Auxiliary electrodes, their use, and manufacturing methods

Abstract

Systems, devices, and methods for electrochemical cells, including auxiliary electrode designs and electrochemical analyzers, and devices including electrochemical cells. [Solution] The electrochemical cell 100 includes a plurality of working electrode zones 104 arranged on the surface of the cell to define a pattern, and at least one auxiliary electrode 102 arranged on the surface. The auxiliary electrode may have a defined interfacial potential.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 068,981, filed on 21 August 2020, and U.S. Provisional Patent Application No. 63 / 118,463, filed on 25 November 2020, both of which are incorporated herein by reference in their entirety.

[0002] Embodiments of the present invention relate to systems, devices, and methods for using auxiliary electrodes in carrying out chemical, biochemical, and biological assays and analyses, and to methods for manufacturing auxiliary electrodes. [Background technology]

[0003] An assay is an analytical technique used in fields such as chemistry, clinical laboratory medicine, pharmacy, environmental biology, and molecular biology to qualitatively evaluate or quantitatively measure the presence, quantity, or functional activity of a target substance (e.g., an analyte). Assay systems can qualitatively and quantitatively evaluate target substances using electrochemical properties and procedures. For example, an assay system can evaluate a target substance by measuring the potential, current, and / or brightness in a sample area containing the target substance, which are generated by an electrochemical process, and then performing various analytical procedures (e.g., potentiometry, coulometry, voltammetry, optical analysis, etc.) on the measurement data.

[0004] Assay systems employing electrochemical properties and procedures may include a sample region (e.g., a well, a well in a multiwell plate, etc.) having 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 resulting data. Depending on the design and configuration of the electrodes, assay systems may be classified as reference systems and non-reference systems. For example, the working electrode is the electrode in the assay system where the reaction of interest is occurring. The working electrode is used in combination with the counter electrode to establish a potential difference, current flow, and / or electric field within the sample region. The potential difference may be divided between the interfacial potentials of the working electrode and the counter electrode. In a non-reference system, the interfacial potential applied to the working electrode (the force that brings about the reaction at the electrode) is uncontrolled or unknown. In a reference system, the sample region includes a reference electrode separate from the working electrode and the counter electrode. The reference electrode has a known potential (e.g., reduction potential) that can be referenced while the reaction is occurring in the sample region.

[0005] One example of these assay systems is the electrochemical luminescence (ECL) immunoassay. The ECL immunoassay involves a process using ECL labels designed to emit light when electrochemically stimulated. Light generation occurs when a voltage is applied to electrodes located within a sample area holding the material under test. The voltage triggers periodic oxidation and reduction reactions, which in turn lead to the generation and emission of light. In ECL, the electrochemical reactions that give rise to ECL are brought about by applying a potential difference between the working electrode and the counter electrode.

[0006] Currently, both reference and non-reference assay systems have drawbacks in the measurement and analysis of target objects. In non-reference assay systems, the unknown nature of the interfacial potential makes it impossible to control the electrochemical process, and it can also be influenced by the design of the assay system. For example, in ECL immunoassays, the interfacial potential applied to the working electrode can be affected by the electrode area (working and / or counter), the composition of the solution, and any surface treatment of the electrode (e.g., plasma treatment). This lack of control has so far been addressed by grading the potential difference from before the start of ECL generation to after the end of ECL generation. In reference systems, the potential is known and can be controlled, but the addition of a reference electrode increases the cost, complexity, and size of the assay system. Furthermore, the addition of a reference electrode can limit the design and placement of the working and / or counter electrodes within the sample area due to the need to accommodate an extra electrode. In addition, both reference and non-reference assay systems can have slow reading times due to the voltage signals required to scan the system. Reference systems can be more expensive because both the counter and reference electrodes must be manufactured.

[0007] Conventional assay systems, devices, and instruments have these and other drawbacks. Therefore, there is a need for systems, devices, and methods that provide a controllable potential for the reference system while reducing the cost, complexity, and size associated with the reference electrode. These drawbacks are addressed by the embodiments described herein. [Overview of the Initiative]

[0008] Embodiments of the present disclosure include systems, devices, and methods for electrochemical cells, including auxiliary electrode designs and electrochemical analyzers, and devices comprising electrochemical cells.

[0009] In one embodiment, the present disclosure provides an electrochemical cell for performing electroanalysis. The electrochemical cell includes a plurality of working electrode zones arranged on the surface of the cell to define a pattern, and at least one auxiliary electrode arranged on the surface. The at least one auxiliary electrode has redox pairs confined on its surface. The at least one auxiliary electrode is positioned substantially equidistant from at least two of the plurality of working electrode zones.

[0010] In another embodiment, an electrochemical cell for performing electrochemical analysis is provided. The electrochemical cell includes a plurality of working electrode zones arranged on the surface of the cell to define a pattern, and at least one auxiliary electrode arranged on the surface and having redox pairs confined on that surface. The redox pairs provide a quantifiable amount of coulombs per unit of surface area of ​​at least one auxiliary electrode through the redox reaction of the redox pairs.

[0011] In another embodiment, an electrochemical cell for performing electrochemical analysis is provided. The electrochemical cell includes a plurality of working electrode zones arranged on the surface of the cell to define a pattern, and at least one auxiliary electrode arranged on the surface and formed of a chemical mixture comprising an oxidizing agent. The at least one auxiliary electrode has a redox pair confined on its surface. The amount of oxidizing agent is sufficient to maintain a predetermined potential throughout the redox reaction of the redox pair.

[0012] In another embodiment, an electrochemical cell for performing electrochemical analysis is provided. The electrochemical cell includes a plurality of working electrode zones arranged on the surface of the cell to define a pattern, and at least one auxiliary electrode arranged on the surface. The auxiliary electrode has a defined interfacial potential.

[0013] In another embodiment, an electrochemical cell for performing electrochemical analysis is provided. The electrochemical cell includes a plurality of working electrode zones arranged on the surface of the cell to define a pattern, and at least one auxiliary electrode arranged on the surface, the at least one auxiliary electrode comprising a first substance and a second substance, the second substance being a redox pair of the first substance.

[0014] In another embodiment, an electrochemical cell for performing electrochemical analysis is provided, which comprises a plurality of working electrode zones arranged on the surface of the cell to define a pattern, and at least one auxiliary electrode arranged on the surface, the at least one auxiliary electrode having redox pairs confined on its surface. When an applied potential is introduced to the cell during electrochemical analysis, the reaction of the species in the redox pairs is the main redox reaction that occurs at the auxiliary electrode.

[0015] In another embodiment, an apparatus for performing electrochemical analysis is provided. This apparatus includes a plate in which a plurality of wells are defined therein, at least one of the plurality of wells comprising a plurality of working electrode zones located on the surface of the cell and defining a pattern, and at least one auxiliary electrode located on the surface and formed of a chemical mixture comprising an oxidizing agent, the at least one auxiliary electrode having a redox pair confined on its surface, the amount of which is sufficient to maintain a defined potential throughout the redox reaction of the redox pair.

[0016] In another embodiment, a method for electrochemical analysis is provided. This method comprises applying a voltage pulse to one or more working electrode zones and at least one auxiliary electrode located in at least one well of a multiwell plate, wherein the one or more working electrode zones define a pattern on the surface of at least one well, and at least one auxiliary electrode is positioned on the surface and has a redox pair confined on that surface, the redox pair being reduced for at least the duration that the voltage pulse is applied.

[0017] In another embodiment, an apparatus is provided for performing electrochemical analysis in a well, comprising a plurality of working electrode zones arranged on a surface adapted to form the bottom of the well, and an auxiliary electrode arranged on the surface, the auxiliary electrode having a potential determined by redox pairs confined on its surface, and one of the plurality of working electrode zones being positioned approximately equidistant from each side wall of the well.

[0018] In another embodiment, a method is provided for performing electrochemical analysis in a well. This method includes applying a first voltage pulse to one or more working electrode zones or counter electrodes in a well of the apparatus, wherein the first voltage pulse causes a first redox reaction in the well and captures first luminescence data from the first redox reaction over a first period of time; and applying a second voltage pulse to one or more working electrode zones or counter electrodes in the well, wherein the second voltage pulse causes a second redox reaction in the well and captures second luminescence data from the second redox reaction over a second period of time.

[0019] The above 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, incorporated herein and forming part of this specification, further illustrate the principles of the various embodiments described herein and enable those skilled in the art to manufacture and utilize the various embodiments described herein. The drawings are not necessarily drawn to scale. [Brief explanation of the drawing]

[0020] [Figure 1A] The following are some figures of electrochemical cells according to embodiments disclosed herein. [Figure 1B] The following are some figures of electrochemical cells according to embodiments disclosed herein. [Figure 1C] The following are some figures of electrochemical cells according to embodiments disclosed herein. [Figure 2A]This document shows a top view of a multiwell plate containing multiple sample regions according to an embodiment disclosed herein. [Figure 2B] This specification shows a multiwell plate for use in an assay device containing multiple sample regions, according to embodiments disclosed herein. [Figure 2C] Figure 1C shows a side view of the sample area of ​​the multiwell plate according to an embodiment disclosed herein. [Figure 3A] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 3B] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 3C] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 3D] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 3E] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 3F] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 4A] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 4B]This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 4C] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 4D] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 4E] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 4F] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 5A] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 5B] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 5C] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 6A] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 6B]This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 6C] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 6D] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 6E] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 6F] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 7A] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 7B] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 7C] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 7D] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 7E]This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 7F] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 8A] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 8B] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 8C] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 8D] This specification shows several design examples of electrodes for use in the electrochemical cells shown in Figures 1A-1C or the multiwell plates shown in Figures 2A-2C, according to embodiments disclosed herein. [Figure 9A] An example of an assay apparatus according to an embodiment disclosed herein is shown. [Figure 9B] An example of an assay apparatus according to an embodiment disclosed herein is shown. [Figure 10A] The decay time of the auxiliary electrode according to the embodiment is shown. [Figure 10B] The decay time of the auxiliary electrode according to the embodiment is shown. [Figure 11] This specification describes a process for performing electrochemical analysis and procedures using pulse waveforms according to embodiments disclosed herein. [Figure 12A] Examples of pulse waveforms according to embodiments disclosed herein are shown. [Figure 12B] Examples of pulse waveforms according to embodiments disclosed herein are shown. [Figure 13] This specification describes a process for performing ECL analysis and procedures using pulse waveforms according to embodiments disclosed herein. [Figure 14A] The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 14B] The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 14C] The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 15A] The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 15B] The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 15C] The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 15D] The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 15E] The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 15F] The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 15G] The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 15H] The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 15I] The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 15J] The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 15K]The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 15L] The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 16] The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 17] The results of an ECL test performed using a pulse waveform according to an embodiment disclosed herein are shown. [Figure 18] This specification describes a process for performing ECL analysis using pulse waveforms according to embodiments disclosed herein. [Figure 19] This specification describes a process for performing ECL analysis using pulse waveforms according to embodiments disclosed herein. [Figure 20] This specification shows a process for manufacturing wells according to embodiments disclosed herein. [Figure 21A] This shows a typical stage in the process of manufacturing a well according to the embodiments disclosed herein. [Figure 21B] This shows a typical stage in the process of manufacturing a well according to the embodiments disclosed herein. [Figure 21C] This shows a typical stage in the process of manufacturing a well according to the embodiments disclosed herein. [Figure 21D] This shows a typical stage in the process of manufacturing a well according to the embodiments disclosed herein. [Figure 21E] This shows a typical stage in the process of manufacturing a well according to the embodiments disclosed herein. [Figure 21F] This shows a typical stage in the process of manufacturing a well according to the embodiments disclosed herein. [Figure 22A] This shows a typical stage in the process of manufacturing a well according to the embodiments disclosed herein. [Figure 22B] The embodiments of the wells relating to this disclosure are shown. [Figure 23A] Some examples of tested electrode configurations according to embodiments disclosed herein are shown. [Figure 23B] Some examples of tested electrode configurations according to embodiments disclosed herein are shown. [Figure 23C] Some examples of tested electrode configurations according to embodiments disclosed herein are shown. [Figure 23D] Some examples of tested electrode configurations according to embodiments disclosed herein are shown. [Figure 24A] The following shows the test results performed on various multiwell plates according to embodiments disclosed herein. [Figure 24B] The following shows the test results performed on various multiwell plates according to embodiments disclosed herein. [Figure 24C] The following shows the test results performed on various multiwell plates according to embodiments disclosed herein. [Figure 25A] The following shows the test results performed on various multiwell plates according to embodiments disclosed herein. [Figure 25B] The following shows the test results performed on various multiwell plates according to embodiments disclosed herein. [Figure 25C] The following shows the test results performed on various multiwell plates according to embodiments disclosed herein. [Figure 26A] The following shows the test results performed on various multiwell plates according to embodiments disclosed herein. [Figure 26B] The following shows the test results performed on various multiwell plates according to embodiments disclosed herein. [Figure 26C] The following shows the test results performed on various multiwell plates according to embodiments disclosed herein. [Figure 26D] The following shows the test results performed on various multiwell plates according to embodiments disclosed herein. [Figure 27A]The following shows the test results performed on various multiwell plates according to embodiments disclosed herein. [Figure 27B] The following shows the test results performed on various multiwell plates according to embodiments disclosed herein. [Figure 27C] The following shows the test results performed on various multiwell plates according to embodiments disclosed herein. [Figure 28] The following shows the test results performed on various multiwell plates according to embodiments disclosed herein. [Figure 29] This specification describes a test performed to optimize the waveform for coating a plasma-treated electrode in comparison to a standard electrode, according to embodiments disclosed herein. [Figure 30] This specification describes a test performed to optimize the waveform for coating a plasma-treated electrode in comparison to a standard electrode, according to embodiments disclosed herein. [Figure 31A] This specification describes a test performed to optimize the waveform for coating a plasma-treated electrode in comparison to a standard electrode, according to embodiments disclosed herein. [Figure 31B] This specification describes a test performed to optimize the waveform for coating a plasma-treated electrode in comparison to a standard electrode, according to embodiments disclosed herein. [Figure 32A] This specification describes a test performed to optimize the waveform for coating a plasma-treated electrode in comparison to a standard electrode, according to embodiments disclosed herein. [Figure 32B] This specification describes a test performed to optimize the waveform for coating a plasma-treated electrode in comparison to a standard electrode, according to embodiments disclosed herein. [Figure 33A] This specification describes a test performed to optimize the waveform for coating a plasma-treated electrode in comparison to a standard electrode, according to embodiments disclosed herein. [Figure 33B]This specification describes a test performed to optimize the waveform for coating a plasma-treated electrode in comparison to a standard electrode, according to embodiments disclosed herein. [Figure 34A] This specification describes a test performed to optimize the waveform for coating a plasma-treated electrode in comparison to a standard electrode, according to embodiments disclosed herein. [Figure 34B] This specification describes a test performed to optimize the waveform for coating a plasma-treated electrode in comparison to a standard electrode, according to embodiments disclosed herein. [Figure 35] This specification describes a test performed to optimize the waveform for coating a plasma-treated electrode in comparison to a standard electrode, according to embodiments disclosed herein. [Figure 36A] This specification describes a test performed to optimize the waveform for coating a plasma-treated electrode in comparison to a standard electrode, according to embodiments disclosed herein. [Figure 36B] This specification describes a test performed to optimize the waveform for coating a plasma-treated electrode in comparison to a standard electrode, according to embodiments disclosed herein. [Figure 37A] This specification describes a test performed to optimize the waveform for coating a plasma-treated electrode in comparison to a standard electrode, according to embodiments disclosed herein. [Figure 37B] This specification describes a test performed to optimize the waveform for coating a plasma-treated electrode in comparison to a standard electrode, according to embodiments disclosed herein. [Figure 38A] An example of an electrochemical cell consistent with the embodiments described herein is shown. [Figure 38B] An example of an electrochemical cell consistent with the embodiments described herein is shown. [Figure 38C] An example of an electrochemical cell consistent with the embodiments described herein is shown. [Figure 39A] An example of an electrochemical cell consistent with the embodiments described herein is shown. [Figure 39B] An example of an electrochemical cell consistent with the embodiments described herein is shown. [Figure 39C] An example of an electrochemical cell consistent with the embodiments described herein is shown. [Figure 39D] An example of an electrochemical cell consistent with the embodiments described herein is shown. [Figure 39E] An example of an electrochemical cell consistent with the embodiments described herein is shown. [Modes for carrying out the invention]

[0021] Specific embodiments of the present invention will be described below with reference to the drawings. The following detailed description is essentially a typical example and is not intended to limit the present invention or its applications and uses. Nor is it intended to be limited to the aforementioned technical field, background art, summary of the invention, or any explicit or implicit theory presented in the following detailed description.

[0022] Embodiments of this 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 pair (e.g., Ag / AgCl) that provides a stable interfacial potential. In certain embodiments, materials, compounds, etc., may be doped to generate the redox pair, but other methods for generating the redox pair are equally conceivable. An auxiliary electrode having a reduction-oxidation pair that determines a stable interfacial potential allows the auxiliary electrode to function as a dual-function electrode; that is, one or more auxiliary electrodes act simultaneously as a counter electrode and a reference electrode. Because the auxiliary electrode acts as a dual-function electrode, the space occupied by the auxiliary electrode in the electrochemical cell is reduced, making it possible to include additional configurations and numbers of working electrode zones in the electrochemical cell.

[0023] In embodiments, the use of one or more auxiliary electrodes also improves the reading time of electrochemical analyzers and devices during electrochemical analysis processes, such as ECL processes. Conventional non-reference ECL systems typically use slow voltage ramps that pass through a voltage providing maximum ECL to provide tolerance for potential fluctuations at the auxiliary electrodes. However, the use of auxiliary electrodes of the present invention, such as auxiliary electrodes with redox pairs, results in improved control over this potential, enabling the use of more efficient and faster waveforms, such as short voltage pulses or fast voltage ramps.

[0024] Figure 1A shows an example of an electrochemical cell 100 according to an embodiment of the present invention. As shown in Figure 1A, the electrochemical cell 100 defines an operating space 101 in which electrical energy is utilized to cause one or more chemical reactions. Within the operating 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 the ionic medium 103. The electrochemical cell 100 can operate by a reduction-oxidation (redox) reaction caused by introducing electrical energy through the auxiliary electrodes 102 and the working electrode zones 104. In some embodiments, the ionic medium 103 may include an electrolyte solution such as water, or another solvent in which ions are dissolved, such as a salt. In some embodiments, as will be described in more detail later, the surface of the ionic medium 103 or the working electrode 102 may contain luminescent species that generate and emit photons during the redox reaction. During the 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 induce a redox reaction in these electrodes.

[0025] As described herein, when 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 reductive-oxidative (redox) pair confined to the surface of the electrode, or (ii) a reductive-oxidative (redox) pair in solution. As described herein, a redox pair includes a pair of elements, chemicals, or compounds that interconvert by a redox reaction, for example, one element, chemical, or compound that is an electron donor and one element, chemical, or compound that is an electron acceptor. An auxiliary electrode having a reductive-oxidative pair that determines a stable interfacial potential can function as a dual-function electrode. That is, one or more auxiliary electrodes 102 may provide functions related to both the counter electrode and the reference electrode in a three-electrode electrochemical system by providing a high current (function of the counter electrode in a three-electrode system) while also providing the ability to determine and control the potential at the working electrode (function of the reference electrode in a three-electrode system). One or more auxiliary electrodes 102 may act as counter electrodes by providing a potential difference with one or more working electrode zones 104 during redox reactions occurring within the electrochemical cell 100 in which the one or more auxiliary electrodes 102 are located. Based on the chemical structure and composition of the one or more auxiliary electrodes 102, one or more auxiliary electrodes 102 may act as reference electrodes for determining the potential difference with one or more working electrode zones 104.

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

[0027] In embodiments, the amount of oxidizing agent in the chemical mixture of one or more auxiliary electrodes 102 is greater than or equal to the amount of oxidizing agent required for the entire redox reaction to occur within the electrochemical cell 100 during a biological, chemical, and / or biochemical assay and / or analysis, such as ECL generation. For example, a sufficient amount of chemical mixture in one or more auxiliary electrodes 102 remains after the redox reaction has occurred for the 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.

[0028] In some embodiments, the amount of oxidizing agent in the chemical mixture of one or more auxiliary electrodes 102 is at least in part based on the ratio of the exposed surface area (also referred to as the 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 surface area) of one or more auxiliary electrodes 102 refers to the two-dimensional (2D) cross-sectional area of ​​the one or more auxiliary electrodes 102 in contact with the ionic medium 103. That is, as shown in Figure 1B, the auxiliary electrodes 102 may be formed in a three-dimensional (3D) shape extending in the Z direction from the bottom surface of the electrochemical cell 100. The exposed surface area of ​​the auxiliary electrodes 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 on the auxiliary electrodes 102, for example, at the interface with the bottom surface 120. Figure 1B shows that the auxiliary electrode 102 is a regularly shaped cylinder, but 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 ​​one or more auxiliary electrode zones 104 that are in contact with the ionic medium 103, similar to the 2D cross-sectional area of ​​the auxiliary electrode 102 described, for example, in Figure 1B. In certain embodiments, the 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. Using these examples, the surface area is less than or equal to the true surface area.

[0029] In some embodiments, one or more auxiliary electrodes 102 may be formed of a chemical mixture containing redox pairs that provide an interfacial potential at or near the standard reduction potential of the redox pair. In some embodiments, one or more auxiliary electrodes 102 may contain silver (Ag) and silver chloride (AgCl), or other suitable metal / halide metal pairs. In some embodiments, one or more auxiliary electrodes 102 formed of an Ag / AgCl mixture can provide an interfacial potential that is at or near the standard reduction potential of Ag / AgCl, which is about 0.22 V. Other examples of chemical mixtures may contain metal oxides with multiple metal oxidation states, such as manganese oxide, or other metal / metal oxide pairs, such as silver / silver oxide, nickel / nickel oxide, zinc / zinc oxide, gold / gold oxide, copper / copper oxide, platinum / platinum oxide, etc. 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 lists examples of redox pair reduction potentials of chemical mixtures that may be contained in one or more auxiliary electrodes 102. Those skilled in the art will recognize that the example reduction potential is an approximation and may vary, for example, by + / - 5.0%, depending on the chemical composition, temperature, impurities in the chemical mixture, or other conditions. [Table 1]

[0030] In some embodiments, the chemical mixture of redox pairs in one or more auxiliary electrodes may be based on a molar ratio of redox pairs within a specified range. In some embodiments, the chemical mixture has a molar ratio of Ag to AgCl, for example, about 1 or more, within a specified range. In some embodiments, one or more auxiliary electrodes 102 may maintain a controlled interfacial potential until all of the one or more chemical parts involved in the redox reaction are oxidized or reduced.

[0031] In some embodiments, one or more auxiliary electrodes 102 have an electrode surface area of ​​approximately 1.56 × 10 -5 ~5.30×10 -4 C / mm 2It may include a redox pair that maintains an interfacial potential of -0.15V to -0.5V while passing the charge. In some embodiments, the one or more auxiliary electrodes 102 may include a redox pair that passes a current of about 0.5 mA to 4.0 mA through the redox reaction of the redox pair and generates ECL in the range of about 1.4V to 2.6V. In some embodiments, the one or more auxiliary electrodes 102 may include a redox pair that passes an average current of about 2.39 mA through the redox reaction and generates ECL in the range of about 1.4V to 2.6V.

[0032] In an embodiment, the one or more auxiliary electrodes 102 may have an amount of oxidizing agent in the redox pair that is greater than or equal to the amount of charge that needs to pass through the auxiliary electrode to complete the electrochemical analysis. In some embodiments, the one or more auxiliary electrodes 102 may include about 3.07×10 -7 ~3.97×10 -7 moles of oxidizing agent. In some embodiments, the one or more auxiliary electrodes 102 may include about 1.80×10 2 ~2.32×10 -7 moles (1.16×10 -7 ~1.5×10 -4 moles / in -4 ) of oxidizing agent per exposed surface area of 1 mm 2 . In some embodiments, the one or more auxiliary electrodes 102 may include at least about 3.7×10 2 moles (2.39×10 -9 moles / in -6 ) of oxidizing agent per total (or overall) exposed surface area of 1 mm of the one or more working electrode zones 104. In some embodiments, the one or more auxiliary electrodes may include at least about 5.7×10 2 moles (3.69×10 2 moles / in -9 ) of oxidizing agent per total (or overall) exposed surface area of 1 mm of the one or more working electrode zones 104.

[0033] ​​​​In some embodiments, one or more auxiliary electrodes 102 may contain a redox pair in which, when a voltage or potential is applied, the species reaction in the redox pair is the main redox reaction that occurs at one or more auxiliary electrodes 102. In some embodiments, the applied potential is less than the specified potential required to reduce water or to perform electrolysis of water. In some embodiments, less than 1 percent of the current is related to the reduction of water. In some embodiments, less than 1 unit current per unit area (exposed surface area) of one or more auxiliary electrodes 102 is related to the reduction of water.

[0034] In embodiments, one or more auxiliary electrodes 102 (and one or more working electrode zones 104) may be formed using any type of manufacturing process, such as printing, vapor deposition, lithography, etching, etc. In embodiments, the form of the metal / metal halide chemical mixture may depend on the manufacturing process. For example, if one or more auxiliary electrodes 102 (and one or more working electrode zones 104) are printed, the chemical mixture may be in the form of an ink or paste. In some embodiments, one or more additional substances may be added to one or more auxiliary electrodes 102 and / or one or more working electrode zones 104 using a doping process.

[0035] The working electrode zone 104 may be a location on the electrode where the reaction of interest may occur. The reaction of interest may be essentially chemical, biological, biochemical, or electrical (or any combination of two or more of these types of reactions). As described herein, the electrode (auxiliary electrode and / or working electrode) may be a continuous / seamless region where the reaction may occur, and the electrode “zone” may be a part (or all) of the electrode where a particular reaction of interest occurs. In certain embodiments, the working electrode zone 104 may comprise the entire electrode, and 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 individual working electrodes. 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 separating a part of a single working electrode. In this example, a single working electrode may be formed of one or more conductive materials, and the working electrode zone may be formed by electrically isolating the region ("zone") of the single working electrode using an insulating material, such as an insulator, thereby creating an electrically isolated working electrode zone. In any embodiment, the working electrode zone 104 may be formed of any type of conductive material, such as a metal, a metal alloy, a carbon compound, or a doped metal, and a combination of conductive and insulating materials.

[0036] In some embodiments, the working electrode zone 104 may be formed of a conductive material. For example, the working electrode zone 104 may include metals such as gold, silver, platinum, nickel, steel, iridium, copper, aluminum, and conductive alloys. 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 carbon-based materials such as carbon, carbon black, graphite 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, as will be described in more detail later, the working electrode zone 104 may be formed of carbon and silver layers manufactured using screen printing of carbon ink and silver ink. In some embodiments, the working electrode zone 104 may be formed of a semiconducting material (e.g., silicon, germanium) or a semiconducting film such as indium tin oxide (ITO) or antimony tin oxide (ATO).

[0037] In embodiments, as will be described in more detail later, one or more auxiliary electrodes 102 and one or more working electrode zones 104 may be formed in 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 electrochemical properties and analyses (e.g., ECL analysis) performed by apparatus and devices including the electrochemical cell. Figure 1C shows an example of an electrode design 150 for an electrochemical cell 100 including multiple working electrode zones. As shown in Figure 1C, the electrochemical cell 100 may include 10 working electrode zones 104 and a single auxiliary electrode 102. Various other examples of electrode designs will be described later with reference to Figures 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, and 8A-8D.

[0038] In some embodiments, the configuration and arrangement of the working electrode zones 104 within the electrochemical cell 100 may be determined according to the adjacency between the working electrode zones 104 and / or the adjacency between the working electrode zones 104 and one or more auxiliary electrodes 102. In some embodiments, adjacency may be defined as the relative number of adjacent working electrode zones 104 and / or one or more auxiliary electrodes 102. In some embodiments, adjacency may be defined as the relative distance between the working electrode zones 104 and / or one or more auxiliary electrodes 102. In some embodiments, adjacency may be defined as the relative distance from the working electrode zones 104 and / or one or more auxiliary electrodes 102 to other features of the electrochemical cell 100, such as the periphery of the electrochemical cell.

[0039] In embodiments relating to this specification, for example, one or more auxiliary electrodes 102 and one or more working electrode zones 104 of each electrochemical cell 100 may be formed such that the ratio of the total exposed surface area of ​​the one or more working electrode zones 104 to the exposed surface area of ​​the one or more auxiliary electrodes 102 is greater than 1, but other ratios (e.g., equal to 1, or less than or greater than 1) are also possible for the electrochemical cell 100. In some embodiments relating to this specification, for example, each of the one or more auxiliary electrodes 102 and / or one or more working electrode zones 104 may be formed in a circular shape having a surface area that substantially defines a circle, but other shapes (e.g., rectangles, squares, ellipses, quat-leaf shapes, or any other regular or irregular geometric shapes) are possible.

[0040] In embodiments relating to this specification, for example, one or more auxiliary electrodes 102 and / or one or more working electrode zones 104 may be formed in a wedge shape having a wedge-shaped surface region, also referred to herein as a trefoil shape. That is, one or more auxiliary electrodes 102 and / or one or more working electrode zones 104 may be formed to have two opposing boundary portions having different dimensions and two side boundary portions connecting the two opposing boundary portions. For example, the two opposing boundary portions may include a wide boundary portion and a narrow boundary portion, with the wide boundary portion having a longer length than the narrow boundary portion. In some embodiments, the wide boundary portion and / or the narrow boundary portion may be blunt, for example, rounded corners at the connection with the side boundary portion. In some embodiments, the wide boundary portion and / or the narrow boundary portion may be sharp, for example, angular corners at the connection with the side boundary portion. In embodiments, the wedge shape described herein may be a substantially trapezoidal shape having rounded or angular corners. In embodiments, the wedge shape described herein may be a substantially triangular shape having flat or rounded vertices and rounded or angular corners. In embodiments, the wedge shape may be used to maximize the usable area on the bottom surface 120 of the electrochemical cell. For example, if the working region 101 of the electrochemical cell is circular, one or more working electrode zones 104 having a wedge shape may be arranged such that the wider boundary is adjacent to the outer periphery of the working region 101 and the narrower boundary is adjacent to the center of the working region 101.

[0041] In some embodiments, the electrochemical cell 100 may be included in an apparatus or device for performing electrochemical analysis. In some embodiments, as described later, the electrochemical cell 100 may form part of a well for an assay device that performs electrochemical analysis, such as an ECL immunoassay. In some embodiments, the electrochemical cell 100 may form a cartridge used in an analytical device or apparatus, such as an ECL cartridge (e.g., those described in U.S. Patents 10,184,884 and 10,935,547), or a flow cell in a flow cytometer. Those skilled in the art will understand that the electrochemical cell 100 may be used in any type of apparatus or device in which a controlled redox reaction is performed.

[0042] Figures 2A–2C show several diagrams of a sample area ("well") 200 including an electrochemical cell (e.g., electrochemical cell 100) with an auxiliary electrode design for use in an assay device for biological, chemical, and / or biochemical analysis according to embodiments of this specification. Those skilled in the art will understand that Figures 2A–2C show examples of wells in an assay device, and that existing components shown in Figures 2A–2C may be removed and / or additional components may be added without departing from the scope of the embodiments described herein.

[0043] As shown in Figure 2A, a top view, the base plate 206 of the multiwell plate 208 (shown in Figure 2B) may include a plurality of wells 200. The base plate 206 may include a surface that forms the bottom of each well 200 and may include one or more auxiliary electrodes 102 and one or more working electrode zones 104 located on and / or inside the surface of the base plate 206 of the multiwell plate 208. As shown in Figure 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 the top surface of the top plate 210 to the base plate 206, and the base plate 206 forms the bottom surface 207 of each well 200. During operation, when a voltage is applied across one or more working electrode zones 104 and one or more auxiliary electrodes 102 located within the wells 200 holding the material under test, luminescence occurs. The applied voltage triggers periodic oxidation and reduction reactions, resulting in the generation and emission of photons (light). The emitted photons can then be measured for analysis of the material being tested.

[0044] Depending on whether the reaction occurring in the working electrode zone 104 accepts or supplies electrons, the reaction in the working electrode zone 104 is reduction or oxidation, respectively. In embodiments, the working electrode zone 104 may be derivatized or modified to immobilize assay reagents, such as binding reagents, onto the electrode. For example, the working electrode zone 104 may be modified to accommodate 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, the working electrode zone 104 may be modified to accommodate non-biological objects such as polymers, elastomers, gels, coatings, ECL tags, redox active species (e.g., tripropylamine, oxalates), inorganic materials, chemical functional groups, chelating agents, linkers, etc., but are not limited to these. The reagent can be immobilized on one or more working electrode zones 104 by various methods, including passive adsorption, specific binding, and / or by the formation of covalent bonds to functional groups present on the electrode surface.

[0045] For example, ECL species that can be induced to emit ECL may adhere to the working electrode zone 104 for analytical measurements to determine the presence of a substance of interest in the fluid in well 200. For example, species that can be induced to emit ECL (ECL-active species) are used as ECL labels. Examples of ECL labels include (i) organometallic compounds in which the metal is a noble metal resistant to corrosion and oxidation, including Ru-containing and Os-containing organometallic compounds such as tris-bipyridyl-ruthenium (RuBpy) moieties, and (ii) luminols and related compounds. Species involved in ECL labeling in the ECL process are referred to herein as ECL colactants. Commonly used colactants include, for example, tertiary amines such as triisopropylamine (TPA), oxalates, and persulfates in the case of ECL from RuBpy, and hydrogen peroxide in the case of ECL from luminol. The light generated by ECL labeling can be used as a reporter signal in diagnostic procedures. For example, the ECL label may be covalently bound to a binder, such as an antibody or nucleic acid probe, and the involvement of the binding reagent in the binding interaction can be monitored by measuring the ECL emitted from the ECL label. Alternatively, the ECL signal from an ECL-active compound may indicate the chemical environment.

[0046] In some embodiments, the working electrode zone 104 and / or the auxiliary electrode 102 (or other components of the well 200) may be treated (e.g., pre-treated) with materials and / or processes that improve the adhesion (e.g., adsorption) of materials used in the electrochemical process (e.g., reagents, ECL species, labels, etc.) to the surface of the working electrode zone 104 and / or the auxiliary electrode. In some embodiments, the working electrode zone 104 and / or the auxiliary electrode 102 (or other components of the well 200) may be treated with a process (e.g., plasma treatment) that causes the surface of the working electrode zone 104 and / or the auxiliary electrode 102 (or other components of the well 200) to exhibit hydrophilicity (also referred to herein as "highly bonded" 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 left untreated or treated using a process that causes the surface of the working electrode zone 104 and / or the auxiliary electrode 102 (or other components of the well 200) to exhibit hydrophobicity (also referred to herein as "standard" or "Std").

[0047] As shown in Figure 2C, a partial side cross-sectional view of the multiwell plate 208 in Figure 2B, a plurality of wells 200 may be included on the multiwell plate 208, three of which are shown in Figure 2C. Each well 200 may be formed by a top plate 210 including one or more side walls 212 that form the boundary of the electrochemical cell 100. The one or more side walls 212 extend from the bottom surface of the top plate 210 to the top surface of the top plate 210. The wells 200 may be adapted to hold one or more fluids 250, such as an ionic medium as described above. In certain embodiments, one or more wells 200 may be adapted to hold gases and / or solids as an alternative or addition to the one or more fluids 250. In embodiments, the top plate 210 may be fixed to the base plate 206 by adhesive 214 or other connecting material or device.

[0048] The multiwell plate 208 may contain any number of wells 200. For example, as shown in Figures 2A and 2B, the multiwell plate 208 may contain 96 wells 200. Those skilled in the art will understand that the multiwell plate 208 may contain any number of wells 200, such as 6, 24, 384, or 1536 wells formed in a regular or irregular pattern. In other embodiments, the multiwell plate 208 may be replaced with a single-well plate or any other apparatus suitable for performing biological, chemical, and / or biochemical analyses and / or assays. The wells 200 are shown in Figures 2A-2C in a circular configuration (consequently forming a cylinder), but other shapes including elliptical, square, and / or other regular or irregular polygons are equally conceivable. Furthermore, the shape and configuration of the multiwell plate 108 may take many forms and are not necessarily limited to the rectangular array shown in these drawings.

[0049] In some embodiments, as described above, the working electrode zone 104 and / or auxiliary electrode 102 used in the multiwell plate 108 may be non-porous (hydrophobic). In some embodiments, the working electrode zone 104 and / or auxiliary electrode 102 may be porous electrodes (e.g., carbon fiber or fibril mats, sintered metals, and metal films deposited on filter membranes, paper, or other porous substrates). When configured as porous electrodes, the working electrode zone 104 and / or auxiliary electrode 102 may be used to filter the solution through the electrodes to i) increase mass transport to the electrode surface (e.g., to increase the dynamics of molecules in the solution binding to molecules on the electrode surface), ii) capture particles on the electrode surface, and / or iii) remove liquid from the wells.

[0050] In the embodiments described above, each of the auxiliary electrodes 102 in the well 200 is formed with a chemical mixture that provides a predetermined potential during reduction of the chemical mixture so that a quantifiable amount of charge is generated through a reduction-oxidation reaction occurring in the well 200. The chemical mixture of the auxiliary electrode 102 includes an oxidizing agent that supports the reduction-oxidation reaction, which may be used in biological, chemical, and / or biochemical assays and / or analyses, such as ECL generation and analysis. In embodiments, the amount of oxidizing agent in the chemical mixture of the auxiliary electrode 102 is greater than or equal to the amount of oxidizing agent required for the amount of charge passing through the auxiliary electrode and / or the amount of charge required to bring about 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 ECL generation. In this regard, a sufficient amount of chemical mixture in the auxiliary electrode 102 remains after the redox reaction has occurred with respect to the initial biological, chemical, and / or biochemical assay and / or analysis, so as to allow one or more additional redox reactions to occur through subsequent biological, chemical, and / or biochemical assays and / or analyses. In other embodiments, the amount of oxidizing agent in the chemical mixture of the auxiliary electrode 102 is at least partially based 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.

[0051] In embodiments, one or more auxiliary electrodes 102 in well 200 may be formed from a chemical mixture containing a redox pair, as described above. In some embodiments, one or more auxiliary electrodes 102 in well 200 may be formed from a chemical mixture containing 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 having multiple metal oxidation states, such as manganese oxide, or other metal / metal oxide pairs, such as silver / silver oxide, nickel / nickel oxide, zinc / zinc oxide, gold / gold oxide, copper / copper oxide, platinum / platinum oxide, etc. In embodiments, the auxiliary electrodes 102 (and working electrode zone 104) may be formed using any type of manufacturing process, such as printing, vapor deposition, lithography, etching, etc. In embodiments, the form of the metal / metal halide chemical mixture may depend on the manufacturing process. For example, if the auxiliary electrodes are printed, the chemical mixture may be in the form of an ink or paste.

[0052] For specific applications such as ECL generation, various embodiments of the auxiliary electrode 102 may be adapted to prevent electrode polarization through 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 using an Ag / AgCl chemical mixture (e.g., ink, paste, etc.) having a specified Ag to AgCl ratio. In embodiments, the amount of oxidizing agent 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, for example, 34 percent, 10 percent AgCl, etc.

[0053] In some embodiments, one or more auxiliary electrodes 102 in the well 200 have a total working electrode area of ​​1 mm² in the well 200. 2 At least approximately 3.7 × 10 -9It may contain a molar amount of oxidizing agent. In some embodiments, one or more auxiliary electrodes 102 in the well 200 have a total working electrode area of ​​1 mm² in the well 200. 2 At least approximately 5.7 × 10 -9 It may contain a molar amount of oxidizing agent.

[0054] In various embodiments, one or more auxiliary electrodes 102 and working electrode zones 104 may be formed with different electrode designs (e.g., different sizes and / or shapes, different numbers of auxiliary electrodes 102 and working electrode zones 104, different arrangements and patterns within the wells, etc.) to improve electrochemical analysis (e.g., ECL analysis) performed by an assay device comprising one or more wells 200, examples thereof will be described later with reference to Figures 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, and 8A-8D. In embodiments relating to this specification, for example, one or more auxiliary electrodes 102 and one or more working electrode zones 104 in each well 200 may be formed 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., equal to 1, or less than or greater than 1) are also possible. In embodiments relating to this specification, for example, each of the auxiliary electrode 102 and / or working electrode zone 104 may be formed in a circular shape having a surface area substantially defining a circle, but other shapes (e.g., rectangle, square, ellipse, quatle, or any other regular or irregular geometric shape) are also possible. In embodiments relating to this specification, for example, the auxiliary electrode 102 and / or working electrode zone 104 may be formed in a wedge shape having a wedge-shaped surface area, where the first side or end of the wedge-shaped surface area adjacent to the side wall of the well 200 is larger than the second side or end of the wedge-shaped surface area adjacent to the center of the well 200. In other embodiments, the second side or end of the wedge-shaped surface area is larger than the first side or end of the wedge-shaped surface. For example, the auxiliary electrode 102 and working electrode zone 104 may be formed in a pattern that maximizes the space available to the auxiliary electrode 102 and working electrode zone 104.

[0055] In some embodiments, one or more auxiliary electrodes 102 and / or one or more working electrode zones 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 being longer than the narrow boundary. In some embodiments, the wide boundary and / or narrow boundary may be blunt, for example, rounded corners at the connection with the side boundaries. In some embodiments, the wide boundary and / or narrow boundary may be sharp, for example, angular corners at the connection with the side boundaries. In embodiments, the wedge shape may be used to maximize the available area on the bottom surface 120 of the electrochemical cell. For example, if the working region 101 of the electrochemical cell is circular, one or more wedge-shaped working electrode zones 104 may be arranged such that the wide boundary is adjacent to the outer periphery of the working region 101 and the narrow boundary is adjacent to the center of the working region 101.

[0056] In embodiments relating to this specification, the auxiliary electrodes 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. Different positional configurations or patterns may improve electrochemical analysis (e.g., ECL analysis) performed by an assay device comprising one or more wells 200, examples of which are described later with reference to Figures 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, and 8A-8D. The auxiliary electrodes 102 and working electrode zones 104 may be arranged within the well according to a desired geometric pattern. For example, the auxiliary electrodes 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 within the total number of working electrode zones 104. This makes it possible to position more working electrode zones adjacent to the auxiliary electrodes 102. For example, as shown in Figures 3A to 3F and described in detail later, the working electrode zones 104 can be formed in a circular or semi-circular shape that minimizes the number of adjacent working electrode zones 104.

[0057] In other examples, as shown in Figures 3A-3F, the auxiliary electrodes 102 and working electrode zones 104 of each well 200 may be formed in a pattern where the number of adjacent working electrode zones 104 is two or less. For example, the working electrode zones 104 may be formed in a circular or semicircular shape adjacent to the parameters of the well (e.g., the side wall 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 other examples, the auxiliary electrodes 102 and working electrode zones 104 of each well 200 may be formed in a pattern where at least one of the working electrode zones 104 is adjacent to three or more other working electrode zones 104 in the total number of working electrode zones 104. For example, as shown in Figures 5A to 5C and described in detail later, 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.

[0058] In embodiments relating to this specification, the auxiliary electrodes 102 and one or more working electrode zones 104 of each well 200 may be formed in a pattern configured to improve the mass transport of material to each of the working electrode zones 104. For example, during orbital or rotational oscillation or mixing, the mass transport of material to the central zone of the well 200 may be relatively slower compared to zones further from the center, and this pattern may be configured to improve mass transport by minimizing or eliminating the number of working electrode zones 104 located in the center of the well 200. That is, during operation, the well 200 undergoes orbital motion or "oscillation" to mix or combine the fluid contained within the well 200. Orbital motion can create vortices within the well 200, resulting in more liquid and faster liquid motion, for example, near the side walls 212 (periphery) of the well 200. For example, as shown in Figures 2A-2F, 3A-3F, 5A-5F, 6A-6F, and 7A-7D, which will be described in detail later, the working electrode zone 104 may be formed in a circular or semicircular shape and located near the periphery of the well 200. Furthermore, due to orbital oscillation, any differences in the concentration of the substance within the well may depend on the radial distance from the center of the well. In a concentric arrangement, since each working electrode zone 104 is at approximately the same distance from the center of the well, it may have a similar concentration of substance even if the concentration of substance is not uniform throughout the well.

[0059] In embodiments relating to this specification, the auxiliary electrodes 102 and one or more working electrode zones 104 of each well 200 may be formed with a pattern configured to reduce the meniscus effect caused by introducing liquid into one or more wells 200 of the multiwell plate 108. For example, as shown in Figure 2C, the fluid 250 in the well 200 may form a curved top surface or meniscus 152 within 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 starting oscillations). Due to the meniscus effect, photons (light) emitted in 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 the substance in the well 200, different liquid level heights may cause different optical effects (e.g., refraction, diffusion, scattering, etc.) to the emitted light, which depends on where the light passes through the liquid and exits. This pattern can mitigate the meniscus effect by positioning each working electrode zone 104 approximately equidistant from each side wall 212 of the well 200. Thus, photons emitted from the working electrode zones 104 travel through the liquid along similar optical paths. In other words, this pattern ensures that all working electrode zones 104 are equally affected by the meniscus effect, minimizing, for example, potentially different effects of the meniscus. Consequently, if the working electrode zones 104 are positioned at different locations relative to the liquid level in the well 200, the emitted light may undergo different optical distortions. For example, as shown in detail in Figures 3A-3F, 4A-4F, 6A-6F, 7A-7F, and 8A-8D, 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 from the working electrode zones 104 undergoes the same optical distortion and can be dealt with equally.

[0060] In embodiments relating to this specification, 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 differences in mass transport to the working electrode zones during mixing of liquids in one or more wells 200 of the multiwell plate 208 (e.g., vortices formed in cylindrical wells using an orbital shaker) (e.g., to provide uniform mass transport). For example, the pattern may be configured to reduce the vortex effect by minimizing or eliminating the number of working electrode zones 104 located in or near the center of each well 200. For example, as shown in detail in Figures 2A-2F, 3A-3F, 5A-5F, 6A-6F, 7A-7D, and 8A, the working electrode zones 104 may be formed in a circular or semicircular shape and located near the periphery of the well 200.

[0061] In embodiments relating to this specification, the auxiliary electrodes 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 the working electrode zones 104, each of which is positioned at approximately equal distances from the sidewall of the well 200, and the auxiliary electrodes 102 may be positioned within the periphery (the entire periphery or only a portion of the periphery) defined by the circular or semicircular pattern of the working electrode zones 104, but other shapes and / or patterns are also conceivable. 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 surrounding the entire or only portion of the periphery of the well 200.

[0062] In other embodiments, for example, the geometric pattern may include a pattern in which the working electrode zone 104 defines a star pattern, and the auxiliary electrode 102 may be positioned between two adjacent working electrode zones 104 defining two adjacent points of the star pattern. For example, the star pattern may be formed by auxiliary electrodes 102 that form the “points” of the star pattern and working electrode zones 104 that form the inner structure of the star pattern. For example, as shown in detail in Figures 5A to 5C, in a five-point star pattern, the auxiliary electrodes 102 may form the five “points” of the star pattern, and the working electrode zones 104 may form the inner “pentagonal” structure. In some embodiments, as shown in detail in Figures 5A to 5C, the star pattern may be defined as one or more concentric circles, and one or more working electrode zones 104 and / or one or more auxiliary electrodes may be arranged in a circular pattern around one or more concentric circles.

[0063] Figures 3A and 3B illustrate embodiments of an electrode design 301 for a well 200 having a circular working electrode zone 104 arranged in an open ring pattern. According to a typical and non-limiting embodiment shown in Figure 3A, the bottom 207 of the well 200 may include a single auxiliary electrode 102. In other embodiments, a plurality of (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 substantially circular shape. In other embodiments, the auxiliary electrode 102 may be formed to have other shapes (e.g., rectangular, square, elliptical, quat-leaf, or any other regular or irregular geometric shape).

[0064] In one embodiment, the well 200 may contain 10 working electrode zones 104. In another embodiment, the well 200 may contain fewer or more than 10 working electrode zones 104 (e.g., 1, 2, 3, 4, etc.). In one embodiment, the working electrode zones 104 may be formed to have a substantially circular shape. In another embodiment, the working electrode zones 104 may be formed to have other shapes (e.g., rectangles, squares, ellipses, quatleaf shapes, or any other regular or irregular geometric shapes).

[0065] The working electrode zones 104 may be arranged adjacent to each other in a semicircular or substantially C-shaped pattern with a distance "D1" from the periphery "P" of the well 200. 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 working electrode zone 104 may be positioned at an equal distance D1 from the periphery P of the well 200, and each working electrode zone 104 is 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 two adjacent working electrode zones 104. In some embodiments, the two working electrode zones 104A, 104B may be spaced far enough apart to form a gap "G". The gap "G" may provide a pitch distance between the two working electrode zones that is greater than the other pitch distance between other working electrode zones. In certain embodiments, the gap G allows electrical traces or contacts to be electrically coupled to the auxiliary electrode 102 without electrically interfering with the working electrode zone 104, thereby maintaining electrical isolation between the auxiliary electrode 102 and the working electrode zone 104. For example, the gap G may be formed at a sufficient distance to allow electrical traces to be formed between adjacent working electrode zones 104 while maintaining electrical isolation. Thus, the size of the gap G may be determined at least in part by the choice of manufacturing method when constructing the electrochemical cell. Accordingly, in embodiments, gaps "G" with larger pitch distances may be 10% or more, 30% or more, 50% or more, or 100% or more larger than the pitch distance D2 between other working electrode zones 104.

[0066] In certain embodiments, distance D1 may not be equal between one or more working electrode zones 104 and the periphery P of the well 200. In further embodiments, distance D2 may not be equal 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, but in other embodiments, distance D3 may differ with respect to one or more working electrode zones 104 when measured from the auxiliary electrode 102. In certain embodiments, as shown, distances D1, D2, D3, and G may be measured from the nearest relative point on the periphery of each feature (e.g., working electrode zone 104, auxiliary electrode 102, or periphery 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. Those skilled in the art will understand that distances can be measured from any relative point on a feature in order to generate repeatable patterns, such as geometric patterns.

[0067] These figures show a single auxiliary electrode 102, but multiple auxiliary electrodes 102 may be included, as shown in Figure 3C. Also, in these figures the auxiliary electrode 102 is shown positioned approximately (or positively) in the center of the well 200, but the auxiliary electrode 102 may be positioned at other locations in the well 200, as shown in Figure 3D. In addition, these figures show 10 working electrode zones 104, but more or fewer working electrode zones 104 may be included, as shown in Figures 3E and 3F.

[0068] The electrochemical cells shown in Figures 3A to 3F may include electrodes made of Ag, Ag / AgCl, carbon, carbon composites and / or other carbon-based materials and / or any other electrode material as described herein.

[0069] In embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may vary. For example, as shown in Table 2A, the sizes of each working electrode zone 104 may be equal, and the size of the auxiliary electrode 102 may vary, for example, by variations in diameter. Those skilled in the art will understand that the dimensions included in Table 2A are approximations and may vary by + / - 5.0% based on conditions such as manufacturing tolerances. [Table 2]

[0070] Table 2A above provides examples of values ​​relating to the shape dimensions of the wells. For example, as described above in paragraph

[0051] , an Ag / AgCl electrode consistent with the embodiments herein contains approximately 3.07 × 10⁻⁶ -7 Moles ~3.97 × 10 -7 It may contain a molar amount of oxidizing agent. In addition to the shape and dimensions shown above, both the working electrode and the auxiliary electrode should be approximately 10 microns (3.937 × 10⁻¹⁶). -4 The thickness may be in inches. Table 2B provides approximate molar values ​​and ranges for the oxidizing agent in the auxiliary electrode per unit area and volume of the auxiliary electrode. Table 2C provides approximate molar values ​​and ranges for the oxidizing agent in the auxiliary electrode per unit area and volume of the working electrode. The values ​​and ranges shown in Tables 2B and 2C are given in inches. Those skilled in the art will recognize that these values ​​can be converted to mm. [Table 3] [Table 4]

[0071] Figures 4A and 4B show non-limiting and typical embodiments of electrode design 401 for a well 200 having non-circular working electrode zones 104 arranged in an open ring pattern within the well, as similarly described above with reference to Figures 3A and 3B. The non-circular working electrode zones 104 shown in Figures 4A and 4B (and Figures 4C-4F) may be wedge-shaped or trefoil-shaped. 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 for the formation of larger working electrode zones 104 within the well 200, and / or more working electrode zones 104 within the well 200. By forming these non-circular shapes, the working electrode zones 104 can 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, since the working electrode zone 104 can be formed to be larger, the working electrode zone 104 can be manufactured more reliably, for example, printed more reliably.

[0072] As shown in Figure 4A, the well 200 may contain a single auxiliary electrode 102. In other embodiments, a plurality of auxiliary electrodes 102 (e.g., 2, 3, 4, 5, etc.) may be included. In embodiments, the auxiliary electrode 102 may be formed to have a substantially circular shape. In other embodiments, the auxiliary electrode 102 may be formed to have other shapes (e.g., rectangle, square, ellipse, quatle leaf, or any other regular or irregular geometric shape).

[0073] In one embodiment, the well 200 may include 10 working electrode zones 104. In other embodiments, there may be fewer or more than 10 working electrode zones 104 (e.g., 1, 2, 3, 4, etc.). Each working electrode zone 104 may be formed to be non-circular, for example, a wedge or trefoil shape with one or more rounded or rounded corners, but in other embodiments, the corners may not be rounded, forming a polygon such as a triangle.

[0074] The working electrode zones 104 may be arranged adjacent to each other in a semicircular or substantially C-shaped pattern with a distance "D1" from the periphery "P" of the well 200. In some embodiments, distance D1 may be the minimum distance between the boundary of a working electrode zone 104 and the periphery P. That is, each working electrode zone 104 may be located at an equal distance D1 from the periphery P of the well 200, and each working electrode zone 104 may be spaced equal to a distance "D2" from each other. In some embodiments, distance D2 may be the minimum distance between the boundaries of two adjacent working electrode zones 104. In some embodiments, two working electrode zones 104A, 104B may be spaced a sufficient distance from each other to form a gap "G". In certain embodiments, distance D1 may not be equal between one or more working electrode zones 104 and the periphery P of the well 200. In further embodiments, distance D2 may not be equal 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 when measured from the auxiliary electrode 102. In certain embodiments, as shown, distances D1, D2, D3, and G may be measured from the nearest neighbor point on the periphery of each feature (e.g., working electrode zone 104, auxiliary electrode 102, or periphery 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. Those skilled in the art will understand that distances may be measured from any relative point on the features in order to generate repeatable patterns, such as geometric patterns.

[0075] These figures show a single auxiliary electrode 102, but multiple auxiliary electrodes 102 may be included, as shown in Figures 4C and 4D. Also, in these figures the auxiliary electrode 102 is shown positioned approximately (or positively) in the center of the well 200, but the auxiliary electrode 102 may be positioned at other locations in the well 200, as shown in Figure 4D. In addition, these figures show 10 working electrode zones 104, but more or fewer working electrode zones 104 may be included, as shown in Figures 4E and 4F.

[0076] In certain embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may be equal. In other embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may vary. In one example, the size of the auxiliary electrode 102 may be constant, and the size of the working electrode zone 104 may vary, for example, due to variations in the radius of the auxiliary electrode 102. Table 3A includes examples of dimensions of the working electrode zone 104 and the auxiliary electrode 102 for embodiments including the wedge-shaped or trefoil-shaped working electrode zone 104 shown in Figures 4A-4F. Those skilled in the art will understand that the dimensions included in Table 3 are approximations and may vary by + / - 5.0% based on conditions such as manufacturing tolerances.

[0077] The electrochemical cells shown in Figures 4A to 4F may include electrodes made of Ag, Ag / AgCl, carbon, carbon composites and / or other carbon-based materials and / or any other electrode material as described herein. [Table 5]

[0078] Table 3A provides examples of values ​​for the dimensions of the trefoil electrode well shape. For example, as described above in paragraph

[0051] , an Ag / AgCl electrode consistent with the embodiments herein contains approximately 3.07 × 10⁻¹⁶ -7 ~3.97×10 -7 It may contain a molar amount of oxidizing agent. In addition to the shape and dimensions presented above, both the working electrode and the auxiliary electrode should be approximately 10 microns (3.937 × 10⁻¹⁶).-4 The thickness may be in inches. Table 3B provides approximate molar values ​​and ranges for the oxidizing agent in the auxiliary electrode per unit area and volume of the auxiliary electrode. Figure 3C provides approximate molar values ​​and ranges for the oxidizing agent in the auxiliary electrode per unit area and volume of the working electrode. The values ​​and ranges presented in Tables 3B and 3C are given in inches. Those skilled in the art will recognize that these values ​​can be converted to millimeters. [Table 6] [Table 7]

[0079] Figures 5A and 5B show non-limiting and typical embodiments of an electrode design 401 for a well 200 having working electrode zones 104 arranged in a star pattern (also referred to herein as a penta pattern), wherein the working electrode zones 104 are circular. As shown in Figure 5A, the well 200 may include five auxiliary electrodes 102, each of which may be formed substantially circularly (but other numbers of auxiliary electrodes, different shapes, etc., are also possible). In this example, the well 200 may also include 10 working electrode zones 104, each of which may be formed substantially circularly. The star pattern is generated by a plurality of working electrode zones 104 arranged in one of the inner and outer circles relative to each other, with each working electrode zone 104 located in the outer circle positioned at the angular midpoint with respect 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 at a distance "R1" from the center of the well 200. Each of the working electrode zones 104 in the outer circle may be spaced at a distance "R2" from the center of the well 200. In the star pattern, each auxiliary electrode 102 may be positioned at an equal distance "D4" from two of the working electrode zones 104 located in the outer circle.

[0080] In certain embodiments, as shown, distances R1, R2, and D4 may be measured from the nearest neighbor point on the periphery of each feature (e.g., working electrode zone 104, auxiliary electrode 102, or periphery P). Those skilled in the art will understand that distances may be measured from any relative point on the feature to generate repeatable geometric patterns.

[0081] These figures show 10 working electrode zones 104, but more or fewer working electrode zones 104 may be included, as shown in Figure 5C. Also, Figures 5A-5C show circular working electrode zones 104, but working electrode zones 104 may be formed to have other shapes (e.g., rectangular, square, elliptical, quatle-shaped, or any other regular or irregular geometric shape). Other embodiments may include electrode configurations of hybrid designs, such as a star pattern including wedge-shaped working electrode zones 104 and / or auxiliary electrodes 102.

[0082] The electrochemical cells shown in Figures 5A to 5F may include electrodes made of Ag, Ag / AgCl, carbon, carbon composites and / or other carbon-based materials and / or any other electrode material as described herein.

[0083] In certain embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may be equal. In other embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may vary. For example, as shown in Table 4A, the size of the working electrode zone 104 may be constant, and the size of the auxiliary electrode 102 may vary by a variation in diameter. Those skilled in the art will understand that the dimensions included in Table 4A are approximations and may vary by + / - 5.0% based on conditions such as manufacturing tolerances. [Table 8]

[0084] Table 4A above provides examples of shape and dimension values ​​for a 10-spot pentaelectrode well. For example, as described above in paragraph

[0051] , an Ag / AgCl electrode consistent with the embodiments herein contains approximately 3.07 × 10 -7 Moles ~3.97 × 10 -7 It may contain a molar amount of oxidizing agent. In addition to the shape and dimensions presented above, both the working electrode and the auxiliary electrode should be 10 microns (3.937 × 10). -4 The thickness may be in inches. Table 4B provides approximate molar values ​​and ranges for the oxidizing agent in the auxiliary electrode per unit area and volume of the auxiliary electrode. Table 4C provides approximate molar values ​​and ranges for the oxidizing agent in the auxiliary electrode per unit area and volume of the working electrode. The values ​​and ranges presented in Tables 4B and 4C are given in inches. Those skilled in the art will recognize that these values ​​can be converted to millimeters. [Table 9] [Table 10]

[0085] Figures 6A and 6B illustrate typical and non-limiting embodiments of electrode design 601 for a well 200 having non-circular (e.g., trefoil or wedge-shaped) working electrode zones 104 arranged in a closed ring pattern. As shown in Figure 6A, the well 200 may include a single auxiliary electrode 102. In other embodiments, a plurality of (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 substantially circular shape. In other embodiments, the auxiliary electrode 102 may be formed to have other shapes (e.g., rectangular, square, elliptical, quadruple-shaped, or any other regular or irregular geometric shape).

[0086] In embodiments, the well 200 may also include 10, or more or fewer, working electrode zones 104. For example, Figures 6A and 6B show an embodiment having 12 working electrode zones 104, Figures 6C and 6D show an embodiment having 11 working electrode zones 104, Figure 6E shows an embodiment having 14 working electrode zones 104, and Figure 6F shows an embodiment having 7 working electrode zones 104. The working electrode zones 104 may be formed to be non-circular, for example, having one or more rounded or R-shaped wedge or triangles, also referred to as trefoil. 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 an adjacent pattern with a distance "D1" from the periphery "P" of the well 200. 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 have an equal distance "D2" between them. In some embodiments, distance D2 may be the minimum distance between the boundaries of two adjacent working electrode zones 104. In certain embodiments, distance D1 may not be equal between one or more working electrode zones 104 and the periphery 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, although in other embodiments, distance D3 may vary for one or more working electrode zones 104 when measured from the auxiliary electrode 102. In some embodiments, distance D3 may be the minimum distance between the boundary of a working electrode zone 104 and the boundary of an auxiliary electrode. In certain embodiments, as shown, distances D1, D2, and D3 may be measured from the nearest neighbor point on the periphery of each feature (e.g., the working electrode zone 104, the auxiliary electrode 102, or the periphery P). Those skilled in the art will understand that distances may be measured from any relative point on the feature in order to generate repeatable patterns, such as geometric patterns.

[0087] These figures show a single auxiliary electrode 102, but multiple auxiliary electrodes 102 may be included, as shown in Figure 6C. Also, in these figures the auxiliary electrode 102 is shown positioned approximately (or positively) in the center of the well 200, but the auxiliary electrode 102 may be positioned at other locations in the well 200, as shown in Figure 6D. In addition, these figures show 10 working electrode zones 104, but more or fewer working electrode zones 104 may be included, as shown in Figures 6E and 6F.

[0088] The electrochemical cells shown in Figures 6A to 6F may include electrodes made of Ag, Ag / AgCl, carbon, carbon composites and / or other carbon-based materials and / or any other electrode material as described herein.

[0089] In certain embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may be equal. In other embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may vary. For example, the size of the auxiliary electrode 102 may be constant, while the size of the working electrode zone 104 may vary, for example, due to variations in the radius of the auxiliary electrode 102. Table 5A includes examples of dimensions for the working electrode zone 104 and the auxiliary electrode 102 relating to embodiments shown in Figures 6A to 6F. Those skilled in the art will understand that the dimensions included in Table 5A are approximations and may vary by + / - 5.0% based on conditions such as manufacturing tolerances. [Table 11]

[0090] Table 5A provides examples of shape and dimension values ​​for closed trefoil electrode wells. For example, as described above in paragraph

[0051] , an Ag / AgCl electrode consistent with the embodiments herein contains approximately 3.07 × 10⁻⁶ -7 Moles ~3.97 × 10 -7 It may contain a molar amount of oxidizing agent. In addition to the shape and dimensions presented above, both the working electrode and the auxiliary electrode should be approximately 10 microns (3.937 × 10⁻¹⁶). -4The thickness may be in inches. Table 5B provides approximate molar values ​​and ranges for the oxidizing agent in the auxiliary electrode per unit area and volume of the auxiliary electrode. Figure 5C provides approximate molar values ​​and ranges for the oxidizing agent in the auxiliary electrode per unit area and volume of the working electrode. The values ​​and ranges presented in Tables 5B and 5C are given in inches. Those skilled in the art will recognize that these values ​​can be converted to millimeters. [Table 12] [Table 13]

[0091] In some embodiments, it may be advantageous to eliminate sharp corners in a trefoil electrode design. For example, Figure 6A shows a trefoil design with sharp corners, while Figure 6B shows a trefoil design with rounded corners. Rounded corners may reduce the area of ​​the working electrode zone 104 by, for example, 1-5%, but may offer greater advantages. For example, sharp corners may hinder the uniform distribution of the solution. Sharp corners may also provide small features that make it difficult to obtain accurate images. Therefore, even if the working electrode zone 104 is smaller, reducing sharp corners may still be advantageous.

[0092] Figures 7A and 7B show typical and non-limiting embodiments of electrode design 701 for a well 200 having a closed ring design with circular electrodes. As shown in Figure 7A, the well 200 may include a single auxiliary electrode 102. In other embodiments, a plurality of auxiliary electrodes 102 (e.g., 2, 3, 4, 5, etc.) may be included. In embodiments, the auxiliary electrode 102 may be formed to have a substantially circular shape. In other embodiments, the auxiliary electrode 102 may be formed to have other shapes (e.g., rectangle, square, ellipse, quatle leaf, or any other regular or irregular geometric shape).

[0093] In one embodiment, the well 200 may include 10 working electrode zones 104. In another embodiment, there may be fewer or more than 10 working electrode zones 104 (e.g., 1, 2, 3, 4, etc.). In one embodiment, the working electrode zones 104 may be formed to have a substantially circular shape. In another embodiment, the working electrode zones 104 may be formed to have other shapes (e.g., rectangles, squares, ellipses, quat-leaf shapes, or any other regular or irregular geometric shapes).

[0094] In a closed ring pattern, the working electrode zones 104 can be arranged in a circular pattern 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, distance D1 may be the minimum distance between the boundary of a working electrode zone 104 and the periphery P. That is, each working electrode zone 104 may be positioned at an equal distance D1 from the periphery P of the well 200, and each working electrode zone 104 may have an equal distance "D2" (also referred to as the working electrode (WE-WE) pitch) between them. In some embodiments, distance D2 may be the minimum distance between the boundaries of two adjacent working electrode zones 104. In certain embodiments, distance D1 may not be equal between one or more working electrode zones 104 and the periphery P of the well 200. In further embodiments, distance D2 may not be equal between two or more working electrode zones 104.

[0095] 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 when 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 periphery of each feature (e.g., the working electrode zone 104, the auxiliary electrode 102, or the periphery P). Those skilled in the art will understand that distances may be measured from any relative point on the features in order to generate repeatable patterns, such as geometric patterns.

[0096] In further examples, the distance from the working electrode zone to the auxiliary electrode (WE-Auxiliary distance) may be measured from the center of the working electrode zone 104 to the center of the auxiliary electrode 102. Examples of WE-Auxiliary distances include 0.088 inches for a 10-spot open concentric design, 0.083 inches for a 10-lobe open concentric design with sharp corners, 0.087 inches for a 10-lobe open concentric design with rounded corners, 0.080 inches for a 10-lobe closed concentric design with sharp corners, 0.082 inches for a 10-lobe closed concentric design with rounded corners, and 0.086 inches for a 10-spot closed concentric design. In a penta design, the WE-Auxiliary distance may be 0.062 inches between the inner working electrode zone 104 and the auxiliary electrode 102, and 0.064 inches between the outer working electrode zone 104 and the auxiliary electrode 102. The value of the WE-Auxiliary distance may vary by 5%, 10%, 15%, and 25%, or more, without departing from the scope of this disclosure. In embodiments, the value of the WE-Auxiliary distance may vary according to the size and configuration of the working electrode zone 104 and the auxiliary zone 102.

[0097] These figures show a single auxiliary electrode 102, but multiple auxiliary electrodes 102 may be included, as shown in Figure 7C. Also, in these figures the auxiliary electrode 102 is shown positioned approximately (or positively) in the center of the well 200, but the auxiliary electrode 102 may be positioned at other locations in the well 200, as shown in Figure 7D. In addition, these figures show 10 working electrode zones 104, but more or fewer working electrode zones 104 may be included, as shown in Figures 7E and 7F.

[0098] The electrochemical cells shown in Figures 7A to 7F may include electrodes made of Ag, Ag / AgCl, carbon, carbon composites and / or other carbon-based materials and / or any other electrode material as described herein.

[0099] In certain embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may be equal. In other embodiments, the sizes of the auxiliary electrode 102 and / or the working electrode zone 104 may vary. For example, as shown in Table 6A, the size of the working electrode zone 104 may be constant, and the size of the auxiliary electrode 102 may vary due to variations in diameter, etc. Those skilled in the art will understand that the dimensions included in Table 6A are approximations and may vary by + / - 5.0% based on conditions such as manufacturing tolerances. [Table 14]

[0100] Table 6A above provides examples of shape and dimension values ​​for closed spot electrode wells. For example, as described above in paragraph

[0051] , an Ag / AgCl electrode consistent with the embodiments herein contains approximately 3.07 × 10⁻⁶ -7 Moles ~3.97 × 10 -7 It may contain a molar amount of oxidizing agent. In addition to the shape and dimensions presented above, both the working electrode and the auxiliary electrode should be approximately 10 microns (3.937 × 10⁻¹⁶). -4The thickness may be in inches. Table 6B provides approximate molar values ​​and ranges for the oxidizing agent in the auxiliary electrode per unit area and volume of the auxiliary electrode. Figure 6C provides approximate molar values ​​and ranges for the oxidizing agent in the auxiliary electrode per unit area and volume of the working electrode. The values ​​and ranges presented in Tables 6B and 6C are given in inches. Those skilled in the art will recognize that these values ​​can be converted to millimeters. [Table 15] [Table 16]

[0101] Tables 2A-2C provide examples of spot size dimensions for the working electrode zone 104 and auxiliary electrode 102. The selection of spot sizes for the working electrode zone 104 and auxiliary electrode 102 can be important for optimizing the results of the ECL process. For example, as described later in paragraphs

[0282] -

[0295] , maintaining an appropriate ratio between the working electrode zone 104 area and the auxiliary electrode 102 area can be important to ensure that the auxiliary electrode 102 has sufficient reducing ability to complete ECL generation with respect to the selected voltage waveform without saturation. In other examples, a larger working electrode zone 104 can provide greater coupling ability and increase the ECL signal. A larger working electrode zone 104 can also be easier to manufacture, as it avoids small features and any manufacturing tolerances are small relative to the overall size. In embodiments, the working electrode zone 104 area can be maximized to increase the ECL signal, coupling ability, and facilitate manufacturing, while being limited by the need to maintain a sufficient insulating dielectric barrier between the working electrode zone 104 and the auxiliary electrode 102.

[0102] Figures 8A–8D show typical and non-limiting embodiments of electrode design 801 for a well 200 having a closed ring design with a circular working electrode zone and complex-shaped auxiliary electrodes 102. As shown in Figure 8A, the well 200 may contain two complex-shaped auxiliary electrodes 102. In other embodiments, as shown in Figure 8D, fewer than (or more than) two auxiliary electrodes 102 may be contained within the well 200. In embodiments, the auxiliary electrodes 102 may be formed to have complex shapes such as “gear”, “cogwheel”, “annular”, “washer”, “oval”, or “wedge”, as described above. For example, as shown in Figure 8B, the inside of the auxiliary electrode 102 may be formed in a circular shape (e.g., “gear”, or “cogwheel”, shape) with an outer semicircular space 802 corresponding to the working electrode zone 104. Similarly, as shown in Figure 8C, for example, the outside of the auxiliary electrode 102 may be formed in a hollow ring shape (e.g., “washer”, shape) with an inner semicircular space 804 corresponding to the working electrode zone 104.

[0103] In one embodiment, the well 200 may contain 10 working electrode zones 104. In another embodiment, fewer or more than 10 (e.g., 1, 2, 3, 4, etc.) working electrode zones 104 may be contained within the well 200. In one embodiment, the working electrode zones 104 may be formed to have a substantially circular shape. In another embodiment, the working electrode zones 104 may be formed to have other shapes (e.g., rectangles, squares, ellipses, quatle-leaf shapes, or any other regular or irregular geometric shapes).

[0104] In some embodiments, the working electrode zone 104 may be arranged in a circular shape between two auxiliary electrodes 102. In this configuration, the outer semicircular space 802 and the inner semicircular space 704 allow the two auxiliary electrodes 102 to partially enclose the working electrode zone. The outside of the two auxiliary electrodes 102 may be spaced at a distance "D1" from the working electrode zone 104, where D1 is measured from the midpoint of the inner semicircular space to the boundary of the working electrode zone 104. In some embodiments, 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, distance D1 may not be equal between one or more working electrode zones 104 and the outside of the two auxiliary electrodes 102. Each of the working electrode zones 104 may be spaced at equal distances "D2" from one another. In some embodiments, distance D2 may be the minimum distance between two adjacent working electrode zones 104. In further embodiments, the distance D2 may not be equal between two or more working electrode zones 104. The inside of the two auxiliary electrodes 102 may have a distance of "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 electrodes. In certain embodiments, the distance D1 may not be equal between one or more working electrode zones 104 and two auxiliary electrodes 102.

[0105] In certain embodiments, as shown, distances D1, D2, and D3 can be measured from the nearest relative point around each feature (e.g., the working electrode zone 104 or the auxiliary electrode 102). Those skilled in the art will understand that distances can be measured from any relative point on the features to generate repeatable geometric patterns.

[0106] The electrochemical cells shown in Figures 8A-8D may include auxiliary electrodes made of Ag / AgCl, carbon, and / or any other auxiliary electrode material as described herein.

[0107] As described above, the electrochemical cell 100 can be used in devices and apparatus for performing electrochemical analysis. For example, the multiwell plate 208 including the wells 200 described above can be used in any type of apparatus that assists in performing biological, chemical, and / or biochemical assays and / or analyses, such as an apparatus for performing ECL analysis. Figure 9 shows a typical assay apparatus 900 in which the multiwell plate 208 including the wells 200 can be used for electrochemical analysis and procedures, according to an embodiment of this specification. Those skilled in the art will understand that Figure 9 shows an example of an assay apparatus and that existing components shown in Figure 9 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.

[0108] As shown in Figure 9, the multiwell plate 208 may be electrically coupled to a plate electrical connector 902. The plate electrical connector 902 may be coupled to a voltage / current source 904. The voltage / current source 904 may be configured to selectively supply a controlled voltage and / or current to the wells 200 of the multiwell plate 208 (e.g., an electrochemical cell 100) via the plate electrical connector 902. For example, the plate electrical connector 1502 may be configured to fit and / or mat with the electrical contacts of the multiwell plate 208, which are coupled to one or more auxiliary electrodes 102 and / or one or more working electrode zones 104, in order to allow voltage and / or current to be supplied to the wells 200 of the multiwell plate 208.

[0109] In some embodiments, the plate electrical connector 902 may be configured to allow one or more wells 200 (including one or more working electrode zones and auxiliary electrodes) to be activated simultaneously, or two or more working electrode zones and / or auxiliary electrodes to be activated individually. In certain embodiments, a device, such as one used to perform scientific analysis, may be electrically coupled to one or more apparatuses (e.g., a plate, a flow cell, 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 make one or more wells 200 selectively addressable, for example, by selectively applying a voltage and / or current to one of the wells 200 and reading a signal from a detector 910. For example, as shown in Figure 9B, a multiwell plate 208 may include 96 wells 200 arranged in rows labeled "A" to "H" and columns labeled "1" to "12". In some embodiments, the plate electrical connector 902 may include a single electrical strip connecting all the wells 200 in one of rows A-H or one of columns 1-12. In this way, all the wells 200 in one of rows A-H or one of columns 1-12 are activated simultaneously and supplied with voltage and / or current, for example, by a voltage / current source 904. Similarly, all the wells 200 in one of rows A-H or one of columns 1-12 are read simultaneously and their signals can be read, for example, by a detector 910.

[0110] In some embodiments, the plate electrical connector 902 may include a matrix of vertical electrical lines 952 and horizontal electrical lines 950, which are individual electrical connections connecting 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 for selectively establishing electrical connections to the vertical electrical lines 952 and horizontal electrical lines 950. Thus, one or more wells 200 in one of rows A-H or one of columns 1-12 may be individually activated and supplied with voltage and / or current by the voltage / current source 904, for example, as shown in Figure 9B. Similarly, one or more wells 200 in one of rows A-H or one of columns 1-12 may be individually read out simultaneously by signals read, for example, by a detector 910. In this example, the one or more wells 200 to be individually activated are selected based on the index of one or more wells 200, for example, well A1, well A2, etc.

[0111] 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 so that one or more of the auxiliary electrodes 102 and / or working electrode zones 104 of each well 200 are selectively addressable, for example, so that a voltage and / or current is selectively applied to one of the individual auxiliary electrodes 102 and / or working electrode zones 104, and a signal is read from the detector 910. Similar to the wells 200 described above, for each well 200, one or more working electrode zones 104 may include separate 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, one or more auxiliary electrodes 102 may include separate 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.

[0112] 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 enable specific wells 200, auxiliary electrodes 102, and / or working electrode zones 104 to be selectively electrically coupled to the voltage / current source 904, and to enable the selective application of voltage and / or current. 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 enable signals to be selectively read from the detector 910 by specific wells 200, auxiliary electrodes 102, and / or working electrode zones 104.

[0113] In certain embodiments, one or more computer systems 906 may be coupled to the voltage / current source 904 to control the supplied voltage and / or current. In other embodiments, the voltage / current source 904 may supply potential and / or current manually, for example, without the assistance of a computer system. The computer system 906 may be configured to control the voltage and / or current supplied to the well 200. Similarly, in embodiments, the computer system 906 may be used to store, analyze, display, transmit, etc., data measured during electrochemical processes and procedures.

[0114] The multiwell plate 208 may be housed within the housing 908. The housing 908 may be configured to support and house the components of the assay apparatus 900. In some embodiments, the housing 908 may be configured to maintain experimental conditions (e.g., airtightness, light shielding, etc.) to accommodate the operation of the assay apparatus 900.

[0115] In some embodiments, the assay apparatus 900 may include one or more detectors 910 for measuring, capturing, storing, and analyzing data related to the electrochemical processes and procedures of the assay apparatus 900. For example, the detectors 910 may include photodetectors 912 (e.g., cameras, photodiodes, etc.), voltmeters, ammeters, potentiometers, temperature sensors, etc. In some embodiments, one or more of the detectors 910 may be incorporated into other components of the assay apparatus 900, such as plate electrical connectors 902, voltage / current sources 904, computer systems 906, housings 908, etc. In some embodiments, one or more of the detectors 910 may be incorporated into the multiwell plate 208. For example, one or more heaters, temperature controllers, and / or temperature sensors may be incorporated into the electrode design of each well 200, as described later.

[0116] In embodiments, 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. One or more photodetectors 912 may be a single detector for detecting sequential emission, or may include multiple detectors and / or sensors for detecting and spatially resolving simultaneous emission in single or multiple wavelengths. The emitted and detected light may be visible light, or it may be emitted as invisible radiation such as infrared or ultraviolet light. One or more photodetectors 912 may be fixed or movable. Emission or other radiation may be manipulated or modified during passage to one or more photodetectors 912 using, for example, (single, multiple, fixed, or movable) lenses, mirrors, and optical fiber optical conductors or optical tubes 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 be used themselves to allow light guidance or transmission.

[0117] As described above, in embodiments, multiple detectors may be used to detect and resolve the simultaneous emission of various optical signals. In addition to the examples already described herein, the detectors may include one or more beam splitters, mirrored lenses (e.g., 50% silver mirrors), and / or other devices for transmitting optical signals to two or more different detectors (e.g., multiple cameras). Embodiments of these multiple detectors may include, for example, configuring one detector (e.g., a camera) to be in a high-gain configuration to capture and quantify low-power signals, and the other to be in a low-gain configuration to capture and quantify high-power signals. In embodiments, the high-power signals may be 2, 5, 10, 100, 1000, or more times greater than the low-power signals. Other examples are similarly conceivable.

[0118] Referring to the beam splitter example described above, a beam splitter with a specific ratio (e.g., a 90:10 ratio with two sensors, although other ratios and / or numbers of sensors are equally possible) may be used to detect and resolve emitted light. In this 90:10 example, 90% of the incident light may be directed to a first sensor using a high-gain configuration for low light levels, and the remaining 10% may be directed to a second sensor using a low-gain configuration for high light levels. In embodiments, the 10% loss of light to the first sensor may be compensated (at least partially) based on various factors, such as the selected sensor / sensor technology, binning technology, etc., to reduce noise.

[0119] In some embodiments, each sensor may be of the same type (e.g., CCD / CMOS), while in other embodiments, they may be of different types (e.g., the first sensor may be a high-sensitivity, high-performance CCD / CMOS sensor, and the second sensor may be a low-cost CCD / CMOS sensor). In other examples, (e.g., for larger sensors) the light may be split such that 90% of the signal is imaged on one half of the sensor and the remaining 10% is imaged on the other half (e.g., 90 / 10 as described above, but other ratios are equally possible). By optimizing the optics of this technique, the dynamic range can be further extended, for example, by applying a 99:1 ratio with multiple sensors, where one sensor (e.g., a camera) is highly sensitive within a first dynamic range, and the lowest sensitivity of the second sensor starts higher than that of the first sensor. When properly optimized, the amount of light each receives is maximized, thus improving overall sensitivity. In these examples, techniques may be used to minimize and / or eliminate crosstalk, for example, by sequentially energizing the working electrode zones. The advantages provided by these examples include the simultaneous detection of low and high light levels, thereby eliminating the need for dual excitation (e.g., multi-pulse method), which can reduce and / or improve ECL readout times.

[0120] In some embodiments, one or more photodetectors 912 may include one or more cameras (e.g., charge-coupled devices (CCDs, complementary metal-oxide-semiconductor (CMOS) image sensors, etc.) that capture images of the wells 200 to capture photons emitted during the operation of the assay apparatus 900. In some embodiments, 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 wells 200, multiple cameras that capture images of all wells 200, or multiple cameras that capture images of a subset of wells 200. In some embodiments, each well 200 of the multiwell plate 200 may include a camera that captures an image of the well 200. In some embodiments, each well 200 of the multiwell plate 200 may include multiple cameras that capture images of a single working electrode zone 104 or a subset of working electrode zones 104 within each well 200. In any embodiment, the computer system 906 may include hardware, software, and combinations thereof that include logic for analyzing images captured by one or more photodetectors 912 and extracting luminance data for ECL analysis. In some embodiments, the computer system 906 may include hardware, software, and combinations thereof that include logic for segmenting and highlighting the image to focus on a portion of the image, such as one or more wells 200 or one or more working electrode zones 104, if the image includes data such as multiple wells 200 or multiple working electrode zones 104. Thus, the assay apparatus 900 can offer flexibility by allowing the photodetector 912 to capture all light from multiple working electrode zones 104, and allowing the computer system 906 to decompose luminescence data for each working electrode zone 104 using imaging processing.Thus, the assay apparatus 900 may operate in various modes, for example, singleplex mode (e.g., one working electrode zone), 10plex mode (e.g., all working electrode zones 104 relating to 10 working electrode zone wells 200), or generally multiplex mode (for example, simultaneously, five working electrode zones 104 relating to multiple 10 working electrode zone wells, or a subset of all working electrode zones contained within a single well 200 or multiple wells 200).

[0121] In some embodiments, 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 a multiwell plate 200 may include a photodiode for detecting and measuring photons emitted within the well 200. In some embodiments, each well 200 of a multiwell plate 200 may include multiple photodiodes for detecting and measuring photons emitted from a single working electrode zone 104 or a subset of working electrode zones 104 within each well 200. Thus, the assay apparatus 900 may operate in various modes. For example, in sequential or "time-resolved" mode, the assay apparatus 900 may apply voltage and / or current to five working electrode zones 104 individually. The photodiodes may then sequentially detect / measure the light entering 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 can be detected and measured by the corresponding photodiode. This is sequentially repeated for each of the five working electrode zones 104. Similarly, in this example, the sequential operation mode may be performed with respect to working electrode zones 104 located in different wells 200, with respect to working electrode zones 104 located in subsets or "sectors" of multiple wells 200, or combinations thereof. Similarly, in some embodiments, the assay apparatus 900 may operate in a multiplex mode in which one or more working electrode zones 104 are simultaneously activated by the application of voltage and / or current, and the emitted photons are detected and measured by multiple photodiodes and multiplexed. The multiplex operation mode may be performed with respect to working electrode zones 104 in the same well 200, with respect to working electrode zones 104 located in different wells 200, with respect to working electrode zones 104 located in subsets or "sectors" of wells 200 in a multiwell plate 208, or combinations thereof.

[0122] In the embodiments described above, the working electrode zone 104 undergoes a natural decay of photon intensity after the voltage supplied to the working electrode zone 104 is removed. That is, when a voltage is applied to the working electrode zone 104, a redox reaction occurs, and photons are emitted at an intensity determined by the applied voltage and the substance undergoing the redox reaction. When the voltage is removed, the substance undergoing the redox reaction continues to emit photons at a decaying intensity for a period of time based on the chemical properties of the substance. Thus, as the working electrode zones 104 are sequentially activated, the assay apparatus 900 (e.g., computer system 906) may be configured to implement a delay when sequentially activating the working electrode zones 104. The assay apparatus 900 (e.g., computer system 906) may determine and implement a delay when sequentially activating the working electrode zones 104 to prevent interference between photons emitted from previously ignited working electrode zones 104 and photons emitted from the currently activated working electrode zone 104. For example, Figure 10A shows the decay of ECL between various voltage pulses, and Figure 10B shows the ECL decay time using a 50 ms pulse. In the example in Figure 10B, intensity data was determined by taking multiple images during and after a 50 ms long 1800 mV voltage pulse. To improve the temporal resolution, an image frame was taken (or a photon was detected) every 17 ms. The 50 ms voltage pulse was imaged in 3 frames (e.g., images 1-3, 17 ms × 3 = 51 ms), as shown in Figure 10B. Any photons emitted after image 3, e.g., an ECL signal, are due to the decay of the intensity of the photons (e.g., ECL) after the working electrode zone 104 is turned off. In Figure 10B, image 4 captures an additional ECL signal after the working electrode zone 104 is turned off, indicating that there may be some small luminescence chemical phenomenon continuing after the driving force (e.g., applied voltage potential) for this chemical phenomenon is deactivated. In other words, since the working electrode zone 104 switches to 0mV 1ms after the end of the 1800mV voltage pulse, the polarization effect is unlikely to affect the delay.In an embodiment, the assay apparatus 900 (e.g., computer system 906) may be configured to utilize such data for different voltage pulses and to delay the sequential activation of the working electrode zone 104. By implementing this delay, the assay apparatus 900 can minimize crosstalk between the working electrode zone 104 and / or the wells 200, have high throughput when performing ECL operation, and more.

[0123] In any embodiment, the use of one or more auxiliary electrodes 102 improves the operation of the assay apparatus 900. In some embodiments, the use of one or more auxiliary electrodes 102 improves the reading time of the detector 910. For example, using Ag / AgCl for one or more auxiliary electrodes 102 improves the ECL reading time for several reasons. For example, the use of an electrode (e.g., auxiliary electrode 102) with a redox pair (Ag / AgCl in this particular embodiment) provides a stable interfacial potential and may allow the electrochemical process to use a voltage pulse rather than a voltage ramp. The use of a voltage pulse improves the reading time because the entire pulse waveform can be applied at a voltage potential that produces ECL throughout the entire duration of the waveform. Tables 7 and 8 below contain the improved reading times (in seconds) for various configurations of the assay apparatus 900 using one or more auxiliary electrodes 102. Examples in these tables are the total reading times for all wells in a 96-well plate (each well containing a single working electrode (or single working electrode zone) or 10 working electrodes (or 10 working electrode zones)). For these reading times, analysis was performed on all working electrodes (or working electrode zones) (1 or 10 depending on the embodiment) in all 96 wells. In Table 7 below, “space” refers to the operating mode in which all working electrode zones 104 are activated simultaneously, images are captured, and processed for resolution. “Time-resolved” refers to the sequential mode as described above. Time-resolved has the additional advantage of allowing adjustments to ECL image acquisition (e.g., adjustment of binning to adjust dynamic range). The “Rectified Plate RT” column contains the reading time for the non-auxiliary electrode (e.g., carbon electrode). The last three columns of the table contain the difference between the reading time for the non-auxiliary electrode and the reading time for the auxiliary electrode (e.g., Ag / AgCl). (Using these examples with 10 working electrode zones per well in both Tables 7 and 8) For time-resolved measurements, the reading time for the subplex is between the reading time for 1 working electrode zone (WE) and 10 WE. For experiment “B”, no improvement in reading time was calculated because the non-auxiliary electrode plate could not operate in time-resolved mode.Table 8 includes similar data for assay apparatus 900, including the photodiode, as described above. Those skilled in the art will understand that the values ​​included in Tables 7 and 8 are approximations and may vary by + / - 5.0% based on conditions such as the operating conditions and parameters of the assay apparatus. [Table 17] [Table 18]

[0124] In relation to Tables 7 and 8, "WE" may refer to either the working electrode or the working electrode zone.

[0125] In contrast, in the case of voltage ramps in ECL applications, there are periods when voltage is applied but ECL does not occur (e.g., part of the beginning and / or end of the ramp). For example, as will be discussed in more detail later, Figures 29 and 30 (using carbon-based and Ag / AgCl-based electrodes, respectively) show a 3-second ramp time (1.0 V / s) applied to the electrodes. In this waveform, there are periods when a potential is applied but no ECL occurs. In other words, when applying a ramp waveform, there is a percentage of the total waveform duration during which no ECL occurs while a potential is applied (e.g., 5%, 10%, 15%, etc.). These percentages can vary based on several factors, including the type of material used to form the electrodes, the relative size and absolute size of the electrodes, etc. Figures 29 and 30 show non-limiting and typical examples of a specific percentage of the time during which no ECL occurred for this particular ramp waveform.

[0126] In any of the embodiments described above, the use of working electrode zones 104 having different sizes and configurations offers various advantages to the assay apparatus 900. For ECL applications, the optimal size and position of the working electrode may depend on the exact nature of the application and the type of photodetector used to detect ECL. In binding assays using binding reagents immobilized on the working electrode, binding performance and binding efficiency and rate generally increase with increasing working electrode zone size. In ECL instruments using imaging detectors (e.g., CCD or CMOS devices), the advantages of a larger 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 in a smaller electrode zone and imaged with fewer imaging device pixels. The position of the working electrode zone 104 may affect the performance of the assay apparatus 900. In some embodiments, the location, size, and shape dimensions of the spot can affect the amount of photon reflection, scattering, or loss on the well sidewall, affecting both the amount of desired light detected and the amount of undesirable light detected as coming from the working electrode zone of interest (e.g., stray light from adjacent working electrode zones or wells). In some embodiments, the performance of the assay apparatus 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, and by having working electrode zones 104 located at a uniform distance from the center of the well 200. In some embodiments, one or more working electrode zones 104 located radially symmetrically within the well 200 can improve the operation of the assay apparatus 900 by ensuring that the light collection and meniscus interaction are the same for all one or more working electrode zones 104 within the well 200, as described above. One or more working electrode zones 104 arranged at a certain distance from each other (e.g., in a circular pattern) allow the assay apparatus to utilize shortened pulse waveforms, e.g., reduced pulse width. In one embodiment, a design in which one or more working electrode zones 104 have one or more auxiliary electrodes 102 as their nearest neighbors (for example, no working electrode zones interposed between them) improves the performance of the assay apparatus 900.

[0127] In embodiments, as briefly described above, the assay apparatus 900 (e.g., computer system 906) may be configured to control the voltage / current source 904 to supply voltage and / or current with pulse waveforms, such as DC, AC, or DC mimicking AC, but other waveforms with different durations, frequencies, and amplitudes (e.g., negative ramp sawtooth, square, rectangular waveforms, etc.) are also possible. These waveforms may also include various duty cycles, such as 10%, 20%, 50%, 65%, 90%, or any other percentage from 0 to 100%. The computer system 906 may selectively control the magnitude and duration of the pulse waveforms, as will be described in more detail later. In embodiments, as described above, the computer system 906 may be configured to selectively supply pulse waveforms to one or more of the wells 200. For example, voltage and / or current may be supplied to all wells 200. Similarly, for example, a pulse waveform may be supplied to selected wells 200 (for example, on an individual basis, or on a sector basis, such as a grouping of subsets of wells, such as 4, 16, etc.). For example, as described above, the wells 200 may be individually addressable, or may be addressable by a group or subset of two or more wells. In an embodiment, the computer system 906 may be configured to selectively supply pulse waveforms to one or more working electrode zones 104 and / or auxiliary electrodes 102 in the manner described above (for example, individually addressable or addressable by a group of two or more auxiliary electrodes). For example, a pulse waveform may be supplied to all working electrode zones 104 in well 200 and / or addressed to one or more selected working electrode zones 104 in well 200. Similarly, for example, a pulse waveform may be supplied to all auxiliary electrodes 102 and / or addressed to one or more selected auxiliary electrodes 102.

[0128] In an embodiment, the pulse waveform supplied by the voltage / current source 904 may be designed to improve the electrochemical analysis and procedures of the assay apparatus 900. Figure 11 shows a flowchart illustrating a process 1100 for operating the assay apparatus using a pulse waveform according to an embodiment of this specification.

[0129] In operation 1102, process 1100 includes applying voltage pulses to one or more working electrode zones 104 or one or more auxiliary electrodes 102 in the well. For example, a computer system 906 may control a voltage / current source 904 to supply voltage pulses to one or more working electrode zones 104 or one or more auxiliary electrodes 102.

[0130] In embodiments, the pulse waveform may include various waveform types such as DC, AC, and DC mimicking AC, but other waveforms with different durations, frequencies, and amplitudes (e.g., negative ramp sawtooth waveform, square waveform, rectangular waveform, etc.) are also possible. These waveforms may also include various duty cycles, e.g., 10%, 20%, 50%, 65%, 90%, or any other percentage from 0 to 100%. Figures 12A and 12B show two examples of pulse waveforms. As shown in Figure 12A, the pulse waveform may be a square wave with a voltage V over time T. Examples of voltage pulses are illustrated by referring to Figures 14A, 14B, 15A-15L, 16, and 17, for example, 1800mV at 500ms, 2000mV at 500ms, 2200mV at 500ms, 2400mV at 500ms, 1800mV at 100ms, 2000mV at 100ms, 2200mV at 100ms, 2400mV at 100ms, 1800mV at 50ms, 2000mV at 50ms, 2200mV at 50ms, and 2400mV at 50ms. As shown in Figure 17, the pulse waveform may be a combination of two waveforms, such as a square wave modulated with a sine wave. Since the resulting ECL signal is also modulated at a sinusoidal frequency, the assay apparatus 900 may include a filter or lock-in circuit to focus on the ECL signal exhibiting a sinusoidal frequency and filter out electronic noise or stray light that does not exhibit a sinusoidal frequency. Figures 12A and 12B show examples of pulse waveforms, but those skilled in the art will understand that the pulse waveform may have any structure in which the potential rises to a predetermined voltage (or voltage range) over a predetermined period of time. Those skilled in the art will understand that the parameters of the voltage pulses and pulse waveforms described herein (e.g., duration, duty cycle, and pulse height in volts) are approximations and may vary by + / - 5.0% based on conditions such as the operating parameters of the voltage / current source.

[0131] In operation 1104, process 1100 includes measuring the potential difference between one or more working electrode zones 104 and one or more auxiliary electrodes 102. For example, the detector 910 may measure the potential difference between the working electrode zones 104 and the auxiliary electrodes 102 in the well 200. In some embodiments, the detector 910 may supply the measured data to the computer system 1506.

[0132] In operation 1106, process 1100 includes performing an analysis based on the measured potential difference and other data. For example, the computer system 906 may perform the analysis on the potential difference and other data. The analysis may be any process or procedure, such as potentiometry, coulometry, voltammetry, or optical analysis (which will be further described below). In embodiments, the use of pulse waveforms allows a particular type of analysis to be performed. For example, a number of different redox reactions may occur in a sample that are activated when the applied potential exceeds a certain level. By using a pulse waveform of a specified voltage, the assay apparatus 900 can selectively activate some of these redox reactions while not activating others.

[0133] In one embodiment, the disclosure provided herein may be applied to a method for performing an ECL assay. Specific examples of methods for performing an ECL assay are provided in U.S. Patents 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.

[0134] In an embodiment, the pulse waveform supplied by the voltage / current source 904 may be designed to improve the ECL emitted during ECL analysis. For example, the pulse waveform may improve the ECL emitted during ECL analysis by providing a stable, constant voltage potential, thereby generating stable and predictable ECL emission. Figure 13 shows a flowchart illustrating a process 1300 for operating an ECL apparatus using a pulse waveform according to an embodiment of this specification.

[0135] In operation 1302, process 1300 includes applying voltage pulses to one or more working electrode zones 104 or auxiliary electrodes 102 in the wells of the ECL apparatus. For example, a computer system 906 may control a voltage / current source 904 to supply voltage pulses to one or more working electrode zones 104 or one or more auxiliary electrodes 102. In embodiments, one or more auxiliary electrodes 102 may include a redox pair in which, when a voltage or potential is applied, the reaction of a species in the redox pair is the primary redox reaction that occurs at one or more auxiliary electrodes 102. In some embodiments, the applied voltage is less than a specified potential required to reduce water or to perform electrolysis of water. In some embodiments, less than 1 percent of the current is related to the reduction of water. In some embodiments, less than 1 percent of the current per unit area (exposed surface area) of one or more auxiliary electrodes 102 is related to the reduction of water.

[0136] In embodiments, the pulse waveform may include various waveforms such as DC, AC, and DC mimicking AC, but other waveforms with different durations, frequencies, and amplitudes (e.g., negative ramp sawtooth waveform, square waveform, rectangular waveform, etc.) are also possible. Figures 12A and 12B above show two examples of pulse waveforms. The pulse waveform may be a square wave with a voltage V over time T. Examples of voltage pulses are illustrated with reference to Figures 14A, 14B, 15A-15L, 16, and 17, for example, 1800mV at 500ms, 2000mV at 500ms, 2200mV at 500ms, 2400mV at 500ms, 1800mV at 100ms, 2000mV at 100ms, 2200mV at 100ms, 2400mV at 100ms, 1800mV at 50ms, 2000mV at 50ms, 2200mV at 50ms, and 2400mV at 50ms. These waveforms may include various duty cycles, such as 10%, 20%, 50%, 65%, 90%, or any other arbitrary percentage from 0 to 100.

[0137] In operation 1304, process 1300 includes capturing luminescence data from an electrochemical cell over a period of time. For example, one or more photodetectors 912 may capture luminescence data emitted from a well 200 and transmit the luminescence data to a computer system 906. In embodiments, the period may be selected so that the photodetectors can capture ECL data. In some embodiments, 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 wells 200. In some embodiments, each well 200 of the multiwell plate 200 may include a camera that captures an image of the well 200. In some embodiments, each well 200 of the multiwell plate 200 may include multiple cameras that capture images of a single working electrode zone 104 or a subset of working electrode zones 104 within each well 200. Therefore, the assay apparatus 900 can offer flexibility by having a camera capture all light from multiple working electrode zones 104, and a computer system 906 using image processing to decompose the luminescence data of each working electrode zone 104. Thus, the assay apparatus 900 may operate in various modes, for example, singleplex mode (e.g., one working electrode zone), 10plex mode (e.g., all working electrode zones 104 with respect to 10 working electrode zone wells 200), or generally multiplex mode (e.g., simultaneously a subset of all working electrode zones contained within a single well or multiple wells 200, such as five working electrode zones 104 simultaneously with respect to 10 working electrode zone wells).

[0138] In some embodiments, the assay apparatus 900 may include a photodiode corresponding to each well 200 of the multiwell plate 200 to detect and measure photons emitted within the wells 200. In some embodiments, the assay apparatus 900 may include multiple photodiodes corresponding to each well 200 of the multiwell plate 200 to detect and measure photons emitted from a single working electrode zone 104 or a subset of working electrode zones 104 within each well 200. Thus, the assay apparatus 900 may operate in various modes. For example, the assay apparatus 900 may apply voltage and / or current individually to one or more working electrode zones 104 in the multiwell plate 208, for example, five working electrode zones 104. The working electrode zones 104 may be located in a single well 200, in different wells 200, or in combination thereof. The photodiodes may then sequentially detect / measure the light entering from each of the five working electrode zones 104. For example, a voltage and / or current may be applied to the first of the five working electrode zones 104, and the emitted photons may be detected and measured by the corresponding photodiodes. This may be repeated sequentially for each of the five working electrode zones 104. Similarly, in this example, the sequential operation mode 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 well 200, or a combination thereof. Similarly, in some embodiments, the assay apparatus 900 may operate in a multiplex mode in which one or more working electrode zones 104 are activated simultaneously by the application of voltage and / or current, and the emitted photons may be detected and measured by multiple photodiodes and multiplexed.The multiplex operating mode may be performed on working electrode zones 104 within the same well 200, on working electrode zones 104 located in different wells 200, on working electrode zones 104 located in a subset or "sector" of wells 200 in a multiwell plate 208, or in combination thereof. Figures 14A, 14B, 15A-15L, 16, and 17 below show some waveform tests used in ECL analysis.

[0139] In embodiments, by applying a pulsed waveform to generate ECL, the reading time and / or exposure time can be improved by faster and more efficient generation, acquisition, observation, and analysis of ECL data. Furthermore, various exposure approaches (e.g., single exposure, double exposure, triple exposure (or more)) with different (or equal) exposure times can be used to improve ECL acquisition, collection, observation, and analysis, for example, by improving dynamic range expansion (DRE), binning, etc. For example, as described above, the use of one or more auxiliary electrodes 102 improves the reading time of the detector 910. For example, the use of Ag / AgCl in one or more auxiliary electrodes 102 improves the ECL reading time for several reasons. For example, the use of an electrode (e.g., auxiliary electrode 102) with a redox pair (Ag / AgCl in this particular embodiment) provides a stable interfacial potential, which can allow the electrochemical analysis process to use a voltage pulse rather than a voltage ramp. The use of a voltage pulse improves the reading time because the entire pulsed waveform can be applied at a voltage potential that generates ECL throughout the entire duration of the waveform. Furthermore, the “time-resolved” or sequential mode has the additional advantage of allowing adjustments to ECL image acquisition (e.g., adjustments to binning to adjust the dynamic range). Also, as described above, the assay apparatus 900 (e.g., computer system 906) may be configured to delay the activation of the continuous working electrode zone 104 using such data for different voltage pulses. By implementing this delay, the assay apparatus 900 can minimize crosstalk between the working electrode zone 104 and / or the wells 200, have high throughput when performing ECL operations, and more.

[0140] In operation 1306, process 1300 includes performing ECL analysis on luminescence data. For example, a computer system 906 may perform ECL analysis on luminescence data. In some embodiments, luminescence data, e.g., signals, emanating from a given target object on the bonding surface of the working electrode zone 104 and / or auxiliary electrode 102, e.g., on the bonding domain, may have a range of values. These values ​​may be correlated with quantitative measurements (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 respect to both techniques and may be used to convert multiple digital signals to provide quantitative results. 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, e.g., techniques for determining concentrations through statistical analysis of bonding across concentration gradients, may be used. Multiple linear arrays of data with concentration gradients can be generated with multiple different specific binding reagents used in different wells 200 and / or different working electrode zones 104. The concentration gradients may consist of separate binding domains representing different concentrations of the binding reagent.

[0141] In embodiments, a control assay solution or reagent, such as a lead buffer, may be used on the working electrode zone of well 200. The control assay solution or reagent may provide uniformity to the nuclear analysis to control signal fluctuations (e.g., fluctuations due to degradation, movement, aging, thermal shift, noise in the electronic circuit, and noise in the photodetector). For example, multiple overlapping working electrode zones 104 (containing the same binding reagent or different binding reagents specific to the same analyte) may be used for the same analyte. In other examples, an analyte of known concentration may be used, or the control assay solution or reagent may be covalently bound to a known amount of ECL label, or a known amount of ECL label in solution may be used.

[0142] In embodiments, the data collected and generated in process 1300 can be used for a variety of purposes. The collected and generated data may be stored in the form of a database consisting of, for example, 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 upon contact with a human DNA sample can be used for signature DNA fingerprints that can be readily 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.) or organisms (e.g., bacteria, viruses, etc.).

[0143] The above describes an exemplary flow of Process Example 1300. The process shown in Figure 13 is merely illustrative, and variations exist without departing from the scope of the embodiments disclosed herein. As described above, the 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 pulse waveforms in combination with auxiliary electrodes offers various advantages to the ECL assay. The auxiliary electrodes allow for more rapid generation of luminescence without the use of a lamp.

[0144] Figures 14A–14C, 15A–15L, 16, and 17 are graphs showing the results of ECL analysis using various pulse waveforms. Figures 15A–15L show raw data for BTI concentration for model binding assays using various pulse waveforms. Figures 15A–15L compare the use of pulse waveforms applied to wells using Ag / AgCl auxiliary electrodes (displayed according to pulse parameters) with the use of a ramp waveform (1.4 V / s for 1 s) applied to wells using carbon electrodes as a control group (displayed as control lot). Figures 14A–14C summarize the performance of model binding assays following various pulse waveforms as shown in Figures 15A–15L. Figures 16 and 17 are described in more detail below. In these tests, model binding assays were used to measure the effect of ECL generation conditions on the amount of ECL generated from a controlled amount of ECL-labeled binding reagent bound to the working electrode zone by a specific binding interaction. In this model system, the ECL-labeled conjugate was an IgG antibody (SULFO-TAG, Meso Scale Diagnostics, LLC) labeled with both biotin and ECL. Various concentrations of this conjugate (referred to as "BTI" or "BTI HC" for high-control BTI) were added to the wells of a 96-well plate with an integrated screen-printed carbon ink working electrode containing a streptavidin immobilization layer in each well. Two types of plates were used: the control plate was an MSD Gold 96-well Streptavidin QuickPlex plate (Meso Scale Diagnostics, LLC) with a screen-printed carbon ink counter electrode, and the test plate was a similar design but with a screen-printed Ag / AgCl auxiliary electrode instead of a counter electrode. The plates were incubated to allow the BTI in the wells to bind to the working electrode via biotin-streptavidin interaction.After incubation, the plates were washed to remove free BTI, ECL Read Buffer (MSD Read Buffer Gold, Meso Scale Diagnostics, LLC) was added, and the plates were analyzed by applying a defined voltage waveform between the working and auxiliary electrodes and measuring the emitted ECL. The Ag:AgCl ratio in the auxiliary electrode ink of the test plates was approximately 50:50. Twelve waveforms were used with four different potentials (1800 mV, 2000 mV, 2200 mV, and 2400 mV) at three different times or pulse widths (500 ms, 100 ms, and 50 ms). One test plate was tested for each waveform. Control plates were tested using the standard lamp waveform.

[0145] For plates tested with each waveform, assay performance data were determined and calculated. Mean, standard deviation, and %CV were calculated for each sample and plotted as data points with error bars. The signals measured for BTI solutions in the range of 0 (blank sample to measure assay background) to 2 nM were linearly fitted (slope, Y-intercept, and R 2 were calculated). The limit of detection was mean background + / −3 *The standard deviation ("stdev") and the linear fit of the titration curve (shown in Figure 14C) were calculated. Signals were also measured for 4, 6, and 8 nM BTI solutions. These signals were divided by the extrapolated signal from the linear fit of the titration curve (this ratio can be used to estimate the binding capacity of the streptavidin layer on the working electrode; a ratio significantly smaller than 1 indicates that the amount of added BTI is close to or greater than the binding capacity). The ratio of the slope from the presented control lot to the slope from each test plate was calculated. Figure 14A shows these calculation results for each pulse waveform. Each of the graphs in Figures 15A–15L shows the average ECL data collected for a ramp voltage applied to a multiwell plate with a carbon counter electrode from the control lot and for different voltage pulses applied to a multiwell plate with an Ag / AgCl auxiliary electrode. Figures 14A–14C provide a summary of the data shown in Figures 15A–15L.

[0146] Furthermore, signal, slope, background, and dark analysis (e.g., the signal that occurs when there is no ECL) were performed. A plot of the 2 nM signal with error bars of 1stdev and the slope were created. Bar graphs of the background and dark and slope with error bars of 1stdev were created. Figure 14B shows these results. As shown in Figures 14A and 14B, a pulse voltage of 1800 mV over 500 ms results in a high average ECL reading. As shown in Figures 14A and 14B, the magnitude and / or duration of the pulse waveform affects the measured ECL signal. The change in the 2 nM signal associated with the waveform reflects the change in the slope. Also, the change in the background reflects the change in the slope. The signal, background, and slope decreased with a decrease in the pulse duration. The signal, background, and slope decreased with an increase in the pulse potential. The change in the signal, background, and slope with a decrease in time decreased with an increase in the pulse potential. There was little change in assay sensitivity due to the simultaneous change in the signal, background, and slope with various pulse potentials and durations. The signal, background, and slope decreased with a decrease in the pulse duration. The signal, background, and slope decreased with an increase in the pulse potential. The change in the signal, background, and slope with a decrease in time decreased with an increase in the pulse potential. There was little change in assay sensitivity due to the simultaneous change in the signal, background, and slope with various pulse potentials and durations.

[0147] Also, for each of the pulse waveforms, the titration curve was analyzed. A plot of the average ECL signal vs. BTI concentration was created. Error bars based on 1stdev were included. The titration curves from the test plates were plotted on the primary y-axis. The titration curves were plotted on the secondary y-axis. The scale of the secondary y-axis was from 0 to 90,000 counts ("cts") of the detected photons. The scale of the primary y-axis was set to the value obtained by dividing 90,000 by the ratio of the slopes. The ratio of the slopes for each test plate to the slope was calculated. Figures 15A - 15L show the results of these calculations for each pulse waveform.

[0148] Regarding background, dark, and dark noise, the dark (1 and 2 cts) and dark noise (2 cts) remained essentially constant across all waveform times tested. The background decreased with decreasing pulse duration. The background decreased with increasing applied pulse potential. The change in background with decreasing time decreased with increasing pulse potential. A background of 1800mV over 50ms was 6±2 cts and was directly above the dark + dark noise.

[0149] As shown in Figures 15A–15L, the %CV was comparable across all test plates and reference signals for all signals (8 replications), excluding the background. The CV for the background increased as the background signal approached the dark and dark noise. The background above 40 cts (16 replications) had good CVs of 55 (3.9%), 64 (5.1%), and 44 (5.4%). Below 40 cts, the CV increased to over 7%. All titrations from the background to 2 nM HC were linearly fitted with an R² value ≥ 0.999.

[0150] Reducing the maximum concentration within the fitting range resulted in a smaller slope and a larger y-intercept. This indicates nonlinearity at the lower end of the titration curve (likely caused by different dilution rates in the test sample). The y-intercepts of other assays were essentially between zero and the measured background. All assays produced sublinear lower signals for 6 nM and 8 nM HC, and this decrease in binding ability was consistent across all assays. All assays produced a 4 nM signal within 2 first denominations of the extrapolated 4 nM signal. After correcting by the ratio of the slope of the control lot to the slope of the test plate, the assay signals were within 3 first denominations of the assay signals from the control lot for 1 nM to 4 nM HC. Below 1 nM HC, the corrected signal was higher than the signal from the control lot. For 0.0125 to 0.5 nM HC, the corrected signals from the test plate were within 3 first denominations of each other. The correction signals for assays performed with the same BTI solution were within 3 stdev of each other at 0.0125 nM to 4 nM HC. As shown in the plot, the performance of assays measured at different pulse potentials and durations was within this range of variation of the performance of the control assay measured with the lamp.

[0151] As can be seen by comparing Figures 15A-15L with Figures 14A and 14B, the signal and slope decreased with decreasing pulse duration (500 ms, 100 ms, and 50 ms). The signal and slope also decreased with increasing pulse potential (1800 mV, 2000 mV, 2200 mV, and 2400 mV). The changes in signal and slope with decreasing pulse duration decreased with increasing pulse potential. The correction factor (ratio of slopes) can compensate for changes in signal with changes in waveform. The calculated detection limits were similar for these waveforms (0.005 nM to 0.009 nM). The calculated detection limit for the 1800 mV, 500 ms pulse waveform was lower (0.004 nM), which is likely due to subtle differences in the fitted and measured background (CV).

[0152] Example 1: ECL measuring instrument

[0153] Referring here in detail to Figures 14A–14C, the ECL measurement was performed in a 96-well plate specifically configured for ECL assay applications by including an integrated screen-printed electrode. The basic structure of the plate is similar to the plate described in U.S. Patent No. 7,842,246 (see, for example, the descriptions of plates B, C, D, and E in Example 6.1), but this design has been improved to incorporate the novel elements of this disclosure. Similar to the aforementioned design, the bottom of the wells is defined by a Mylar sheet having a screen-printed electrode on its top surface that provides an integrated working electrode and counter electrode surface (or, in some embodiments of the invention, a novel working electrode and auxiliary electrode) within each well. A patterned screen-printed dielectric ink layer printed on the working electrode defines one or more exposed working electrode zones within each well. Conductive through-holes penetrating the Mylar to screen-printed electrical contacts on the bottom surface of the Mylar sheet provide the electrical contacts necessary to connect an external electrical energy source to the electrode.

[0154] ECL measurements on specially configured plates were performed using an ECL plate reader designed to accept the plate, connect to electrical contacts on the plate, apply electrical energy to the contacts, and image the ECL generated within the wells. For some measurements, improved software was used to allow customization of the timing and shape of the applied voltage waveform.

[0155] A typical plate reader is the MESO SECTOR S 600. 1768274699823_0 QUICKPLEX SQ 120 1768274699823_1 Available from Scale Diagnostics, LLC, the plate reader is described in U.S. Patent No. 6,977,722 and U.S. Patent Application No. 62 / 874,828, titled “Assay Apparatuses, Methods and Reagents,” filed July 16, 2019, each of which is incorporated herein by reference in its entirety. Other device examples are described in U.S. Patent Application No. 16 / 513,526, titled “Graphical User Interface System,” filed July 16, 2019, and U.S. Patent Application No. 16 / 929,757, titled “Assay Apparatuses, Methods and Reagents,” filed July 15, 2020, each of which is incorporated herein by reference in its entirety.

[0156] Example 2: High-speed pulsed ECL measurement

[0157] A model binding assay was used to demonstrate the use of a fast pulsed voltage waveform in combination with an Ag / AgCl auxiliary electrode to generate an ECL signal and to compare it with the performance observed with conventional combinations of slow voltage ramps and carbon counter electrodes. The model binding assay was performed in a 96-well plate in which each well had an integrated screen-printed carbon ink working electrode region supporting a streptavidin immobilization layer. These screen-printed plates had either a screen-printed carbon ink counter electrode (MSD Gold 96-Well Streptavidin Plate, Meso Scal Diagnostics, LLC.) or a plate with a similar electrode design except for the use of a screen-printed Ag / AgCl ink auxiliary electrode. In this model system, the ECL-labeled binding reagent was an IgG antibody labeled with both biotin and ECL (SULFO-TAG, Meso Scal Diagnostics, LLC.). Various concentrations of this binding reagent (referred to as "BTI" or "BTI HC" in the case of high-control BTI) in 50 μL aliquots were added to the wells of the 96-well plate. The binding reagent was incubated with agitation for a sufficient time to be depleted from the assay solution by binding to immobilized streptavidin on the working electrode. The plate was washed to remove the assay solution and then filled with ECL read buffer (MSD Read Buffer T 2X, Meso Scal Diagnostics, LLC). A standard waveform (100 ms ramp at 3200 mV–4600 mV) was applied to the plate with the counter electrode. Twelve constant voltage pulse waveforms at four different potentials (1800 mV, 2000 mV, 2200 mV, 2400 mV) and three different time or pulse widths (500 ms, 100 ms, and 50 ms) were evaluated on the plate with the Ag / AgCl auxiliary electrode. One plate was tested for each waveform. Figures 14A, 14B, and 15A–15L are graphs showing the results of the ECL analysis obtained from this study.

[0158] For plates tested with each waveform, assay performance data was determined and calculated. For each sample, the mean, standard deviation, and %CV were calculated. Figures 15A–15L show plots of the mean signal-paired reagent concentration, with the signal from the standard waveform plotted on a different y-axis from the signal from the potential pulse. Data points in the lower linear region of the plot, i.e., BTI concentrations in the range of 0 (blank sample for measuring assay background) to 0.1 nM, are fitted to a straight line, and the slope, standard error of the slope, Y-intercept, standard error of the Y-intercept, and R are calculated. 2 The value was calculated. All linear fits are in R 2 The result was ≥0.999. Figures 14A and 14B show the 2nM mean signal, 0nM (assay background) mean signal, and mean dark signal (empty well) for each test condition, along with 1stdev error bars. Both figures also show the slope calculated for each condition. The detection limit provided for the concentration of BTI was calculated based on the mean Y-intercept of the background + 3 * standard deviation ("stdev") and the linear fit of the titration curve. The standard errors in the slope and Y-intercept, and the standard deviation of the background, were propagated into the error of the detection limit. Based on the volume of BTI per well and the number of ECL labels per BTI molecule (~0.071), the detection limit could be expressed in terms of the number of moles of ECL label required to produce a detectable signal (plotted in Figure 14E).

[0159] Figures 14C and 14D show that the ECL signal from the BTI on the electrode, generated by a 500 ms pulse waveform at a potential of 1800 mV, is equivalent to the signal generated by a conventional 1000 ms ramp waveform, but in half the time. Figure 14C shows that for a given pulse potential, ECL decreases as the pulse time decreases to less than 500 ms, but comparison with Figure 14D shows that the corresponding decrease in the assay background signal remains significantly above the camera signal for the dark image of the empty well (i.e., the image without ECL excitation). This result indicates that very short pulses can be used to significantly reduce the time required to perform ECL measurements while maintaining overall sensitivity.

[0160] The detection limit calculated for a standard waveform (1000 ms ramp) using a carbon counter electrode is 2.4 ± 2.6 atmoles (10) of ECL-labeled material. -18 The result was (moles). Figure 14E shows that the estimated detection limits for different excitation conditions tend to increase with decreasing pulse time, but this is significantly less than what would be expected from a linear relationship. For example, the estimated detection limit for a 100 ms pulse at 2000 mV was nearly twice as high as the detection limit for a 1000 ms ramp, but for one-tenth the time. In addition, the increase in the detection limit with decreasing pulse time was not always statistically significant. The detection limits for pulses of "1800 mV, 500 ms", "2000 mV, 500 ms", "2000 mV, 100 ms", and "2200 mV, 500 ms" using the Ag / AgCl auxiliary electrode were within the error range of the detection limit for the standard waveform (1000 ms ramp) using the carbon counter electrode.

[0161] Figure 16 shows a graph illustrating the results of ECL analysis using pulse waveforms with a lead buffer solution, e.g., lead buffer T. For the tests, Ag / AgCl Std 96-1 IND plates printed with 50:50 ink were used. For the tests, aliquots of MSD T4x (Y0140365) were diluted with molecular-grade water to prepare T3x, T2x, and T1x. Ag / AgCl Std 96-1 IND plates were filled with 150 μL aliquots of these solutions, with T4x in two adjacent rows of well 200, T3x in two adjacent rows of well 200, T2x in two adjacent rows of well 200, and T1x in two adjacent rows of well 200, as shown in Figure 9B. These solutions were allowed to be immersed covered on a bench for 15 minutes ± 0.5 minutes. Each plate was measured with waveforms of 1800mV for 100ms, 1800mV for 300ms, 1800mV for 1000ms, and 1800mV for 3000ms. The average ECL signal and average integrated current were calculated for 24 replicas for each condition, and plots of the average values ​​against MSD T concentrations (4, 3, 2, and 1) were created.

[0162] As shown in Figure 16, the ECL signal and integrated current increased with increasing lead buffer T concentration. The ECL signal and integrated current also increased with increasing pulse duration. The lead buffer ECL signal increased linearly between T1x and T3x, but not between 3x and 4x. The integrated current increased linearly between T1x and T4x.

[0163] Figure 17 shows a graph illustrating the results of another ECL analysis using pulse waveforms. The tests used Ag / AgCl Std 96-1 IND plates printed with 50:50 ink. The test methods described above for Figures 14A and 14B were used with different, longer pulse waveforms. One plate was measured with each of the following waveforms: 1800mV for 3000ms, 2200mV for 3000ms, 2600mV for 3000ms, and 3000mV for 3000ms. The average ECL signal and average integrated current were calculated for 24 copies for each condition, and plots of the average value versus the lead buffer T concentration (4, 3, 2, and 1) were created.

[0164] As shown in Figure 17, the ECL signal increased with increasing concentration of read buffer T for pulse potentials of 1800mV, 2200mV, and 2600mV. For the 3000mV pulse, the ECL signal decreased between T1x and T2x, and then increased through T4x. The integrated current increased with increasing concentration of T for all pulse potentials. The integrated current for the 2600mV and 3000mV pulses was somewhat linear between T1x and T3x, but at T4x, the increase in current was less than linear with respect to the concentration of read buffer T.

[0165] Example 3: Reduction capacity of Ag / AgCl auxiliary electrode

[0166] To determine the reduction ability of the auxiliary electrode, i.e., the amount of reduction charge that can pass through the electrode while maintaining a controlled potential, an assay plate having an integrated screen-printed carbon ink working electrode and a screen-printed Ag / AgCl auxiliary electrode (as described in Example 2) was used. To evaluate the ability in relation to the requirements of an ECL experiment using pulsed ECL measurements, the total charge passing through the auxiliary electrode was measured when an ECL read buffer containing TPA was present while applying a pulsed voltage waveform between the working electrode and the auxiliary electrode. Two types of experiments were conducted. In the first experiment (shown in FIG. 16), a voltage pulse (1800 mV) near the optimal potential for ECL generation was applied and held for different times (100 to 3000 ms). In the second experiment (FIG. 17), different pulse potentials (2200 to 3000 mV) were held for a fixed time (3000 ms). In both experiments, the resistance to changes in concentration or co-reactant and electrolyte in the read buffer composition was evaluated by testing each voltage and time condition in a situation where the components of MSD Read Buffer T were present at 1 to 4 times the nominal working concentration of TPA. Each point in the graph represents the average of 24 replicate measurements.

[0167] The Ag / AgCl auxiliary electrode assists in the oxidation of TPA at the working electrode under the experimentally applied potential until the charge passing through the auxiliary electrode consumes all the accessible oxidant (AgCl) within the auxiliary electrode. Figure 16 shows that the charge passing through the auxiliary electrode using 1800mV increases approximately linearly with respect to pulse duration and TPA concentration, demonstrating that the electrode capacity is sufficient to support pulses as long as 3000ms at 1800mV, even in the presence of higher-than-typical TPA concentrations. Figure 17 shows an experiment designed to determine the capacity of the auxiliary electrode by increasing the potential until the charge passing through the electrode reaches its maximum value, using the longest pulse (3000ms) from Figure 16. Data points collected using 3000mV show that the charge increased linearly with respect to the ECL read buffer concentration up to approximately 30mC of the total charge. Around 45mC, the total charge flattens out, indicating depletion of the oxidant in the Ag / AgCl auxiliary electrode. A charge of 30 mC corresponds to 3.1 × 10⁻⁶ of the oxidizing agent in the Ag / AgCl auxiliary electrode. -7 Equivalent to 45 mC in moles, 4.7 × 10⁻⁶ mC is the amount of oxidizing agent in the Ag / AgCl auxiliary electrode. -7 It is equal to a mole.

[0168] Furthermore, reduction capacity tests were conducted to determine the differences in reduction capacity due to spot patterns and auxiliary electrode sizes. Four different spot patterns were tested using a reduction capacity waveform of 2600 mV, 4000 ms and a standardized test solution. The four spot patterns tested were a 10-spot penta pattern (Figure 5A), a 10-spot open pattern (Figure 1C), a 10-spot closed pattern (Figure 7A), and a 10-spot open trefoil pattern (Figure 4A). The results for each of the penta, open, closed, and open trefoil patterns are reproduced in Tables A, B, C, and D below. As shown in Tables A-C, increasing the auxiliary electrode (indicated as CE) area in the three different patterns increases the total measured charge (e.g., reduction capacity). As shown in Table D, multiple tests with the same auxiliary electrode area yield nearly similar measured charges. Therefore, maximizing the auxiliary electrode area may help increase the total reduction capacity of the Ag / AgCl auxiliary electrode in multiple different spot patterns. [Table 19] [Table 20] [Table 21] [Table 22]

[0169] Experiments were also conducted to determine the amount of AgCl accessible to the redox reaction under various experimental conditions. The experiments used electrodes printed with an Ag / AgCl ink film approximately 10 microns thick. Different electrode portions, ranging from 0% to 100% contact with the solution, were exposed to the solution, and the amount of charge that passed through was measured. The experimental results show that the amount of charge that passed through increased roughly linearly with increasing percentage of the electrode in contact with the solution. This indicates that reduction is not strong or does not occur at all in the electrode portions not directly in contact with the test solution. Furthermore, the total amount of charge that passed through the experimental electrode (2.03E+18e-) is approximately equivalent to the total amount of electrons available in the experimental electrode, based on the total amount of Ag / AgCl in the printed electrode. This indicates that, at a thickness of 10 microns and 100% solution contact, all or almost all of the available AgCl may be accessible to the redox reaction. Therefore, for films thinner than 10 microns, all or almost all of the available AgCl can be accessed during the reduction reaction.

[0170] In an embodiment, the pulse waveform supplied by the voltage / current source 904 may be designed to allow the ECL instrument to capture different luminescence data over time in order to improve ECL analysis. Figure 18 shows a flowchart illustrating another process 1800 for operating the ECL instrument using the pulse waveform according to an embodiment of this specification.

[0171] In operation 1802, process 1800 includes applying voltage pulses to one or more working electrode zones 104 or auxiliary electrodes 102 in the wells of the ECL apparatus, the voltage pulses causing a reduction-oxidation reaction in the wells. For example, a computer system 906 may control a voltage / current source 904 to supply one or more voltage pulses to one or more working electrode zones 104 or auxiliary electrodes 102.

[0172] In embodiments, a voltage pulse may be configured to induce a reduction-oxidation reaction between one or more working electrode zones 104 and one or more auxiliary electrodes 102. As described above, based on a predetermined chemical composition of one or more auxiliary electrodes 102 (e.g., a mixture of Ag:AgCl), one or more auxiliary electrodes 102 may act as a reference electrode for determining the potential difference with one or more working electrode zones 104 and as a counter electrode for the working electrode zones 104. For example, a predetermined chemical mixture (e.g., the ratio of elements and alloys in the chemical composition) may provide an interfacial potential during the reduction of the chemical mixture such that a quantifiable amount of charge is generated through the reduction-oxidation reaction occurring in well 200. That is, the amount of charge passing through during the redox reaction can be quantified, for example, by measuring the current in the working electrode zone 104. In some embodiments, as AgCl is consumed, the interfacial potential at the auxiliary electrode 102 shifts to the negative side relative to the reduction potential of water, so that one or more auxiliary electrodes 102 can define the total amount of charge that can pass through the applied potential difference. As a result, the potential of the working electrode zone 104 shifts to a lower potential (maintaining the applied potential difference), and the oxidation reaction that occurred during the reduction of AgCl is turned off.

[0173] In embodiments, the pulse waveform may include various waveform types such as DC, AC, and DC mimicking AC, but other waveforms with different durations, frequencies, and amplitudes (e.g., negative ramp sawtooth waveform, square waveform, rectangular waveform, etc.) are also possible. Figures 12A and 12B above show two examples of pulse waveforms. The pulse waveform may be a square wave having a voltage V over time T. Examples of voltage pulses are also illustrated with reference to Figures 14A, 14B, 15A-15L, 16, and 17, and include, for example, 1800mV at 500ms, 2000mV at 500ms, 2200mV at 500ms, 2400mV at 500ms, 1800mV at 100ms, 2000mV at 100ms, 2200mV at 100ms, 2400mV at 100ms, 1800mV at 50ms, 2000mV at 50ms, 2200mV at 50ms, 2400mV at 50ms, etc. These waveforms may include various duty cycles, such as 10%, 20%, 50%, 65%, 90%, or any other percentage from 0 to 100.

[0174] In operation 1804, process 1800 includes capturing first luminescence data from a first reduction-oxidation reaction over a first period. In operation 1806, process 1800 includes capturing second luminescence data from a second reduction-oxidation reaction over a second period, the first period not having the same duration as the second period. For example, one or more photodetectors 910 may capture first and second luminescence data emitted from well 200 and transmit the first and second luminescence data to computer system 906. For example, in an embodiment, well 200 may contain a substance of interest that requires different periods with respect to photodetector 912 to capture luminescence data. Thus, photodetector 912 may capture ECL data over two different periods. For example, one of the periods may be a short period (e.g., a short camera exposure time for the light generated from ECL) and the other may be a longer period. These periods may be affected, for example, by photosaturation during ECL generation. From there, depending on the captured photons, the assay apparatus 900 may use long exposure, short exposure, or a combination of the two. In some embodiments, the assay apparatus 900 may use long exposure, or a combination of long and short exposure. In some embodiments, if the captured photons exceed the dynamic range of the photodetector 912, the assay apparatus 900 may use short exposure. By adjusting / optimizing these, the dynamic range may be increased by an order of magnitude or two. For example, short exposure may be followed by 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), a single well, two or more wells, or a sector, or two or more sectors). In these examples, using longer exposure may be advantageous, except when the exposure is saturated. In that case, for example, shorter exposure may be used.The dynamic range can be improved by making these adjustments (for example, manually or with the help of hardware, firmware, software, algorithms, computer-readable media, computing devices, etc.). In other examples, a first short pulse (e.g., 50 ms, but other durations are possible) may be applied to an electrode or a set of two or more electrodes, and then a second longer pulse (e.g., 200 ms, but other durations are possible) may be applied to each electrode or set of electrodes. Another approach may involve reading the entire plate (e.g., 96 wells) with one or more first short pulses (e.g., 50 ms, but other durations are possible), and then reading the entire plate a second time with a second longer pulse (e.g., 200 ms, but other durations are possible). In other examples, a long pulse may be applied first, followed by short pulses, multiple short pulses, and / or long pulses being applied and / or alternately. In addition to one or more discrete pulses, composite or hybrid functions using these durations or other durations may be used to determine and / or model the response in, for example, the transition region (e.g., during the transition between pulses). Furthermore, in the above example, longer pulses may be used first, before shorter pulses. The waveform and / or capture window may also be adjusted to improve the dynamic range.

[0175] Furthermore, if additional information is known about one or more individual working electrodes and / or working electrode zones (for example, if a particular working electrode zone is known to contain a high concentration of analyte), exposure times can be optimized to prevent camera saturation by using this information before reading and / or sampling. Using the example of high concentration analytes mentioned above, shorter exposure times can be used because the signal is expected to be high in the dynamic range (and vice versa for electrodes where a low signal is expected), and therefore exposure time, pulse duration, and / or pulse intensity can be customized and / or optimized for individual wells, electrodes, etc., for example, to improve the overall reading time. Alternatively, pixels from one or more ROIs can be sampled sequentially to obtain an ECL curve over time, which can be further used to determine how to truncate the exposure time and extrapolate the ECL generation curve beyond saturation. In other examples, the camera may initially be set to perform short exposures, and then the signal intensity from these short exposures may be investigated. This information can then be used to adjust the binning of the final exposure. In other examples, instead of adjusting binning, other parameters such as waveform, capture window, or other current-based techniques may be adjusted.

[0176] Additional techniques may be employed to keep the waveform and / or exposure constant. For example, if the intensity of pixels within one or more ROIs is measured and pixel saturation is observed, other aspects of ECL generation and / or measurement (e.g., current-ECL correlation, dark mask schemes that observe a dark mask region around the ROI, which may be used to update the estimated ECL with respect to the saturated electrode and / or a portion of the electrode) may be employed to optimize the readout and / or readout time. These solutions avoid the need for fast analysis and / or reaction times to adjust the duration of the waveform and / or exposure over relatively short periods (e.g., milliseconds). This is because, for example, ECL generation and / or capture may be performed in the same and / or similar manner, and analysis may be performed last.

[0177] Other techniques may also be used to improve the dynamic range. For example, when applied to electrochemical luminescence (ECL) applications, since the ECL label fluoresces, pre-emission and / or pre-exposure may be performed to obtain information about the extent of labeling present in one or more wells, working electrodes, working electrode zones, etc. The information obtained from pre-emission and / or pre-exposure may be used to optimize exposure and / or pulse duration to achieve additional improvements in dynamic range and / or readout time. In other embodiments, particularly with respect to ECL, a correlation may exist between the current and one or more electrodes and the ECL signal, so the signal signature may indicate the camera's exposure time and / or applied waveform (e.g., waveform stop, waveform decrease, waveform increase, etc.). This can be further optimized by improving the accuracy and update speed of current measurement and optimizing the current path to provide a better correlation between the current and the ECL signal.

[0178] Further improvements in dynamic range may be achieved with respect to specific imaging devices according to a particular embodiment. 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 points in time within one or more exposure ranges to optimize exposure time. For example, the ROI (e.g., a working electrode and / or part or all of the working electrode zone) may have a specific sampling percentage (e.g., 1%, 5%, 10%, etc., but other percentages are also possible) of a fixed or variable number of pixels or electrode areas. In this example, the pixels and / or sampling 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 and typical example, a subset of pixels may be sampled over a sampling period. If the signal from that subset tends to be high, the exposure time may be reduced (e.g., from 3 seconds to 1 second, but longer or shorter durations are also possible). Similarly, if the signal tends to be low, a longer exposure time may be used (e.g., 3 seconds, but other durations are also possible). These adjustments can be made manually or with the assistance of hardware, firmware, software, algorithms, computer-readable media, computing devices, etc. In other embodiments, the ROIs may be selected to be distributed in a manner that avoids any possible ring effects. This can occur, for example, due to non-uniformity of light around the working electrode zone (e.g., a bright ring is formed around the outer periphery of the working electrode zone, with a dark spot in the center). To address this, an ROI may be selected that samples both bright and dark regions (e.g., a pixel sequence from edge to edge, random sampling of pixels from both regions, etc.). Furthermore, pixels may be continuously sampled with respect to one or more working electrode zones to determine the ECL generation curve over time. This sampled data may then be used to extrapolate the ECL generation curve with respect to points where saturation is exceeded.

[0179] 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., duration), and / or waveform type (e.g., square, sawtooth, etc.). The use of different pulse waveforms may be advantageous when multiple types of electroactive species that require different activation potentials and can emit light at different wavelengths are used as ECL labels. For example, such ECL labels may be complexes based on ruthenium, osmium, hassium, iridium, etc.

[0180] In operation 1808, process 1800 includes performing ECL analysis on first and second luminescence data. For example, a computer system 906 may perform ECL analysis on the luminescence data. These values ​​may be correlated with quantitative measurements (e.g., ECL intensity) to provide analog signals. 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 respect to both techniques and may be used to convert multiple digital signals to provide quantitative results. 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 for determining concentrations 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 different working electrode zones 104. The concentration gradient may consist of separate binding domains presenting different concentrations of the binding reagent.

[0181] In embodiments, a control assay solution or reagent, such as a lead buffer, may be used on the working electrode zone of well 200. The control assay solution or reagent may provide uniformity to each analysis to control signal fluctuations (e.g., fluctuations due to degradation, movement, aging, thermal shift, electronic circuit noise, and noise from the photodetector device of the multiwell plate 208). For example, multiple redundant working electrode zones 104 (containing the same binding reagent or different binding reagents specific to the same analyte) may be used for the same analyte. In other examples, an analyte of known concentration may be used, or the control assay solution or reagent may be covalently bound to a known amount of ECL label, or a known amount of ECL label in solution may be used.

[0182] In embodiments, the data collected and generated in process 1800 can be used for a variety of purposes. The collected and generated data may be stored in the form of a database consisting of, for example, 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 in contact with a human DNA sample can be used for signature DNA fingerprints that can 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., bacteria, viruses, etc.), etc.

[0183] In this embodiment, the process 1800 described above includes capturing luminescence data over two periods, but the process 1800 may be used to capture luminescence data over any number of periods, for example, three, four, five, etc. In this embodiment, different pulse waveforms may be used for some or all of the periods. In this embodiment, the pulse waveforms may differ in amplitude (e.g., voltage), duration (e.g., duration), and / or waveform type (e.g., square, sawtooth, etc.).

[0184] The above describes a typical flow of Process Example 1800. The process shown in Figure 18 is merely a typical example, and variations exist without departing from the scope of the embodiments disclosed herein. The steps may be performed in a different order than described, additional steps may be performed, and / or fewer steps may be performed.

[0185] In the embodiment, different configurations of pulse waveforms supplied by the voltage / current source 904 may be used together to improve the ECL emitted during ECL analysis. Figure 19 shows a flowchart illustrating another process 1900 for operating the ECL apparatus using pulse waveforms according to the embodiment of this specification.

[0186] In operation 1902, process 1900 includes applying a first voltage pulse to one or more working electrode zones 104 or auxiliary electrodes 102 in the well of the ECL apparatus, the first voltage pulse causing a first reduction-oxidation reaction in the well. In operation 1904, process 1900 includes capturing first luminescence data from the first reduction-oxidation reaction over a first period of time.

[0187] In operation 1906, process 1900 includes applying a second voltage pulse to one or more working electrode zones or auxiliary electrodes in the well, the second voltage pulse causing a second reduction-oxidation reaction in the well. In operation 1908, process 1900 includes capturing second luminescence data from the second reduction-oxidation reaction over a second period, the first period not having the same duration as the second period.

[0188] In embodiments, the voltage level (amplitude or magnitude) or pulse width (or duration) of the first and / or second voltage pulses may be selected to induce a first reduction-oxidation reaction, and the first luminescence data corresponds to the first reduction-oxidation reaction that occurs. In embodiments, the voltage level (amplitude or magnitude) or pulse width (or duration) of the first and / or second voltage pulses may be selected to induce a second reduction-oxidation reaction, and the second luminescence data corresponds to the second reduction-oxidation reaction that occurs. In embodiments, the magnitude of at least one of the first and second voltage pulses may be selected at least in part on the chemical composition of the counter electrode.

[0189] In operation 1910, process 1900 includes performing ECL analysis on the first and second luminescence data. For example, a computer system 906 may perform ECL analysis on the luminescence data. In some embodiments, the luminescence data, e.g., signals, emanating from a given target object on the bonding surface of the working electrode zone 104 and / or auxiliary electrode 102, e.g., the bonding domain, may have a range of values. These values ​​may be correlated with quantitative measurements (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 or not an analyte is present. Statistical analysis may be used with respect to both techniques and may be used to convert multiple digital signals to provide quantitative results. 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, e.g., techniques for determining concentrations by statistical analysis of bonding on a concentration gradient, may be used. Multiple linear data arrays with concentration gradients can be generated for multiple different specific binding reagents used in different wells 200 and / or different working electrode zones 104. The concentration gradients may consist of separate binding domains that present different concentrations of the binding reagent.

[0190] In embodiments, a control assay solution or reagent, such as a lead buffer, may be used on the working electrode zone of well 200. The control assay solution or reagent may provide uniformity to each analysis to control signal fluctuations (e.g., fluctuations due to degradation, movement, aging, thermal shift, electronic circuit noise, and noise from the photodetector device of the multiwell plate 208). For example, multiple redundant working electrode zones 104 (containing the same binding reagent or different binding reagents specific to the same analyte) may be used for the same analyte. In other examples, an analyte of known concentration may be used, or the control assay solution or reagent may be covalently bound to a known amount of ECL label, or a known amount of ECL label in solution may be used.

[0191] In embodiments, the data collected and generated in process 1900 can be used for a variety of purposes. The collected and generated data may be stored in the form of a database consisting of, for example, 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 in contact with a human DNA sample can be used for signature DNA fingerprints that can 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., bacteria, viruses, etc.), etc.

[0192] The above describes a typical flow of Process Example 1900. The process shown in Figure 19 is merely a typical example, and variations exist without departing from the scope of the embodiments disclosed herein. The steps may be performed in a different order than described, additional steps may be performed, and / or fewer steps may be performed.

[0193] 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 supplied to all working electrode zones 104 and / or auxiliary electrodes 102 in one or more wells 106 of a multiwell plate 108. Similarly, for example, a voltage pulse may be supplied to a selected (or "addressable") set of working electrode zones 104 and / or auxiliary electrodes 102 in one or more wells 106 of a multiwell plate 208 (e.g., per zone, per well, per sector (e.g., a group of two or more wells)).

[0194] The systems, devices, and methods described herein may be applied to a variety of situations. For example, the systems, devices, and methods may be applied to improve various aspects of ECL measurement and reader devices. Typical plate readers include those described above and throughout this application, for example, in paragraph

[0174] .

[0195] For example, by applying one or more voltage pulses to generate ECL as described herein, reading and / or exposure times can be improved by more rapid and efficient generation, collection, observation, and analysis of ECL data. Furthermore, improved exposure times (e.g., single exposure, double (or more) exposures using different (or equal) exposure times) contribute to improved ECL generation, collection, observation, and analysis by improving materials of interest that, in some embodiments, require individual periods to capture luminescence data, such as dynamic range expansion (DRE), binning, etc. Thus, emitted photons can be captured as ECL data over multiple different periods, which may be affected, for example, by the level of photosaturation during ECL generation. Dynamic range can be improved by implementing various multi-pulse and / or multi-exposure schemes. For example, short exposures may be followed by long exposures (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), a single well, two or more wells, or a sector, or two or more sectors, etc.). In these examples, using longer exposures may be advantageous as long as the exposure does not saturate. For example, when performing short and long exposures, if saturation occurs during the long exposure, that exposure may be discarded and the short exposure may be used. If neither exposure saturates, the longer exposure may be used to provide better sensitivity. In that case, for example, the short exposure may be used. By making these adjustments (manually or with the help of hardware, firmware, software, algorithms, computer-readable media, computing devices, etc.), the dynamic range may be improved, as detailed above.

[0196] Furthermore, the systems, devices, and methods described herein can be utilized in various ways to enable the optimization of software, firmware, and / or control logic for hardware equipment such as the reader described above. For example, the systems, devices, and methods described herein enable faster and more efficient generation, collection, observation, and / or analysis of ECL, so that the equipment can be optimized with improved software, firmware, and / or control logic, and the cost of hardware required to perform ECL analysis can be reduced (e.g., less expensive lenses, fewer and / or less expensive motors to drive the equipment). The examples provided herein are merely typical examples, and additional improvements to these equipment are also conceivable.

[0197] In the embodiments described above, the wells 200 of the multiwell plate 208 may contain one or more fluids (e.g., reagents) for performing ECL analysis. For example, the fluids may include ECL colactant (e.g., TPA), lead buffer, preservatives, additives, excipients, carbohydrates, proteins, detergents, polymers, salts, biomolecules, inorganic compounds, lipids, etc. In some embodiments, the chemical properties of the fluid in the wells 200 during the ECL process can alter the electrochemical / ECL generation. For example, the relationship between the ion concentration of the fluid and the electrochemical / ECL generation may depend on different types of liquids, lead buffers, etc. In embodiments, one or more auxiliary electrodes may provide a constant interfacial potential regardless of the current passing through them, as described above. That is, a current-vs-potential plot would result in an infinite current at a fixed potential.

[0198] In some embodiments, the fluid used (for example, in well 200 of a multiwell plate 208) may contain an ionic compound such as NaCl (e.g., a salt). In some embodiments, for example, a higher concentration of NaCl in the fluid contained in well 200 may improve the control of ECL generation through the ECL process. For example, a current-to-potential plot of an auxiliary electrode 102 having a redox pair such as 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 well 200. When Ag+ is reduced, it is necessary to balance the charge equilibrium in the redox pair of the auxiliary electrode 102, 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-to-potential curve, which, for example, affects the reference potential at the interface of the auxiliary electrode 102 containing Ag / AgCl with respect to the current passing through it. Thus, in embodiments, the concentration of ions such as salt can be modified and controlled to maximize the current generated with respect to the applied voltage.

[0199] In some embodiments, the volume of fluid in the well 200 during the ECL process can alter the electrochemical / ECL generation. In some embodiments, the relationship of the fluid volumes in the well 200 may depend on the design of the electrochemical cell 100. For example, working electrode zones 104 and auxiliary electrodes 102 separated by a relatively thick fluid layer may exhibit more ideal electrochemical behavior, such as a spatially consistent interfacial potential. Conversely, working electrode zones 104 and auxiliary electrodes 102 separated by a relatively thin fluid layer covering both may exhibit non-ideal electrochemical behavior due to a spatial gradient of the interfacial potential across both electrodes. In some embodiments, the design and layout of one or more working electrode zones 104 and one or more auxiliary electrodes 102 may maximize the spatial distance between the working electrode zones 104 and the auxiliary electrodes 102. For example, as shown in Figure 3A, the working electrode zones 104 and auxiliary electrodes 102 may be arranged to maximize the spatial distance D1. The spatial distance can be maximized by reducing the number of working electrode zones 104, reducing the exposed surface area of ​​the working electrode zones 104, and reducing the exposed surface area of ​​the auxiliary electrodes 102. Although not described, spatial distance maximization can be applied to the designs shown in Figures 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, and 8A-8D.

[0200] In embodiments, the multiwell plate 208 described above may form part of one or more kits for use in an assay apparatus when performing an assay, such as an ECL assay. The kit may include an assay module, such as the multiwell plate 208, and at least one assay component selected from a group consisting of conjugate reagents, enzymes, enzyme substrates, and other reagents useful for performing the assay. Examples include whole cells, cell surface antigens, intracellular particles (e.g., organelles or membrane fragments), viruses, prions, mites and their fragments, viroids, antibodies, antigens, haptens, fatty acids, nucleic acids (and synthetic analogs), proteins (and synthetic analogs), lipoproteins, polysaccharides, lipopolysaccharides, glycoproteins, peptides, polypeptides, enzymes (e.g., phosphorylases, phosphatases, esterases, transglutaminases, transferases, oxidases, reductases, dehydrogenases, glycosidases, protein-processing enzymes (e.g., proteases, kinases, protein phosphatases, ubiquitin-protein ligases, etc.), nucleic acids These include, but are not limited to, processing enzymes (e.g., polymerases, nucleases, integrases, ligases, helicases, telomerases, etc.), enzyme substrates (e.g., substrates of 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, luminescence labels (preferably electrochemiluminescence labels), electrochemical luminescence coreactants, pH buffers, blocking agents, preservatives, stabilizers, cleaning agents, degreasing agents, hygroscopic agents, lead buffers, etc. Such assay reagents may be unlabeled or labeled (preferably luminescence labeled, most preferably electrochemiluminescent labeled).In some embodiments, the kit may include an ECL assay module, for example, a multiwell plate 208, and at least one assay component selected from the group consisting of (a) at least one luminescence label (preferably an electrochemiluminescence label), (b) at least one electrochemical luminescence coreactant, (c) one or more binding reagents, (d) pH buffer, (e) one or more blocking reagents, (f) preservatives, (g) stabilizers, (h) enzymes, (i) washing agents, (j) desiccants, and (k) hygroscopic agents.

[0201] Figure 20 shows a flowchart illustrating a process 2000 for manufacturing a well containing a working electrode and an auxiliary electrode, according to an embodiment of this specification. For example, process 2000 may be used to manufacture one or more wells 200 of a multiwell plate 208 containing one or more working electrode zones 104 and one or more auxiliary electrodes 102.

[0202] In operation 2002, process 2000 includes forming one or more working electrode zones 104 on a substrate. In embodiments, one or more working electrodes may be formed using any type of manufacturing process, such as screen printing, three-dimensional (3D) printing, vapor deposition, lithography, etching, and combinations thereof. In embodiments, one or more working electrode zones 104 may be formed as a multilayer structure that can be vapor-deposited and patterned.

[0203] In embodiments, one or more working electrodes may be a continuous / seamless region in which a reaction may occur, and an electrode “zone” may be a portion (or all) of an electrode in which a reaction of particular interest occurs. In certain embodiments, a 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, a working electrode zone may be formed by individual working electrodes. In this example, a working electrode zone may be configured as a single working electrode formed of one or more conductive materials. In other examples, a working electrode may be formed by isolating a portion of a single working electrode. In this example, a single working electrode may be formed of one or more conductive materials, and a working electrode zone may be formed by electrically isolating a region ("zone") of the single working electrode using an insulating material, such as a dielectric. In any embodiment, a working electrode may be formed of any type of conductive material, such as metals, alloys, carbon compounds, and combinations of conductive and insulating materials.

[0204] In operation 2004, process 2000 includes forming one or more auxiliary electrodes 102 on a substrate. In embodiments, one or more auxiliary electrodes may be formed using any type of manufacturing process, e.g., screen printing, three-dimensional (3D) printing, vapor deposition, lithography, etching, and combinations thereof. In embodiments, the auxiliary electrodes 102 may be formed as a multilayer structure that can be vapor-deposited and patterned. In embodiments, one or more auxiliary electrodes may be formed of a chemical mixture that provides an interfacial potential during the reduction of the chemical mixture so that a quantifiable amount of charge is generated through a reduction-oxidation reaction occurring in a well. One or more auxiliary electrodes may include an oxidizing agent that supports the reduction-oxidation reaction, which may be used in biological, chemical, and / or biochemical assays and / or analyses, e.g., ECL generation and analysis. In embodiments, the amount of oxidizing agent in the chemical mixture of one or more auxiliary electrodes is greater than or equal to 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, e.g., ECL generation. In this regard, a sufficient amount of the chemical mixture in one or more auxiliary electrodes remains even after a redox reaction has occurred with respect to the initial biological, chemical, and / or biochemical assay and / or analysis, so that one or more additional redox reactions can occur through subsequent biological, chemical, and / or biochemical assays and / or analyses. In other embodiments, the amount of oxidizing agent in the chemical mixture of one or more auxiliary electrodes is at least partially based 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.

[0205] For example, one or more auxiliary electrodes may be formed from a mixture of silver (Ag) and silver chloride (AgCl), or other suitable metal / metal halide pairs. Examples of other chemical mixtures may include metal oxides with multiple metal oxidation states, such as manganese oxide, or other metal / metal oxide pairs, such as silver / silver oxide, nickel / nickel oxide, zinc / zinc oxide, gold / gold oxide, copper / copper oxide, platinum / platinum oxide, etc.

[0206] In operation 2006, the process includes forming an electrical insulating material for electrically insulating one or more auxiliary electrodes from one or more working electrodes. In embodiments, the electrical insulating material may be formed using any type of manufacturing process, such as screen printing, 3D printing, vapor deposition, lithography, etching, and combinations thereof. The electrical insulating material may include a dielectric.

[0207] In operation 2008, process 2000 includes forming additional electrical components on the substrate. In embodiments, one or more auxiliary electrodes may be formed using any type of manufacturing process, such as screen printing, 3D printing, vapor deposition, lithography, etching, and combinations thereof. Additional electrical components may include through holes, electrical traces, electrical contacts, etc. For example, through holes may be formed in the working electrode zone 104, the auxiliary electrode 102, and the layers or materials forming the electrical insulating material, so that electrical contacts with the working electrode zone 104 and the auxiliary electrode 102 can be made without causing short circuits with other electrical components. For example, one or more additional insulating layers may be formed on the substrate to support electrical traces that are through-connected while isolating the electrical traces.

[0208] In embodiments, additional electrical components may include electric heaters, temperature controllers, and / or temperature sensors. These electric heaters, temperature controllers, and / or temperature sensors may assist in electrochemical reactions, such as ECL reactions, and electrode performance may be temperature-dependent. For example, screen-printed resistance heaters may be integrated into the electrode design. Resistance heaters may be powered and controlled by built-in or external temperature controllers and / or temperature sensors. These are self-regulating and assembled to generate a specific temperature when a constant voltage is applied. Ink may assist in temperature control during assays or plate readouts. Ink (and / or heaters) may also be useful when a temperature increase is desired during an assay (for example, in assays using PCR components). Temperature sensors may be printed on electrodes (working and / or auxiliary electrodes) to provide actual temperature information.

[0209] Figures 21A to 21F show non-limiting examples of the process for forming a working electrode zone 104 and an auxiliary electrode 102 in one or more wells 200 according to embodiments of this specification. Although Figures 21A to 21F show the formation of two wells (as shown in Figure 22A), those skilled in the art will understand that the process shown in Figures 21A to 21F can be applied to any number of wells 200. Also, although Figures 21A to 21F show the formation of the auxiliary electrode 102 and the working electrode zone 104 in an electrode design similar to the electrode design 701 shown in Figures 7A to 7F, those skilled in the art will understand that the process shown in Figures 21A to 21F can be used in the electrode designs described herein.

[0210] The process for manufacturing the auxiliary electrode 102, the working electrode zone 104, and other electrical components may be carried out using a screen printing process, as described later, in which various materials are formed using ink or paste. In embodiments, the auxiliary electrode 102 and the working electrode zone 104 may be formed using any type of manufacturing process, such as 3D printing, vapor deposition, lithography, etching, and combinations thereof.

[0211] As shown in Figure 21A, the first conductive layer 2102 can be printed on the substrate 2100. In embodiments, the substrate 2100 can 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 can 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, and conductive alloys. 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, graphite 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.

[0212] The substrate 2100 may include one or more through-holes or other types of electrical connections (e.g., traces, electrical contacts) for connecting components of the substrate 2100 and providing locations where electrical connections can be made to 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 possible. For example, through-holes may be formed in the working electrode zone 104, the auxiliary electrode 102, and the layers or materials forming the electrical insulating material, so that electrical contacts with the working electrode zone 104 and the auxiliary electrode 102 can be made without causing short circuits with other electrical components. For example, one or more additional insulating layers may be formed on the substrate to support electrical traces that are through-and coupled while isolating the electrical traces.

[0213] As shown in Figure 21B, the second conductive layer 2108 can be printed on the first conductive layer 2102. In embodiments, the second conductive layer 2108 can be formed from a mixture of silver (Ag) and silver chloride (AgCl), or from a chemical mixture containing other suitable metal / metal halide pairs. Other examples of chemical mixtures may include metal oxides as described above. In some embodiments, the second conductive layer 2108 can be formed to approximate the dimensions of the first conductive layer 2102. In some embodiments, the second conductive layer 2108 can be formed to dimensions greater than or smaller than the first conductive layer 2102. The second conductive layer 2108 can be formed by printing the second conductive layer 2108 using an Ag / AgCl chemical mixture (e.g., ink, paste, etc.) having a defined Ag to AgCl ratio. In embodiments, the amount of oxidizing agent in the auxiliary electrode chemical mixture is at least partially based on the Ag to AgCl ratio in the auxiliary electrode chemical mixture. In embodiments, the chemical mixture of the auxiliary electrode having Ag and AgCl comprises about 50 percent or less, for example, 34 percent, 10 percent AgCl. 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.

[0214] As shown in Figure 21C, the first insulating layer 2110 can be printed on the second conductive layer 2108. The first insulating layer 2110 can be formed from any kind of mixed circular material, such as a dielectric, polymer, glass, etc. The first insulating layer 2110 may form two auxiliary electrodes 102 by being formed in a pattern that exposes two portions ("spots") of the second conductive layer 2108. The exposed portions may correspond to the desired shape and size of the auxiliary electrodes 102. In embodiments, the auxiliary electrodes 102 can be formed in any number, size, and shape as described in the electrode designs described above with reference to, for example, Figures 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, 8A-8D, and 38A-39E.

[0215] As shown in Figures 21D and 21E, a third conductive layer 2112 may be printed on the insulating layer 2110, followed by a fourth conductive layer 2114 printed on the third conductive layer 2112. In embodiments, the third conductive layer 2112 may be formed of a metal, such as Ag. In embodiments, the fourth conductive layer 2114 may be formed of a composite material, such as a carbon composite. Other examples of the first conductive layer 2102 may include metals such as gold, silver, platinum, nickel, steel, iridium, copper, aluminum, and conductive alloys. 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, graphite 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 above in the electrode design with reference to, for example, Figures 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, 8A-8D, and 38A-39E.

[0216] As shown in Figure 21F, a second insulating layer 2116 can be printed on the fourth conductive layer 2114. The second insulating layer 2116 can be formed of any type of insulating material, such as a dielectric. The second insulating layer 2116 may be formed in a pattern that exposes 20 portions ("spots") of the fourth conductive layer 2114, thereby forming 10 working electrode zones 104 for each well 200, as shown in Figure 22A. The second insulating layer 2116 may be formed to expose auxiliary electrodes 102. Thus, the printing or deposition of the second insulating layer 2116 can control the size and / or area of ​​the working electrode zones 104, as well as the size and / or area of ​​the auxiliary electrodes 102. The exposed portions may correspond to the desired shape and size of the working electrode zones 104 and auxiliary electrodes 102. In embodiments, the working electrode zone 104 may be formed in any number, size, and shape as described in the electrode design described above with reference to, for example, Figures 3A-3F, 4A-4F, 5A-5C, 6A-6F, 7A-7F, 8A-8D, and 38A-39E. In certain embodiments, one or more of the described layers may be formed in a specific order to minimize contamination of the layers (e.g., carbon-based layers).

[0217] In the method described above, conductivity between the auxiliary electrodes 102 is maintained by a conductive layer 2108 masked by an insulating layer 2110. This design allows conductive connections between the auxiliary electrodes 102 to pass beneath the working electrode zone 104. Figure 22B shows a further embodiment of the well 200, which is manufactured by a manufacturing method somewhat similar to that described above with respect to Figures 21A-F and 22A. As shown in Figure 22B, the working electrode zone 104 may be arranged in a circular pattern with gaps, for example, in a C-shape. Each well 200 may have, for example, 10 working electrode zones. In further embodiments, any suitable number of working electrode zones may be included. The gaps in the pattern of the working electrode zone 104 allow a conductive trace 2120 to pass between the auxiliary electrodes 102 of two wells 200. Since the conductive trace 2120 passes between the auxiliary electrodes 102 and does not straddle them, the auxiliary electrodes 102, the working electrode zone 104, and the conductive trace 2120 can be printed on the same layer during the manufacturing process. For example, in an embodiment including individually addressable working electrode zones 104, each of the auxiliary electrodes 102, working electrode zones 104, and conductive traces 2120 may be printed as individual features on the same layer of the substrate. The C-shaped electrode design shown in Figure 22B is not limited to use in a double-well layout. Other layouts including different numbers of wells are also consistent with the 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, and may have multiple conductive traces 2120 connecting the auxiliary electrodes 102 of each well 200 in the layout.

[0218] Figures 24A-24C, 25A-25C, 26A-26D, 27A-27C, 28, and 29 show the test results performed on various multiwell plates according to the embodiments of this specification. The tests included two different test lots. Each of the two different test lots included four different configurations of multiwell plates: Standard ("Std") 96-1 plate, Std 96ss plate (small spot plate), Std 96-10 plate, and Std 96ss "BAL". The Std 96-1 plate, as shown in Figure 23A, contains 96 wells 106, with one working electrode zone in each well 106. The Std 96ss plate, as shown in Figure 23B, contains 96 wells 106, with one working electrode zone in each well 106. The Std 96-10 plate, as shown in Figure 23C, contains 96 wells 106, with each well 106 having 10 working electrode zones. The Std 96ss "BAL" has two auxiliary electrodes and a single working electrode zone, as shown in Figure 23D. In each test lot, three sets of multi-well plates of each configuration were screen printed using different Ag / AgCl inks to produce different ratios of Ag / AgCl chemical mixtures, as shown in Table 8. Each of the plates described above was configured to have two auxiliary electrodes per well. The "BAL" configuration was configured to have auxiliary lines of smaller dimensions compared to the other configurations. [Table 23]

[0219] This test also included a presentation control, which included a working electrode zone and counter electrode made of carbon, labeled as the presentation control in the figure.

[0220] Tests were performed with test solutions using the electrode design described above to generate voltammetry, ECL trace (ECL intensity versus applied potential difference), and integrated ECL signal measurements. The test solutions included three solutions: a 1 μM TAG solution in T1x (TAG refers to an ECL label or species that emits photons when electrically excited), a 1 μM TAG solution in T2x, and MSD Free TAG 15,000 ECL (Y0260157). The 1 μM TAG solution in T1x included a stock solution of 5.0 mM Tris(2,2'-bipyridine)ruthenium(II) chloride (Y0420016) and MSD T1x (Y0110066). The 1 μM TAG solution in T2x contained 5.0 mM Tris(2,2'-bipyridine)ruthenium(II) chloride stock solution (Y0420016) and MSD T2x (Y0200024). The test solution also contained a read buffer solution containing MSD T1x (Y0110066). Measurements were performed under the following conditions for voltammetry, ECL tracing, and Free TAG 15,000 ECL testing and MSD T1x ECL signal.

[0221] Voltammetry was measured using a standard three-electrode configuration (working electrode, reference electrode, and counter electrode) on one plate of each Ag / AgCl ink, and one plate from the Std 96-1, Std 96ss, and Std 96-10 inventory. Reducing voltammetry was measured on the counter electrode. For reducing voltammetry, wells were filled with 150 μL of 1 μM TAG in T1x or 1 μM TAG in T2x and allowed to stand for at least 10 minutes. Waveforms were applied to the Ag / AgCl plates from 0.1V to 1.0V and then to 0.1V at 100 mV / s. Waveforms were applied to the presentation control from 0V to 3V and then to 0V at 100 mV / s. Three replicate wells of each solution were measured and averaged.

[0222] Oxidative voltammetry was measured at the working electrode. For oxidative voltammetry, wells were filled with 150 μL of 1 μM TAG in T1x or 1 μM TAG in T2x and allowed to stand for at least 10 minutes. Waveforms were applied to Ag / AgCl from 0V to 2V and then to 0V at 100 mV / s. Waveforms were applied to the presentation control from 0V to 2V and then to 0V at 100 mV / s. Three replicate wells of each solution were measured and averaged.

[0223] For ECL traces, one plate was measured for each Ag / AgCl ink, and one plate was measured from the Std 96-1, Std 96ss, and Std 96-10 inventory. Six wells were filled with 150 microliters (μL) of 1 micromolar (μM) TAG in T1x, and six wells were filled with 1 mM TAG in T2x. The plates were allowed to stand for at least 10 minutes. ECL was measured using a proprietary video system with the following parameters: Ag / AgCl: 0V–3000mV at 3000ms, using 120 consecutive 25ms frames (e.g., image exposure length), and presentation control: 2000mV–5000mV at 3000ms, using 25ms frames. Six replicated wells of each solution were averaged with respect to ECL intensity versus potential and current versus potential.

[0224] For the integrated ECL signal, six plates of each AgCl, and six plates from the Std 96-1, Std 96ss, and Std 96-10 inventory were measured, two plates being MSD T1x and four plates being "Free TAG 15,000 ECL". Each plate was filled with 150 μL of either "Free TAG 15,000 ECL" or MSD T1x and allowed to stand for at least 10 minutes. ECL was measured using a MESO QUICKPLEX SQ 120 instrument ("SQ120") with a waveform of 0V–3000mV at 3000ms for AgCl. ECL was also measured using the SQ120 with a waveform of 2000mV–5000mV at 3000ms for the presentation control. Intra-plate and inter-plate values ​​were calculated. The test results are described below.

[0225] Figures 24A-24C show the results of ECL measurements performed on the Std 96-1 plate. Figure 24A is a graph showing the voltammetry measurements for the Std 96-1 plate. In particular, Figure 24A shows the average voltammogram for the Std 96-1 plate. As shown in Figure 24A, an increase in current occurred between the T1x and T2x solutions. The oxidation curves were similar for the three Ag / AgCl ink plates and the control plate. The onset of oxidation was approximately 0.8V vs. Ag / AgCl. The peak potential was approximately 1.6V vs. Ag / AgCl. A shift in reduction occurred when CE was changed from carbon to Ag / AgCl. The onset of reduction of water at carbon was approximately -1.8V vs. Ag / AgCl. The onset of reduction of AgCl was approximately 0V vs. Ag / AgCl. An increase in the total reduction of AgCl occurred with increasing AgCl content in the Ag / AgCl ink. A small shoulder appeared at -0.16V in the reduction voltammetry with Ag / AgCl, and the current increased between the T1x and T2x solutions. These results indicate that increasing the concentration of the lead buffer from T1x to T2x increases the oxidation current. By incorporating AgCl into the auxiliary electrode, the onset of reduction shifted to the expected 0V relative to the carbon reference electrode. Increasing the amount of AgCl in the ink increased the total amount of AgCl reduction without affecting the slope of the current-potential curve.

[0226] Figures 24B and 24C are graphs showing ECL measurements for the Std 96-1 plate. In particular, Figures 24B and 24C show the average ECL and current traces for the Std 96-1 plate with either the T1x or T2x solution, as shown in Figure 24A. As illustrated, the three Ag / AgCl ink plates yielded similar ECL traces. The onset of ECL occurred at ca. 1100 mV in both the T1x and T2x solutions. Peak potentials occurred at 1800 mV in the T1x solution and 1900 mV in the T2x solution. The ECL intensity returned to baseline at ca. 2500 mV. The three Ag / AgCl ink plates yielded similar current traces, except for a drop in current at the ink ratio 1 (90 / 10 Ag:AgCl) with T2x at the end of the waveform. On the production plate, the onset of ECL shifted to approximately 3100 mV, and the peak potential shifted to approximately 4000 mV. The relative shift in ECL on the production plate corresponded to the shift in the onset of the reduction current measured by reference voltammetry. The total width of the ECL trace at the half-maximum on the production plate was wider than on the Ag / AgCl ink plate, which correlated with a lower slope of the reduction current measured by reference voltammetry.

[0227] As shown in Figure 24C, the total current flowing during the waveform with a 90:10 ratio is smaller than that for other inks. This indicated that the 90:10 ratio could limit the amount of oxidation that could occur at the working electrode. For experiments where more current could flow than in the FT of T2x using this waveform, a 50:50 ratio was chosen to ensure sufficient reducing capacity. As demonstrated by the tests, the Ag / AgCl ink provides a controlled potential for reduction at the auxiliary electrode 102. With Ag / AgCl, the auxiliary electrode 102 shifts the ECL reaction to a potential where TPA oxidation occurs when measured using a true Ag / AgCl reference electrode.

[0228] Regarding the auxiliary electrode 102, the amount of AgCl accessible within the auxiliary electrode 102 must be such that it is not completely consumed during the ECL measurement. For example, 1 mole of AgCl is required for every 1 mole of electrons passing through during oxidation at the working electrode. If the amount of AgCl is less than this, a loss of control of the interfacial potential in the working electrode zone 104 will occur. Loss of control refers to a situation in which the interfacial potential is not maintained within a specific range during the chemical reaction. One purpose of having a controlled interfacial potential is to ensure consistency and reproducibility of readings between wells, between plates, between screen lots, etc.

[0229] Table 10 shows the intra-plate and inter-plate FT and T1x values ​​of the Std 96-1 plate determined from ECL measurements. As shown in Table 10, the three Ag / AgCl ink plates yielded comparable values. The production plate yielded higher FT and T1x ECL signals. These higher signals may be due to a decrease in the effective run plate due to a lower slope of the reducing voltammetry. [Table 24]

[0230] Figures 25A-25C show the results of ECL measurements performed on Std96ss plates. Figure 25A is a graph showing voltammetry measurements for Std96ss plates. In particular, Figure 25A shows the average voltammogram of Std96ss plates. As shown in Figure 25A, an increase in current occurred between T1x and T2x solutions. The oxidation curves were similar for the three Ag / AgCl ink plates and the control plate. Oxidation initiation occurred at approximately 0.8V vs. Ag / AgCl. The peak potential occurred at approximately 1.6V vs. Ag / AgCl. A shift in reduction occurred when the auxiliary electrode was changed from carbon to Ag / AgCl. Reduction of water in carbon initiation occurred at approximately -1.8V vs. Ag / AgCl. Reduction of AgCl initiation occurred at approximately 0V vs. Ag / AgCl. The total reduction of AgCl increased with increasing AgCl content in the Ag / AgCl ink. In the reduction voltammetry with Ag / AgCl, a small shoulder appeared at -0.16V, and the current increased between the T1x and T2x solutions.

[0231] Figures 25B and 25C are graphs showing ECL measurements for Std96ss plates. In particular, Figures 25B and 25C show the average ECL and current traces for Std96ss plates with either T1x or T2x solution, as shown in Figure 10A. As illustrated, the three Ag / AgCl ink plates yielded very similar ECL traces. ECL onset occurred at approximately 1100mV in both T1x and T2x solutions. Peak potential occurred at 1675mV in the T1x solution and 1700mV in the T2x solution. ECL intensity returned to baseline at approximately 2175mV. The three Ag / AgCl ink plates yielded similar current traces. In the production plate, ECL onset shifted to approximately 3000mV, and peak potential shifted to approximately 3800mV. The relative shift of the ECL on the production plate corresponded to the shift in the onset of the reduction current measured by reference voltammetry. The total width of the ECL trace at the half-maximum on the production plate was wider than that of the Ag / AgCl ink plate, which correlated with a lower slope of the reduction current in reference voltammetry. The results shown in Figures 25A–25C are consistent with those in Figures 24A–24C, indicating that the changes resulting from the use of the Ag / AgCl electrode are robust across different electrode configurations.

[0232] Table 11 shows the intra-plate and inter-plate FT and T1x values ​​for Std96ss plates determined from ECL measurements. As shown in Table 11, the three Ag / AgCl ink plates yielded comparable values. The production plate yielded higher FT and T1x ECL signals. These higher signals may be due to a decrease in the effective run plate due to a lower slope of the reducing voltammetry. The higher background signal in the production plate may be due to a non-standard waveform in the reader used in that experiment. [Table 25]

[0233] Figures 26A-26D show the results of ECL measurements performed on Std96ss BAL plates. Figure 26A is a graph showing voltammetry measurements for Std96ss BAL plates. In particular, Figure 26A shows the average voltammogram of Std96ss BAL plates. As shown in Figure 26A, an increase in current occurred between the T1x and T2x solutions. The oxidation curves were similar for the three Ag / AgCl ink plates and the presenting control. The onset of oxidation occurred at approximately 0.8V vs. Ag / AgCl. The peak potential occurred at approximately 1.6V vs. Ag / AgCl. With increasing AgCl content in the Ag / AgCl ink, an increase in the total reduction of AgCl occurred. A small shoulder occurred at -0.16V in the reduction voltammetry in Ag / AgCl, and the current increased between the T1x and T2x solutions. The overall auxiliary electrode current was reduced compared to the Std96ss plate configuration due to the smaller electrode area. The slope of the current-to-potential plot was smaller than that of the Std96ss plate configuration.

[0234] Figure 26B is a graph showing Std96ss versus Std96ss BAL with a T2x solution at an ink ratio of 3. As shown in Figure 26B, the oxidation peak current (approximately -0.3 mA) was similar for both of these formats. For most of the reduction current, Std96ss BAL was at a higher negative potential than Std96ss.

[0235] Figures 26C and 26D are graphs showing ECL measurements for Std96ss BAL plates. In particular, Figures 26C and 26D show the mean ECL and current traces of Std96ss BAL plates with either T1x or T2x solution. As shown, the three plates with Ag / AgCl counter electrodes yielded similar ECL traces. ECL onset occurred at approximately 1100mV in both T1x and T2x solutions. Peak potential occurred at 1750mV in the T1x solution and 1800mV in the T2x solution. ECL intensity returned to baseline at approximately 2300mV. While ECL onset was similar to the Std96ss plate, the peak potential and return to baseline shifted to later potentials than the Std96ss plate. The difference between the Std96ss plate and the Std96ss BAL plate may be due to a decrease in the effective ramp plate due to a lower slope of reducing voltammetry at the small counter electrode. The three plates with Ag / AgCl counter electrodes yielded similar current traces, except for a decrease in current at 90 / 10 Ag:AgCl with T2x solution at the end of the waveform. Different behavior of ink ratio 1 with T2x solution was also observed in the Std96-1 plate format. The results shown in Figures 26A–26D are consistent with the results in Figures 24A–24C and 25A–25C, demonstrating that the changes resulting from the use of Ag / AgCl electrodes are robust across different electrode configurations.

[0236] Table 12 shows the intra-plate and inter-plate FT and T1x values ​​for Std96ss BAL plates determined from ECL measurements. As shown in Table 12, the ECL signal is higher than that of the Std96ss plate configuration. This higher signal may be due to a decrease in the effective ramp plate due to a smaller slope of reducing voltammetry at the small counter electrode. A decrease in the FT signal occurred with increasing AgCl content in the ink. [Table 26]

[0237] Figures 27A-27C show the results of ECL measurements performed on Std96-10 plates. Figure 27A is a graph showing voltammetry measurements for Std96-10. In particular, Figure 27A shows the average voltammogram of the Std96-10 plate. As shown in Figure 27A, an increase in current occurred between the T1x and T2x solutions. The oxidation curves were similar for the three plates with Ag / AgCl counter electrodes and for the presentation control. Oxidation initiation occurred at approximately 0.8V vs. Ag / AgCl. The peak potential occurred at approximately 1.6V vs. Ag / AgCl. A higher oxidation current was present in the presentation control. When the auxiliary counter electrode was changed from carbon to Ag / AgCl, a shift to reduction occurred. Reduction initiation of water occurred at approximately -1.8V vs. Ag / AgCl. Reduction initiation of AgCl occurred at approximately 0V vs. Ag / AgCl. As the AgCl content of the Ag / AgCl ink increased, the total amount of AgCl reduced also increased. In the reduction voltammetry with Ag / AgCl, a small shoulder appeared at -0.16V, and the current increased between the T1x and T2x solutions.

[0238] Figures 27B and 27C are graphs showing ECL measurements for Std96-10 plates. In particular, Figures 27B and 27C show the mean ECL and current traces for Std96-10 plates with either T1x or T2x solution. As shown, the three plates with Ag / AgCl counter electrodes yielded similar ECL traces. ECL onset occurred at approximately 1100 mV in both T1x and T2x solutions. Peak potential occurred at 1700 mV in the T1x solution and 1750 mV in the T2x solution. ECL intensity returned to baseline at approximately 2250 mV. The three plates with Ag / AgCl counter electrodes yielded similar current traces. In the production plate, ECL onset shifted to approximately 3000 mV, and the peak potential shifted to approximately 3800 mV. The relative shift in ECL in the production plate corresponded to the shift in the onset of the reduction current measured by reference voltammetry. The full width of the half-maximum ECL trace on the production plate was wider than in the case of Ag / AgCl ink, which correlated with a lower slope of the reduction current in the reference voltammetry. The results shown in Figures 27A–27C are consistent with the results in Figures 24A–24C, 25A–25C, and 26A–26D, indicating that the changes resulting from the use of the Ag / AgCl electrode are robust across different spot sizes.

[0239] Table 13 shows the intra-plate and inter-plate FT and T1x values ​​for Std96-10 plates determined from ECL measurements. As shown in Table 13, the three plates with Ag / AgCl counter electrodes yielded comparable values. The production plate yielded lower FT and T1x ECL signals. The reason for the lower signal in the production plate is unknown, but it may be related to the high oxidation current measured by reference voltammetry. [Table 27]

[0240] As shown in the test results described above and in Figure 28, the auxiliary electrode with Ag / AgCl shifted the ECL in the non-reference system to a potential corresponding to the oxidation measured in the reference system, i.e., a system with another reference electrode. In the case of the auxiliary electrode composed of Ag / AgCl, the onset of ECL occurred at a potential difference of 1100 mV. The peak of ECL occurred at potential differences (plate type average) of 1833 mV for the Std96-1 plate, 1688 mV for the Std96ss plate, 1775 mV for the Std96ss BAL plate, and 1721 mV for the Std96-10 plate. The onset of the oxidation current occurred at 0.8 V versus Ag / AgCl. The peak oxidation current occurred at approximately 1.6 V versus Ag / AgCl.

[0241] Furthermore, as shown by the test results, the three ink formulations were tested within a certain range of Ag:AgCl ratios, and variations in the amount of AgCl were detectable in the referenced reduced voltammetry. All three formulations yielded comparable ECL traces. When ECL was measured in T2x solution, there were some differences in the current-to-potential plots. Current capacity appeared to be limited for Std96-1 and Std96ss BAL with an Ag:AgCl ratio of 90 / 10, and these plate types had the largest working electrode-to-counter electrode area ratio. The FT signals were comparable for all three formulations except the 96ss BAL plate type.

[0242] In the above example, the working electrode area of ​​the Std96-1 plate is 0.032171 square inches. The working electrode area of ​​the Std96ss plate is 0.007854 square inches. The auxiliary electrode area of ​​Std96-1 and Std96sspr was estimated to be 0.002464 square inches. The auxiliary electrode area of ​​the Std96ss BAL plate was designed to be 0.0006459 square inches. The area ratios may be Std96-1:12.16, Std96ss:2.968, and Std96ss BAL:12.16. The ratio of peak reduction currents in the Std96ss plate and the Std96ss BAL plate indicates that the auxiliary electrode area of ​​the Std96ss BAL plate was reduced to 0.0007938 square inches. The ECL trace suggests that this reduction in counter electrode area is a necessary approach to unify the traces on Std96-1 and Std96ss BAL plates.

[0243] Example 4: Effect of the area ratio between the working electrode and the auxiliary electrode on the performance of the Ag / AgCl auxiliary electrode

[0244] Four different multi-well plate configurations were tested, each having a different ratio of working electrode to auxiliary electrode area in each well, as shown by the exposed working electrode area 10 and auxiliary electrode area 102 in the electrode patterns shown in Figures 23A-D. The first "Std96-1 plate" (Figure 23A) has wells with large working electrode areas, bounded by two auxiliary electrode strips (defined by dielectric ink patterned on the working electrode), and has the same electrode configuration as the plates used in Examples 2 and 3. The second "Std96ss plate" (Figure 23B) is similar to the first, except that the dielectric ink on the working electrode area is patterned to expose only a small circular exposed working electrode area in the center of the well (providing a small spot or "ss" area). The third "Std96-10" (Figure 23C) is similar to the first, except that the dielectric ink on the working electrode area exposes 10 small circular exposed working electrode areas, providing a "10-spot" pattern of working electrode area in each well. The fourth "Std96ss BAL" (Figure 23D) has a smaller exposed working electrode area than the Std96ss pattern, but the ratio of the working electrode area to the counter electrode area is similar to that of the Std96-1 configuration, and the area of ​​the exposed auxiliary electrode is significantly reduced so as to maintain balance between these areas. For each configuration, the total area of ​​the exposed working electrode, the total area of ​​the exposed auxiliary electrode, and the area ratio of the working electrode to the counter electrode are provided in Table 14. To evaluate the effect of Ag / AgCl ink on auxiliary electrode performance, each electrode configuration was manufactured using auxiliary electrodes made with three different inks having different Ag to AgCl ratios as described in Table 15. The Std96-1, Std96ss, and Std96-10 configurations were also compared to "control" or "presentation control" plates (MSD96 well, MSD96 Well Small Spot, and MSD 96 Well 10 Spot Plates, Meso Scale Diagnostics, LLC), which are similar plates that have a conventional carbon ink counter electrode instead of an Ag / AgCl auxiliary electrode. [Table 28] [Table 29]

[0245] Different electrode configurations were evaluated by cyclic voltammetry in the presence of ECL read buffers (MSD read buffer T at 1x and 2x nominal working concentration), and by the use of tris(2,2'-bipyridine)ruthenium(II) chloride ("TAG") solution in these read buffers for ECL measurements. Voltammetry was performed using a standard three-electrode configuration (working electrode, reference electrode, and counter electrode) with a 3M KCl Ag / AgCl reference electrode. Oxidation of the ECL read buffer at working electrode 104 was measured by circulating from 0V to 2V at a scan rate of 100mV / s, using working electrode 104 and auxiliary electrode 102 as the working electrode and counter electrode for voltammetry, respectively. Reduction of the ECL read buffer at auxiliary electrode 102 was measured by circulating from -0.1V to -1V at a scan rate of 100mV / s, using auxiliary electrode 102 and working electrode 104 as the working electrode and counter electrode for voltammetry, respectively. To measure the reduction of the ECL read buffer at the carbon counter electrode in the "control" plate, a wider voltage range was required, and the voltage circulated from 0V to -3V at a scan rate of 100mV / s. The wells were filled with 150μL of ECL read buffer and allowed to stand for at least 10 minutes before voltammetry measurements. Each solution was measured in triple wells, and the voltammetry data were averaged.

[0246] The integrated ECL signal of the TAG solution was measured using a MESO QUICKPLEX SQ 120 instrument ("SQ120") with waveforms of a 0V to 3000mV ramp over 3000ms (for the test plate with the Ag / AgCl auxiliary electrode) and a 2000mV to 5000mV ramp over 3000ms (for the control plate with the carbon ink counter electrode). All wells were filled with 150 μL of MSD Free Tag ("FT", TAG solution in MSD read buffer T 1X designed to provide approximately 15,000 signals within the ECL signal unit of the SQ120 instrument), and the plates were allowed to stand for at least 10 minutes. Two duplicate plates of T1x (96 wells per plate) were used to measure the background signal in the absence of TAG, and four duplicate plates of FT were used to measure the ECL signal generated from TAG. After normalization with respect to the area of ​​the exposed working electrode, a value proportional to the integrated ECL intensity over the duration of the applied waveform is reported by the instrument. For each solution and electrode configuration, the intra-plate and inter-plate mean and standard deviation were calculated for the entire well.

[0247] To measure ECL intensity as a function of time during ECL measurement, ECL measurements were performed from TAG solutions using an improved MSD plate reader with a proprietary video system. The same waveforms and procedures as for integrated signal measurement were used, but ECL was imaged as a series of continuous 120×25ms frames captured over a 3000ms waveform, and a higher density TAG (1μM TAG in MSD read buffers T1X and 2X) was used. Each frame was background-corrected using images captured before the start of the waveform. The ECL intensity of each exposed working electrode area (or "spot") in the image was calculated by summing the intensities measured for each pixel within the area defined by the spot. For images with multiple spots in a well, the intensity values ​​of the spots in the well were averaged. The instrument also measured the current passing through the well as a function of time during the ECL experiment. For each solution and electrode configuration, the mean and standard deviation of ECL intensity and current were calculated based on data from six replicated wells.

[0248] Voltammetry data for Std96-1, Std96ss, Std96ss BAL, and Std96-10 plates are shown in Figures 24A, 25A, 26A, and 27A, respectively. The oxidation current at the working electrode 104 in this three-electrode configuration is largely independent of the properties of the auxiliary or counter electrode, and in all examples, oxidation of the lead buffer begins at approximately 0.8 V, with the peak current occurring at approximately 1.6 V. The oxidation current increases from 1X lead buffer to 2X lead buffer with increasing concentration of tripropylamine ECL coriactant, and the peak and integrated oxidation currents increase roughly proportionally to the exposed working surface area (as shown in Table 14). The slight differences observed between the currents of the test plate and the control plate in some examples are likely related to differences in the carbon ink batches used to manufacture the working electrodes.

[0249] The reduction current measured with the auxiliary electrode or counter electrode 102 showed an initiation of reduction at approximately 0V for the Ag / AgCl auxiliary electrode (related to the reduction of AgCl to Ag), compared to approximately 3100mV for the carbon ink counter electrode (likely related to the reduction of water). An increase in the slope of the current initiation and the total integrated current was observed with 2X lead buffer T compared to 1X concentration, but this increase was small and may be related to the high ion concentration at 2X. For a given combination of Ag / AgCl ink and lead buffer formulations, the reduction current measured with auxiliary electrodes in Std96-1, Std96ss, and Std96-10 electrode configurations was largely independent of the electrode configuration, as the shape and dimensions of the auxiliary electrodes in these configurations were identical. As the proportion of AgCl in the Ag / AgCl ink increased from 10% (ratio 1) to 34% (ratio 2) and 50% (ratio 3), the slope of the reduction initiation potential and reduction initiation current did not change significantly, indicating that the electrode potential is relatively low-sensitivity to the proportion of AgCl. However, as AgCl increases, the peak potential shifts to the negative side, and the integrated current increases roughly in proportion to the proportion of AgCl in the ink, indicating that an increase in AgCl is associated with an increase in reducing ability. Comparing the reducing current in 96ss versus 96ss BAL configurations (Figure 26B), the shape and peak potential are roughly the same, but the peak and integrated current of the 96ss BAL decrease roughly in proportion to the decrease in auxiliary electrode area.

[0250] The ECL intensity from 1 μM TAG in MSD read buffer T 1X is provided as a function of applied potential in Figures 24B, 25B, 26C, and 27B for Std96-1, Std96ss, Std96ss BAL, and Std96-10 electrode configurations, respectively. Similar plots for 1 μM TAG in MSD read buffer T 2X are provided in Figures 24C, 25C, 26D, and 27C, respectively. All plots also provide plots of the associated current through the electrodes as a function of potential. In each test electrode configuration, the ECL traces generated using auxiliary electrodes with three different Ag / AgCl ink formulations were generally superimposed, demonstrating that even the lowest AgCl percentage (10%) Ag / AgCl formulation had sufficient reducing capacity to complete ECL generation. For the measurement of TAG in MSD read buffer T 1X with Ag / AgCl, the current traces were also generally superimposed. However, with the MSD lead buffer T 2X, particularly in the configurations with the lowest ratio of Ag / AgCl auxiliary electrode area to working electrode area (96-1 and 96ss BAL configurations), the current measured using the ink with the lowest AgCl content diverged at high potentials, showing a decrease in current with increasing potential. This divergence occurred at a potential near the endpoint of the ECL peak and therefore did not significantly affect the ECL trace, but it suggests that 10% AgCl ink is close to the boundary of sufficient reducing capacity to complete ECL generation using the selected waveform, lead buffer, and electrode configuration.

[0251] Subtle changes in the peak shape in the ECL trace were observed with variations in electrode configuration. For all configurations and both lead buffer concentrations, ECL formation initiated at approximately 3100 mV with a carbon ink counter electrode and 1100 mV with an Ag / AgCl auxiliary electrode. The initiation potential with the Ag / AgCl auxiliary electrode was significantly closer to the approximately 800 mV initiation potential observed in a three-electrode system with an Ag / AgCl reference electrode. While the initiation potential was relatively independent of the electrode configuration, slight differences were observed in the potential at which peak ECL intensity occurred. In the Std96-1 configuration, peak ECL with the Ag / AgCl auxiliary electrode occurred at approximately 1800 mV and 1900 mV for 1X and 2X lead buffer formulations, respectively. For the carbon counter electrode, the peaks were at 4000 and 4100 mV. As the ratio of the working electrode area to the auxiliary / counter electrode area decreased, the peak potential decreased. This effect occurs because the current required at the working electrode to achieve peak ECL can be achieved at a lower current density in the auxiliary / counter electrode, i.e., with a lower potential drop. In the Std96-10 configuration, peak ECL with the Ag / AgCl auxiliary electrode occurs at approximately 1700mV and 1750mV for 1X and 2X lead buffer formulations, respectively. In the Std96ss configuration, which has the lowest electrode area ratio, peak ECL with the Ag / AgCl auxiliary electrode occurs at approximately 1675mV and 1700mV for 1X and 2X lead buffer formulations, respectively. The shape of the ECL curve can be more consistent between configurations with varying working electrode areas by balancing the auxiliary electrode area to maintain a constant ratio. The Std96ss BAL configuration has the working electrode area of ​​the Std96ss configuration, but the auxiliary electrode area is reduced so that the electrode area ratio matches that of the Std96-1 configuration. In the Std96ss BAL configuration, peak ECL using the Ag / AgCl auxiliary electrode occurred at approximately 1750mV and 1800mV for 1X and 2X read buffer formulations, respectively, which is higher than the values ​​observed in the Std966 configuration and closer to the values ​​observed in the Std96-1 configuration.The difference in peak potential between the Std96-1 and Std96ss BAL configurations may simply indicate that the actual area ratio achieved during printing of the Std96ss plate may be lower than the target in the screen printing design. The ECL traces and currents of 1 μM TAGs in the MSD read buffer T 2X for the three electrode configurations are compared in Figure 28.

[0252] The integrated ECL signal results for Std96-1, Std96ss, Std96ss BAL, and Std96-10 electrode configurations are provided in Tables 16, 17, 18, and 19, respectively. Each table provides results for three different Ag / AgCl auxiliary electrode compositions and control carbon counter electrode conditions (Ag:AgCl=”n / a”). The tables provide the start potential (Vi), end potential (Vf), and duration (T) of the ramp waveform used for that condition, as well as the average integrated ECL signal measured with respect to the TAG solution (FT), and the background signal measured with respect to the base buffer used with the TAG solution (T1X) when TAG was absent. The coefficient of variation (CV) is also provided for variations within and between plates. Tables (16-19) show that the integrated signal was largely independent of the electrode configuration and auxiliary / counter electrode ink composition. No clear trend in CV associated with electrode configuration or composition was observed, and the conditions with the highest CV were generally associated with a single outlier well or plate. Despite sharing the same working electrode shape dimensions, a slightly higher signal was observed with the Std96ss BAL configuration than with the Std96ss configuration. The current required at the working electrode during ECL generation was higher with a smaller Std96ss BAL auxiliary electrode, and the auxiliary electrode was in the region of the current-voltage curve with a low slope (Figure 26B). As a final result, the effective voltage ramp rate at the working electrode was slower, increasing the time it took for ECL to be generated. [Table 30] [Table 31] [Table 32] [Table 33]

[0253] Examples of voltage pulses are described above with reference to 12A, 12B, 14A, 14B, 15A-15L, 16, and 17. In embodiments, the magnitude and duration of the pulse waveform may be tailored to the chemical mixture of the auxiliary electrode 102 and / or the configuration of the working electrode zone 104. Figures 14A, 14B, 15A-15L, 16, and 17 are graphs showing tests performed to optimize the waveform of a high-coupling versus standard plate. The tests were performed with various configurations relating to the working electrode zone 104 formed of carbon, the counter electrode formed of carbon, and the auxiliary electrode 102 formed of Ag / AgCl in various ratios. In these tests, the voltage was sloped to determine the potential value that maximizes ECL. The graphs show how the high-coupling versus standard electrode affects how and at what point in the curve ECL is generated by the change in potential. The test results may be used to determine the optimal magnitude and / or duration of the pulse waveform.

[0254] Specifically, in the tests, FT ECL traces were performed on uncoated standard ("Std") and high-bonding ("HB") 96-1, 96ss, and 96-10 plates, as shown in Figures 8A-8D. FT at 300k was measured for 12 different SI plate types, Std and HB96-1, 96ss, and 96-10 presentation control plates, and Std and HB96-1, 96ss, and 96-10 with an ink ratio of 3 where the Ag:AgCl ratio was 50:50. Five waveforms were performed for each plate type (four copies per well). The waveforms for the production plate ranged from 2000mV to 5000mV at 3000ms (1.0V / s), 2000ms (1.5V / s), 1500ms (2.0V / s), 1200ms (2.5V / s), and 1000ms (3.0V / s). The waveforms for the Ag / AgCl plate ranged from 0mV to 3000mV at 3000ms (1.0V / s), 2000ms (1.5V / s), 1500ms (2.0V / s), 1200ms (2.5V / s), and 1000ms (3.0V / s). The production plate and Ag / AgCl plate were measured using an ECL system with a video system to capture luminescence data. To create the graphs shown in Figures 14A, 14B, 15A-15L, 16, and 17, the ECL intensity at each potential was determined using macros, and four copies were averaged. A plot of the average ECL value versus potential was created.

[0255] Based on the tests performed, the ECL peak voltage was determined for each of the production and test plates, as shown in Table 20. The ECL peak voltage can be used to set the magnitude of the pulse waveform in the ECL process. [Table 34]

[0256] As shown in Figures 26, 27, 28A, 28B, 29, 30, 31, 32A, and 32B, and further as shown in Table 21, the ramp rate altered the measured ECL. As the ramp rate increased, the intensity increased and the signal decreased. As the ramp rate increased, the width of the ECL peak increased. Baseline intensity was defined as the average intensity over the first 10 frames. The start potential was defined as the potential where the ECL intensity was greater than twice the average baseline. Return to baseline was defined as the potential where the ECL intensity was less than twice the baseline. Width was defined as the potential difference between the return potential and the start potential.

[0257] Regarding the Ag / AgCl auxiliary electrode 102, in the case of a carbon counter electrode, the width increased from 175mV to 525mV between 1.0V / s and 3.0V / s. The largest change was observed in the case of HB96-1. The smallest change was observed in Std96ss. In the case of an Ag / AgCl counter electrode, the width increased from 375mV to 450mV between 1.0V / s and 3.0V / s. [Table 35]

[0258] Regarding the Ag / AgCl auxiliary electrode 102, in the case of a carbon counter electrode, the width increased from 175mV to 525mV between 1.0V / s and 3.0V / s. The largest change was observed in the case of HB96-1. The smallest change was observed in Std96ss. In the case of an Ag / AgCl counter electrode, the width increased from 375mV to 450mV between 1.0V / s and 3.0V / s.

[0259] Example 5: Effects of working electrode composition and ramp rate on ECL generation using Ag / AgCl auxiliary electrodes

[0260] In this experiment, plates were prepared in 96-1, 96ss, and 96-10 configurations, as described in Example 4. Test plates with Ag / AgCl auxiliary electrodes ("Ag / AgCl") used the 50% AgCl Ag / AgCl mixture shown in Example 4 to provide more than sufficient reducing capacity for ECL generation using the selected electrode configuration. A control plate ("Carbon") with a conventional carbon counter electrode instead of the Ag / AgCl electrode was also prepared. For each combination of electrode configuration and auxiliary / counter electrode composition, plates were prepared with working electrodes having standard carbon ink electrodes ("Standard" or "Std") or carbon electrodes treated with oxygen plasma after printing ("High Bonding" or "HB"), as used in the examples above.

[0261] Using these plates, ECLs were generated from TAG (a solution referred to as "300k Free Tag" or "300k FT") dissolved in MSD read buffer T 1X at a concentration that provided an ECL signal of approximately 300,000 ECLs when analyzed on a Std96-1 plate with an MSD SECTOR Imager plate reader. In this example, analysis was performed using a video capture system (as described in Example 4) to measure the ECL time course during the ECL experiment. ECLs were generated using a 3V ramp waveform of 0V–3V for plates with Ag / AgCl auxiliary electrodes and 2V–5V for plates with carbon counter electrodes. The effect of ramp speed was evaluated by testing each plate / electrode condition at five different ramp durations (ramp speeds): 3.0 s (1.0 V / s), 2.0 s (1.5 V / s), 1.5 s (2.0 V / s), 1.2 s (2.5 V / s), and 1.0 s (3.0 V / s). Plots of ECL intensity versus applied potential for a control plate with a carbon counter electrode at the five different ramp speeds are provided in Figures 29, 31A, 32A, 33A, and 34A, respectively. Similar plots for a test plate with an AgCl auxiliary electrode are provided in Figures 30, 31B, 32B, 33B, and 34B. Traces of the control and test plates are plotted together in Figure 35 for the 1.0 V / s ramp plate.

[0262] In all ramp plates and electrode configurations, the onset of ECL was at a lower potential with the HB working electrode than with the Std working electrode, which is due to the lower onset potential of TPA oxidation (~0.6V with HB and ~0.8V with Std, relative to the Ag / AgCl reference electrode). Regarding the control plate with a carbon counter electrode, the onset of ECL in the HB96-1 plate was at a higher potential than in other HB electrode configurations, which is likely due to the higher reduction potential at the counter electrode required to support the high current required for the large-area working electrode of the 96-1 type. This large shift in onset potential was not observed when an Ag / AgCl auxiliary electrode was used, indicating that the potentials at these electrodes are not sensitive to this change in current density. Figures 36A and 36B plot the integrated ECL intensity across the waveform as a function of ramp plate, showing that the integrated ECL intensity decreases with ramp plate as the time spent in the voltage region where ECL is generated decreases. Figures 37A and 37B plot the ECL onset potential as a function of ramp plate and show that, compared to the case with a carbon counter electrode, the Ag / AgCl auxiliary electrode provides an ECL onset potential that is less sensitive to electrode configuration and ramp plate.

[0263] Figure 35 plots the ECL traces of the test plate (Ag / AgCl) and control plate (carbon) at a 1.0 V / s ramp plate (colored curves). This plot also shows the cyclic voltammetry current vs. voltage traces for the oxidation of TPA in MSD lead buffer T 1X at the Std and HB carbon working electrodes (black curves). This plot indicates that the higher ECL onset potential at Std versus HB is associated with a higher onset potential for TPA oxidation. The higher sensitivity of HB versus Std regarding the effect of electrode configuration on the ECL onset potential is thought to be due to the significantly higher TPA oxidation current observed at the HB electrode near the ECL onset potential. Table 22 shows the applied potentials that provide the maximum ECL intensity for each plate type measured at a 1.0 V / s waveform. For the Ag / AgCl auxiliary electrode, the ECL peak potential correlated with the working electrode vs. counter electrode area ratio, with 96-1 > 96-10 > 96ss. Similar to the ECL onset potential on the HB plate, the Ag / AgCl auxiliary electrode minimized the effect of the electrode area ratio on the ECL peak potential and shift on the HB plate. [Table 36]

[0264] Various experiments were conducted on assay plates using Ag / AgCl auxiliary electrodes and working electrodes in various configurations. Some of these results are described herein. Experiments were conducted to determine the difference in ECL signal intensity with changes in the working electrode-to-auxiliary electrode ratio at different BTI concentrations and electrode configurations. For all configurations tested, an increase in ECL response intensity with increasing ratio was observed for concentric open-spot configurations (e.g., as shown in Figures 3A and 3B), concentric closed-spot configurations (e.g., as shown in Figures 7A and 7B), concentric open trefoil configurations (e.g., as shown in Figures 4A and 4B), and concentric penta configurations (e.g., as shown in Figures 5A and 5B). This result was observed when the increase in ratio was due to a change in auxiliary electrode size or a change in working electrode size.

[0265] In another experiment, differences in ECL signal intensity with varying incubation times were observed for different BTI concentrations and electrode configurations. For all configurations tested, including the concentric open-spot configuration (e.g., shown in Figures 3A and 3B), the concentric open-trefoil configuration (e.g., shown in Figures 4A and 4B), and the concentric penta configuration (e.g., shown in Figures 5A and 5B), an increase in the ECL signal was observed for incubation times of 2 or 3 hours compared to 1 hour. An increase in ECL signal intensity was also observed for incubation times of 3 hours compared to 2 hours. In further experiments, differences in %CV with incubation time were observed between various electrode configurations with different BTI concentrations. The configurations tested were the concentric open-spot configuration (e.g., shown in Figures 3A and 3B), the concentric open-trefoil configuration (e.g., shown in Figures 4A and 4B), and the concentric penta configuration (e.g., shown in Figures 5A and 5B). In the concentric open spot arrangement, a decrease in %CV was observed with increasing incubation time. In the concentric open trefoil arrangement, an increase in %CV was observed with increasing incubation time from 1 hour to 2 hours. In the concentric penta arrangement, an increase in %CV was observed with increasing incubation time from 1 hour to 2 hours, and from 2 hours to 3 hours.

[0266] In another experiment, gain differences were observed between different spots in an electrochemical cell with different working electrode zone to auxiliary electrode zone ratios in different electrode configurations. The test configurations were a non-concentric 10-spot arrangement, a concentric open-spot arrangement (as shown in Figures 3A and 3B), and a concentric open trefoil arrangement (as shown in Figures 4A and 4B). The results summarized in Table 23 show that the spread between minimum and maximum gain decreases in the concentric open arrangement compared to the non-concentric layout. Therefore, a concentric working electrode zone arrangement may offer advantages in maintaining consistent gain across all spots or positions within the well. [Table 37]

[0267] In embodiments, a concentric, nearly equidistant electrode configuration can offer specific advantages to the ECL procedure, as described above and throughout. Due to the symmetry of these designs (see, for example, Figures 1C, 3A–3F, and 6A–7F), each spot or working electrode zone is similarly affected by the overall geometric dimensions of the well. For example, as described with respect to Figure 2C, the meniscus effect in the fluid filling the well is roughly equal for each of the concentrically arranged working electrode zones. This is because the meniscus is a radial effect, and the concentrically arranged working electrode zones are located approximately equidistant from the center of the well. In addition, as mentioned above, the mass transport effect can be equalized between different working electrode zones. Due to the mass transport effect over time during orbital or rotational oscillations, the distribution of material in the well can depend on its distance from the center of the well. Thus, concentrically arranged working electrode zones help to reduce or minimize the variations that may occur due to the non-uniform material distribution throughout the well. Furthermore, since each working electrode zone is located approximately equidistant from the auxiliary electrode, any voltammetry effects that might occur otherwise can be reduced or minimized.

[0268] The above disclosure provides an electrochemical cell including a working electrode zone and auxiliary electrodes. Various designs are presented and described. In several examples, electrode arrangements (e.g., concentric and equidistant arrangements) and the advantages provided thereby are described. In further examples, electrode compositions (e.g., Ag, Ag / AgCl, and / or any other materials described throughout (e.g., metal oxides, metal / metal oxide pairs, etc.)) and the advantages provided thereby are described. It is understood that the scope of the embodiments described herein also includes examples of various electrode arrangements used with electrodes of other materials (e.g., carbon, carbon composites, and / or other carbon-based materials, etc.) (e.g., as shown in Figures 3A-8D). The advantages arising from the electrochemical cell electrode arrangements and shape dimensions described herein can be realized in embodiments including electrodes of any of the materials described herein. Furthermore, the advantages arising from electrochemical cells in which electrodes are formed using Ag, Ag / AgCl, and / or any other material disclosed throughout this specification (e.g., metal oxides, metal / metal oxide pairs, etc.) as described herein can be realized in embodiments including other working electrode zone arrangements (for example, see Figures 3A-4E of U.S. Patent No. 7,842,246 issued November 30, 2010, which is incorporated herein by reference in its entirety). Examples of such electrochemical cells using non-concentric electrode arrangements formed from various materials such as metal oxides, metal / metal oxide pairs, etc. (e.g., Ag and / or Ag / AgCl) are shown in Figures 38A-39E.

[0269] Figures 38A–39E show an electrochemical cell including a working electrode, a working electrode zone, and a counter electrode or auxiliary electrode. The electrodes shown may comprise any of the various electrode materials described herein, including at least Ag / AgCl, and other chemical mixtures including metal oxides having multiple metal oxidation states, such as manganese oxide, or other metal / metal oxide pairs, such as silver / silver oxide, nickel / nickel oxide, zinc / zinc oxide, gold / gold oxide, copper / copper oxide, platinum / platinum oxide, etc. In certain embodiments, the auxiliary / counter electrodes shown in Figures 38A–39E include Ag / AgCl according to the embodiments described herein.

[0270] Figure 38A shows a well 300 according to another embodiment of the present invention. The well 300 has a wall 302 having an inner surface 304, auxiliary / counter electrodes 306A and 306B, and a working electrode 310 having a working electrode zone 312.

[0271] Figure 38B shows a well 330 according to the embodiment, which has a plurality of working electrode zones 336.

[0272] Figure 38C shows a well 360 according to the embodiment, which has a plurality of working electrode zones 366.

[0273] Figure 39A shows a well 400 according to yet another embodiment of the present invention. The well 400 has a wall 402 having an inner surface 404, auxiliary / counter electrodes 406A and 406B, a working electrode 410, and a boundary 416 defining a group 420 of working electrode zones 418 of the working electrode 410.

[0274] Figure 39B shows a well 430 according to the embodiment. The well 430 includes a wall 431 having an inner surface 432. A boundary 440 separates the auxiliary / vs-auxiliary electrodes 434A and 434B from the working electrode 444.

[0275] Figure 39C shows a well 460 according to the embodiment, where a boundary 470 separates the auxiliary / counter electrodes 464A and 464B from the working electrode 474. The well 460 includes a wall 461 having an inner surface 462. The working electrode 474 has a plurality of working electrode zones 476.

[0276] Figure 39D shows a well 480 according to the present invention, having a wall 482 with an inner surface 484, auxiliary / counter electrodes 488A and 488B, boundary 492, working electrode 494, boundaries 498A and 498B, and working electrode zones 499A and 499B.

[0277] Figure 39E shows a well 4900 according to the present invention. The well 4900 has a wall 4902 having an inner surface 4903, auxiliary / counter electrodes 4904A and 4904B, gaps 4906A and 4906B for exposing a support, and a boundary 4908 having a plurality of holes 4912 for exposing a working electrode zone 4910.

[0278] Further embodiments include the following:

[0279] Embodiment 1 is an electrochemical cell for performing electrochemical analysis, the electrochemical cell comprising a plurality of working electrode zones arranged on the surface of the cell to define a pattern, and at least one auxiliary electrode arranged on the surface, the at least one auxiliary electrode having a redox pair confined on its surface and positioned at substantially equidistant from at least two of the plurality of working electrode zones.

[0280] Embodiment 2 is the electrochemical cell of Embodiment 1, wherein during electrochemical analysis, the auxiliary electrode has a potential determined by the redox pair.

[0281] Embodiment 3 is an electrochemical cell of Embodiment 2, with a potential in the range of approximately 0.1 volts (V) to approximately 3.0 V.

[0282] Embodiment 4 is the electrochemical cell of Embodiment 3, with a potential of approximately 0.22V.

[0283] Embodiment 5 is an electrochemical cell of Embodiment 1, wherein the multiple working electrode zones have a total exposed area, and at least one auxiliary electrode has an exposed surface area, and when the total exposed area of ​​the multiple working electrode zones is divided by the exposed surface area of ​​at least one auxiliary electrode, an area ratio greater than 1 is determined.

[0284] Embodiment 6 is an electrochemical cell of Embodiment 1, wherein the pattern minimizes the number of working electrode zones adjacent to each other with respect to each of the multiple working electrode zones.

[0285] Embodiment 7 is an electrochemical cell of Embodiment 6, wherein the number of adjacent working electrode zones is 2 or less.

[0286] Embodiment 8 is the electrochemical cell of Embodiment 1, wherein at least one of the plurality of working electrode zones is adjacent to three or more other working electrode zones of the plurality of working electrode zones.

[0287] Embodiment 9 is an electrochemical cell of Embodiment 1, wherein the pattern is configured to provide uniform mass transport of a substance to each of a plurality of working electrode zones under rotational oscillation conditions.

[0288] Embodiment 10 is an electrochemical cell of Embodiment 1, and the pattern comprises a geometric pattern.

[0289] Embodiment 11 is an electrochemical cell according to any of Embodiments 1 to 10, wherein each of the multiple working electrode zones defines a circle having a surface area that defines a circle.

[0290] Embodiment 12 is an electrochemical cell according to any of Embodiments 1 to 11, wherein the multiple working electrode zones comprise multiple electrically isolated regions formed on a single electrode.

[0291] Embodiment 13 is the electrochemical cell of Embodiment 1, wherein the redox pair comprises a mixture of silver (Ag) and silver chloride (AgCl).

[0292] Embodiment 14 is the electrochemical cell of Embodiment 13, wherein the mixture of Ag and AgCl comprises about 50 percent or less AgCl.

[0293] Embodiment 15 is the electrochemical cell of Embodiment 14, wherein the mixture has an Ag to AgCl molar ratio within a specified range.

[0294] Embodiment 16 is the electrochemical cell of Embodiment 15, where the molar ratio is approximately equal to or greater than 1.

[0295] Embodiment 17 is the electrochemical cell of Embodiment 13, in which, during electrochemical analysis, the auxiliary electrode has a potential determined by the redox pair, which is approximately 0.22 volts (V).

[0296] Embodiment 18 is an electrochemical cell according to any of Embodiments 1 to 17, and the electrochemical analysis comprises electrochemical luminescence (ECL) analysis.

[0297] Embodiment 19 is an electrochemical cell of any of Embodiments 1 to 18, wherein the electrochemical analysis involves the reduction or oxidation of one or more chemical moieties, and at least one auxiliary electrode is configured to maintain a controlled interfacial potential until all of the chemical moieties are oxidized or reduced.

[0298] Embodiment 20 is an electrochemical cell according to any of Embodiments 1 to 19, and the electrochemical cell is part of a flow cell.

[0299] Embodiment 21 is an electrochemical cell according to any of Embodiments 1 to 19, where the electrochemical cell is part of a plate.

[0300] Embodiment 22 is an electrochemical cell according to any of Embodiments 1 to 19. The electrochemical cell is part of the cartridge.

[0301] Embodiment 23 is an electrochemical cell for performing electrochemical analysis, the electrochemical cell comprising a plurality of working electrode zones arranged on the surface of the cell to define a pattern, and at least one auxiliary electrode arranged on the surface, the at least one auxiliary electrode having a redox pair confined on its surface, the redox pair providing a quantifiable amount of coulombs per unit of surface area of ​​the at least one auxiliary electrode by the redox reaction of the redox pair.

[0302] Embodiment 24 is the electrochemical cell of Embodiment 23, wherein during electrochemical analysis, the auxiliary electrode has a standard reduction potential determined by a redox pair.

[0303] Embodiment 25 is the electrochemical cell of Embodiment 24, and its standard reduction potential is in the range of approximately 0.1 volts (V) to approximately 3.0 V.

[0304] Embodiment 26 is the electrochemical cell of Embodiment 25, with a standard reduction potential of approximately 0.22 volts.

[0305] Embodiment 27 is the electrochemical cell of Embodiment 23, wherein the amount of oxidizing agent in the redox pair is greater than or equal to the amount of charge that needs to pass through the auxiliary electrode to complete the electrochemical analysis.

[0306] Embodiment 28 is the electrochemical cell of Embodiment 27, wherein at least one auxiliary electrode is approximately 3.07 × 10⁻⁶ -7 ~3.97×10 -7 It contains a molar amount of oxidizing agent.

[0307] Embodiment 29 is the electrochemical cell of Embodiment 27, wherein at least one auxiliary electrode has an auxiliary electrode area of ​​1 mm². 2 Approximately 1.80 x 10 -7 ~2.32 × 10 -7 It contains a molar amount of oxidizing agent.

[0308] Embodiment 30 is an electrochemical cell of Embodiment 27, wherein at least one auxiliary electrode has a total working electrode area of ​​1 mm² in the well. 2At least approximately 3.7 × 10 -9 It contains a molar amount of oxidizing agent.

[0309] Embodiment 31 is an electrochemical cell of Embodiment 27, wherein at least one auxiliary electrode has a total working electrode area of ​​1 mm² in the well. 2 At least approximately 5.7 × 10 -9 It contains a molar amount of oxidizing agent.

[0310] Embodiment 32 is an electrochemical cell of Embodiment 23, in which the redox pair passes a current of approximately 0.5 to 4.0 mA through the redox reaction of the redox pair to generate electrochemical luminescence (ECL) in the range of approximately 1.4 V to 2.6 V.

[0311] Embodiment 33 is an electrochemical cell of Embodiment 23, in which the redox pair passes an average current of about 2.39 mA through the redox reaction to generate electrochemical luminescence (ECL) in the range of about 1.4 V to 2.6 V.

[0312] Embodiment 34 is an electrochemical cell of Embodiment 23, and the redox pair has an electrode surface area of ​​1 mm². 2 Approximately 1.56 x 10 -5 ~5.30×10 -4 The interface potential of -0.15 to -0.5V is maintained while allowing the charge of C to pass through.

[0313] Embodiment 35 is an electrochemical cell of Embodiment 23, wherein the multiple working electrode zones have a total exposed area, and at least one auxiliary electrode has an exposed surface area, and when the total exposed area of ​​the multiple working electrode zones is divided by the exposed surface area of ​​at least one auxiliary electrode, an area ratio greater than 1 is determined.

[0314] Embodiment 36 is an electrochemical cell of Embodiment 23, wherein the pattern minimizes the number of working electrode zones adjacent to each other with respect to each of the working electrode zones among a plurality of working electrode zones.

[0315] Embodiment 37 is an electrochemical cell of Embodiment 23, wherein the number of adjacent working electrode zones is 2 or less.

[0316] Embodiment 38 is an electrochemical cell of Embodiment 23, wherein at least one of the plurality of working electrode zones is adjacent to three or more other working electrode zones of the plurality of working electrode zones.

[0317] Embodiment 39 is an electrochemical cell of Embodiment 23, wherein the pattern is configured to provide uniform mass transport of a substance to each of a plurality of working electrode zones under rotational oscillation conditions.

[0318] Embodiment 40 is an electrochemical cell of Embodiment 23, and the pattern comprises a geometric pattern.

[0319] Embodiment 41 is an electrochemical cell according to any of Embodiments 23 to 40, wherein each of the multiple working electrode zones defines a circle having a surface area that defines a circle.

[0320] Embodiment 42 is an electrochemical cell according to any of Embodiments 23 to 41, wherein the multiple working electrode zones comprise multiple electrically isolated regions formed on a single electrode.

[0321] Embodiment 43 is the electrochemical cell of Embodiment 1, wherein the redox pair comprises a mixture of silver (Ag) and silver chloride (AgCl).

[0322] Embodiment 44 is the electrochemical cell of Embodiment 43, wherein the mixture of Ag and AgCl comprises about 50 percent or less AgCl.

[0323] Embodiment 45 is the electrochemical cell of Embodiment 43, wherein the mixture has an Ag to AgCl molar ratio within a specified range.

[0324] Embodiment 46 is the electrochemical cell of Embodiment 45, where the molar ratio is approximately equal to or greater than 1.

[0325] Embodiment 47 is the electrochemical cell of Embodiment 43, in which the auxiliary electrode has a standard reduction potential during electrochemical analysis, and the standard reduction potential is approximately 0.22 volts (V).

[0326] Embodiment 48 is an electrochemical cell according to any of Embodiments 23 to 47, and the electrochemical analysis comprises electrochemical luminescence (ECL) analysis.

[0327] Embodiment 49 is an electrochemical cell of any of Embodiments 23 to 48, wherein the electrochemical analysis involves the reduction or oxidation of one or more chemical moieties, and at least one auxiliary electrode is configured to maintain a controlled interfacial potential until all of the chemical moieties are oxidized or reduced.

[0328] Embodiment 50 is an electrochemical cell according to any of Embodiments 23 to 49, and the electrochemical cell is part of a flow cell.

[0329] Embodiment 51 is an electrochemical cell according to any of Embodiments 23 to 49, where the electrochemical cell is part of the plate.

[0330] Embodiment 52 is an electrochemical cell according to any of Embodiments 23 to 49, and the electrochemical cell is part of a cartridge.

[0331] Embodiment 53 is an electrochemical cell for performing electrochemical analysis, the electrochemical cell comprising a plurality of working electrode zones located on the surface of the cell and defining a pattern, and at least one auxiliary electrode located on the surface and formed of a chemical mixture comprising an oxidizing agent, the at least one auxiliary electrode having a redox pair confined on its surface, the amount of which is sufficient to maintain a defined potential throughout the redox reaction of the redox pair.

[0332] Embodiment 54 is the electrochemical cell of Embodiment 53, in which, during electrochemical analysis, the auxiliary electrode has a potential determined by the redox pair.

[0333] Embodiment 55 is an electrochemical cell of Embodiment 54, with a potential in the range of approximately 0.1 volts (V) to approximately 3.0 V.

[0334] Embodiment 56 is an electrochemical cell of Embodiment 55, with a potential of approximately 0.22V.

[0335] Embodiment 57 is the electrochemical cell of Embodiment 53, wherein the amount of oxidizing agent is greater than or equal to the amount of charge that must pass through at least one auxiliary electrode to complete the electrochemical analysis.

[0336] Embodiment 58 is an electrochemical cell of Embodiment 53, wherein at least one auxiliary electrode is approximately 3.07 × 10⁻⁶ -7 ~3.97×10 -7 It contains a molar amount of oxidizing agent.

[0337] Embodiment 59 is an electrochemical cell of Embodiment 53, wherein at least one auxiliary electrode has an auxiliary electrode area of ​​1 mm². 2 Approximately 1.80 x 10 -7 ~2.32 × 10 -7 It contains a molar amount of oxidizing agent.

[0338] Embodiment 60 is an electrochemical cell of Embodiment 53, wherein at least one auxiliary electrode has a total working electrode area of ​​1 mm². 2 At least approximately 3.7 × 10 -9 It contains a molar amount of oxidizing agent.

[0339] Embodiment 61 is an electrochemical cell of Embodiment 53, wherein at least one auxiliary electrode has a total working electrode area of ​​1 mm². 2 At least approximately 5.7 × 10 -9 It contains a molar amount of oxidizing agent.

[0340] Embodiment 62 is an electrochemical cell of Embodiment 53, in which the redox pair passes a current of approximately 0.5 to 4.0 mA through the redox reaction of the redox pair to generate electrochemical luminescence (ECL) in the range of approximately 1.4 V to 2.6 V.

[0341] Embodiment 63 is an electrochemical cell of Embodiment 53, in which the redox pair passes an average current of about 2.39 mA through the redox reaction to generate electrochemical luminescence (ECL) in the range of about 1.4 V to 2.6 V.

[0342] Embodiment 64 is an electrochemical cell of Embodiment 53, and the redox pair has an electrode surface area of ​​1 mm². 2 Approximately 1.56 x 10 -5 ~5.30×10 -4 The interface potential of -0.15 to -0.5V is maintained while allowing the charge of C to pass through.

[0343] Embodiment 65 is an electrochemical cell of Embodiment 53, wherein the multiple working electrode zones have a total exposed area, and at least one auxiliary electrode has an exposed surface area, and when the total exposed area of ​​the multiple working electrode zones is divided by the exposed surface area of ​​at least one auxiliary electrode, an area ratio greater than 1 is determined.

[0344] Embodiment 66 is an electrochemical cell of Embodiment 53, wherein the pattern minimizes the number of working electrode zones adjacent to each other with respect to each of the working electrode zones among a plurality of working electrode zones.

[0345] Embodiment 67 is an electrochemical cell of Embodiment 53, wherein the number of adjacent working electrode zones is 2 or less.

[0346] Embodiment 68 is an electrochemical cell of Embodiment 53, wherein at least one of the plurality of working electrode zones is adjacent to three or more other working electrode zones of the plurality of working electrode zones.

[0347] Embodiment 69 is an electrochemical cell of Embodiment 53, wherein the pattern is configured to provide uniform mass transport of a substance to each of a plurality of working electrode zones under rotational oscillation conditions. ...

Claims

1. An electrochemical cell for performing electroanalysis, The cell surface comprises a plurality of working electrode zones that define a pattern, At least one auxiliary electrode disposed on the surface and having a redox pair confined on that surface Equipped with, An electrochemical cell wherein the at least one auxiliary electrode is positioned at approximately equidistant from at least two of the plurality of working electrode zones.

2. The electrochemical cell according to claim 1, wherein, during the electrochemical analysis, the auxiliary electrode has a potential determined by the redox pair.

3. The electrochemical cell according to claim 2, wherein the potential is in the range of about 0.1 volts (V) to about 3.0 V.

4. The electrochemical cell according to claim 2, wherein the potential is approximately 0.1 volts (V) to approximately 3.0 V.

5. The electrochemical cell according to claim 1, wherein the pattern minimizes the number of working electrode zones adjacent to each of the working electrode zones among the plurality of working electrode zones.

6. The electrochemical cell according to claim 1, wherein the pattern is configured to provide uniform mass transport of a substance to each of the plurality of working electrode zones under rotational oscillation conditions.

7. The electrochemical cell according to claim 1, wherein each of the plurality of working electrode zones defines a circle having a surface area that defines a circle.

8. The at least one auxiliary electrode is positioned approximately in the center of the electrochemical cell. The plurality of working electrode zones include 10 working electrode zones spaced approximately equidistant from at least one auxiliary electrode. The electrochemical cell according to claim 7, wherein the two working electrode zones have a larger pitch distance between them than the remaining working electrode zone.

9. The electrochemical cell according to claim 1, wherein the redox pair comprises a mixture of silver (Ag) and silver chloride (AgCl).

10. The electrochemical cell according to claim 9, wherein the mixture of Ag and AgCl comprises about 50 percent or less AgCl.

11. The electrochemical cell according to claim 10, wherein the mixture has a molar ratio of Ag to AgCl within a specified range.

12. During the electrochemical analysis, the auxiliary electrode has a potential determined by the redox pair. The electrochemical cell according to claim 9, wherein the potential is approximately 0.22 volts (V).

13. The electrochemical cell according to claim 1, wherein the electrochemical analysis comprises electrochemical luminescence (ECL) analysis.

14. An electrochemical cell for performing electrochemical analysis, The cell surface comprises a plurality of working electrode zones that define a pattern, At least one auxiliary electrode disposed on the surface and having a defined interfacial potential An electrochemical cell equipped with the following features.

15. The electrochemical cell according to claim 14, wherein the amount of oxidizing agent in the at least one auxiliary electrode is equal to or greater than the amount of charge that needs to pass through the at least one auxiliary electrode to complete the electrochemical analysis.

16. The aforementioned at least one auxiliary electrode is approximately 3.07 × 10 －７ ~3.97 x 10 －７ The electrochemical cell according to claim 15, comprising a molar amount of an oxidizing agent.

17. The aforementioned at least one auxiliary electrode has an auxiliary electrode area of ​​1 mm². ２ Approximately 1.80 x 10 －７ ~2.32 x 10 －７ The electrochemical cell according to claim 15, comprising a molar amount of an oxidizing agent.

18. The at least one auxiliary electrode has a total working electrode area of ​​1 mm² within the well. ２ At least approximately 3.7 x 10 －９ The electrochemical cell according to claim 15, comprising a molar amount of an oxidizing agent.

19. The electrochemical cell according to claim 14, wherein the plurality of working electrode zones have a total exposed area, the at least one auxiliary electrode has an exposed surface area, and when the total exposed area of ​​the plurality of working electrode zones is divided by the exposed surface area of ​​the at least one auxiliary electrode, an area ratio greater than 1 is determined.

20. The electrochemical cell according to claim 14, wherein the at least one auxiliary electrode comprises a mixture of silver (Ag) and silver chloride (AgCl).

21. The electrochemical cell according to claim 20, wherein the mixture of Ag and AgCl comprises about 50 percent or less AgCl.

22. The electrochemical cell according to claim 20, wherein the mixture has a molar ratio of Ag to AgCl within a specified range.

23. The electrochemical cell according to claim 22, wherein the molar ratio is approximately equal to or greater than 1.

24. The electrochemical cell according to claim 14, wherein the electrochemical cell is part of a flow cell.

25. The electrochemical cell according to claim 14, wherein the electrochemical cell is part of a plate.

26. The electrochemical cell according to claim 14, wherein the electrochemical cell is part of a cartridge.

27. An apparatus for performing chemical analysis, The plate comprises a plate with multiple wells defined inside, and at least one of the multiple wells is The cell surface comprises a plurality of working electrode zones that define a pattern, Displaced on the surface, at least one auxiliary electrode formed of a chemical mixture comprising an oxidizing agent and Equipped with, The apparatus wherein at least one auxiliary electrode has a redox pair confined on its surface, and the amount of the oxidizing agent is sufficient to maintain a predetermined potential throughout the redox reaction of the redox pair.

28. The apparatus according to claim 27, wherein the redox pair is subjected to a current of approximately 0.5 to 4.0 mA through the redox reaction of the redox pair in order to generate electrochemical luminescence (ECL) in the range of approximately 1.4 V to 2.6 V.

29. The apparatus according to claim 27, wherein the redox pair passes an average current of about 2.39 mA through a redox reaction to generate electrochemical luminescence (ECL) in the range of about 1.4 V to 2.6 V.

30. The redox pair maintains an interfacial potential of -0.15 to -0.5 V while passing a charge of about 1.56×10 ２ to 5.30×10 －５ C per 1 mm －４ of electrode surface area. The device according to claim 27.

31. The apparatus according to claim 27, wherein the number of adjacent working electrode zones is two or less.

32. The apparatus according to claim 27, wherein at least one of the plurality of working electrode zones is adjacent to three or more other working electrode zones among the plurality of working electrode zones.

33. The apparatus according to claim 27, wherein the pattern comprises a geometric pattern.

34. A method for electrochemical analysis, The method comprises applying voltage pulses to one or more working electrode zones and at least one auxiliary electrode located within at least one well of a multiwell plate. The one or more working electrode zones define a pattern on the surface of at least one well. The at least one auxiliary electrode is disposed on the surface and has a redox pair confined on that surface, A method by which the redox pair is reduced at least for the duration during which the voltage pulse is applied.

35. The method according to claim 34, wherein the luminescence data is captured during the duration of the voltage pulse.

36. The method according to claim 35, wherein the luminescence data is captured for at least 50 percent of the duration of the voltage pulse.

37. The method according to claim 35, wherein the luminescence data is captured for at least 75 percent of the duration of the voltage pulse.

38. The method according to claim 35, wherein the luminescence data is captured for at least 100 percent of the duration of the voltage pulse.

39. The method according to claim 34, wherein the duration of the voltage pulse is approximately 200 milliseconds (ms) or less.

40. The method according to claim 39, wherein the duration of the voltage pulse is approximately 100 ms.

41. The method according to claim 39, wherein the duration of the voltage pulse is approximately 50 ms.

42. The method according to claim 34, wherein the voltage pulse is applied simultaneously to the one or more working electrodes and the at least one auxiliary electrode.

43. The method according to claim 34, wherein the voltage pulse is sequentially applied to the one or more working electrodes and the at least one auxiliary electrode.

44. The method according to claim 34, wherein the voltage pulse is applied to an addressable subset of the one or more working electrode zones.

45. The magnitude of the voltage pulse is selected at least partially based on the chemical composition of the at least one auxiliary electrode. The method according to claim 34, further comprising the above.

46. A computer-readable medium storing instructions for causing one or more processors to perform the method described in claim 34.

47. An apparatus for performing electrochemical analysis in a well, Multiple working electrode zones are arranged on a surface adapted to form the bottom of the well, An auxiliary electrode disposed on the aforementioned surface and having a potential determined by a redox pair confined on that surface, Equipped with, The apparatus wherein one of the plurality of working electrode zones is positioned at approximately equidistant from each side wall of the well.

48. The apparatus according to claim 47, wherein the plurality of working electrode zones comprises a plurality of electrically isolated regions formed on a single electrode.

49. The apparatus according to claim 47, wherein the electrochemical analysis comprises electrochemical luminescence (ECL) analysis.

50. A method for performing electrochemical analysis, Applying a first voltage pulse to one or more working electrode zones or counter electrodes in a well of the apparatus, wherein the first voltage pulse causes a first redox reaction in the well, To capture first luminescence data from the first redox reaction over a first period, Applying a second voltage pulse to one or more working electrode zones or the counter electrode in the well, wherein the second voltage pulse causes a second redox reaction in the well. To capture second luminescence data from the second redox reaction over a second period, A method for providing this.

51. The method according to claim 50, further comprising performing electrochemical luminescence analysis on the first luminescence data and the second luminescence data.

52. The method according to claim 50, wherein at least one of the first voltage pulse and the second voltage pulse is applied to an addressable subset of the one or more working electrode zones.

53. The method according to claim 50, further comprising selecting the magnitude of at least one of the first voltage pulse and the second voltage pulse based at least in part on the chemical composition of the counter electrode, wherein the counter electrode is an auxiliary electrode.

54. The method according to claim 50, wherein the first duration of the first period is not equal to the second duration of the second period.

55. The method according to claim 54, wherein the first duration and the second duration are selected to improve the dynamic range of the electrochemical luminescence analysis performed on the first luminescence data and the second luminescence data.

56. The method according to claim 54, wherein the first luminescence data is captured for a first duration of the first voltage pulse.

57. The method according to claim 54, wherein one of the first duration or the second duration is about 200 milliseconds (ms) or less.

58. The method according to claim 57, wherein one of the first duration or the second duration is about 100 ms.

59. The method according to claim 57, wherein one of the first duration or the second duration is about 50 ms.

60. The method according to claim 50, wherein the counter electrode comprises an auxiliary electrode.

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