of each well 200 may be formed at the bottom of the well 200 according to different positional configurations or patterns. The different positional configurations or patterns may improve an electrochemical analysis (e.g., ECL analysis) performed by an assay device including one or more of the wells 200, examples of which are described below with reference to Figures 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, and 8A-8D. The auxiliary electrode 102 and working electrode zones 104 may be arranged 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 working electrode zones 104 adjacent to each other for each of the working electrode zones 104 among the total number of working electrode zones 104. This allows more working electrode zones to be arranged adjacent to the auxiliary electrode 102. For example, as shown in FIGS. 3A-3F and described in more detail below, the working electrode zones 104 may be formed in a circular or semi-circular shape that minimizes the number of working electrode zones 104 adjacent to one another.

[0050] 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 working electrode zones 104 adjacent to each other is two or less. For example, the working electrode zones 104 may be formed in a circular or semicircular shape adjacent to a parameter of the well (e.g., 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 of the 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 among the total number of working electrode zones 104. For example, as shown in Figures 5A-5C and described in more 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.

[0051] In embodiments 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 improve mass transport of material to each of the working electrode zones 104. For example, during orbital rocking or mixing, mass transport of material to a central zone of the well 200 may be relatively slow compared to zones away 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 at the center of the well 200. That is, during operation, the well 200 undergoes an orbital motion or "rocking" to mix or combine fluids contained within the well 200. The orbital motion may create vortices within the well 200, resulting in more liquid and faster liquid movement, for example, 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 more detail below, the working electrode zone 104 may be circular or semicircular in shape and located near the periphery of the well 200.

[0052] In embodiments 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 the introduction of liquid into one or more of the wells 200 of the multi-well plate 108. For example, as shown in FIG. 2C, the fluid 250 in the well 200 may form a curved top surface or meniscus 252 in the well 200. The curved top surface may be caused by several factors, such as surface tension, electrostatic effects, and fluid motion (e.g., due to actuated rocking). Due to the meniscus effect, photons (light) emitted during luminescence undergo different optical effects (e.g., refraction, diffusion, scattering, etc.) based on the optical path of the photons through the liquid. That is, when light is emitted from a material in the well 200, different liquid level heights may cause different optical effects (e.g., refraction, diffusion, scattering, etc.) on the emitted light, which depends on where the light exits through the liquid. This pattern may mitigate the meniscus effect by positioning each of the working electrode zones 104 approximately equidistant from each sidewall 212 of the well 200. Thus, photons emitted from the working electrode zones 104 travel a similar optical path through the liquid. That is, this pattern ensures that all working electrode zones 104 are equally affected by the meniscus effect, e.g., minimizing potentially different effects of the meniscus. Thus, if the working electrode zones 104 are positioned at different positions relative to the liquid level in the well 200, the emitted light may experience different optical distortions. For example, as shown in Figures 3A-3F, 4A-4F, 6A-6F, 7A-7F, and 8A-8D, which are described in more detail below, the working electrode zones 104 may be formed in a circular or semicircular shape and located near the periphery of the well 200. As a result, the light emitted at the working electrode zones 104 may experience the same optical distortions and be equally addressed.

[0053] In embodiments according to the present disclosure, the auxiliary electrodes 102 and one or more working electrode zones 104 of each well 200 may be formed in a pattern configured to minimize differential mass transport to the working electrode zones during mixing of liquid within one or more of the wells 200 of the multiwell plate 208 (e.g., vortexes formed in a cylindrical well using an orbital shaker). For example, the pattern may 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 more detail below, the working electrode zones 104 may be formed in a circular or semicircular shape and located near the periphery of the well 200.

[0054] In embodiments 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 is disposed at an approximately equal distance from a sidewall of the well 200, and the auxiliary electrode 102 may be disposed within (the entire perimeter or only a portion of) a perimeter defined by the circular or semicircular pattern of working electrode zones 104, although other shapes and / or patterns are contemplated as well. 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 that surrounds the entire perimeter or only a portion of the perimeter of the well 200.

[0055] In other embodiments, 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 by the auxiliary electrodes 102 forming the "points" of the star pattern and the working electrode zones 104 forming the inner structure of the star pattern. For example, as shown in Figures 5A-5C, which are described in more detail below, 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 the inner "pentagonal" structure. In some embodiments, as shown in Figures 5A-5C, which are described in more detail below, the star pattern may be defined as one or more concentric circles, and the one or more working electrode zones 104 and / or one or more auxiliary electrodes may be disposed in a circular pattern around the one or more concentric circles.

[0056] 3A and 3B show an embodiment of an electrode design 301 of a well 200 having circular working electrode zones 104 arranged in an open ring pattern. According to an exemplary and non-limiting embodiment shown in FIG. 3A, the bottom 207 of the well 200 may include a single auxiliary electrode 102. In other embodiments, multiple (e.g., 2, 3, 4, 5, etc.) auxiliary electrodes 102 may be included in the well 200. 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, quatrefoil, or any other regular or irregular geometric shape).

[0057] In embodiments, the well 200 may include ten working electrode zones 104. In other embodiments, fewer or more than ten (e.g., 1, 2, 3, 4, etc.) working electrode zones 104 may be included in the well 200. 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., rectangular, square, oval, quatrefoil, or any other regular or irregular geometric shape).

[0058] The working electrode zones 104 may be disposed in a semicircular or generally "C-shaped" pattern adjacent to each other at a distance "D1" from the perimeter "P" of the well 200. In some embodiments, the distance D1 may be the minimum distance between the border of the working electrode zones 104 and the perimeter P. That is, each of the working electrode zones 104 may be disposed at 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 two adjacent working electrode zones 104. In some embodiments, the two working electrode zones 104A, 104B may be spaced apart from each other by a sufficient distance to form a gap "G". 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 separation of the auxiliary electrode 102 and the working electrode zone 104. For example, gap G may be formed with a sufficient distance to allow electrical traces to be formed between adjacent working electrode zones 104.

[0059] 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 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, while in other embodiments, the distance D3 may be different for one or more of the working electrode zones 104 as measured from the auxiliary electrode 102. In certain embodiments, the distances D1, D2, D3, and G may be measured from the nearest relative points on the perimeter of the respective features (e.g., the working electrode zone 104, the auxiliary electrode 102, or the perimeter P) as shown. 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. Those skilled in the art will appreciate that distances may be measured from any relative point on the feature to generate a repeatable pattern, for example a geometric pattern.

[0060] Although these figures show a single auxiliary electrode 102, multiple auxiliary electrodes 102 may be included, as shown in Figure 3C. Also, 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 in other locations in the well 200, as shown in Figure 3D. Additionally, although these figures show 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.

[0061] In embodiments, the size of the auxiliary electrode 102 and / or the working electrode zone 104 may vary. For example, as shown in Table 2, the size of each of the working electrode zones 104 may be equal, and the size of the auxiliary electrode 102 may vary, such as by varying diameter. One of ordinary skill in the art will appreciate that the dimensions included in Table 2 are approximate and may vary + / - 5.0% based on conditions such as manufacturing tolerances.

[0062] [Table 2]

[0063] 4A and 4B show non-limiting and exemplary embodiments of an electrode design 401 for a well 200 having non-circular working electrode zones 104 arranged in an open ring pattern within the well, as also described above with reference to FIGS. 3A and 3B. In embodiments, the non-circular working electrode zones 104 may allow for improved area utilization within the well 200. The use of non-circular working electrode zones 104 may allow larger working electrode zones 104 to be formed within the well 200 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. As a result, the ratio of working electrode zones 104 to auxiliary electrodes 102 may be maximized. In addition, because the working electrode zones 104 may be formed larger, the working electrode zones 104 may be more reliably manufactured, e.g., printed.

[0064] 4A, the well 200 may include a single auxiliary electrode 102. In other embodiments, multiple (e.g., 2, 3, 4, 5, etc.) auxiliary electrodes 102 may be included. 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, quatrefoil, or any other regular or irregular geometric shape).

[0065] In an embodiment, the well 200 may include ten working electrode zones 104. In other embodiments, fewer or more than ten (e.g., 1, 2, 3, 4, etc.) working electrode zones 104 may be included. Each of the working electrode zones 104 may be formed to have a non-circular shape, e.g., a wedge or trefoil shape with one or more rounded or radiused corners, although in other embodiments, the corners are not rounded and form a polygon, e.g., a triangle.

[0066] The working electrode zones 104 may be arranged in a semicircular or generally "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 border of the working electrode zones 104 and the perimeter P. That is, each of the working electrode zones 104 may be located at 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". In some embodiments, the distance D2 may be the minimum distance between the borders of two adjacent working electrode zones 104. In some embodiments, the two working electrode zones 104A, 104B may be spaced apart from each other 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 working electrode zones 104. The auxiliary electrode 102 may be positioned at the center of the C-shaped pattern at an equal distance "D3" 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 from 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., working electrode zone 104, auxiliary electrode 102, or 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 appreciate that distances may be measured from any relative point on the feature to generate a repeatable pattern, e.g., a geometric pattern.

[0067] Although these figures show a single auxiliary electrode 102, multiple auxiliary electrodes 102 may be included, as shown in Figure 4C. Also, 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 in other locations in the well 200, as shown in Figure 4D. Additionally, although these figures show 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.

[0068] In certain embodiments, the auxiliary electrode 102 and / or the working electrode zone 104 may be equal in size. In other embodiments, the auxiliary electrode 102 and / or the working electrode zone 104 may vary in size. In one example, the size of the auxiliary electrode 102 may be constant and the size of the working electrode zone 104 may vary, such as by varying the radius of the auxiliary electrode 102. Table 3 includes example dimensions of the working electrode zone 104 and the auxiliary electrode 102 for the embodiment shown in Figures 4A-4F. One of ordinary skill in the art will appreciate that the dimensions included in Table 3 are approximate and may vary by + / - 5.0% based on conditions such as manufacturing tolerances.

[0069] [Table 3]

[0070] 5A and 5B show a non-limiting exemplary embodiment of an electrode design 401 of a well 200 having working electrode zones 104 arranged in a star pattern, where the working electrode zones 104 are circular. As shown 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 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 is created by multiple working electrode zones 104 arranged in one of an inner circle and an outer circle relative to each other, with each working electrode zone 104 located in the outer circle being located at the angular midpoint 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 on 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 on the outer circle.

[0071] In certain embodiments, as shown, distance R1, distance R2, and distance 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). One 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.

[0072] Although these figures show ten working electrode zones 104, as shown in Figure 5C, a greater or lesser number of working electrode zones 104 may be included. Also, although Figures 5A-5C show circular working electrode zones 104, the working electrode zones 104 may be formed having other shapes (e.g., rectangular, square, oval, quatrefoil, or any other regular or irregular geometric shape). Other embodiments may include hybrid design electrode configurations, such as, for example, a star pattern including wedge-shaped working electrode zones 104 and / or auxiliary electrodes 102.

[0073] In certain embodiments, the auxiliary electrode 102 and / or working electrode zone 104 may be equal in size. In other embodiments, the auxiliary electrode 102 and / or working electrode zone 104 may vary in size. In one example, the size of the working electrode zone 104 may be constant and the size of the auxiliary electrode 102 may vary due to diameter variations, as shown in Table 4. One of ordinary skill in the art will appreciate that the dimensions included in Table 4 are approximate and may vary, for example, by + / - 5.0% based on conditions such as manufacturing tolerances.

[0074] [Table 4]

[0075] 6A and 6B show exemplary, non-limiting embodiments of an electrode design 601 for a well 200 having non-circular working electrode zones 104 arranged in a closed ring pattern. As shown in FIG. 6A, the well 200 may include a single auxiliary electrode 102. In other embodiments, multiple (e.g., 2, 3, 4, 5, etc.) auxiliary electrodes 102 may be included in the well 200. 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, quatrefoil, or any other regular or irregular geometric shape).

[0076] In an embodiment, the well 200 may also include ten working electrode zones 104. The working electrode zones 104 may be formed to have a non-circular shape, for example, a wedge shape or a triangle shape with one or more rounded or R corners. In a closed ring pattern, the working electrode zones 104 may be arranged in a circle surrounding the periphery of the well 200 such that each is adjacent to the periphery "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 periphery P. That is, each of the working electrode zones 104 may be positioned at an equal distance D1 from the periphery P of the well 200, and each of the working electrode zones 104 may be equally spaced apart from each other 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 certain embodiments, the distance D1 may not be equal between one or more of the 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 at an equal distance "D3" from each of the working electrode zones 104, while in other embodiments, the distance D3 may vary for one or more of the working electrode zones 104 as measured from 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 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 appreciate that the distances may be measured from any relative point on the feature to generate a repeatable pattern, e.g., a geometric pattern.

[0077] Although these figures show a single auxiliary electrode 102, multiple auxiliary electrodes 102 may be included, as shown in Figure 6C. Also, 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 in other locations in the well 200, as shown in Figure 6D. Additionally, although these figures show 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.

[0078] In certain embodiments, the auxiliary electrode 102 and / or the working electrode zone 104 may be equal in size. In other embodiments, the auxiliary electrode 102 and / or the working electrode zone 104 may vary in size. In one example, the size of the auxiliary electrode 102 may be constant and the size of the working electrode zone 104 may vary, such as by varying the radius of the auxiliary electrode 102. Table 5 includes example dimensions of the working electrode zone 104 and the auxiliary electrode 102 for the embodiment shown in Figures 6A-6F. One of ordinary skill in the art will appreciate that the dimensions included in Table 5 are approximate and may vary by + / - 5.0% based on conditions such as manufacturing tolerances.

[0079] [Table 5]

[0080] 7A and 7B show exemplary, non-limiting embodiments of an electrode design 701 for a well 200 having a closed ring design with a circular electrode. As shown in FIG. 7A, the well 200 may include a single auxiliary electrode 102. In other embodiments, multiple (e.g., 2, 3, 4, 5, etc.) auxiliary electrodes 102 may be included. 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, quatrefoil, or any other regular or irregular geometric shape).

[0081] In embodiments, the well 200 may include ten working electrode zones 104. In other embodiments, fewer or more than ten (e.g., 1, 2, 3, 4, etc.) working electrode zones 104 may be included. 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., rectangular, square, oval, quatrefoil, or any other regular or irregular geometric shape).

[0082] In a closed ring pattern, the working electrode zones 104 may be arranged in a circle surrounding the periphery of the well 200 such that each of the working electrode zones 104 is adjacent to the periphery "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 periphery P. That is, each of the working electrode zones 104 may be arranged at an equal distance D1 from the periphery P of the well 200, and each of the working electrode zones 104 may be equally spaced 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 boundary 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 periphery P of the well 200. In further embodiments, the distance D2 may be unequal between two or more working electrode zones 104.

[0083] The auxiliary electrode 102 may be positioned at the center of the ring 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, the distance D3 may vary for one or more of the working electrode zones 104 as measured from 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 appreciate that the distances may be measured from any relative point on the feature to generate a repeatable pattern, e.g., a geometric pattern.

[0084] Although these figures show a single auxiliary electrode 102, multiple auxiliary electrodes 102 may be included, as shown in Figure 7C. Also, 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 in other locations in the well 200, as shown in Figure 7D. Additionally, although these figures show 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.

[0085] In certain embodiments, the auxiliary electrodes 102 and / or working electrode zones 104 may be equal in size. In other embodiments, the auxiliary electrodes 102 and / or working electrode zones 104 may vary in size. In one example, the size of the working electrode zones 104 may be constant and the size of the auxiliary electrodes 102 may vary, such as by variation in diameter, as shown in Table 6. One of ordinary skill in the art will appreciate that the dimensions included in Table 6 are approximate and may vary, for example, by + / - 5.0%, based on conditions such as manufacturing tolerances.

[0086] [Table 6]

[0087] 8A-8D show exemplary and non-limiting embodiments of electrode designs 801 for wells 200 having a closed ring design with a circular working electrode zone and a complex shaped auxiliary electrode 102. As shown in FIG. 8A, wells 200 may include two complex shaped auxiliary electrodes 102. In other embodiments, fewer (or more) than two auxiliary electrodes 102 may be included in wells 200, as shown in FIG. 8D. In embodiments, auxiliary electrodes 102 may be formed to have complex shapes, such as, for example, "gear", "tooth", "washer" shapes, "oval" shapes, "wedge" shapes, etc., as described above. For example, as shown in FIG. 8B, the interior of auxiliary electrode 102 may be formed in a circular shape (e.g., "gear" or "tooth" shape) with an outer semicircular space 802 corresponding to working electrode zone 104. Similarly, as shown in FIG. 8C, for example, auxiliary electrode 102 may be formed in a hollow ring shape (e.g., "washer" shape) with an inner semicircular space 804 corresponding to working electrode zone 104.

[0088] In embodiments, the well 200 may include ten working electrode zones 104. In other embodiments, fewer or more than ten (e.g., 1, 2, 3, 4, etc.) working electrode zones 104 may be included within the well 200. 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., rectangular, square, oval, quatrefoil, or any other regular or irregular geometric shape).

[0089] In an embodiment, the working electrode zone 104 may be arranged in a circle between the two auxiliary electrodes 102. In this configuration, the outer semicircular space 802 and the inner semicircular space 804 allow the two auxiliary electrodes 102 to partially surround the working electrode zone. The outside 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 border of the working electrode zone 104. In some embodiments, the distance D1 may be the minimum distance between the outside of the two auxiliary electrodes 102 and the working electrode zone 104. In certain embodiments, the distance D1 may not be equal between one or more of the working electrode zones 104 and the outside of the two auxiliary electrodes 102. Each of the working electrode zones 104 may be equally spaced a distance "D2" from each other. In some embodiments, the distance D2 may be the minimum distance between two adjacent working electrode zones 104. In further embodiments, the distance D2 may be unequal between two or more working electrode zones 104. The inside 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 two auxiliary electrodes 102.

[0090] In certain embodiments, as shown, distance D1, distance D2, and distance D3 may be measured from the nearest relative point on the perimeter of the respective feature (e.g., working electrode zone 104 or auxiliary electrode 102). One 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.

[0091] As described above, the electrochemical cell 100 may be used in devices and apparatus for performing electrochemical analyses. For example, the multi-well plate 208 containing the wells 200 described above may be used in any type of apparatus that aids in performing biological, chemical, and / or biochemical assays and / or analyses, such as an apparatus for performing ECL analyses. Figures 9A-9B show a general assay apparatus 900 in which the multi-well plate 208 containing the wells 200 may be used for electrochemical analyses and procedures, according to embodiments herein. Those skilled in the art will appreciate that Figures 9A-9B show one example of an assay apparatus, and that existing components shown in Figures 9A-9B may be omitted and / or additional components may be added to the assay apparatus 900 without departing from the scope of the embodiments described herein.

[0092] 9A-9B, 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 provide a controlled voltage and / or current to the wells 200 (e.g., electrochemical cells 100) of the multiwell plate 208 via the plate electrical connector 902. For example, the plate electrical connector 902 may be configured to match 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 104 to allow a voltage and / or current to be provided to the wells 200 of the multiwell plate 208.

[0093] In some embodiments, the plate electrical connector 902 may be configured to allow one or more wells 200 (including one or more of the working electrode zones and auxiliary electrodes) to be activated simultaneously, 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., plates, flow cells, etc.). The coupling between the device and one or more apparatuses may include the entire surface of the apparatus (e.g., the entire bottom surface 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, such as by selectively applying a voltage and / or current to one of the wells 200 and reading a signal 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, the plate electrical connector 902 may include a single electrical strip connecting all of the wells 200 in one of the rows A-H or one of the columns 1-12. In this manner, all of the wells 200 in one of the rows A-H or one of the columns 1-12 may be simultaneously activated and supplied with a voltage and / or current, for example, by the voltage / current source 904. Similarly, all of the wells 200 in one of the rows A-H or one of the columns 1-12 may be simultaneously read out and a signal may be read, for example, by the detector 910.

[0094] In some embodiments, the plate electrical connector 902 may include a matrix of vertical electrical lines 952 and horizontal electrical lines 950 that are individual electrical connections connecting the individual wells 200 in rows A-H and columns 1-12. The plate electrical connector 902 (or power / current source 904) may include switches or other electrical connection devices that selectively establish electrical connections to the vertical electrical lines 952 and horizontal electrical lines 950. In this manner, one or more wells 200 in one of the rows A-H or one of the columns 1-12 may be individually activated and provided with a voltage and / or current by the voltage / current source 904, for example as shown in FIG. 9B. Similarly, one or more wells 200 in one of the rows A-H or one of the columns 1-12 may be individually and simultaneously read out, for example 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 an index of the one or more wells 200, for example well A1, well A2, etc.

[0095] 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 of each of the wells 200 to be selectively addressable, for example to allow a 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 individual electrical contacts that allow the plate electrical connector 902 to be electrically coupled 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 individual electrical contacts that allow the plate electrical connector 902 to be electrically coupled to each of the one or more auxiliary electrodes 102 of the well 200.

[0096] Although not shown, the plate electrical connector 902 (or other components of the assay apparatus 900) may include any number of electrical components, such as electrical lines, switches, multiplexers, transistors, etc., to allow particular wells 200, auxiliary electrodes 102, and / or working electrode zones 104 to be selectively electrically coupled to a voltage / current source 904 and to allow voltages and / or currents to be selectively applied. Similarly, although not shown, the plate electrical connector 902 (or other components of the assay apparatus 900) may include any number of electrical components, such as electrical lines, switches, multiplexers, transistors, etc., to allow signals to be selectively read from the detector 910 by particular wells 200, auxiliary electrodes 102, and / or working electrode zones 104.

[0097] In certain embodiments, one or more computer systems 906 may be coupled to the voltage / current sources 904 to control the voltage and / or current supplied. In other embodiments, the voltage / current sources 904 may supply the potential and / or current without the assistance of a computer system, e.g., 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 used to store, analyze, display, transmit, etc., data measured during electrochemical processes and procedures.

[0098] The multi-well 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.

[0099] 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.

[0100] In embodiments, the one or more photodetectors 912 may be, for example, a film, a photomultiplier tube, a photodiode, an avalanche photodiode, a charge-coupled device ("CCD"), or other photodetectors or cameras. The one or more photodetectors 912 may be a single detector for sequentially detecting emitted light, or may include multiple detectors for detecting and spatially resolving simultaneous emissions at single or multiple wavelengths of light. The light emitted and detected may be visible light, or may be emitted as non-visible radiation, for example, infrared or ultraviolet. The one or more photodetectors 912 may be fixed or movable. The emitted light or other radiation may be manipulated or modified during passage to the one or more photodetectors 912 using, for example, lenses (single, multiple, fixed, or movable), mirrors, and fiber optic light guides or light conduits located 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 used to enable light guiding or transmission.

[0101] 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 of the multiwell plate 208, a single camera that captures images of a subset of the wells 200, multiple cameras that capture images of all wells 200, or multiple cameras that capture images of a subset of the wells 200. In some embodiments, each well 200 of the multiwell plate 208 may include a camera that captures images of the wells 200. In some embodiments, each well 200 of the multiwell plate 208 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 to analyze an image captured by the one or more photodetectors 912 and extract brightness data for performing an ECL analysis. In some embodiments, the computer system 906 may include hardware, software, and combinations thereof including logic to segment and enhance the image 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., where the image includes data for multiple wells 200, multiple working electrode zones 104, etc. Thus, the assay device 900 may provide flexibility by allowing the photodetector 912 to capture all light from multiple working electrode zones 104 and the computer system 906 to resolve the luminescence data for each working electrode zone 104 using imaging processing.In this manner, the assay device 900 may operate in various modes, such as singleplex mode (e.g., one working electrode zone), 10plex mode (e.g., all working electrode zones 104 for a 10 working electrode zone well 200), or generally in multiplex mode (e.g., a subset of all working electrode zones contained within a single well 200 or multiple wells 200 simultaneously, such as, for example, five working electrode zones 104 for a multiple 10 working electrode zone well).

[0102] 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 208 may include a photodiode for detecting and measuring photons emitted within the well 200. In some embodiments, each well 200 of the multiwell plate 208 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. In this manner, the assay device 900 may operate in a variety of 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 a first of the five working electrode zones 104, and the emitted photons may be detected and measured by the corresponding photodiode. This is 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 different wells 200, for working electrode zones 104 located in a subset or "sector" of a plurality 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 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 in the same well 200, for working electrode zones 104 located in different wells 200, for working electrode zones 104 located in a subset or "sector" of a well 200 in a multi-well plate 208, or combinations thereof.As used herein, the term "sector" may refer to wells 200 that are addressable in a group. A sector may include one well or multiple wells in any configuration. A sector of wells includes one or more wells that are contiguous with one another, such as, for example, 2x1, 2x2, 3x3, 4x4, 8x8, and / or any other size or shape well array, and is simultaneously addressable via electrical communication between the sides of the wells in a multiwell plate or via electrical communication disposed via hardware designed to contact the multiwell plate.

[0103] In the above-described embodiment, the working electrode zone 104 undergoes a natural decay in the intensity of the 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 material undergoing the redox reaction. When the voltage is removed, the material undergoing the redox reaction continues to emit photons at a decaying intensity for a period of time based on the chemical properties of the material. In this manner, when the working electrode zones 104 are sequentially activated, the assay device 900 (e.g., computer system 906) may be configured to implement a delay in sequentially activating the working electrode zones 104. The assay device 900 (e.g., computer system 906) may determine and implement a delay in sequentially activating the working electrode zones 104 to prevent a previously fired working electrode zone 104 from interfering with photons emitted from a currently activated working electrode zone 104. For example, FIG. 10A shows the decay of ECL during various voltage pulses, and FIG. 10B shows the ECL decay time using a 50 ms pulse. In the example of FIG. 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 was taken (or photons were detected) every 17 ms. The 50 ms voltage pulse was imaged in 3 frames (e.g., images 1-3, 17 ms x 3 times = 51 ms), as shown in FIG. 10B. Any photons, e.g., ECL signal, emitted after image 3 is due to a decay in the intensity of the photons (e.g., ECL) after the working electrode zone 104 is turned off. In FIG. 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 inactivated. 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 with respect to the different voltage pulses to delay activation of sequential 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.

[0104] 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, the use of Ag / AgCl for one or more auxiliary electrodes 102 improves the 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 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 duration of the waveform. Tables 7 and 8 below include the 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 times 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, all (1 or 10 depending on the embodiment) working electrodes (or working electrode zones) in all 96 wells were analyzed. In Table 7 below, "spatial" refers to the mode of operation where 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 the ECL image collection (e.g., adjusting the binning to adjust the dynamic range). The "Rectification Plate RT" column contains the read times of the non-auxiliary electrodes (e.g., carbon electrodes). The last three columns of the table contain the difference between the read times of the non-auxiliary electrodes and the auxiliary electrodes (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 times will be between the read times of 1 working electrode zone (WE) and 10 WE. For the "B" experiments, no read time improvement was 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 appreciate 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. [Table 7] [Table 8]

[0105] With respect to Tables 7 and 8, "WE" can refer to either the working electrode or the working electrode zone.

[0106] In contrast, with a voltage ramp in an ECL application, there is a period of time during which voltage is applied but ECL does not occur (e.g., part of the beginning of the ramp and / or part of the end of the ramp). With this waveform, there is a period of time during which ECL does not occur even though a potential is applied. In other words, when applying a ramp waveform, there is a percentage of the total waveform duration during which ECL does not occur while the potential is applied (e.g., 5%, 10%, 15%, etc.). These percentages vary based on several factors, including the type of material used to form the electrodes, the relative and absolute sizes of the electrodes, etc.

[0107] In any of the above-described embodiments, the use of working electrode zones 104 having 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 the ECL. In binding assays using binding reagents immobilized on the working electrode, the binding performance and efficiency and speed of binding generally increase with the size of the working electrode zone. For ECL instruments using imaging detectors (e.g., CCD or CMOS devices), the advantages of a large working electrode zone in binding performance and efficiency may be offset by the improved sensitivity of these devices in terms of the total number of photons when light is generated at a smaller electrode zone and imaged at a smaller number of imaging device pixels. The location of the working electrode zone 104 may affect the performance of the assay device 900. In some embodiments, the location, size, and geometry of the spot can affect the amount of reflection, scattering, or loss of photons on the well sidewall, affecting 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 can be improved by having a design in which there is no working electrode zone 104 located at the center of the well 200, as well as having the 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 symmetrical positions within the well 200 can improve the operation of the assay device 900 by having the light collection and meniscus interaction be the same for all of the one or more working electrode zones 104 within the well 200, as described above. One or more working electrode zones 104 positioned at a fixed distance (e.g., in a circular pattern) can allow the assay device to utilize shortened pulse waveforms, e.g., reduced pulse widths. In an embodiment, a design in which one or more working electrode zones 104 have nearest neighbors as one or more auxiliary electrodes 102 (eg, no intervening working electrode zones between them) improves the performance of the assay device 900.

[0108] 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 in a pulsed waveform, e.g., direct current, alternating current, direct current mimicking alternating current, etc., although other waveforms of different duration, frequency, and amplitude are contemplated (e.g., negative ramp sawtooth waveform, square waveform, rectangular waveform, 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 of the pulse waveform and the duration of the pulse waveform, as described in more detail below. In embodiments, as described above, the computer system 906 may be configured to selectively provide a pulse waveform to one or more of the wells 200. For example, a 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., on an individual basis or on a sector basis, such as a grouping of a subset of wells, e.g., 4, 16, etc.). For example, as described above, the wells 200 may be individually addressable or addressable in groups of two or more wells. In an embodiment, the computer system 906 may be configured to selectively provide the pulse waveform to one or more of the working electrode zones 104 and / or auxiliary electrodes 102 (e.g., individually addressable or addressable in groups of two or more auxiliary electrodes) in a manner as described above. 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.

[0109] 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 flow chart illustrating a process 1100 for operating an assay device with a pulse waveform according to an embodiment herein.

[0110] 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 may control the voltage / current source 904 to provide the voltage pulse to the one or more working electrode zones 104 or the one or more auxiliary electrodes 102.

[0111] In embodiments, the pulse waveform may include various waveform types, such as DC, AC, DC mimicking AC, although other waveforms of different durations, frequencies, and amplitudes are contemplated (e.g., negative ramp sawtooth waveform, square waveform, rectangular waveform, 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 examples of pulse waveforms. As shown in Figure 12A, the pulse waveform may be a square wave having a voltage V over a time T. Examples of voltage pulses include, for example, 1800mV in 500ms, 2000mV in 500ms, 2200mV in 500ms, 2400mV in 500ms, 1800mV in 100ms, 2000mV in 100ms, 2200mV in 100ms, 2400mV in 100ms, 1800mV in 50ms, 2000mV in 50ms, 2200mV in 50ms, 2400mV in 50ms, etc. Figures 12A and 12B show examples of pulse waveforms, however, one skilled in the art will understand that the pulse waveform can have any structure in which the potential rises to a defined voltage (or voltage range) over a predefined period of time. Those skilled in the art will understand that the parameters of the voltage pulses and pulse waveforms described herein (e.g., duration, duty cycle, and pulse height in volts) are approximate and can vary by + / - 5.0% based on conditions such as the operating parameters of the voltage / current sources.

[0112] In operation 1104, the process 1100 includes measuring a potential difference between the one or more working electrode zones 104 and the one or more auxiliary electrodes 102. For example, the detector 910 may 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 may provide the measured data to the computer system 906.

[0113] In operation 1106, the process 1100 includes performing an analysis based on the measured potential difference and other data. For example, the computer system 906 may perform an analysis on the potential difference and other data. The analysis may be any process or procedure, such as, for example, potentiometry, coulometry, voltammetry, optical analysis (described further below), etc. In an embodiment, the use of a pulse waveform allows a particular type 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 particular level. By using a pulse waveform of specified voltage, the assay device 900 can selectively activate some of these redox reactions and not others.

[0114] In one embodiment, the disclosure provided herein can be applied to the method for carrying out ECL assay.Specific examples of the method for carrying out 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, 6,214,552 and 7,842,246, and published PCT applications WO87 / 06706 and WO98 / 12539, which are incorporated herein by reference.

[0115] In an embodiment, the pulse waveform provided by the voltage / current source 904 may be designed to improve the ECL emitted during an ECL analysis. For example, the pulse waveform may improve the ECL emitted during an ECL analysis by providing a stable and constant voltage potential to generate a stable and predictable ECL emission. Figure 13 shows a flow chart illustrating a process 1300 for operating an ECL device with a pulse waveform, according to an embodiment herein.

[0116] 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 may control a voltage / current source 904 to provide a voltage pulse to the one or more working electrode zones 104 or the one or more auxiliary electrodes 102. In embodiments, the one or more auxiliary electrodes 102 may include a redox couple where the reaction of a species in the redox couple is the primary redox reaction occurring at the one or more auxiliary electrodes 102 when a voltage or potential is applied. In some embodiments, the voltage applied is less than a 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 of the current per unit area (exposed surface area) of the one or more auxiliary electrodes 102 is associated with water reduction.

[0117] In embodiments, the pulse waveform may include various waveforms, such as DC, AC, DC mimicking AC, although other waveforms of different durations, frequencies, and amplitudes are contemplated (e.g., negative ramp sawtooth waveform, square waveform, rectangular waveform, etc.). Figures 12A and 12B above show two examples of pulse waveforms. The pulse waveform may be a square wave having a voltage V over a time T.

[0118] In operation 1304, the process 1300 includes capturing luminescence data from the electrochemical cell over a period of time. For example, the one or more photodetectors 912 may capture luminescence data emitted from the wells 200 and communicate the luminescence data to the computer system 906. In an embodiment, the period of time may be selected to allow the photodetectors to capture ECL data. In some embodiments, the one or more photodetectors 912 may include a single camera that captures images of all wells 200 of the multiwell plate 208, or multiple cameras that capture images of a subset of the wells 200. In some embodiments, each well 200 of the multiwell plate 208 may include a camera that captures images of the wells 200. In some embodiments, each well 200 of the multiwell plate 208 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 may provide flexibility in that a camera may capture all the light from multiple working electrode zones 104 and a computer system 906 may use image processing to resolve the luminescence data for each working electrode zone 104. In this manner, the assay device 900 may 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 for a 10 working electrode zone well 200), or generally in multiplex mode (e.g., a subset of all working electrode zones contained within a single well or multiple wells 200 simultaneously, such as five working electrode zones 104 simultaneously for a 10 working electrode zone well).

[0119] In some embodiments, each well 200 of the multiwell plate 208 may include a photodiode to detect and measure photons emitted within the well 200. In some embodiments, each well 200 of the multiwell plate 208 may include multiple photodiodes 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. In this manner, the assay device 900 may operate in various modes. For example, the assay device 900 may apply a voltage and / or current individually to one or more of the working electrode zones 104, for example, five working electrode zones 104, in the multiwell plate 208. The working electrode zones 104 may be located within a single well 200, may be located within different wells 200, or a combination thereof. The photodiode 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 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 the well 200, or combinations thereof. Similarly, in some embodiments, the assay device 900 may be operated in a multiplex mode, in which one or more working electrode zones 104 are simultaneously activated 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 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 any combination thereof.

[0120] In embodiments, by applying a pulse waveform to generate ECL, readout time and / or exposure time may be improved by more rapid and efficient generation, collection, observation, and analysis of ECL data. Also, various exposure approaches (e.g., single exposure, double exposure, triple exposure (or more)) that can use different exposure times (or equal exposure times) may be used to improve ECL acquisition, collection, observation, and analysis, for example, by improving dynamic range extension (DRE), binning, etc. For example, as described above, the utilization of one or more auxiliary electrodes 102 improves the readout time of the detector 910. For example, the use of Ag / AgCl in one or more auxiliary electrodes 102 improves the readout time of ECL 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 a voltage pulse rather than a voltage ramp. The use of a voltage pulse improves readout time because the entire pulse waveform may be applied at a voltage potential that generates ECL throughout the duration of the waveform. Additionally, the "time resolved" or sequential mode has the added advantage of allowing adjustments to the ECL image collection (e.g., adjusting the binning to adjust the dynamic range, etc.). Also, as described above, the assay device 900 (e.g., computer system 906) may be configured to use such data regarding the different voltage pulses to 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.

[0121] In operation 1306, the process 1300 includes performing an ECL analysis on the luminescence data. For example, the computer system 906 may perform an ECL analysis on the luminescence data. In some embodiments, the luminescence data, e.g., signals, resulting from a given target object 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 ​​may be correlated with a quantitative measurement (e.g., ECL intensity) to provide an analog signal. In other embodiments, a digital signal (yes or no signal) may be obtained from each working electrode zone 104 to indicate the presence or absence of an analyte. Statistical analysis may be used with both techniques and may be used 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 may be used separately or in combination. Other statistical methods may be used, such as techniques to determine concentration through statistical analysis of binding across a concentration gradient. Multiple linear arrays of data with a concentration gradient can be generated with multiple different specific binding reagents being used in different wells 200 and / or at different working electrode zones 104. The concentration gradient can be made up of distinct binding domains representing different concentrations of the binding reagent.

[0122] In an embodiment, a control assay solution or reagent, such as a read buffer, may be used on the working electrode zone of the well 200. The control assay solution or reagent may provide uniformity to the nuclear analysis to control for signal variations (e.g., variations due to degradation, perturbations, aging of the multiwell plate 208, thermal shifts, noise in the electronics, and noise in the light detection device, etc.). For example, multiple overlapping working electrode zones 104 for the same analyte (containing the same binding reagent or different binding reagents specific to the same analyte) may be used. In other examples, a known concentration of analyte may be used, 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 is used.

[0123] In an embodiment, the data collected and generated in the process 1300 may be used in a variety of applications. The collected and generated data may be stored, for example, in the form of a database consisting of a collection of clinical or research information. The collected and generated data may be used for rapid forensic or personal identification. For example, the use of multiple nucleic acid probes when touching a human DNA sample may be used for a signature DNA fingerprint that may be easily used to identify clinical or research samples. The collected and generated data may be used to identify symptoms (e.g., disease, radiation levels, etc.), the presence of organisms (e.g., bacteria, viruses, etc.), etc.

[0124] The above describes an exemplary flow of the example process 1300. The process shown in FIG. 13 is merely exemplary, and variations exist without departing from the scope of the embodiments disclosed herein. As mentioned 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 the ECL assay. The auxiliary electrode allows luminescence to be generated more quickly without the use of a lamp.

[0125] In embodiments, the pulse waveform provided by the voltage / current source 904 can be designed to enable the ECL device to capture different luminescence data over time to improve ECL analysis. Figure 14 shows a flow chart illustrating another process 1800 for operating an ECL device with a pulse waveform according to embodiments herein.

[0126] In operation 1802, the process 1800 includes applying a voltage pulse to one or more working electrode zones 104 or auxiliary electrodes 102 in a well of the ECL device, the voltage pulse causing a reduction-oxidation reaction in the well. For example, the computer system 906 may control the voltage / current source 904 to provide one or more voltage pulses to the one or more working electrode zones 104 or auxiliary electrodes 102.

[0127] In embodiments, the voltage pulse may be configured to induce a reduction-oxidation reaction between the one or more working electrode zones 104 and the one or more auxiliary electrodes 102. As described above, based on a predetermined chemical composition of the one or more auxiliary electrodes 102 (e.g., a mixture of Ag:AgCl), the one or more auxiliary electrodes 102 may operate as a reference electrode to determine a potential difference with the one or more working electrode zones 104 and as a counter electrode for the working electrode zones 104. For example, the predetermined chemical mixture (e.g., the ratio of elements and alloys in the chemical composition) may 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 in the well 200. That is, the amount of charge passed during the redox reaction is quantifiable, for example, by measuring the current at the working electrode zone 104. In some embodiments, the one or more auxiliary electrodes 102 may define the total amount of charge that may pass with an applied potential difference, since as AgCl is consumed, the interfacial potential at the auxiliary electrode 102 shifts negatively relative to the reduction potential of water. This causes the potential of the working electrode zone 104 to shift to a lower potential (maintaining the applied potential difference) and turns off the oxidation reaction that occurred during the reduction of AgCl.

[0128] In embodiments, the pulse waveform may include various waveform types, such as DC, AC, DC mimicking AC, although other waveforms of different durations, frequencies, and amplitudes are also contemplated (e.g., negative ramp sawtooth waveform, square waveform, rectangular waveform, etc.). Figures 12A and 12B above show two examples of pulse waveforms. The pulse waveform may be a square wave having a voltage V over a time T.

[0129] At operation 1804, the process 1800 includes capturing first luminescence data from the first reduction-oxidation reaction over a first time period. At operation 1806, the process 1800 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. For example, one or more photodetectors 910 may capture first and second luminescence data emitted from the well 200 and communicate the first and second luminescence data to the computer system 906. For example, in an embodiment, the well 200 may contain a substance of interest that requires different time periods for the photodetector 912 to capture luminescence data. Thus, the 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 the ECL) and one of the time periods may be a longer time period. These 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 sum of a long and short exposure. In some embodiments, if the captured photons exceed the dynamic range of the light detector 912, the assay device 900 may use a short exposure. By adjusting / optimizing these, it may be possible to increase the dynamic range by one or two orders of magnitude. 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 (in 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, except when 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 may be improved. In another example, a first short pulse (e.g., 50 ms, but other durations are also possible) may be applied to an electrode or set of two or more electrodes, followed by a second, longer pulse (e.g., 200 ms, but other durations are also possible) 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, but other durations are also possible), and then reading the entire plate a second time with a second, longer pulse (e.g., 200 ms, but other durations are also possible). In addition to one or more discrete pulses, composite or hybrid functions may be used with these or other durations, for example, to determine and / or model responses in transition regions (e.g., during the transition between pulses). Furthermore, in the above example, a long pulse may be used first, before a short pulse. The waveform and / or acquisition window may also be adjusted to improve dynamic range.

[0130] 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 concentration analyte), this information can be used prior to reading and / or sampling to optimize exposure times to prevent camera saturation. Using the high concentration analyte example above, a shorter exposure time can be used since the signal is expected to be high in the dynamic range (and vice versa for electrodes where a low signal is expected), and thus the exposure time, pulse duration, and / or pulse strength can be customized and / or optimized for individual wells, electrodes, etc., for example, to improve overall read times. 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.

[0131] Additional techniques may also be used where the waveform and / or exposure are held constant. For example, the intensity of pixels within one or more ROIs may be measured and if pixel saturation is observed, other aspects of ECL generation and / or measurement may be used to optimize the readout and / or readout time (e.g., current-ECL correlation, dark mask methods of observing dark mask regions around the ROI that may be used to update the estimated ECL for saturated electrodes and / or portions of electrodes, etc.). These solutions avoid the need for fast analysis and / or reaction times to adjust the waveform and / or exposure duration in relatively short periods of time (e.g., milliseconds), for example, because ECL generation and / or capture may be performed in the same and / or similar manner, and analysis performed last.

[0132] Other techniques for improving the dynamic range may also be used. For example, when applied to electrochemiluminescence (ECL) applications, since the ECL label fluoresces, a pre-illumination and / or pre-exposure may 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 the pre-illumination and / or pre-exposure may be used to optimize the exposure and / or pulse duration to achieve additional improvements in the dynamic range and / or read time. In other embodiments, particularly as it pertains to ECL, since a correlation may exist between the current and one or more of the electrodes and the ECL signal, the signal signature may inform the camera exposure time and / or the applied waveform (e.g., stop waveform, decrease waveform, increase waveform, etc.). This may 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.

[0133] Additional improvements in dynamic range may be realized with respect to a particular imaging device 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 out at different times within one or more exposure ranges to optimize exposure times. For example, an ROI (e.g., a portion or the entire 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., but other percentages are also contemplated). In this example, the pixels and / or sample percentage may be read out early during exposure. Depending on the signal read out 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, typical example, if 1% of the pixels of a single working electrode zone show a strong signal for a particular exposure time (e.g., 3 seconds, but other periods longer or shorter than 3 seconds are also possible), the exposure period may be reduced (e.g., 2 seconds, but other periods longer or shorter than 3 seconds are also possible), and vice versa (e.g., if the signal is weak, the period may be increased). These adjustments may be made manually or with the aid of hardware, firmware, software, algorithms, computer readable media, computing devices, etc.

[0134] 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 may 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.

[0135] In operation 1808, the process 1800 includes performing an ECL analysis on the first luminescence data and the second luminescence data. For example, the computer system 906 may perform an ECL analysis on the luminescence data. These values ​​may be correlated with a quantitative measurement (e.g., ECL intensity) to provide an analog signal. In other embodiments, a digital signal (yes or no signal) may be obtained from each working electrode zone 104 to indicate whether an analyte is present or not. Statistical analysis may be used with both techniques and used 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 may be used separately or in combination. Other statistical methods may be used, such as techniques to determine concentration by statistical analysis of binding on a concentration gradient. Multiple linear data arrays with concentration gradients may be generated with multiple different specific binding reagents used in different wells 200 and / or at different working electrode zones 104. The concentration gradient may be composed of individual binding domains that present different concentrations of the binding reagent.

[0136] In an embodiment, a control assay solution or reagent, such as a read buffer, may be used 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 of the multiwell plate 208, thermal shifts, noise in the electronics, and noise in the photodetection device, etc.). For example, multiple redundant working electrode zones 104 for the same analyte (containing the same binding reagent or different binding reagents specific to the same analyte) may be used. In other examples, a known concentration of analyte may be used, 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 is used.

[0137] In an embodiment, the data collected and generated in the process 1800 may be used for various applications. The collected and generated data may be stored in the form of a database consisting of a collection of clinical or research information, for example. The collected and generated data may be used for rapid forensic or personal identification. For example, the use of multiple nucleic acid probes when touching a human DNA sample may be used for a signature DNA fingerprint that may be easily used to identify clinical or research samples. The collected and generated data may be used to identify the presence of symptoms (e.g., disease, radiation levels, etc.), organisms (e.g., viruses, etc.), etc.

[0138] In an embodiment, although the process 1800 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 be used for some or all of the periods. In an embodiment, the pulse waveforms may differ in amplitude (e.g., voltage), duration (e.g., period), and / or waveform type (e.g., square, sawtooth, etc.).

[0139] The above describes an exemplary flow of an example process 1800. The process as shown in FIG. 14 is merely exemplary, 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.

[0140] In embodiments, different configurations of pulse waveforms provided by voltage / current source 904 can be used together to improve the ECL emitted during an ECL analysis. Figure 15 shows a flow chart illustrating another process 1900 for operating an ECL device with a pulse waveform according to embodiments herein.

[0141] At operation 1902, the process 1900 includes applying a first voltage pulse to one or more working electrode zones 104 or auxiliary electrodes 102 in a well of the ECL device, the first voltage pulse causing a first reduction-oxidation reaction in the well. At operation 1904, the process 1900 includes capturing first luminescence data from the first reduction-oxidation reaction over a first period of time.

[0142] At operation 1906, the 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. At operation 1908, the 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.

[0143] In an embodiment, a 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 an embodiment, a 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 an 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.

[0144] 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 may perform an ECL analysis on the luminescence data. In some embodiments, the luminescence data, e.g., signal, resulting from a given target object 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 ​​may be correlated with a quantitative measurement (e.g., ECL intensity) to provide an analog signal. In other embodiments, a digital signal (yes or no signal) may be obtained from each working electrode zone 104 to indicate whether the analyte is present or not. Statistical analysis may be used with both techniques and may be used 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 may be used separately or in combination. Other statistical methods may be used, such as techniques to determine concentration by statistical analysis of binding on a concentration gradient. With multiple different specific binding reagents used in different wells 200 and / or at different working electrode zones 104, multiple linear data arrays can be generated having a concentration gradient. The concentration gradient can be made up of distinct binding domains that exhibit different concentrations of binding reagent.

[0145] In an embodiment, a control assay solution or reagent, such as a read buffer, may be used 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 of the multiwell plate 208, thermal shifts, noise in the electronics, and noise in the photodetection device, etc.). For example, multiple redundant working electrode zones 104 for the same analyte (containing the same binding reagent or different binding reagents specific to the same analyte) may be used. In other examples, a known concentration of analyte may be used, 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 is used.

[0146] In an embodiment, the data collected and generated in the process 1900 may be used for various applications. The collected and generated data may be stored, for example, in the form of a database consisting of a collection of clinical or research information. The collected and generated data may be used for rapid forensic or personal identification. For example, the use of multiple nucleic acid probes when touching a human DNA sample may be used for a signature DNA fingerprint that may be easily used to identify clinical or research samples. The collected and generated data may be used to identify the presence of symptoms (e.g., disease, radiation levels, etc.), organisms (e.g., viruses, etc.), etc.

[0147] The above describes an exemplary flow of an example process 1900. The process as shown in FIG. 15 is merely exemplary, 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.

[0148] In any of the processes 1300, 1800, and 1900 described above, 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 provided to all working electrode zones 104 and / or auxiliary electrodes 102 in one or more wells 200 of a multiwell plate 208. Similarly, for example, a voltage pulse may be provided to a selected (or "addressable") set of working electrode zones 104 and / or auxiliary electrodes 102 in one or more wells 200 of a multiwell plate 208 (e.g., zone-by-zone, well-by-well, sector-by-sector (e.g., a group of one or more wells), etc.).

[0149] 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 measurements and reader devices. An exemplary plate reader is the MESO SECTOR S600 ( 1721192825909_0 No. 6,977,722 and U.S. Provisional Patent Application No. 62 / 874,828, entitled “Assay Apparutuses, Methods and Reagents” by Krivoy et al., filed July 16, 2019, each of which is incorporated herein by reference in its entirety, and plate readers described in International Patent Publication No. WO2021 / 011630. Other exemplary devices are described in U.S. Pat. No. 10,936,163, 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 Apparutuses, Methods and Reagents," by Krivoy et al., filed July 15, 2020, each of which is incorporated herein by reference in its entirety.

[0150] For example, by applying one or more voltage pulses to generate ECL as described herein, readout and / or exposure times may be improved by more rapid and efficient generation, collection, observation, and analysis of ECL data. Improved exposure times (e.g., single exposure, double (or more) exposures with different (or equal) exposure times) also help improve ECL generation, collection, observation, and analysis, for example by improving dynamic range extension (DRE), binning, etc., for example, in embodiments, for substances of interest that require different time periods to capture luminescence data. Thus, emitted photons may be captured as ECL data over multiple different time periods, which may be affected, for example, by the light saturation level during ECL generation. 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, exposure of two or more single working electrodes or working electrode zones (in 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 long exposure, so long as the exposure is not saturated, in which case, for example, a short exposure may be used. Making these adjustments (manually or with the aid of hardware, firmware, software, algorithms, computer readable media, computing devices, etc.) may improve dynamic range, as detailed above.

[0151] Additionally, the systems, devices, and methods described herein may be leveraged in various ways to enable optimization of software, firmware, and / or control logic for hardware instruments, such as, for example, 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 cost hardware 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 representative, and additional improvements to these instruments are contemplated.

[0152] In embodiments such as those described above, the wells 200 of the multi-well plate 208 may contain one or more fluids (e.g., reagents) for performing an ECL analysis. For example, the fluids may include ECL reactants (e.g., TPA), lead buffers, preservatives, additives, excipients, carbohydrates, proteins, detergents, polymers, salts, biomolecules, inorganic compounds, lipids, etc. In some embodiments, the chemical properties of the fluids in the wells 200 during the ECL process may change the electrochemistry / ECL generation. For example, the relationship between ion concentration of the fluid and electrochemistry / ECL generation may depend on different liquid types, lead buffers, etc. In embodiments, the one or more auxiliary electrodes may provide a constant interfacial potential regardless of the current passing therethrough, as described above. That is, a plot of current versus potential results in infinite current at a fixed potential.

[0153] In some embodiments, the fluid used (e.g., in the wells 200 of the multi-well plate 208) may include an ionic compound, such as, for example, NaCl (e.g., a salt). In some embodiments, for example, a higher concentration of NaCl 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, for example, Ag-AgCl, has a defined slope. In some embodiments, this slope depends on the composition and concentration of the 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 in 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 may affect, for example, the reference potential at the interface of the auxiliary electrode 102 containing Ag-AgCl versus the current passing through it. Thus, in embodiments, the concentration of ions, such as, for example, salts, may be modified and controlled to maximize the current generated for an applied voltage.

[0154] In embodiments, the volume of fluid in the well 200 during the ECL process may change the electrochemistry / ECL generation. In some embodiments, the relationship of the volumes of fluid in the well 200 may 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 may have a more ideal electrochemical behavior, e.g., 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 may have a non-ideal electrochemical behavior due to a spatial gradient of the interfacial potential across both electrodes. In some embodiments, the design and layout of the working electrode zone(s) 104 and the auxiliary electrode 102 may maximize the spatial distance between the working electrode zone 104 and the auxiliary electrode 102. For example, as shown in FIG. 3A, the working electrode zone 104 and the auxiliary electrode 102 may 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.

[0155] In an embodiment, the multi-well 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 multi-well 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, mites and fragments thereof, viroids, antibodies, antigens, haptens, fatty acids, nucleic acids (and synthetic analogues), proteins (and synthetic analogues), 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 phosphatases, ubiquitin-protein ligases, etc.), nucleic acids, and the like. These include, but are not limited to, processing enzymes (e.g., polymerases, nucleases, integrases, ligases, helicases, telomerases, etc.), enzyme substrates (e.g., substrates for the enzymes mentioned above), second messengers, intracellular 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 reactants, pH buffers, blocking agents, preservatives, stabilizing agents, detergents, drying agents, moisture absorbents, 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 reactant; (c) one or more binding reagents; (d) a pH buffer; (e) one or more blocking reagents; (f) a preservative; (g) a stabilizing agent; (h) an enzyme; (i) a detergent; (j) a desiccant; and (k) a moisture absorbent.

[0156] 16 shows a flow chart illustrating a process 2000 for manufacturing wells including working and auxiliary electrodes according to an embodiment herein. For example, the process 2000 may be used to manufacture one or more of the wells 200 of a multiwell plate 208 that include one or more working electrode zones 104 and one or more auxiliary electrodes 102.

[0157] In operation 2002, the 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, 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.

[0158] In embodiments, the working electrode or electrodes may be continuous / unbroken areas where reactions may occur, and an electrode "zone" may be a portion (or entirety) of an electrode where a particular reaction of interest occurs. In certain embodiments, the working electrode zone may comprise the entire working electrode, and in other embodiments, multiple working electrode zones may be formed within and / or on a single working electrode. For example, the 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 of one or more conductive materials. In other examples, the working electrodes may be formed by isolating portions of a single working electrode. In this example, the single working electrode may be formed of one or more conductive materials, and the working electrode zones may be formed by electrically isolating areas ("zones") of the single working electrode using insulating materials, such as, for example, dielectrics. In any embodiment, the working electrodes may be formed of any type of conductive material, such as, for example, metals, alloys, carbon compounds, and combinations of conductive and insulating materials.

[0159] In operation 2004, the process 2000 includes forming one or more auxiliary electrodes 102 on the substrate. In an embodiment, the one or more auxiliary electrodes may be formed using any type of manufacturing process, such as screen printing, three-dimensional (3D) printing, deposition, lithography, etching, and combinations thereof. In an embodiment, the auxiliary electrodes 102 may be formed as a multi-layer structure that may be deposited and patterned. In an embodiment, the one or more auxiliary electrodes may be formed with a chemical mixture that provides an interfacial potential during reduction of the chemical mixture such that a quantifiable amount of charge is generated through the reduction-oxidation reaction occurring in the well. The one or more auxiliary electrodes include an oxidizing agent that supports a reduction-oxidation reaction that may be used during biological, chemical, and / or biochemical assays and / or analyses, such as, for example, ECL generation and analysis. In an 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 a redox reaction has occurred for an initial biological, chemical, and / or biochemical assay and / or analysis to allow one or more additional redox reactions to occur throughout a subsequent biological, chemical, and / or biochemical assay and / or analysis. In other embodiments, the amount of oxidant in the chemical mixture of the one or more auxiliary electrodes is based at least in part on a ratio of the exposed surface area of ​​each of the multiple working electrode zones to the exposed surface area of ​​the auxiliary electrode.

[0160] For example, the auxiliary electrode(s) may be formed of 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 having 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.

[0161] In operation 2006, the process includes forming an electrically insulating material to electrically insulate 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, deposition, lithography, etching, and combinations thereof. The electrically insulating material can include a dielectric.

[0162] In operation 2008, the process 2000 includes forming additional electrical components on the substrate. In an embodiment, the one or more auxiliary electrodes may be formed using any type of manufacturing process, such as screen printing, 3D printing, deposition, lithography, etching, and combinations thereof. The additional electrical components may include through holes, electrical traces, electrical contacts, and the like. For example, through holes are formed in the layers or materials forming the working electrode zone 104, the auxiliary electrode 102, and the electrical insulating material such that electrical contacts may be made with the working electrode zone 104 and the auxiliary electrode 102 without creating shorts with other electrical components. For example, one or more additional insulating layers may be formed on the substrate to support the electrical traces that are coupled through while isolating the electrical traces.

[0163] In embodiments, the additional electrical components may include an electrical heater, a temperature controller, and / or a temperature sensor. The electrical heater, the temperature controller, and / or the temperature sensor may assist in electrochemical reactions, such as ECL reactions, and electrode performance may be temperature dependent. For example, a screen-printed resistive heater may be integrated into the electrode design. The resistive heater may be powered and controlled by an internal or external temperature controller and / or temperature sensor. They are self-regulating and are constructed to generate a specific temperature when a constant voltage is applied. The ink may assist in controlling the temperature during the assay or during plate readout. The ink (and / or heater) may also be useful when an increase in temperature is 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.

[0164] 17A-17F illustrate non-limiting examples of a process for forming a working electrode zone 104 and an auxiliary electrode 102 in one or more wells 200, according to embodiments herein. Although FIGS. 17A-17F illustrate the formation of two wells (as shown in FIG. 18A), one skilled in the art will understand that the process illustrated in FIGS. 17A-17F may be applied to any number of wells 200. Also, although FIGS. 17A-17F illustrate the formation of an auxiliary electrode 102 and a working electrode zone 104 in an electrode design similar to electrode design 701 illustrated in FIGS. 7A-7F, one skilled in the art will understand that the process illustrated in FIGS. 17A-17F may be used with any electrode design described herein.

[0165] The process of fabricating the auxiliary electrode 102, working electrode zone 104, and other electrical components may be performed using a screen printing process, as described below, in which various materials are formed using inks or pastes. In embodiments, the auxiliary electrode 102 and working electrode zone 104 may be formed using any type of fabrication process, such as 3D printing, deposition, lithography, etching, and combinations thereof.

[0166] As shown in FIG. 17A, a first conductive layer 2102 may be printed on the 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, and the like. Other examples of the first conductive layer 2102 may include metals coated with oxides (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 fibers, and mixtures thereof. Other examples of the first conductive layer 2102 may include conductive carbon polymer composites.

[0167] The substrate 2100 may 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 2012. 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 layers or materials forming the working electrode zone 104, the auxiliary electrode 102, and the electrically insulating material such that electrical contacts may be made with the working electrode zone 104 and the auxiliary electrode 102 without creating shorts with other electrical components. For example, one or more additional insulating layers may be formed on the substrate to support the electrical traces that are coupled through while isolating the electrical traces.

[0168] As shown in FIG. 17B, the second conductive layer 2108 may be printed on the first conductive layer 2102. In embodiments, the second conductive layer 2108 may 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 may be formed to approximate dimensions of the first conductive layer 2102. In some embodiments, the second conductive layer 2108 may be formed to dimensions larger or smaller than the first conductive layer 2102. The second conductive layer 2108 may be formed by printing the second conductive layer 2108 with an Ag-AgCl chemical mixture (e.g., ink, paste, etc.) having a defined Ag to AgCl ratio. In embodiments, 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 an 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.

[0169] As shown in FIG. 17C, 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 miscellaneous material, such as a dielectric, polymer, glass, etc. 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 a desired shape and size of the auxiliary electrodes 102. In an embodiment, 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, and 8A-8D.

[0170] As shown in FIG. 17D and FIG. 17E, the third conductive layer 2112 may be printed on the insulating layer 2110, followed by printing the 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 material. Other examples of the first conductive layer 2102 may include metals, such as gold, silver, platinum, nickel, steel, iridium, copper, aluminum, conductive alloys, and the like. Other examples of the first conductive layer 2102 may include metals coated with oxides (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 fibers, 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-hole 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, and 8A-8D.

[0171] As shown in FIG. 17F, 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, e.g., a dielectric. The second insulating layer 2116 may be formed in a pattern that exposes a plurality of portions (e.g., 20 portions) (also referred to throughout as "spots") of the fourth conductive layer 2114 to form ten working electrode zones 104 for each well 200, as shown in FIG. 18A. The exposed portions may correspond to the desired shape and size of the working electrode zones 104. In embodiments, the working electrode zones 104 may be formed in any number, size, and shape, as described in the electrode designs discussed above with reference to, e.g., FIGS. 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, and 8A-8D. In certain embodiments, one or more of the described layers may be formed in a particular order to minimize contamination of the layers (eg, carbon-based layers, etc.).

[0172] In the manner described above, electrical conductivity between the auxiliary electrodes 102 is maintained by the conductive layer 2108 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. 18B shows a further embodiment of a well 200 as fabricated by a fabrication method somewhat similar to that described above with respect to FIGS. 17A-17F and FIG. 22A. As shown in FIG. 18B, the working electrode zones 104 may be arranged in a circular pattern with gaps, for example in 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 working electrode zones 104 allow the conductive traces 2120 to pass between the auxiliary electrodes 102 of the two wells 200. Because the conductive trace 2120 passes between and does not span the auxiliary electrodes 102, the auxiliary electrodes 102, the working electrode zone 104, and the conductive trace 2120 may be printed on the same layer during the manufacturing process. For example, in an embodiment including an individually addressable working electrode zone 104, each of the auxiliary electrodes 102, the working electrode zone 104, and the conductive trace 2120 may be printed as individual features on the same layer of the substrate. The C-shaped design of the electrodes shown in FIG. 18B 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.

[0173] In embodiments, electrochemical cells described herein may be provided with sector-addressable wells. As discussed throughout, electrochemical cells consistent with the present disclosure include working and auxiliary electrodes arranged according to a particular positioning and patterning. In embodiments, a multiwell assay plate may include one or more wells that are addressable as a group, e.g., as a sector. Thus, multiple wells of a sector (and one or more individual spots therein) may be simultaneously addressed, e.g., electrically actuated or excited. A multiwell assay plate may include multiple well sectors, and the wells of each sector may be simultaneously addressable. An embodiment consistent with a sector-addressable multiwell assay plate is described below with respect to other sectors in a multiwell assay plate in Figures 20A and 20B, 21A-21D, 22A-22O, and 23A-23PP. In embodiments, as described below, the working electrodes of a given sector are in electrical communication with each other, facilitating simultaneous addressability.

[0174] In some embodiments, multi-assay plates containing individually isolated working electrode zones may be sector addressable by specific hardware and / or software arrangements in the device designed to facilitate electrical connection between the multi-well assay plate and a plate reader system.

[0175] 20A and 20B show electrochemical cells having working electrode zones according to embodiments encompassed herein. FIG. 20A shows a single spot electrochemical cell 1000, and FIG. 20B shows a multi-spot electrochemical cell 1001. The electrochemical cell 1000 includes a single working electrode zone 1002, at least one auxiliary electrode zone 1003, and an electrochemical cell boundary 1004. The electrochemical cell 1001 includes multiple working zones 1002, at least one auxiliary electrode zone 1003, and an electrochemical cell boundary 1004. In an example, the electrochemical cell 1001 of FIG. 20B may include four working electrode zones 1002 and two auxiliary electrode zones 1003. In other embodiments, a fewer or greater number of working electrode zones 1002 (e.g., 1, 2, 3, 6, 7, 8, etc.) may alternatively be provided. The following description of the electrochemical cell refers to both a four working electrode zone design electrochemical cell 1001 (also referred to throughout as a four-spot design and a one-spot design) and a single working electrode zone electrochemical cell 1000. However, it is understood that the devices, systems, and methods disclosed herein in connection with electrode electrochemical cells are not limited to the particular single and four-electrode zone designs, and may be applied to other patterns and locations of electrode zones, including at least those disclosed herein, as appropriate.

[0176] As discussed above, a working electrode zone may comprise an entire electrode, and in other embodiments, multiple working electrode zones may be formed in and / or on a single electrode or two or more electrodes. For example, in electrochemical cell 1001 and electrochemical cell 1000 formed by well electrode structure 3101 described below with reference to FIG. 22A, multiple working electrode zones 1002 may be formed by individually isolating surface zones, regions, or portions of a single working electrode to form working electrode zones 1002. In such embodiments, multiple working electrode zones 1002 sharing the same working electrode may be in electrical communication with each other. In this example, a single working electrode may be formed by one or more conductive materials, and the working electrode zones 1002 may be formed by electrically isolating regions ("zones") of a single working electrode using an insulating material, such as a dielectric. In an embodiment, as described in more detail below, a multi-well assay plate may include several well sectors in which each of the working electrode zones 1002 in the sectors are in electrical communication by having a common working electrode. In further embodiments, a well sector may include multiple working electrode zones 1002 that are not in direct electrical communication through the assay plate. In such embodiments, sector addressability may be achieved through simultaneous energization of multiple working electrode zones 1002 by a plate reader or other system configured to use the assay plates discussed herein.

[0177] Similarly, the auxiliary electrode zones 1003 of the electrochemical cell 1000 / 10001 may be formed from individually separated surface areas ("zones") of a larger continuous auxiliary electrode pattern or structure. Thus, the multiple auxiliary electrode zones 1003 of a continuous auxiliary electrode pattern or structure may each be in electrical communication with one another. As described below, the multiple auxiliary electrode zones 1003 may be separated by sectors in a multi-well assay plate and / or may be in electrical communication with all other auxiliary electrode zones 1003 of the assay plate.

[0178] Figures 21A-21D show a portion of a multiwell assay plate having wells containing electrochemical cells according to embodiments disclosed herein. Figure 21A shows a perspective top view of a multiwell plate 2000. Figure 21A 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 being defined by a well area as described in more detail below. Figure 21A also shows a base plate 3010 that includes a substrate 3100 as shown in Figure 21B.

[0179] 21B and 21C show the substrate 3100 and its top surface 3180. In the exemplary multi-well assay plate 2000 shown in FIG. 21A, the top surface 3180 fits onto the top plate 3001. FIG. 21B and 21C show various elements visible on the top surface 3180 of the substrate 3100 that combine to form a plurality of sector electrode structures 3150 and well electrode structures 3101. Further elements and additional description of the sector electrode structures 3150 and well electrode structures 3101 are provided below. The sector electrode structures 3150 and well electrode structures 3101 serve to define an electrochemical cell 1000 / 1001 (FIG. 20A / 20B) that comprises a plurality of working electrode zones 1002 and at least one auxiliary electrode zone 1003. FIG. 21B shows a well electrode structure 3101 defining a one-spot electrochemical cell 1000, and FIG. 21C shows a well electrode structure 3101 defining a four-spot electrochemical cell 1001.

[0180] Figure 21D shows the substrate 3100 and its bottom surface 3210. The bottom surface 3210 of the substrate 3100 features a number of working electrode contacts 3204 that form part of the sector electrode structure 3150 and the well electrode structure 3101, as described below. Figure 21D further shows an auxiliary electrode contact pattern 3291.

[0181] 21A-21C show a multiwell plate 2000 having 384 wells 3003 in a 16×24 arrangement. As discussed herein, the 384 wells may be arranged into 24 sectors of 16 (4×4) wells each. The following description makes specific reference in many places to this 384-well plate arrangement. However, the disclosure is not limited to this particular arrangement and encompasses any other suitable well sector arrangement. For example, in a 384-well plate, more sectors with fewer wells (e.g., 48 sectors of 8 (2×4) wells, 96 sectors of 4 (1×4 or 2×2) wells, etc.), as well as fewer sectors with more wells (e.g., 16 sectors of 24 (6×4) wells, 12 sectors of 32 (8×4) wells, 6 sectors of 64 (8×8) wells, etc.) are also consistent with embodiments herein. Additionally, the sectorized multi-well assay plates described herein may include a greater or lesser number of wells total, e.g., 96 wells. Features described herein with respect to the 24 sector / 16 well arrangement may be utilized or used to facilitate assay plates having alternative sector arrangements. Additionally, the multi-well assay plate 2000 described herein includes square wells with rounded corners. This shape is merely one example, and further embodiments may include wells of any suitable shape, as discussed herein.

[0182] Each well 3003 corresponds to a well electrode structure 3101. Furthermore, the wells 3003 and well electrode structures 3101 may be grouped into sectors defined by sector electrode structures 3150, as described below. In further embodiments, any suitable number of wells 3003 and well electrode structures 3101 may be provided. Also, the multi-well plate 2000 shown in Figures 21A-21C is only one example of use of the well electrode structures 3101 described herein. The well electrode structures 3101 described herein may be used to form electrochemical cells 1000 / 1001 for a variety of applications including, for example, cartridge readers, plate-based analyzers, lateral flow based test devices, and the like.

[0183] In embodiments, the well electrode structures 3101 may be formed on the substrate 3100 in a variety of ways, for example, via sequential screen printing processes, stencils, etching, deposition, lithography, and / or other methodologies for forming electrodes. In these examples, the well electrode structures 3101 may be printed layer-by-layer on the substrate 3100, although other methodologies may be used. In embodiments, the electrodes described throughout may be implemented on one or more circuits, such as, for example, a printed circuit board (PCB). Thus, in the following description, a 384-well plate using 24 sectors of 16 wells arranged in a 4×4 format fabricated via a screen printing process may be described, although other fabrication techniques and other well / sector arrangements as described herein are within the scope of the present disclosure. Additionally, different layering processes may be used in conjunction with each other as appropriate, for example, some layers may be screen printed and other layers may be printed via a stencil, etc.

[0184] Additionally, the conductive and non-conductive layers described below may comprise any suitable material, such as conductive materials such as carbon, carbon composites, silver, gold, silver chloride, and various non-conductive insulating dielectric materials (e.g., dielectric inks, polymer-based coatings and films, etc.), as appropriate. In the embodiments described below, specific materials are described for specific layers. These are provided merely as examples, and other materials may be used without departing from the scope of the present disclosure.

[0185] 22A-22O show sector electrode structures 3150 and well electrode structures 3101, as well as aspects of a lamination process. The lamination process, described below with respect to FIGS. 22A-22O, includes a screen printing process. In a particular screen printing process, the screen printing may use a screen or mesh that is partially made impermeable with a shielding material. The screen is placed over a substrate, and ink or other substances are forced through the screen to form a pattern on the substrate. Screen printing is just one manufacturing method, and other lamination printing processes may be used to print the patterns and layers described below.

[0186] In the following description of the lamination process used to create the substrate 3100, various dimensions are discussed. As described below with respect to FIGS. 22A-22O, nominal dimensions are discussed. 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 manufacturing tolerances that do not interfere with the described functionality of the various structures. For example, approximate dimensions may vary by 10% or less, 5% or less, 3% or less, 2% or less, and / or 1% or less.

[0187] The dimensions described below with respect to FIGS. 22A-22O are selected to allow for placement of all of the required features within the allowed space without interference between the features. Interference can refer to physical interference, two features crossing in an unintentional manner, as well as electrical interference, e.g., two features electrically affecting each other in an unintentional manner. The dimensions described below are non-limiting and exemplary, and are selected with consideration of manufacturing tolerances and limitations. Such considerations relate to both manufacturing tolerances in the manufacture of a single layer, e.g., tolerances involved in the manufacture of various screens and templates for printing, as well as the tolerances involved in printing one or more features with a screen or template. The tolerances considered also relate to manufacturing tolerances across multiple layers, e.g., print-to-print registration tolerances involved in the alignment of one layer with a subsequent layer. These types of manufacturing errors require consideration of the possibility of tolerance or error stack-up. For example, to meet a requirement that two features maintain a specific separation distance 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 selected to take into account possible print-to-print registration errors.

[0188] FIG. 22A shows an electrode pattern for a portion of a top surface 3180 of a substrate 3100. The depicted portion shows several well electrode structures 3101 and several sector electrode structures 3150 features disposed on the top surface 3180 of the substrate 3100. Features belonging to one well electrode structure 3101 are shown outlined with a dashed border. Features belonging to one sector electrode structure 3150 are also shown with a dashed border. As described below, the well electrode structures 3101 and sector electrode structures 3150 features are patterned on the top surface 3180 and bottom surface 3210 (shown in FIG. 22C) of the substrate 3100. Each sector electrode structure 3150 includes one or more well electrode structures 3101 shown in more detail in FIG. 22B. Each of the sector electrode structures 3150 includes one or more electrode bus bars 3190 and a portion of an auxiliary electrode pattern 3191 spanning the substrate 3100, as described in more detail below.

[0189] 22B shows an expanded view of several well electrode structures 3101. Each of the well electrode structures 3101 includes an electrode zone group 3104 that includes multiple working electrode zones 1002 and one or more auxiliary electrode zones 1003. The working electrode zones 1002 are formed by isolating a portion of a working electrode busbar 3190 at the surface of the substrate 3100, as described below. The auxiliary electrode zones 1003 are formed by isolating a portion of an auxiliary electrode pattern 3191, as described below.

[0190] The electrode group 3104 is disposed within a well area 3106 defined by a well perimeter 3105. The well perimeter 3105 corresponds to the electrochemical cell boundary 1004. When the substrate 3100 is attached to the top plate 3001, the well area 3106 is configured to correspond to the top plate opening 3002 to form the bottom of the well 3003.

[0191] As shown in Figures 22A and 22B, the working electrode zones 1002 of each well electrode structure 3101 of the sector electrode structure 3150 are in electrical communication with each other (via the working electrode busbar 3190), and the auxiliary electrode zones 1003 of each well electrode structure 3101 of the sector electrode structure 3150 are in electrical communication with each other (via the auxiliary electrode pattern 3191). This structure allows all working electrode zones 1002 of the sector electrode structure 3150 to be addressed, excited, and / or energized in parallel or simultaneously. Thus, the interconnected auxiliary electrode zones 1003 may function as counter or ground electrodes for all excited working electrode zones 1002. Figure 22A shows a 4 x 4 sector of 16 wells. In an embodiment, a 384-well plate may be arranged in sectors in a 6 x 4 sector arrangement. This patterning is merely exemplary and a greater or lesser number of wells may be included in a sector.

[0192] 22C shows an electrode contact pattern for a portion of the bottom surface 3210 of the substrate 3100. The electrode contact pattern is arranged according to sector electrode structures 3150. Each sector electrode structure 3150 includes a working electrode contact 3204 and a portion of an auxiliary electrode contact pattern 3291 patterned on the bottom surface 3210 of the substrate 3100. Each of the working electrode contacts 3204 corresponds to an electrode bus bar 3190 of the sector electrode structure 3150. The auxiliary electrode contact pattern 3291 corresponds to the auxiliary electrode pattern 3191.

[0193] The portion of the sector electrode structure 3150 patterned on the top surface 3180 of the substrate 3100 is connected to the portion of the sector electrode structure 3150 on the bottom surface 3210 of the substrate by a number of vias (e.g. holes filled with a conductor). The vias include two different types: working electrode vias 3205 and auxiliary electrode vias 3206. The working electrode vias 3205 connect the working electrode contacts 3204 to the respective electrode bus bars 3190 on the top surface 3180 of the substrate 3100. As shown in FIG. 21C, four pairs of working electrode vias 3205 (one for each electrode bus bar 3190 of the sector electrode structure 3150) may connect the working electrode contacts 3204 to the respective electrode bus bars 3190. The auxiliary electrode vias 3206 connect the auxiliary electrode contact pattern 3291 to the auxiliary electrode pattern 3191 on the top surface 3180 of the substrate 3100. The auxiliary electrode vias 3206 may be provided in rows, for example one row of auxiliary electrode vias 3206 provided between each row of sector electrode structures 3150. Each row of auxiliary electrode vias 3206 may include three pairs of auxiliary electrode vias 3206.

[0194] In some embodiments, the vias may be provided in a different pattern or amount than that shown in FIG. 22C. For example, the vias may be provided singly rather than in pairs. In further embodiments, a greater or lesser number of vias may be provided. The vias described herein provide the function of connecting the auxiliary electrode contact pattern 3291 to the auxiliary electrode pattern 3191 and connecting the working electrode contacts 3204 to the respective electrode bus bars 3190. Any suitable number of vias that can perform this function may be within the scope of the present disclosure.

[0195] For illustrative purposes, FIG. 22C further shows an example pattern of pin contacts that may be used to electrically connect with a multi-well assay plate utilizing substrate 3100. When used in a plate reader or other instrument, the contact pins of the instrument are positioned to contact working electrode contact 3204 and auxiliary electrode pattern 3191. The instrument is configured to electrically address various wells and working electrode zones of a multi-well assay plate via the contact pins. FIG. 22C shows an example pattern of pin contacts (3971, 3972, 3973) showing where the contact pins of an exemplary instrument may contact bottom surface 3210 of substrate 3100. FIG. 22C shows the pin contact arrangement for a single well sector, showing working electrode pin contact 3971 configured to contact working electrode contact 3204 and auxiliary electrode pin contact 3972 configured to contact auxiliary electrode contact pattern 3291. A 384-well multiwell assay plate consistent with embodiments described herein may be configured for use with plate readers having different contact pin arrangements (e.g., for use with plates of multiple different types and designs). For example, some plate readers may include additional electrode pins used for electrode contact in alternative plate designs. Such pins may contact the substrate at additional working electrode pin contacts 3973. In embodiments, a multiwell assay plate consistent with embodiments herein may include cutouts 3295 (e.g., spaces, gaps, and / or voids) in the auxiliary electrode contact pattern 3291 to accommodate the arrangement of such additional working electrode pin contacts 3973 without contacting the additional working electrode pins with the auxiliary electrode contact pattern 3291. As discussed above, a multiwell assay plate consistent with embodiments herein may further include a different number or arrangement of wells. Additionally, a multiwell assay plate consistent with embodiments herein may be configured with different electrode contact patterns to accommodate readers or systems having alternative contact pin arrangements.

[0196] 22A-22C, each sector electrode structure 3150 includes at least a plurality of electrode bus bars 3190, a portion of an auxiliary electrode pattern 3191, a portion of an auxiliary electrode contact pattern 3291, a working electrode contact 3204, and a plurality of working electrode vias 3205. Auxiliary electrode vias 3206 are not included in each sector electrode structure 3150, and there may be fewer auxiliary electrode vias 3206 than in the sector electrode structures 3150. Each well electrode structure 3101 includes an electrode group 3104 (including one or more working electrode zones 1002 and one or more auxiliary electrode zones 1003). The well electrode structures 3101 may form an electrochemical cell 1001 as described herein.

[0197] 22D-22M show the individual layers associated with the construction of the sector electrode structure 3150 and the well electrode structure 3101 according to embodiments disclosed herein.

[0198] 22D and 22DD show a pattern of holes formed in the substrate. FIG. 22D shows a plan view and FIG. 22DD shows a perspective view. Holes 3115 and 3116 are formed in the substrate 3100. Holes 3115 / 3116 may be formed by laser cutting, micro drilling, or any other suitable method. Holes 3115 are formed in pairs for redundancy purposes, one pair corresponding to each pair of working electrode vias 3205. Holes 3116 are also formed in pairs, one pair corresponding to each pair of auxiliary electrode vias 3206. The pairs of holes 3115 may be formed in rows or columns, with each row or column including six sets of four pairs of holes, each set of four pairs corresponding to a sector electrode structure 3150, each pair corresponding to a working electrode bus bar 3190 (not shown) and a working electrode contact 3204 (not shown). The substrate 3100 may include four rows or columns of holes 3115, with each row or column including six sets of four pairs of holes, providing a total of 24 sets of holes of four pairs each. The substrate 3100 may further include holes 3116 arranged in pairs corresponding to the auxiliary electrode patterns 3191 and auxiliary electrode contact patterns 3291. The substrate 3100 may include nine or more pairs of holes 3116 corresponding to the auxiliary electrode patterns 3191 and auxiliary electrode contact patterns 3291. As described below, the auxiliary electrode contact patterns 3191 and auxiliary electrode contact patterns 3291 are continuous across the top surface 3180 and bottom surface 3210 of the substrate 3100, respectively. Thus, any number of suitable numbers and patterns of holes 3116 (e.g., single, paired, triple, etc., as well as more or less rows and / or columns of holes 3116) may be provided to connect the auxiliary electrode patterns 3191 and auxiliary electrode contact patterns 3291.

[0199] 22E shows a pattern of a layer applied to the bottom surface 3210 of the substrate 3100 to fill holes 3115 to form the working electrode via 3205 and the auxiliary electrode via 3206. A conductive layer is applied to the bottom surface 3210 of the substrate 3100. The conductive layer flows through and fills holes 3115 and 3116 to form the conductive working electrode via 3205 and the conductive auxiliary electrode via 3206. The conductive layer is arranged to form the working electrode contact base 3284 and the auxiliary electrode contact base 3281. In an embodiment, the conductive layer in FIG. 22E 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.

[0200] The conductive layer forms working electrode contact bases 3284. Each working electrode contact base 3284 is elongated dogbone shaped with enlarged circular ends and a bridge or connector between the circular ends. Each working electrode contact base 3284 corresponds to a sector electrode structure 3150 and contains four pairs of holes 3115 which, when filled with the conductive layer, form working electrode vias 3205 corresponding to electrode bus bars 3190 of the sector electrode structures. More specifically, a total of 24 working electrode contact bases 3284 are provided on the bottom surface 3210 of the substrate 3100.

[0201] The conductive layer further forms an auxiliary electrode contact pattern base 3281. The auxiliary electrode contact pattern base 3281 forms a continuous base for the auxiliary electrode contact pattern 3291 across the bottom surface 3210 of the substrate 3100. The auxiliary electrode contact pattern base 3281 includes a plurality of regularly spaced cutouts 3295 that represent gaps or voids in the pattern base 3281. The auxiliary electrode contact pattern base 3281 may include a 6×7 (or 7×6) pattern of cutouts 3295. Each cutout 3295 represents a gap in the pattern base 3281 and may be fully or partially surrounded by the auxiliary electrode contact pattern base 3281. As used herein, "fully surrounded" may refer to instances where the entire perimeter of the cutout 3295 is occupied or bordered by the auxiliary electrode contact pattern 3291. Thus, a straight line drawn from any point on the cutout 3295 in any direction will intersect the auxiliary electrode contact pattern 3291. For example, cutout 3295A is completely surrounded. As used herein, "partially surrounded" may refer to instances where a portion of the perimeter of cutout 3295 is occupied or bordered by auxiliary electrode contact pattern 3291. For example, partially surrounded may refer to instances where more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, and / or more than 99% (and less than 100%) of the perimeter is occupied or bordered by the surroundings. For example, cutout 3295B is partially surrounded. Cutout 3295 may be in the shape of a pinched ellipse (or half of a pinched ellipse) with straight sides and rounded ends, with a depression (circular or other shape) along each of the straight sides (optionally in the center of each straight side). At the end of the auxiliary electrode contact pattern base 3281 a cutout 3295 may be formed as one half of a sandwiched ellipse and partially enclosed by the auxiliary electrode contact pattern base 3281. A recess in the side of the sandwiched ellipse may be formed to accommodate the auxiliary electrode pin contact 3972, for example to provide a larger contact area to allow for possible registration errors during contact.The cutout 3295 may accommodate the location of the working electrode contact base 3284 (i.e., by a first plurality of cutouts 3295) and provide a non-conductive region 3285, also referred to as a separation zone 3285 (e.g., by a second plurality of cutouts 3295), which may be contacted by an additional electrode contact pinpoint 3973 as described above. In further embodiments, alternative shapes of the cutout 3295 are also contemplated that still provide clearance for the working electrode contact base 3284 and the additional electrode contact pinpoint 3973.

[0202] The working electrode contact base 3284 disposed in all or a portion of the cutout 3295 is configured to provide a contact for establishing electrical communication with a first or active set of one or more working electrode contact pins to enable energization of the working electrode bus bar 3190. The non-conductive region 3285 disposed in all or a portion of the cutout 3295 is configured to provide a contact for isolating a second or inactive set of one or more working electrode contact pins to prevent the inactive set of one or more working electrode contact pins from energizing any feature of the multi-well assay plate. In an embodiment, the non-conductive region 3285 and the working electrode contact base 3284 may be arranged in alternating rows of the cutout 3295, for example as shown in FIG. 22E. In further embodiments, the non-conductive region 3285 and the working electrode contact base 3284 may be arranged in different patterns to properly accommodate the active and inactive working electrode contact pins. As used herein, the "active" and "inactive" states of the working electrode contact pins refer to the states of the electrode pins determined according to contact with the multiwell assay plate 2000. The working electrode contact base 3284 is configured to place the set of working electrode contact pins in an active state such that they are in electrical communication with the working electrode bus bar 3190, allowing the energization of the working electrode bus bar 3190 when the active working electrode contact pins are activated (e.g., supplied with a voltage) by the assay system. The non-conductive region 3285 places the set of working electrode contact pins in an inactive state such that they are separated from and do not electrically communicate with any electrodes of the multiwell assay plate 2000, thereby preventing the inactive working electrode contact pins from energizing either side of the multiwell assay plate 2000 even when activated (e.g., supplied with a voltage) by the assay system.

[0203] Thus, the multiwell assay plate 2000 employing the auxiliary electrode contact pattern base 3281 may be configured to enable use with an assay system designed or configured with an alternate contact pin pattern required by the multiwell assay plate 2000. The auxiliary electrode contact pattern 3281 is configured to render one or more working electrode contact pins of the assay system inactive and render one or more working electrode contact pins of the assay system active. In embodiments, the auxiliary electrode contact pattern 3281 may be further configured to render one or more auxiliary electrode contact pins of the assay system inactive and render one or more auxiliary electrode contact pins of the assay system active.

[0204] Both ends of the working electrode contact base 3284 are spaced apart from the auxiliary electrode contact pattern base 3281 by a nominal dimension 3905 of 0.040". The nominal dimension 3905 is well within the screen printing tolerances to ensure that the working electrode contact base 3284 does not overlap the auxiliary electrode contact pattern base 3281.

[0205] 22F shows a carbon layer applied to the bottom surface of the substrate to form working electrode contacts 3204 and auxiliary electrode contact pattern 3291. The carbon layer also serves to ensure that vias 3205 and 3206 are completely filled, for example if a previously applied conductive layer did not completely fill holes 3115 and 3116. A conductive layer is applied to the bottom surface 3210 of the substrate 3100. The conductive layer is made of carbon and configured to provide working electrode contacts 3204, each working electrode contact 3204 containing four pairs of working electrode vias 3205. More specifically, a total of 24 working electrode contacts 3204 may be disposed on the bottom surface 3210 of the substrate 3100. The carbon conductive layer also provides the auxiliary electrode contact pattern 3291.

[0206] Both ends of the working electrode contact 3204 are spaced from the auxiliary electrode contact pattern 3291 by a nominal dimension 3909 of 0.015". The nominal dimension 3909 is well within the screen printing tolerances to ensure that the working electrode contact 3204 does not overlap the auxiliary electrode contact pattern 3291.

[0207] The carbon layer shown in FIG. 22F is configured to cover the silver conductive layer of FIG. 22E. The carbon layer of FIG. 22F is configured to extend beyond the edge / boundary of the silver conductive layer of FIG. 22E. The nominal extension of the working electrode contact base 3284 of the working electrode contact 3204 is about 0.010″, and the nominal extension of the auxiliary electrode contact pattern 3291 over the auxiliary electrode contact pattern base 3281 is about 0.015″. These values ​​exceed the print-to-print registration tolerance of 0.008″, thus ensuring complete coverage of the silver conductive layer of FIG. 22E by the carbon layer of FIG. 22F while still being within the tolerance limits of the screen printing process for layer-to-layer registration. Thus, the carbon layer providing the working electrode contact 3204 and the auxiliary electrode contact pattern 3291 are defined by the same shapes as described above for the working electrode contact base 3284 and the auxiliary electrode contact pattern base 3281.

[0208] 22G and 22GG show a conductive layer pattern made of silver that is applied to the top surface 3180 of the substrate 3100 to form a working electrode busbar base 3990 and an auxiliary electrode contact pattern base 3980. The working electrode busbar base 3990 provides a foundation for the working electrode busbar 3190, as described in more detail below. Each working electrode busbar base 3990 corresponds to a pair of working electrode vias 3205 and is configured to extend laterally in both directions from the corresponding working electrode via 3205. The working electrode busbar base 3990 may be any suitable shape. In an embodiment, the working electrode busbar base 3990 is generally rectangular and includes a series of extensions at regular intervals along its length that give the rectangle greater width. Thus, the long side of the generally rectangular working electrode busbar base 3990 is notched. The enlarged extensions may correspond to individual well electrode structures 3101, as described in more detail below.

[0209] The working electrode busbar bases 3990 are provided in 24 rows of four working electrode busbar bases 3990 per row for a total of 96 working electrode busbar bases 3990. The working electrode busbar bases 3990 may be grouped into sector electrode structures 3150. Each sector electrode structure 3150 may include four working electrode busbar bases 3990, one from each of four consecutive rows. Thus, the substrate 3100 may include a 4×6 pattern of sector electrode structures 3150. Four pairs of working electrode vias 3205 corresponding to the four working electrode busbar bases 3990 of the sector electrode structures 3150 may correspond to a single working electrode contact 3204 (one shown filled in black) located on the bottom surface 3210 of the substrate 3100. Thus, the four working electrode busbar bases 3990 of the sector electrode structure 3150 may be in electrical communication with each other, allowing each of the four working electrode busbar bases 3990 to be addressed simultaneously.

[0210] The auxiliary electrode pattern base 3980 is a continuous pattern of conductive silver. The auxiliary electrode pattern base 3980 comprises a generally rectangular sheet of conductive silver covering the entire substrate 3100 with a plurality of openings 3981 configured to accommodate working electrode busbar bases 3990. Each working electrode busbar base 3990 has an opening 3981 corresponding to the auxiliary electrode pattern base 3980. The shape of each opening 3981 is a larger version of the notched rectangle of the working electrode busbar base 3990.

[0211] In embodiments, the conductive silver may flow into holes 3115 / 3116 and bond to the conductive silver applied to the bottom surface 3210 of substrate 3100 to complete the formation of vias 3205 and 3206. In further embodiments, any other suitable conductive material may be used in place of the conductive silver, such as, for example, other metals (e.g., silver, platinum, nickel, steel, iridium, copper, aluminum, etc.), conductive alloys, etc.

[0212] 22G and 22GG show a conductive silver layer applied to a substrate 3100 to form a four-spot embodiment of a sector multiwell assay plate 2000 as described herein. In further embodiments, alternative conductive silver layers may be applied, for example to form a second multiwell assay plate 2000. The conductive silver layer of the one-spot embodiment may have similar characteristics to the four-spot embodiment, with alternative dimensions. A one-spot conductive silver layer is shown, for example, in FIG. 23FFF.

[0213] Figure 22H shows a conductive carbon layer pattern applied to the top surface of the working electrode busbar base 3990 to form the working electrode busbar carbon layer 3991. The conductive layer shown in Figure 22H that forms the working electrode busbar carbon layer 3991 is configured to extend beyond the working electrode busbar base 3990. The nominal dimensions take into account possible registration errors between the conductive layer of Figure 22H and the conductive layer of Figure 22G. Thus, the shape of the working electrode busbar carbon layer 3991 corresponds to the notched rectangle of the working electrode busbar base 3990.

[0214] Figure 22H shows a conductive carbon layer applied to a substrate 3100 to form a four-spot embodiment of the sector multiwell assay plate 2000 described herein. In further embodiments, alternative conductive carbon layers may be applied, for example to form a one-spot embodiment of the sector multiwell assay plate 2000. The conductive carbon layer of the one-spot embodiment may have similar features to the four-spot embodiment, with alternative dimensions. A one-spot conductive carbon layer is shown, for example, in Figure 23HHH.

[0215] FIG. 22I shows a conductive layer of silver chloride (AgCl) applied to the top surface 3180 of the substrate 3100 to form the auxiliary electrode pattern 3191. The auxiliary electrode pattern 3191 overlaps and extends beyond the auxiliary electrode pattern base 3980. The nominal dimensions account for possible registration errors between the conductive layer of FIG. 22I and the conductive layer of FIG. 22G. Thus, the auxiliary electrode pattern 3191 corresponds to the shape of the auxiliary electrode pattern base 3980 and includes a generally rectangular outline with a plurality of notched rectangular openings 3982 (corresponding to the openings 3981) to accommodate the working electrode bus bar 3190.

[0216] FIG. 22I shows a conductive AgCl layer applied to the substrate 3100 to form a four-spot embodiment of the sector multiwell assay plate 2000 described herein. In further embodiments, alternative conductive AgCl layers may be applied, for example to form a one-spot embodiment of the sector multiwell assay plate 2000. The conductive AgCl layer of the one-spot embodiment may have similar features to the four-spot embodiment, with alternative dimensions. A one-spot conductive silver layer is shown, for example, in FIG. 23JJJ.

[0217] 22J shows a second conductive carbon layer pattern applied to the top surface of the working electrode busbar carbon layer 3991 to form the working electrode busbar 3190. The conductive carbon layer shown in FIG. 22J that forms the working electrode busbar 3190 is configured to extend beyond the working electrode busbar carbon layer 3991. The nominal dimensions allow for possible registration errors between the conductive layer of FIG. 22J and the conductive layer of FIG. 22G. Thus, the shape of the working electrode busbar 3190 corresponds to the notched rectangle of the working electrode busbar carbon layer 3991.

[0218] Figure 22J shows a second conductive carbon layer applied to the substrate 3100 to form a four-spot embodiment of the sector multiwell assay plate 2000 described herein. In further embodiments, an alternative second conductive carbon layer may be applied, for example to form a one-spot embodiment of the sector multiwell assay plate 2000. The second conductive carbon layer of the one-spot embodiment may have similar features to the four-spot embodiment, with alternative dimensions. A one-spot second conductive carbon layer is shown, for example, in Figure 23LLL.

[0219] FIG. 22K shows a dielectric layer applied to the top surface 3180 of the substrate 3100 to physically separate the portions of the substrate surface that form the working electrode zone 3102 and the auxiliary electrode zone 3103 in a four-spot embodiment. FIG. 22K shows a dielectric layer applied to a four-spot well embodiment consistent with FIG. 20B. Each well electrode structure 3101 of the substrate 3100 includes a dielectric mask 3195. Thus, each sector electrode structure 3150 may include 16 dielectric masks 3195. In further embodiments, the sector electrode structure 3150 may include a greater or lesser number of well electrode structures 3101 and may include a greater or lesser number of dielectric masks 3195 accordingly. Each dielectric mask 3195 includes a rectangular core 3954 having a pair of extensions 3950, four tabs 3951, four working electrode openings 3196, and a pair of recesses 3953. The rectangular core 3954 includes extensions 3950 on two opposing sides that correspond to the indentations in the working electrode bus bar 3190 onto which the dielectric mask 3195 is laminated. The remaining two opposing sides of the rectangular core 3954 include indentations 3953. Each corner of the rectangular core 3954 further includes a tab 3951 that extends outwardly from the core. Finally, each dielectric mask 3195 includes a number of working electrode openings 3196. As shown in FIG. 22K, each of the dielectric masks 3195 may include four working electrode openings 3196 to form the four working electrode zones 1002 of the four-spot electrochemical cell 1001.

[0220] When the top plate 3001 is adhered to the substrate 3100, the openings 3002 in the top plate 3001 define wells 3003. FIG. 22L shows the substrate 3100 and the defined wells 3003. Each opening 3002 in the top plate 3001 corresponds to a well electrode structure 3101. The openings 3002 include well walls 3004 that define the wells 3003. Also shown is an adhesive edge 3005 that surrounds the wells 3003 and is provided for adhering the top plate 3001 to the substrate 3100. Each well 3003 includes multiple (in this case four) working electrode zones 3102 and one or more (in this case two) auxiliary electrode zones 3103 that remain exposed after application of the dielectric mask 3195. For example, in an embodiment as shown in FIG. 22L, the ratio of the working electrode zone area to the auxiliary electrode zone area is about 3.24 (as used herein, about indicates that the ratio may vary by less than 5%, 3%, 1%).

[0221] FIG. 22M shows a dielectric layer applied to the top surface 3180 of the substrate 3100 to physically separate the portions of the substrate surface that form the working electrode zone 3102 and the auxiliary electrode zone 3103 in a one-spot embodiment. FIG. 22M shows a dielectric layer applied to a one-spot well embodiment consistent with FIG. 20A. Each one-spot well electrode structure 3301 of the substrate 3100 includes a dielectric mask 3175. Thus, each sector electrode structure 3550 may include 16 dielectric masks 3175. In further embodiments, the sector electrode structure 3150 may include a greater or lesser number of well electrode structures 3101 and may include a greater or lesser number of dielectric masks 3175 accordingly. Each dielectric mask 3175 is similar to the dielectric mask 3195 and includes a rectangular core 3754 having a pair of extensions 3750, four tabs 3751, a working electrode opening 3176, and a pair of recesses 3753. The rectangular core 3754 includes extensions 3750 on two opposing sides that correspond to the indentations in the working electrode bus bar 3190 onto which the dielectric mask 3175 is laminated. The remaining two opposing sides of the rectangular core 3754 include indentations 3753. Each corner of the rectangular core further includes a tab 3751 that extends outwardly from the core. Finally, each dielectric mask 3175 includes a single working electrode opening 3176. As shown in FIG. 22M, each of the dielectric masks 3175 includes one working electrode opening 3176 to form one working electrode zone 1002 of the one-spot electrochemical cell 1000.

[0222] When the top plate 3001 is adhered to the substrate 3100, the openings 3002 in the top plate 3001 define wells 3003. FIG. 22N shows the substrate 3100 and the defined wells 3003. Each opening 3002 in the top plate 3001 corresponds to a well electrode structure 3301. The openings 3002 include well walls 3004 that define the wells 3003. Also shown is an adhesive edge 3005 that surrounds the wells 3003 and is provided for adhering the top plate 3001 to the substrate 3100. Each well 3003 includes one working electrode zone 3102 and one or more (in this case four) auxiliary electrode zones 3103 that remain exposed after application of the dielectric mask 3175. For example, in an embodiment as shown in FIG. 22N, the ratio of working electrode zone area to auxiliary electrode zone area is about 3.78 (as used herein, about indicates that the ratio may vary by less than 5%, 3%, 1%).

[0223] The well electrode structures 3101 / 3301 are configured such that the working electrode zones 3102 and auxiliary electrode zones 3103 of a particular sector electrode structure 3150 are in electrical communication with each other's working electrode zones 3102 and auxiliary electrode zones 3103. The working electrode zones 3102 of different sector electrode structures 3150 are isolated from each other. The auxiliary electrode zones 3103 of different sector electrode structures 3150 are in electrical communication with each other.

[0224] As mentioned above, the layers of the substrate 3100 are constructed with nominal dimensions selected to achieve the functionality described above. The nominal dimensions may be selected to allow all of the various features and aspects of the substrate 3100 to be located close to one another without compromising the isolation characteristics discussed herein. The nominal dimensions are selected to accommodate manufacturing tolerances and increase the likelihood that the manufactured substrate will meet the functional requirements discussed herein. The nominal dimensions discussed herein are merely exemplary and provide one example of dimensioning to manufacture the substrate 3100 having the characteristics discussed herein. In further embodiments, alternative nominal dimensions may be used to provide the required functionality without departing from the scope of the present disclosure.

[0225] Figure 22O shows a cross section of the substrate 3100 after each of the layers described above in the one spot embodiment have been added. Figure 22O shows the dielectric layer 5000 (corresponding to the dielectric mask 3175 described with reference to Figure 22N), the holes 3115 (corresponding to the holes 3115 described with reference to Figure 22D), the first bottom conductive layer 5001 (corresponding to the working electrode contact base 3284 and auxiliary electrode contact pattern base 3281 described with reference to Figure 22E), the second bottom conductive layer 5002 (corresponding to the working electrode contact 3204 and auxiliary electrode contact pattern 3291 described with reference to Figure 22F), the second bottom conductive layer 5003 (corresponding to the working electrode contact 3204 and auxiliary electrode contact pattern 3291 described with reference to Figure 22G), the second bottom conductive layer 5004 (corresponding to the working electrode contact 3204 and auxiliary electrode contact pattern 3291 described with reference to Figure 22H), the second bottom conductive layer 5005 (corresponding to the working electrode contact 3204 and auxiliary electrode contact pattern 3291 described with reference to Figure 22H), the second bottom conductive layer 5006 (corresponding to the working electrode contact 3204 and auxiliary electrode contact pattern 3291 described with reference to Figure 22H), and the second bottom conductive layer 5007 (corresponding to the working electrode contact 3204 and auxiliary electrode contact pattern 3291 described with reference to Figure 22H). FIG. 22G shows a first electrode trace conductive layer 5003 (corresponding to the working electrode bus bar base 3990 and auxiliary electrode pattern base 3980 described with respect to FIG. 22G), a first working electrode conductive layer 5004 (corresponding to the working electrode bus bar carbon layer 3991 feature described with respect to FIG. 22H), a second working electrode conductive layer 5005 (corresponding to the working electrode bus bar 3190 of FIG. 22J), and an auxiliary electrode conductive layer 5006 (corresponding to the auxiliary electrode pattern 3191 described with respect to FIG. 22I). Example thickness values ​​for these layers are shown in Table 9 below. The listed values ​​are merely examples and may vary by 1%, 5%, 10%, etc. based on manufacturing process tolerances. In further examples, alternative values ​​including values ​​that vary by 1%, 5%, 10%, 15%, 20%, or more may be used without departing from the scope of the present disclosure. [Table 9]

[0226] 23A-23PP show aspects of a substrate structure including multiple working electrode structures (forming multiple individually addressable electrode electrochemical cells) according to embodiments disclosed herein, and various patterns (e.g., formed by screens) that may be used in one or more processes to form these structures (e.g., screen printing processes to print the various layers required for the working electrode structures and the resulting printed patterns using the respective screens).

[0227] 23A, 23B, and 23BB respectively show a first bottom conductive screen 4001, a bottom conductive layer 5001 resulting from using the first bottom conductive screen 4001 in printing a first conductive layer on the bottom side of a substrate, and a perspective view of the bottom conductive layer 5001. The pattern in FIG. 23B corresponds to the features shown in FIG. 22E. FIG. 23A shows the first bottom conductive screen 4001. The first bottom conductive screen pattern 4101 is provided on the first bottom conductive screen 4001, which may be manufactured, for example, from stainless steel, nylon, polyester, etc. The first bottom conductive screen pattern 4101 is configured to mask the substrate and allow screen printing ink to pass through the first bottom conductive screen pattern 4101 to create the bottom conductive layer 5001 and form the working electrode contact base 3284 and the auxiliary electrode contact pattern base 3281, as shown in FIG. 23B. Further details of the bottom conductive layer 5001 created through the use of the first bottom conductive screen 4001 are described above with respect to FIG. 22E. As shown in FIG. 23A, the first bottom conductive screen pattern 4101 can be configured to print the working electrode contact base 2384 and auxiliary electrode contact pattern base 3281 corresponding to 384 wells of a 16-well by 24-well plate. Further embodiments may include screens configured to print the via tab screen pattern 4191 across smaller plates (such as 96-well plates) and / or multiple plates (such as 2, 3, 4, or more 384-well plates). Exemplary dimensions for the first bottom conductive screen 4001 are further shown in FIG. 23A. The illustrated dimensions are nominal dimensions provided merely as examples.

[0228] 23C, 23D, and 23DD respectively show a second screen (second bottom conductive screen) 4003, a second printed pattern 4004 (corresponding to the second bottom conductive layer 5002) resulting from using the second screen 4003 in printing the second bottom conductive layer 5002 on the bottom surface 3210 of the substrate 3100, and a perspective view of the second bottom conductive layer 5002. The pattern in FIG. 23D corresponds to the features shown in FIG. 22F. FIG. 23C shows a second screen pattern (second bottom conductive screen pattern) 4103. The second screen pattern 4103 is provided on a second screen 4003, which may be made of, for example, stainless steel, nylon, polyester, etc. The second screen 4003 is configured to mask the substrate and allow screen printing ink to pass through the second screen pattern 4103 to deposit the bottom conductive layer 5002 and form the working electrode contacts 3204 and auxiliary electrode contact pattern 3291 as shown in FIG. 23D. Further details of the second printed pattern 4004 created through the use of the second screen 4003 are described above with respect to FIG. 22F. As shown in FIG. 23C, the second screen pattern 4103 can be configured to print the working electrode contacts 3204 and auxiliary electrode contact pattern 3291 corresponding to 384 wells of a 16 well by 24 well plate. Further embodiments may include screens configured to print the second printed pattern 4004 across smaller plates (such as, for example, a 96 well plate) and / or multiple plates (such as, for example, 2, 3, 4, or more 384 well plates). Exemplary dimensions for the first bottom conductive screen 4001 are further shown in FIG. 23C. The dimensions shown are nominal dimensions provided as an example only.

[0229] 23E, 23F, and 23FF respectively show a third screen (busbar base screen) 4005, a third printed pattern (busbar trace base pattern, corresponding to the first electrode trace conductive layer 5003) 4006 resulting from using the third screen 4005 in printing the first electrode trace conductive layer 5003 on the top surface 3180 of the substrate 3100, and a perspective view of the first electrode trace conductive layer 5003. The pattern in FIG. 23F corresponds to the features shown in FIG. 22G and FIG. 22GG. FIG. 23E shows a third screen pattern (busbar base screen pattern) 4104. The third screen pattern 4104 is provided on a third screen 4005, which may be manufactured from, for example, stainless steel, nylon, polyester, etc. The third screen 4005 is configured to mask the substrate and allow the screen printing ink to pass through the third screen pattern 4104 to form the working electrode busbar base 3990 and the auxiliary electrode pattern base 3980, as shown in FIG. 23F. Further details of the third printed pattern 4006 created through the use of the third screen 4005 are described above with respect to FIG. 22G and FIG. 22GG. As shown in FIG. 23E, the third screen pattern 4104 can be configured to print the working electrode busbar base 3990 and the auxiliary electrode pattern base 3980 corresponding to 384 wells of a 16 well by 24 well plate. Further embodiments may include a screen configured to print the third pattern 4006 across smaller plates (such as, for example, a 96 well plate) and / or multiple plates (such as, for example, 2, 3, 4, or more 384 well plates).

[0230] Figures 23E, 23F and 23FF show patterns and layers for forming a four spot embodiment of the sector multiwell assay plate 2000 described herein. In further embodiments, alternative patterns and layers may be used, for example to form a one spot embodiment of the sector multiwell assay plate 2000. Figure 23FFF shows a one spot first electrode trace conductive layer 5003. In the one spot embodiment, the first electrode trace conductive layer 5003 may have similar features as the four spot embodiment, with alternative dimensions.

[0231] 23G, 23H, and 23HH respectively show a fourth screen (busbar base screen) 4007, a fourth printed pattern (busbar base pattern) 4008 resulting from using the fourth screen 4007 in printing the first working electrode conductive layer 5004 on the top surface of the substrate, and a perspective view of the first working electrode conductive layer 5004. The pattern in FIG. 23H corresponds to the features shown in FIG. 22H. FIG. 23G shows a fourth screen pattern (busbar base screen pattern) 4106. The fourth screen pattern 4106 is provided on the fourth screen 4007, which may be manufactured, for example, of stainless steel, nylon, polyester, etc. The fourth screen 4007 is configured to mask the substrate 3100 and allow screen printing ink to pass through the fourth screen pattern 4106 to form the first working electrode conductive layer 5004 and form the working electrode busbar carbon layer 3991, as shown in FIG. 23H. Further details of the fourth printed pattern 4008 created through the use of the fourth screen 4007 are described above with respect to Figure 22H. As shown in Figure 23G, the fourth screen pattern 4106 can be configured to print a working electrode busbar carbon layer 3991 corresponding to 384 wells of a 16 well by 24 well plate. Further embodiments may include a screen configured to print the fourth pattern 4008 across smaller plates (such as a 96 well plate) and / or multiple plates (e.g., 2, 3, 4, or more 384 well plates).

[0232] Figures 23G, 23H, and 23HH show patterns and layers for forming a four-spot embodiment of the sector multiwell assay plate 2000 described herein. In further embodiments, alternative patterns and layers may be used, for example, to form a one-spot embodiment of the sector multiwell assay plate 2000. Figure 23HHH shows a one-spot first working electrode conductive layer 5004. In the one-spot embodiment, the first working electrode conductive layer 5004 may have similar features as the four-spot embodiment, with alternative dimensions.

[0233] 23I, 23J, and 23JJ respectively show a fifth screen (auxiliary electrode pattern screen) 4009, a fifth printed pattern (auxiliary electrode pattern) 4010 resulting from using the fifth screen 4009 in printing the auxiliary electrode conductive layer 5006 on the top surface 3180 of the substrate 3100, and a perspective view of the auxiliary electrode conductive layer 5006. The pattern in FIG. 23J corresponds to the features shown in FIG. 22I. FIG. 23J shows a fifth screen pattern (auxiliary electrode screen pattern) 4108. The fifth screen pattern 4108 is provided on the fifth screen 4009, which may be manufactured, for example, of stainless steel, nylon, polyester, etc. The fifth screen 4009 is configured to mask the substrate and allow screen printing ink to pass through the fifth screen pattern 4108 to form the auxiliary electrode conductive layer 5006 and form the auxiliary electrode pattern 3191. Further details of the fifth printed pattern 4010 created through use of the fifth screen 4009 are described above with respect to Figure 22I. As shown in Figure 23J, the fifth screen pattern 4108 can be configured to print an auxiliary electrode pattern 3191 corresponding to 384 wells of a 16 well by 24 well plate. Further embodiments may include screens configured to print the fifth pattern 4010 across smaller plates (such as, for example, a 96 well plate) and / or multiple plates (e.g., 2, 3, 4 or more 384 well plates).

[0234] Figures 23I, 23J, and 23JJ show patterns and layers for forming a four-spot embodiment of the sector multiwell assay plate 2000 described herein. In further embodiments, alternative patterns and layers may be used, for example to form a one-spot embodiment of the sector multiwell assay plate 2000. Figure 23JJJ shows a one-spot auxiliary electrode conductive layer 5006. In the one-spot embodiment, the auxiliary electrode conductive layer 5006 may have similar features as the four-spot embodiment, with alternative dimensions.

[0235] 23K, 23L, and 23LL respectively show a sixth screen (busbar screen) 4011, a sixth printed pattern (busbar pattern) 4012 resulting from using the sixth screen in printing the second working electrode conductive layer 5005 on the top surface 3180 of the substrate 3100, and a perspective view of the second working electrode conductive layer 5005. The pattern in FIG. 23L corresponds to the features shown in FIG. 22J. FIG. 23K shows a sixth screen pattern (busbar screen pattern) 4110. The sixth screen pattern 4110 is provided on a sixth screen 4011, which may be manufactured, for example, of stainless steel, nylon, polyester, or the like. The sixth screen 4011 is configured to mask the substrate and allow screen printing ink to pass through the sixth screen pattern 4110 to create the second working electrode conductive layer 5005 and form a plurality of working electrode busbars 3190, as shown in FIG. 23L. Further details of the sixth printed pattern 4012 created through the use of the sixth screen 4011 are described above with respect to Figure 22I. As shown in Figure 23K, the sixth screen pattern 4110 can be configured to print a plurality of working electrode bus bars 3190 corresponding to the 384 wells of a 16 well by 24 well plate. Further embodiments may include screens configured to print the sixth pattern 4012 across smaller plates (such as, for example, a 96 well plate) and / or multiple plates (e.g., 2, 3, 4, or more 384 well plates).

[0236] Figures 23K, 23L, and 23LL show patterns and layers for forming a four-spot embodiment of the sector multiwell assay plate 2000 described herein. In further embodiments, alternative patterns and layers may be used, for example to form a one-spot embodiment of the sector multiwell assay plate 2000. Figure 23LLL shows a one-spot second working electrode conductive layer 5005. In the one-spot embodiment, the second working electrode conductive layer 5005 may have similar features as the four-spot embodiment, with alternative dimensions.

[0237] 23M, 23N, and 23NN respectively show a seventh screen (four spot dielectric screen) 4013, a seventh printed pattern (four spot dielectric pattern) 4014 resulting from using the seventh screen 4013 in printing a four spot dielectric layer 5010 on the top surface 3180 of the substrate 3100, and a perspective view of the four spot dielectric layer 5010. The pattern in FIG. 23N corresponds to the features shown in FIG. 22K. FIG. 23M shows a seventh screen pattern (four spot dielectric screen pattern) 4112. The seventh screen pattern 4112 is patterned on the seventh screen 4013, which may be made of, for example, stainless steel, nylon, polyester, etc. The seventh screen 4013 is configured to mask the substrate and allow the screen printing ink (e.g., a dielectric material) to pass through the seventh screen pattern 4112 to create a dielectric layer as shown in FIG. 23N, providing surface insulation to the substrate 3100. Further details of the seventh printed pattern 4014 created through the use of the seventh screen 4013 are described above with respect to Figures 22K and 22I. As shown in Figure 23M, the seventh printed pattern 4014 can be configured to provide a four spot dielectric layer 5010 corresponding to the 384 wells of a 16 well by 24 well plate. Further embodiments may include screens configured for smaller plates (such as, for example, a 96 well plate) and / or multiple plates (e.g., 2, 3, 4 or more 384 well plates).

[0238] 23O, 23P, and 23PP respectively show an eighth screen (one spot dielectric screen) 4015, an eighth printed pattern (one spot dielectric pattern) 4014 resulting from using the eighth screen 4015 in printing a one spot dielectric layer 5000 on the top surface 3180 of the substrate 3100, and a perspective view of the one spot dielectric layer 5000. The pattern in FIG. 23O corresponds to the features shown in FIG. 22M. FIG. 23O shows an eighth screen pattern (one spot dielectric screen pattern) 4114. The eighth screen pattern 4114 is patterned on the eighth screen 4015, which may be made of, for example, stainless steel, nylon, polyester, etc. The eighth screen 4015 is configured to mask the substrate and allow the screen printing ink (e.g., a dielectric material) to pass through the eighth screen pattern 4114 to create the dielectric layer 5000 as shown in FIG. 23P, providing surface insulation to the substrate 3100. Further details of the eighth printed pattern 4016 created through the use of the eighth screen 4015 are described above with respect to Figures 22M and 22N. As shown in Figure 23O, the eighth printed pattern 4016 can be configured to provide a one spot dielectric layer 5000 corresponding to 384 wells of a 16 well by 24 well plate. Further embodiments may include screens configured for smaller plates (such as, for example, a 96 well plate) and / or multiple plates (e.g., 2, 3, 4, or more 384 well plates).

[0239] In an embodiment, the present invention may be embodied as a computer program product that may include computer readable storage medium(s) and / or computer readable storage device. Such computer readable storage medium or device may store computer readable program instructions for causing a processor to execute one or more methodologies described herein. In one embodiment, the computer readable storage medium or device includes a tangible device capable of holding and storing instructions for use by an instruction execution device. Examples of computer readable storage medium or device may include, but are not limited to, electronic storage devices, magnetic storage devices, optical storage devices, electromagnetic storage devices, semiconductor storage devices, or any suitable combination thereof, such as, but not limited to, computer diskettes, hard disks, random access memories (RAMs), read only memories (ROMs), erasable programmable read only memories (EPROMs or flash memories), static random access memories (SRAMs), portable compact disk read only memories (CD-ROMs), digital versatile disks (DVDs), memory sticks, and the like. The computer readable medium may comprise both a computer readable storage medium (as described above) or a computer readable transmission medium, which may include, for example, coaxial cables, copper wire, and fiber optics. The computer readable transmission medium may be in the form of acoustic or light waves, such as those generated in radio frequency, infrared, radio, or other media including radio, magnetic, or electromagnetic waves.

[0240] The term "computer system" as used in this application may include various combinations of fixed and / or portable computer hardware, software, peripherals, mobile, and storage devices. A computer system may include multiple separate components networked or otherwise linked to execute cooperatively, or may include one or more stand-alone components. The hardware and software components of the computer system of this application may include and be included in fixed and portable devices, such as desktops, laptops, and / or servers. A module may be a device, software, program, or system component that implements some "function," which may be embodied as software, hardware, firmware, electronic circuitry, etc.

[0241] Further embodiments of the present disclosure include at least the following:

[0242] Embodiment 1 is 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 being defined by a well area; a base plate having a top surface and a bottom surface, the top surface mating with the top plate; and a plurality of sector electrode structures forming a plurality of well electrode structures, each of the plurality of sector electrode structures comprising at least one working electrode bus bar deposited on the top surface and a portion of an auxiliary electrode pattern deposited on the top surface, a working electrode contact patterned on the bottom surface in electrical communication with the at least one working electrode bus bar, and an auxiliary electrode contact pattern in communication with the auxiliary electrode pattern.

[0243] Embodiment 2 is the plate of embodiment 1, wherein at least one working electrode bus bar of a first sector electrode structure is configured to be energized separately from the energization of at least one working electrode bus bar of a remaining one of the plurality of sector electrode structures.

[0244] Embodiment 3 is the plate of embodiment 1 or 2, wherein at least one working electrode bus bar of each sector electrode structure includes multiple working electrode bus bars configured to be energized simultaneously.

[0245] Embodiment 4 is the plate of embodiments 1-3, wherein the top surface further comprises an adhesive layer corresponding to the well pattern on the top surface, with no adhesive in the well regions.

[0246] Embodiment 5 is the plate of embodiments 1-4, wherein the working electrode contact is in electrical communication with at least one working electrode bus bar through at least one via.

[0247] Example 6 is the plate of Example 5, wherein the at least one via includes a plurality of vias respectively corresponding to a plurality of working electrode bus bars included in the at least one working electrode bus bar.

[0248] Example 7 is the plate of Example 6, wherein the plurality of vias includes two vias connecting each of the plurality of working electrode contacts to each of the plurality of working electrode bus bars.

[0249] Embodiment 8 is the plate of any of Embodiments 1 to 13, wherein the auxiliary electrode is disposed approximately in the center of the well region, and the working electrodes are disposed in a circle approximately equidistant from the auxiliary electrode.

[0250] Example 9 is the plate of Examples 1-8, wherein each well electrode structure includes four working electrode zones formed from one of the at least one working electrode busbars.

[0251] Embodiment 10 is the plate of embodiments 1-9, wherein each well electrode structure includes two auxiliary electrode zones formed from the auxiliary electrode pattern.

[0252] Example 11 is the plate of Examples 1-8, wherein each well electrode structure includes one working electrode zone formed from one of the at least one working electrode busbars.

[0253] Example 12 is the plate of Examples 1-8 and Example 11, wherein each well electrode structure includes four auxiliary electrode zones formed from the auxiliary electrode pattern.

[0254] Embodiment 13 is the plate of embodiments 1-12, wherein the top surface of the substrate further comprises an insulating layer arranged in a pattern exposing the working electrode zone and the auxiliary electrode zone of each well electrode structure and covering the remainder of the top surface of the substrate within each well.

[0255] Embodiment 14 is the plate of embodiments 1 to 13, wherein the plurality of sector electrode structures includes 24 sector electrode structures.

[0256] Example 15 is the plate of Examples 1-14, wherein the well pattern includes a 16x24 arrangement of 384 wells organized into 24 sector electrode structures containing 16 wells each.

[0257] Embodiment 16 is a method of using a multi-well assay plate, the multi-well assay plate including a plurality of wells arranged in a well pattern and a plurality of sector electrode structures forming a plurality of well electrode structures corresponding to the plurality of wells, each of the plurality of sector electrode structures including at least one working electrode bus bar deposited on a top surface forming a plurality of working electrode zones, a portion of an auxiliary electrode pattern deposited on the top surface forming a plurality of auxiliary electrode zones, a working electrode contact patterned on a bottom surface in electrical communication with the at least one working electrode bus bar, and an auxiliary electrode contact pattern in communication with the auxiliary electrode pattern, the method including generating an electrical potential between the plurality of working electrode zones formed by the at least one working electrode bus bar associated with a selected sector electrode structure and the plurality of auxiliary electrode zones associated with the selected sector electrode structure, maintaining substantial electrical isolation between the plurality of working electrode zones of the selected sector electrode structure and the remaining working electrode zones of the remaining sector electrode structures, and measuring a response to the electrical potential.

[0258] Example 17 is the method of example 16, in which generating the potential and measuring the response occur substantially simultaneously.

[0259] Example 18 is the method of Examples 16-17, further comprising, after measuring the response, generating successive potentials on each of the remaining sector electrode structures in sequence relative to previously unenergized working electrode zones of the remaining sector electrode structures, maintaining substantial electrical isolation between the currently unenergized sector electrode structures and the currently energized sector electrode structures, and measuring a plurality of responses to the successive potentials.

[0260] Example 19 is the method of Examples 16-18, wherein the multiwell assay plate further includes a plurality of working electrode contacts deposited on a bottom surface of the multiwell assay plate electrically connected to corresponding working electrode bus bars, and an auxiliary electrode contact pattern deposited on the bottom surface electrically connected to the auxiliary electrode pattern, and generating the electric potential includes contacting the working electrode contacts to one or more working electrode contact pins, contacting the auxiliary electrode pattern to one or more auxiliary electrode contact pins, and applying a voltage across the one or more working electrode contact pins and the one or more auxiliary electrode contact pins.

[0261] Example 20 is the method of Examples 16-19, further comprising depositing a biological sample in at least one well of the plurality of wells.

[0262] Embodiment 21 is a method of manufacturing a multi-well assay plate including a plurality of wells, the method including: 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 and providing a plurality of working electrode contact bases and an auxiliary electrode contact pattern base; applying a second conductive layer of material to the first side of the substrate, the second conductive layer overlying the first conductive layer and forming a plurality of working electrode contacts and an auxiliary electrode contact pattern; applying a third conductive layer of material to a second side of the substrate, the third conductive layer forming a plurality of working electrode bus bar bases and an auxiliary electrode pattern base; and applying a fourth conductive layer of material to the second side of the substrate. the fourth conductive layer includes forming a plurality of working electrode busbar carbon layers; applying a fifth conductive layer of material to a second side of the substrate overlying the fourth conductive layer to form the plurality of working electrode busbars; applying a sixth conductive layer of material to the second side of the substrate overlying the auxiliary electrode pattern base, the sixth conductive layer forming an auxiliary electrode pattern; applying an insulating layer of material to the second side of the substrate, the insulating layer exposing the plurality of auxiliary electrode zones and the plurality of working electrode zones and insulating a remainder of the plurality of wells; and adhering the substrate to a top plate having top plate openings defining wells of a multi-well assay plate arranged in the well pattern, each well defined by a well area.

[0263] Example 22 is the method of example 21, in which the plurality of vias includes a working electrode via connecting a working electrode contact to a working electrode bus bar and an auxiliary electrode via connecting an auxiliary electrode contact pattern to an auxiliary electrode pattern.

[0264] Embodiment 23 is the method of embodiments 21 and 22, wherein the plurality of working electrode zones includes four working electrode zones corresponding to each well.

[0265] Embodiment 24 is the method of embodiments 21 and 22, wherein the multiple working electrode zones include one working electrode zone corresponding to each well.

[0266] Example 25 is the method of Examples 21-24, in which adhering the substrate to the top plate includes applying an adhesive to a second side of the substrate outside the well region.

[0267] Embodiment 26 is a substrate including a plurality of electrochemical cells for performing electrochemical analysis, the plurality of electrochemical cells including one or more working electrode zones disposed on a surface of the cells and at least one auxiliary electrode disposed on a surface of the cells, each of the one or more working electrode zones being in electrical communication with each other.

[0268] Embodiment 27 is the substrate of embodiment 26, wherein the plurality of electrochemical cells are arranged in sectors, each sector including a group of electrochemical cells, and one or more working electrode zones in the group of electrochemical cells of a sector are in electrical communication with each other.

[0269] Embodiment 28 is the substrate of embodiments 26 and 27, wherein the working electrode zone or zones in each sector are separated from the working electrode zones in each of the other sectors.

[0270] Embodiment 29 is the substrate of embodiments 26 to 28, where the substrate is a part of a plate.

[0271] Embodiment 30 is the substrate of any of embodiments 26 to 28, and the substrate is a part of a cartridge.

[0272] Embodiment 31 is the substrate of any of embodiments 26 to 28, and the substrate is a part of a flow cell.

[0273] Embodiment 32 is the substrate of any one of embodiments 26 to 31, wherein the electrochemical analysis includes electrochemiluminescence (ECL) analysis.

[0274] Embodiment 33 is 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 being defined by a well area, a base plate having a top surface and a bottom surface, the top surface mating with the top plate, and a plurality of sector electrode structures forming a plurality of well electrode structures. Each of the plurality of sector electrode structures forming the plurality of well electrode structures includes at least one working electrode bus bar deposited on the top surface and a portion of an auxiliary electrode pattern deposited on the top surface. The assay plate further includes at least one working electrode contact patterned on the bottom surface in electrical communication with the at least one working electrode bus bar, and an auxiliary electrode contact pattern in electrical communication with the auxiliary electrode pattern. The auxiliary electrode contact pattern includes a first plurality of cutouts and a second plurality of cutouts. The first plurality of cutouts is configured to accommodate at least one working electrode contact, and the second plurality of cutouts is configured to generate a plurality of separation zones.

[0275] Embodiment 34 is the multi-well assay plate of embodiment 33, wherein the at least one working electrode contact is configured to be in electrical contact with the first plurality of working electrode pins, and the plurality of isolation zones are configured to electrically isolate the second plurality of electrode pin contacts from the auxiliary electrode pattern and the at least one working electrode bus bar.

[0276] Embodiment 35 is the multi-well assay plate of embodiment 33 or 34, wherein each cutout of the first plurality of cutouts and the second plurality of cutouts represents a gap in the auxiliary electrode pattern and is surrounded by the auxiliary electrode pattern.

[0277] Embodiment 36 is the multi-well assay plate of any of Embodiments 33 to 35, wherein the first plurality of cutouts and the second plurality of cutouts are arranged in an alternating array.

[0278] Embodiment 37 is a multi-well assay plate of any of embodiments 33 to 36, wherein at least one working electrode bus bar of a first sector electrode structure is configured to be energized separately from the energization of at least one working electrode bus bar of a remaining one of the plurality of sector electrode structures.

[0279] Embodiment 38 is the multi-well assay plate of embodiments 33-37, wherein at least one working electrode bus bar of each sector electrode structure includes multiple working electrode bus bars configured to be energized simultaneously.

[0280] Embodiment 39 is the multi-well assay plate of any of Embodiments 33-38, wherein the top surface further comprises an adhesive layer corresponding to the well pattern on the top surface, and the well regions are free of adhesive.

[0281] Embodiment 40 is the multi-well assay plate of any of Embodiments 33-39, wherein the working electrode contacts are in electrical communication with at least one working electrode bus bar through at least one via.

[0282] Embodiment 41 is a multi-well assay plate of any of embodiments 33 to 40, wherein the auxiliary electrode zone electrically connected to the auxiliary electrode pattern is positioned approximately in the center of the well area, and the working electrode zone electrically connected to at least one working electrode bus bar is positioned in a circle approximately equidistant from the auxiliary electrode.

[0283] Embodiment 42 is the multi-well assay plate of any of embodiments 33-41, wherein each well electrode structure includes four working electrode zones formed from one of the at least one working electrode busbars.

[0284] Embodiment 43 is the multi-well assay plate of any of Embodiments 33 to 42, wherein each well electrode structure includes two auxiliary electrode zones formed from the auxiliary electrode patterns.

[0285] Embodiment 44 is the multi-well assay plate of any of embodiments 33-43, wherein each well electrode structure includes one working electrode zone formed from one of the at least one working electrode busbars.

[0286] Embodiment 45 is the multi-well assay plate of any of Embodiments 33-44, wherein each well electrode structure includes four auxiliary electrode zones formed from the auxiliary electrode pattern.

[0287] Embodiment 46 is a multi-well assay plate of any of embodiments 33 to 45, wherein the top surface of the substrate further comprises an insulating layer arranged in a pattern exposing the working electrode zone and auxiliary electrode zone of each well electrode structure and covering the remainder of the top surface of the substrate within each well.

[0288] Embodiment 47 is 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 sector electrode structures forming a plurality of well electrode structures corresponding to the plurality of wells, each including at least one working electrode bus bar deposited on the top surface of a substrate of the multi-well assay plate forming a plurality of working electrode zones, and a portion of the auxiliary electrode pattern deposited on the top surface forming a plurality of auxiliary electrode zones; an auxiliary electrode contact pattern in communication with the auxiliary electrode pattern, the auxiliary electrode contact pattern including a first plurality of cutouts and a second plurality of cutouts; a plurality of working electrode contacts deposited on the bottom surface of the multi-well assay plate within the first plurality of cutouts and electrically connected to the corresponding working electrode bus bar; and a plurality of separation zones disposed within the second plurality of cutouts. The method includes generating an electrical potential between a plurality of working electrode zones formed by at least one working electrode bus bar associated with a selected sector electrode structure and a plurality of auxiliary electrode zones associated with the selected sector electrode structure, maintaining substantial electrical isolation between the plurality of working electrode zones of the selected sector electrode structure and remaining working electrode zones of the remaining sector electrode structures, and measuring a response to the electrical potential.

[0289] Example 48 is the method of example 47, in which generating the potential and measuring the response occur substantially simultaneously.

[0290] Example 49 is the method of example 48, further comprising, after measuring the response, generating successive potentials on each of the remaining sector electrode structures in sequence relative to previously unenergized working electrode zones of the remaining sector electrode structures, maintaining substantial electrical separation between the currently unenergized sector electrode structures and the currently energized sector electrode structures, and measuring a plurality of responses to the successive potentials.

[0291] Example 50 is the method of example 48 or 49, wherein generating the electric potential includes contacting the plurality of working electrode contacts with one or more active working electrode contact pins, contacting the plurality of separation zones with one or more inactive working electrode contact pins, contacting the auxiliary electrode pattern with one or more auxiliary electrode contact pins, and applying a voltage across the one or more working electrode contact pins and the one or more auxiliary electrode contact pins.

[0292] Embodiment 51 is the method of any of embodiments 48 to 50, wherein the method is carried out for electrochemiluminescence (ECL) analysis.

[0293] Embodiment 52 is a method of manufacturing a multi-well assay plate including a plurality of wells, the method including 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 and providing a plurality of working electrode contact bases and an auxiliary electrode contact pattern base, applying a second conductive layer of material to the first side of the substrate, the second conductive layer overlying the first conductive layer and forming a plurality of working electrode contacts and an auxiliary electrode contact pattern having a first plurality of cutouts and a second plurality of cutouts, the working electrode contacts being formed within the first plurality of cutouts and the separation zones being formed within the second plurality of cutouts, applying a third conductive layer of material to a second side of the substrate, the third conductive layer filling the plurality of working electrode busbar bases and the auxiliary electrode pattern base, and applying a second conductive layer of material to the second side of the substrate, the second conductive layer overlying the first conductive layer and forming a plurality of working electrode contacts and an auxiliary electrode contact pattern having a first plurality of cutouts and a second plurality of cutouts, the working electrode contacts being formed within the first plurality of cutouts and the separation zones being formed within the second plurality of cutouts. applying a fourth conductive layer of material to a second side of the substrate overlying the fourth conductive layer to form a plurality of working electrode busbars, the fourth conductive layer forming a carbon layer; applying a fifth conductive layer of material to a second side of the substrate overlying the fourth conductive layer to form a plurality of working electrode busbars, the sixth conductive layer forming an auxiliary electrode pattern; applying an insulating layer of material to the second side of the substrate overlying the auxiliary electrode pattern base, the sixth conductive layer forming an auxiliary electrode pattern; applying an insulating layer of material to the second side of the substrate, the insulating layer exposing the plurality of auxiliary electrode zones and the plurality of working electrode zones and insulating a remainder of the plurality of wells; and adhering the substrate to a top plate having top plate openings defining wells of a multi-well assay plate arranged in the well pattern, each well defined by a well area.

[0294] Example 53 is the method of example 52, in which the plurality of vias includes a working electrode via connecting a working electrode contact to a working electrode bus bar and an auxiliary electrode via connecting an auxiliary electrode contact pattern to an auxiliary electrode pattern.

[0295] Embodiment 54 is the method of embodiment 52 or 53, in which the multiple working electrode zones include four working electrode zones corresponding to each well.

[0296] Embodiment 55 is the method of embodiment 53 or 54, in which the multiple working electrode zones include one working electrode zone corresponding to each well.

[0297] Example 56 is the method of any of Examples 53-55, in which adhering the substrate to the top plate includes applying an adhesive to a second side of the substrate outside the well region.

[0298] Embodiment 57 is a substrate having a top surface and a bottom surface, the substrate includes a plurality of electrochemical cells disposed on the top surface for performing electrochemical analysis, each including one or more working electrode zones disposed on a surface of the cell, each in electrical communication with one another, and at least one auxiliary electrode zone disposed on the surface of the cell, and an auxiliary electrode contact pattern disposed on the bottom surface and in electrical communication with the at least one auxiliary electrode, the auxiliary electrode contact pattern including a first plurality of cutouts and a second plurality of cutouts, the first plurality of cutouts configured to receive at least one working electrode contact in electrical communication with the one or more working electrode zones, and the second plurality of cutouts configured to generate a plurality of separation zones.

[0299] Embodiment 58 is the substrate of embodiment 57, wherein the plurality of electrochemical cells are arranged in sectors, each sector including a group of electrochemical cells, and one or more working electrode zones in the group of electrochemical cells of a sector are in electrical communication with each other.

[0300] Embodiment 59 is the substrate of embodiment 58, wherein the working electrode zone or zones in each sector are separated from the working electrode zones in each of the other sectors.

[0301] Embodiment 60 is the substrate of embodiment 58 or 59, wherein the substrate is part of a plate, cartridge, or flow cell.

[0302] Embodiment 61 is the substrate of any of embodiments 57 to 60, wherein the electrochemical analysis comprises electrochemiluminescence (ECL) analysis.

[0303] The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the present invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural, unless the context clearly indicates otherwise. It is also understood that as used herein, the terms "comprise" and / or "comprising" specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0304] The above-described embodiments are exemplary, and the present invention should not be construed as being limited to these specific embodiments. It should be understood that the various embodiments disclosed herein may be combined in different combinations than those specifically shown in the description and accompanying drawings. It should also be understood that, depending on the example, certain operations or events of any of the processes or methods described herein may be performed in a different order, or may be added, merged, or removed altogether (e.g., not all operations or events described to perform a method or process are required). In addition, although certain features of the embodiments herein have been described as being performed by a single module or unit for clarity, it should be understood that the features and functions described herein may be performed by any combination of units or modules. Thus, various changes and modifications may be employed by those skilled in the art without departing from the spirit or scope of the present invention.

[0305] Although various embodiments according to the present disclosure have been described above, it should be understood that they have been presented by way of illustration and example, not limitation. As will be apparent to those skilled in the art, various changes in form and detail may be made without departing from the spirit and scope of the present disclosure. Thus, the breadth and scope of the present disclosure should not be limited by any of the exemplary embodiments described above, but should be defined only according to the appended claims and their equivalents. It is also understood that each feature of each embodiment described herein and each reference cited herein may be used in combination with the features of any other embodiment. In other words, the multi-well plate aspect may be used in any combination with other methods described herein, or the methods may be used individually. All patents and publications described herein are hereby incorporated by reference in their entirety.

Claims

1. 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 being defined by a well area; a base plate including a substrate having a top surface and a bottom surface, the top surface mating with the top plate; a plurality of sector electrode structures forming a plurality of well electrode structures, each including at least one working electrode busbar deposited on said top surface and a portion of an auxiliary electrode pattern deposited on said top surface; at least one working electrode contact patterned on the bottom surface in electrical communication with the at least one working electrode bus bar; an auxiliary electrode contact pattern disposed on the bottom surface in electrical communication with the auxiliary electrode pattern, the auxiliary electrode contact pattern including a first plurality of cutouts and a second plurality of cutouts; Equipped with A multi-well assay plate, wherein the first plurality of cutouts is configured to accommodate the at least one working electrode contact and the second plurality of cutouts is configured to generate a plurality of separation zones.

2. 2. The multi-well assay plate of claim 1, wherein the at least one working electrode contact is configured to be in electrical contact with a first plurality of working electrode pins and the plurality of isolation zones are configured to isolate a second plurality of electrode pin contacts from the auxiliary electrode pattern and the at least one working electrode bus bar.

3. 2. The multi-well assay plate of claim 1, wherein each of the first and second plurality of cutouts represents a gap in the auxiliary electrode pattern and is surrounded by the auxiliary electrode pattern.

4. The multi-well assay plate of claim 1 , wherein the first plurality of cutouts and the second plurality of cutouts are arranged in an alternating array.

5. 2. The multi-well assay plate of claim 1, wherein the at least one working electrode bus bar of a first sector electrode structure is configured to be energized separately from the energization of the at least one working electrode bus bar of a remaining one of the plurality of sector electrode structures.

6. 10. The multi-well assay plate of claim 1, wherein the at least one working electrode bus bar of each sector electrode structure comprises a plurality of working electrode bus bars configured to be energized simultaneously.

7. 10. The multi-well assay plate of claim 1, wherein the top surface further comprises an adhesive layer corresponding to the well pattern on the top surface, the well regions being free of adhesive.

8. 10. The multi-well assay plate of claim 1, wherein the working electrode contacts are in electrical communication with the at least one working electrode bus bar through at least one via.

9. 2. The multi-well assay plate of claim 1, wherein an auxiliary electrode zone electrically connected to the auxiliary electrode pattern is disposed approximately in the center of the well area, and a working electrode zone electrically connected to the at least one working electrode bus bar is disposed in a circle approximately equidistant from the auxiliary electrode.

10. 10. The multi-well assay plate of claim 1, wherein each well electrode structure includes four working electrode zones formed from one of said at least one working electrode busbar.

11. 10. The multi-well assay plate of claim 1, wherein each well electrode structure comprises two auxiliary electrode zones formed from said auxiliary electrode pattern.

12. 10. The multi-well assay plate of claim 1, wherein each well electrode structure includes one working electrode zone formed from one of the at least one working electrode busbars.

13. 2. The multi-well assay plate of claim 1, wherein each well electrode structure comprises four auxiliary electrode zones formed from said auxiliary electrode pattern.

14. 2. The multi-well assay plate of claim 1, wherein the top surface of the substrate further comprises an insulating layer arranged in a pattern exposing working and auxiliary electrode zones of each well electrode structure and covering the remainder of the top surface of the substrate within each well.

15. 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; a plurality of sector electrode structures forming a plurality of well electrode structures corresponding to the plurality of wells, each including at least one working electrode bus bar deposited on a top surface of a substrate of the multi-well assay plate forming a plurality of working electrode zones and a portion of an auxiliary electrode pattern deposited on the top surface forming a plurality of auxiliary electrode zones; an auxiliary electrode contact pattern in communication with the auxiliary electrode pattern, the auxiliary electrode contact pattern including a first plurality of cutouts and a second plurality of cutouts; a plurality of working electrode contacts deposited on a bottom surface of the multi-well assay plate within the first plurality of cutouts and electrically connected to corresponding working electrode bus bars; a plurality of separation zones disposed within the second plurality of cutouts; and said method comprising: generating an electrical potential between the plurality of working electrode zones defined by the at least one working electrode bus bar associated with a selected sector electrode structure and the plurality of auxiliary electrode zones associated with the selected sector electrode structure; maintaining substantial electrical isolation between the plurality of working electrode zones of the selected sector electrode structure and remaining working electrode zones of remaining sector electrode structures; measuring a response to said potential; A method comprising:

16. 16. The method of claim 15, wherein generating the potential and measuring the response occur substantially simultaneously.

17. After measuring said response, sequentially for the previously unpowered working electrode zones of the remaining sector electrode structures, generating a continuous potential on each of said remaining sector electrode structures; maintaining substantial electrical isolation between currently non-energized sector electrode structures and currently energized sector electrode structures; measuring a plurality of responses to said successive potentials; The method of claim 16 further comprising:

18. Generating the electrical potential includes: contacting the plurality of working electrode contacts with one or more active working electrode contact pins; contacting the plurality of separation zones with one or more inactive working electrode contact pins; contacting the auxiliary electrode pattern with one or more auxiliary electrode contact pins; applying a voltage across said one or more working electrode contact pins and said one or more auxiliary electrode contact pins; 17. The method of claim 16, comprising:

19. 17. The method of claim 16, wherein the method is performed for electrochemiluminescence (ECL) analysis.

20. 1. A method for manufacturing 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 holes to form a plurality of vias and providing a plurality of working electrode contact bases and an auxiliary electrode contact pattern base; applying a second conductive layer of material to the first side of the substrate, the second conductive layer overlying the first conductive layer and forming a plurality of working electrode contacts and an auxiliary electrode contact pattern having a first plurality of cutouts and a second plurality of cutouts, the working electrode contacts being formed within the first plurality of cutouts and isolation zones being formed within the second plurality of cutouts; applying a third conductive layer of material to a second side of the substrate, the third conductive layer forming a plurality of working electrode busbar bases and an auxiliary electrode pattern base; applying a fourth conductive layer of material to the second side of the substrate, the fourth conductive layer forming a plurality of working electrode busbar carbon layers; applying a fifth conductive layer of material to the second side of the substrate overlying the fourth conductive layer to form a plurality of working electrode bus bars; applying a sixth conductive layer of material to the second side of the substrate overlying the auxiliary electrode pattern base, the sixth conductive layer forming an auxiliary electrode pattern; applying an insulating layer of material to the second side of the substrate, the insulating layer exposing a plurality of auxiliary electrode zones and a plurality of working electrode zones and insulating a remainder of the plurality of wells; adhering the substrate to a top plate having top plate openings defining the wells of the multi-well assay plate arranged in a well pattern, each well being defined by a well area; The method includes:

21. 21. The method of claim 20, wherein the plurality of vias includes a working electrode via connecting the working electrode contact to the working electrode bus bar and an auxiliary electrode via connecting the auxiliary electrode contact pattern to the auxiliary electrode pattern.

22. 21. The method of claim 20, wherein the plurality of working electrode zones includes four working electrode zones corresponding to each well.

23. 22. The method of claim 21, wherein the multiple working electrode zones include one working electrode zone corresponding to each well.

24. 22. The method of claim 21, wherein adhering the substrate to the top plate comprises applying an adhesive to the second side of the substrate outside of the well area.

25. A substrate having a top surface and a bottom surface, a plurality of electrochemical cells disposed on the top surface for performing electrochemical analyses, each comprising: one or more working electrode zones disposed on a surface of the cell, each of the working electrode zones being in electrical communication with each other; at least one auxiliary electrode zone arranged on said surface of said cell; a plurality of electrochemical cells including: an auxiliary electrode contact pattern disposed on the bottom surface and in electrical communication with the at least one auxiliary electrode, the auxiliary electrode contact pattern including a first plurality of cutouts and a second plurality of cutouts, the first plurality of cutouts configured to accommodate at least one working electrode contact in electrical communication with the one or more working electrode zones, and the second plurality of cutouts configured to generate a plurality of separation zones; A substrate comprising:

26. 26. The substrate of claim 25, wherein the plurality of electrochemical cells are arranged in sectors, each sector comprising a group of electrochemical cells, the one or more working electrode zones in the group of electrochemical cells of a sector being in electrical communication with each other.

27. 27. The substrate of claim 26, wherein the one or more working electrode zones in each sector are separated from the working electrode zones in each of the other sectors.

28. The substrate of claim 26 , wherein the substrate is part of a plate, a cartridge, or a flow cell.

29. The substrate of claim 25 , wherein the electrochemical analysis comprises an electrochemiluminescence (ECL) analysis.

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