Closed loop pH control using a differential sensor

Abstract

The present invention relates to closed loop pH control using a differential sensor. A closed loop system and method for controlling pH. The system includes a working electrode, a counter electrode, a reference electrode, a first ion sensitive field effect transistor (ISFET), a second ISFET, and an electronic controller. The working electrode, the counter electrode, the reference electrode, and a first sensing terminal of the first ISFET can be immersed in an active solution. A second sensing terminal of the second ISFET can be immersed in a reference solution. The electronic controller is configured to apply a first amount of current or voltage to the working electrode and determine a differential voltage between the first ISFET and the second ISFET. The electronic controller is further configured to set a second amount of current or voltage to reduce a difference between the differential voltage and a target voltage. The electronic controller is further configured to apply the second amount of current or voltage to the working electrode.

Description

TECHNICAL FIELD

[0001] The present disclosure relates generally to closed-loop pH control. More specifically, the present disclosure relates to closed-loop pH control employing a differential ion-sensitive field effect transistor (ISFET) scheme. BACKGROUND

[0002] pH is a factor that plays an important role in binding interactions between biomolecules, enzyme activity, chemical modifications such as protection / deprotection of functional groups, chemical / biochemical reaction kinetics, and visualization of pH-sensitive reporter molecules. Because pH can act as a universal switch or controller for various types of processes, precise control of pH, especially for parallel control of multiple conditions, can provide great opportunities in various applications.

[0003] Currently, the pH of a sample is changed by replacing the entire buffer with a target pH or adding acid or base to the solution. This approach is time-consuming, error-prone, and in many cases results in significant dilution of the sample. If the sample volume is small or multiple rounds of pH changes are needed during the course of an assay or reaction, currently available technologies do not provide a good solution. Therefore, there is still a need for a technical solution that can control pH at flexible temporal and spatial target values and minimal dilution factors for a variety of research and industrial applications.

[0004] Reported methods include electronic pH control described in US 10379080, the entire contents of which are hereby incorporated by reference. Similar pH control schemes can be used in various design formats, especially in array format, in order to perform highly multiplexed, independent measurements and reactions in parallel in the same sample solution. When an array of electrodes is used to locally control the microenvironment near each of these electrodes, “cross-talk” or “bleed-over” between different sites is a common problem. This problem is addressed by spacing the individual sites apart or with a buffer reagent added to the bulk solution. The former approach results in reduced array density (larger device size), the latter approach requires that the rate of the electrochemical reaction be high enough to overcome the buffering capacity of the bulk solution. In practice, this means applying higher voltages or currents, or using higher concentrations of electroactive molecules. These measures can lead to side reactions involving other components of the reaction system. Here, the present disclosure describes how closed-loop control can be used to combat these problems. In particular, several embodiments of closed-loop control in a high-density array of individually addressable electrodes are described. SUMMARY

[0005] The present disclosure provides a closed loop system for controlling pH that, in some embodiments, includes a feedback electrode set and an electronic controller. The feedback electrode set includes a working electrode, a counter electrode, a reference electrode, a first ion-sensitive field effect transistor (ISFET), and a second ISFET. The working electrode, the counter electrode, and the reference electrode are submersible in an active solution. The first ISFET includes a first sensing terminal submersible in the active solution. The second ISFET includes a second sensing terminal submersible in a reference solution. The electronic controller is coupled to the feedback electrode set. The electronic controller is configured to apply a first amount of current or voltage to the working electrode. The electronic controller is further configured to determine a differential voltage between the first ISFET and the second ISFET. The electronic controller is further configured to determine a difference between the differential voltage and a target voltage. The electronic controller is further configured to set a second amount of current or voltage to reduce the difference between the differential voltage and the target voltage. The electronic controller is further configured to apply the second amount of current or voltage to the working electrode.

[0006] The present disclosure also provides a closed loop method for controlling pH that includes submerging a working electrode, a counter electrode, a reference electrode, and a first sensing terminal of a first ion-sensitive field effect transistor (ISFET) in an active solution. The method also includes submerging a second sensing terminal of a second ISFET in a reference solution. The working electrode, the counter electrode, the reference electrode, the first ISFET, and the second ISFET are included in a feedback electrode set. The method also includes applying, with an electronic controller, a first amount of current or voltage to the working electrode. The method further includes determining, with the electronic controller, a differential voltage between the first ISFET and the second ISFET. The method also includes determining, with the electronic controller, a difference between the differential voltage and a target voltage. The method also includes setting, with the electronic controller, a second amount of current or voltage to reduce the difference between the differential voltage and the target voltage. The method further includes applying, with the electronic controller, the second amount of current or voltage to the working electrode. BRIEF DESCRIPTION OF DRAWINGS

[0007] The accompanying drawings, where like reference numerals refer to identical or functionally similar elements throughout the separate views, which are incorporated in and constitute part of the specification, illustrate embodiments and serve to further explain the principles of, and expound upon the various features of, the embodiments.

[0008] Figure 1 is a schematic of one example of modulating solution pH via oxidation / reduction of a redox active species under closed loop control using a surface patterned electrode, according to some embodiments.

[0009] Figure 2 is a graph of one example of changing solution pH via oxidation / reduction of a redox species under closed loop control.

[0010] Figure 3A is a diagram of one example of a pH control device with an external counter electrode and reference electrode according to some embodiments.

[0011] Figure 3B is a diagram of one example of a pH control device with a counter electrode and reference electrode disposed on a substrate according to some embodiments.

[0012] Figure 4A and 4B is a top view of an example of a pH control electrode of different shapes according to some embodiments.

[0013] Figure 4C is a side view of one example of a pH control electrode with a sensing element and a working electrode disposed on the same location according to some embodiments.

[0014] Figure 4D is a side view of one example of a pH control electrode with a multi-layer stack according to some embodiments.

[0015] Figure 4E is a top view of a pH control electrode with a counter electrode patterned around a working electrode according to some embodiments.

[0016] Figure 5A is a diagram of one example of a p-channel ion-sensitive field effect transistor (ISFET) according to some embodiments.

[0017] Figure 5B is a diagram of one example of an n-channel ISFET according to some embodiments.

[0018] Figure 6 is a diagram of one example of a closed loop system for controlling pH according to some embodiments.

[0019] Figures 7A to 7E is a diagram of one example of a load according to some embodiments.

[0020] Figure 8 is a block diagram of one example of an electronic controller according to some embodiments.

[0021] Figure 9 is a flow diagram of one example of a method for controlling pH according to some embodiments.

[0022] Figure 10 is a diagram of one example of an array of feedback electrode groups according to some embodiments.

[0023] Figure 11 is a diagram of one example of an array of feedback and non-feedback electrode groups arranged in different sections according to some embodiments.

[0024] Figure 12 is a diagram of one example of an array of feedback and non-feedback electrode groups interspersed with one another according to some embodiments.

[0025] The system and method components have been represented in the drawings by conventional symbols, showing only those specific details that are pertinent to understanding the embodiments so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having the benefit of the description herein. DETAILED DESCRIPTION

[0026] DETAILED DESCRIPTION

[0027] Figure 1 is a schematic diagram illustrating one example of modulating solution pH via oxidation / reduction of a redox active species under closed loop control using surface patterned electrodes. In Figure 1 , a pH sensing electrode measures the initial pH value, from which the amount of current or voltage to be applied to the working electrode is determined. When current or voltage is applied to the working electrode, the electrochemical oxidation and / or reduction of a pH modulating reagent, such as a quinone, introduces a local pH change through the generation or consumption of protons and the balance between the buffer capacity of the buffer solution. This local pH change creates a pH modulation zone with a very short vertical distance from the electrode surface, for example, a few nanometers to a few millimeters, which enables pH dependent chemical / biochemical reactions to occur only within this volume. The size of the modulation zone depends in part on the buffer capacity of the solution. For example, the size of the modulation zone is more compact in stronger buffers. Monitoring the actual pH during the modulation process by the pH sensing electrode enables continuous changes in the electrical output through closed loop control, which enables faster and more accurate pH control, among other things. In some embodiments, pH control is performed in unbuffered solutions.

[0028] The reversible electrochemical oxidation / reduction of pH modulating reagents, such as quinone derivatives, hydrazine derivatives, or water, has proven to enable fast pH changes in a localized region. The pH modulation limit depends on the pKa and oxidation / reduction potential of the specific pH modulating reagent and their concentrations. Figure 2 is a graph illustrating one example of closed loop control of pH modulation by oxidation of 2,5-dimethyl-l,4-hydroquinone (DMHQ) and reduction of 2,5-dimethyl-l,4-benzoquinone (DMBQ) in 1 mM phosphate buffer on an indium tin oxide electrode. When an anodic current is applied to the indium tin oxide electrode, the generation of protons overcomes the buffer capacity and the pH of the solution becomes more acidic, and vice versa. The pH values shown in Figure 2 are determined by a pre-calibrated iridium oxide sensing electrode patterned on the surface. As Figure 2 shown in , closed loop control achieves the target pH value in an accurate and fast manner.

[0029] Electrochemical pH modulation under closed-loop control uses a set of electrodes, such as a working electrode, a sensing element, a counter electrode, and a reference electrode. The reference electrode provides a stable potential reference for the measurement. In some embodiments, the sensing element is used as the reference electrode when it has good stability and is placed in a stable pH solution. In addition, in some embodiments, multiple working electrodes and sensing elements share the counter electrode and the reference electrode. In some embodiments, external counter and reference electrodes are used. For example, Figure 3A is a pH control device with external counter and reference electrodes. In other embodiments, surface-patterned on-chip counter and reference electrodes are used. For example, Figure 3B is a pH control device with counter and reference electrodes placed on a substrate.

[0030] The working electrode and the sensing element can have various shapes and sizes. For example, Figure 4A and 4B are top views of pH control electrodes of different shapes. The sensing element needs to be physically separated from the working electrode to avoid cross-talk or short-circuiting. In some embodiments, the sensing element is located in the same plane as the working electrode with a small gap between them to provide the physical separation. For example, Figure 4C is a side view of a pH control electrode with a sensing element and a working electrode located in the same plane. The gap between the sensing element and the working electrode can be, for example, 1 nanometer to 100 micrometers. In other embodiments, the sensing element is placed on top of the working electrode with an insulating layer between them to provide the physical separation. For example, Figure 4D is a side view of a pH control electrode with a multi-layer stack.

[0031] In some embodiments, the counter electrode is patterned around the working electrode, which minimizes the diffusion effects and helps to have a more well-defined shape of the pH modulation zone, especially with unbuffered solutions to control the pH. For example, Figure 4E is a top view of a pH control electrode with a counter electrode patterned around the working electrode.

[0032] The sensing element includes an ion-sensitive field effect transistor (ISFET). The ISFET is a specific type of chemically sensitive field effect transistor (chemFET) that has sensitivity to ion concentrations in a solution. The ISFET is similar to a metal-oxide-semiconductor field-effect transistor (MOSFET) and has a source terminal (S), a drain terminal (D), and a body (or bulk) connection. However, instead of a metal gate electrode, the ISFET has an ion-sensitive region immersed in the solution and a separate reference electrode. Figure 5A is an example of a p-channel ISFET. Figure 5BISFET is an example of an n-channel ISFET. For the p-channel case, the electrical channel is primarily formed by holes, and when the device is on, most of the current is composed of holes flowing through the channel. For the n-channel case, the electrical channel is primarily formed by electrons, and when the device is on, most of the current is composed of electrons flowing through the channel. In both cases, the threshold voltage of the ISFET depends on the ion concentration in the solution. Modulation of the threshold voltage, in turn, modulates the current through the ISFET and the voltage at the source or drain terminal. The ISFET is configured to be sensitive to ions, such as hydrogen ions, and thus to solution pH.

[0033] Figure 6 FIG. 1 is an example of a closed-loop system 100 for controlling pH. In Figure 6 , multiple ISFETs are used in a differential manner to read and control ion concentration and pH in a closed-loop system. Figure 6 The closed-loop system 100 shown in FIG. 1 includes a feedback electrode set 102 and an electronic controller 104. Figure 6 The feedback electrode set 102 shown in FIG. 1 includes a working electrode 106, a counter electrode 108, a reference electrode 110, a first ISFET 112, and a second ISFET 114. These electrodes are composed of materials including, for example, metal oxides, glassy carbon, graphene, metals, gold, silver, platinum, conductive polymers, silver chloride, standard hydrogen, mercury drop, saturated calomel, or combinations thereof. In some embodiments, these electrodes are patterned on a support including, for example, a glass slide, a plastic plate, a silicon wafer, a glass wafer, a quartz wafer, a flexible plastic sheet, a polymer layer, paper, or combinations thereof.

[0034] The sensing region SA1 of the first ISFET 112 (an instance of a "first sensing terminal") is immersed in an active solution 116. The active solution 116 includes, for example, a buffered solution, an unbuffered solution, an aqueous solution, an organic solution, or a combination thereof. The active solution 116 contains one or more redox active species including, for example, quinones, catechols, aminophenols, hydrazines, any derivatives thereof, or any combination thereof. The sensing region SA2 of the second ISFET 114 (an instance of a "second sensing terminal") is immersed in a reference solution 118. In some embodiments, the active solution 116 is physically separated from the reference solution 118 to eliminate an interface between the two. For example, the active solution 116 and the reference solution 118 can be contained in separate containers. In other embodiments, the active solution 116 is isolated from the reference solution 118 to minimize interference between the two. For example, the active solution 116 and the reference solution 118 can be contained in separate containers that are connected by, for example, a channel that limits interaction and interference between the two solutions. As a further example, the active solution 116 and the reference solution 118 can be contained in a single container but are separated by a distance sufficient that they do not interfere with each other. In some embodiments, the reference solution 118 is maintained at a desired pH level so that the output of the second ISFET 114 is consistent. The reference solution 118 can be allowed to naturally drift based on environmental effects or (actively or passively) maintained at a desired pH level.

[0035] The drain terminal D1 of the first ISFET 112 is coupled to the electronic controller 104. The drain terminal D1 of the first ISFET 112 (an instance of a "first drain terminal") is also coupled to a first load 120. The first load 120 can be used to bias the first ISFET 112 and / or facilitate conversion from current to voltage. This configuration is similar to a common-source amplifier using a MOSFET. In some embodiments, the first load 120 is coupled between the drain terminal D1 of the first ISFET 112 and a supply voltage (e.g., VDD). In some embodiments, as shown in FIG. 1A, the first load 120 includes a current source with a fixed current. Figure 7A In other embodiments, as shown in FIG. 1B, the first load 120 includes a p-channel MOSFET that acts as a current source. Figure 7B In other embodiments, as shown in FIG. 1C, the first load 120 includes a p-channel MOSFET that acts as a current source. Figure 7C In other embodiments, as shown in FIG. 1D, the first load 120 includes a p-channel MOSFET that acts as a current source. Figure 7D In other embodiments, as shown in FIG. 1E, the first load 120 includes a resistor. In other embodiments, the first load 120 includes a resonant circuit. For example, as shown in FIG. 1F, the first load 120 includes a resonant circuit that includes a capacitor and an inductor. Figure 7EAs shown in FIG. 1, the first load 120 can include capacitors, resistors, and inductors coupled in a parallel configuration. In other embodiments, the first load 120 includes other combinations of transistors and / or passive elements.

[0036] The drain terminal D2 of the second ISFET 114 is coupled to the electronic controller 104. The drain terminal D2 of the second ISFET 114 ("an instance of a second drain terminal") is also coupled to a second load 122. In some embodiments, the second load 122 is coupled between the drain terminal of the second ISFET 114 and a supply voltage (e.g., VDD). The second load 122 includes any type of load as described above for the first load 120. In some embodiments, the second load 122 is the same type of load as the first load 120. For example, the first load 120 and the second load 122 can each include a single resistor.

[0037] The source terminal S1 of the first ISFET 112 ("an instance of a first source terminal") is coupled to the source terminal S2 of the second ISFET 114 ("an instance of a second source terminal"). In some embodiments, the source terminal S1 of the first ISFET 112 and the source terminal S2 of the second ISFET 114 are coupled to a current source 124 for biasing, as shown in FIG. 1. In other embodiments, the source terminal S1 of the first ISFET 112 and the source terminal S2 of the second ISFET 114 are coupled to a reference terminal having a pseudo-differential configuration for biasing. In other embodiments, the source terminal S1 of the first ISFET 112 and the source terminal S2 of the second ISFET 114 are coupled to a load impedance for biasing. Figure 6

[0038] In some embodiments, to improve device linearity and avoid body effects, as shown in FIG. 1, the body terminals of the first ISFET 112 and the second ISFET 114 are coupled to the source terminals, respectively. In other embodiments, the voltage at the body terminals is configured based on other voltages in the closed loop system 100 using a body control circuit (e.g., a reference terminal or a supply voltage). Figure 6

[0039] Figure 8 is a block diagram of an instance of the electronic controller 104. Figure 8 The electronic controller 104, as shown in FIG. 2, includes an electronic processor 202 (e.g., one or more microprocessors, application specific integrated circuits (ASICs), systems on a chip (SoCs), or other electronic controllers), a memory 204, an input / output interface 206, a user interface 208, and a bus 210. In some embodiments, the electronic controller 104 is configured differently than the electronic controller 104 shown in FIG. 1. Figure 8 ​​The configuration shown in FIG. 1 includes fewer or additional components. For example, in practice, the electronic controller 104 can include additional components such as one or more power sources, one or more sensors, and the like. These additional components are not shown here for ease of explanation.

[0040] The input / output interface 206 includes routines for transferring information between components within the electronic controller 104 and components external to the electronic controller 104. The input / output interface 206 is configured to send and receive data via one or more wired couplings (e.g., wires, optical fibers, and the like), wirelessly, or a combination thereof.

[0041] The user interface 208 includes, for example, one or more input mechanisms (e.g., a touchscreen, a keyboard, buttons, knobs, and the like), one or more output mechanisms (e.g., a display, a printer, a speaker, and the like), or a combination thereof. In some embodiments, the user interface 208 includes a touch-sensitive interface (e.g., a touchscreen display) that displays visual output generated by software applications executed by the electronic processor 202. The visual output includes, for example, graphical indicators, lights, colors, text, images, graphical user interfaces (GUIs), combinations of the foregoing, and the like. The touch-sensitive interface also receives user input using detected physical contact (e.g., detected capacitance or resistance). In some embodiments, the user interface 208 is separate from the electronic controller 104.

[0042] The bus 210 connects the various components of the electronic controller 104, including, for example, the memory 204, to the electronic processor 202. The memory 204 includes, for example, read-only memory (ROM), random access memory (RAM), electronically erasable programmable read-only memory (EEPROM), other non-transitory computer-readable media, or a combination thereof. In some embodiments, the memory 204 is included in the electronic processor 202. The electronic processor 202 is configured to retrieve computer-readable instructions and data from the memory 204 and execute the computer-readable instructions to implement the functionality described herein.

[0043] Figure 6 The first ISFET 112 shown in FIG. 1 detects the ion concentration in the active solution 116 proximate to its sensing area SAl and outputs a voltage to the electronic controller 104 via its drain terminal Dl. Figure 6The second ISFET 114 shown in FIG. 1 detects the ion concentration in the reference solution 118 proximate to its sensing area SA2 and outputs a voltage via its drain terminal D2 to the electronic controller 104. The electronic controller 104 is configured to determine the differential voltage between the first ISFET 112 and the second ISFET 114. In some embodiments, the electronic controller 104 determines the differential voltage between the drain terminal Dl of the first ISFET 112 and the drain terminal D2 of the second ISFET 114. The differential voltage reduces (or eliminates) system drift or interference caused by, for example, temperature variations, time variations, and coupling from other nodes (e.g., power supply or clock nodes) in the closed loop system 100.

[0044] The working electrode 106 is disposed in the active solution 116 proximate to the sensing area SAi. The electronic controller 104 is also configured to apply a current or voltage to the working electrode 106 to adjust the ion concentration (and pH) in the active solution 116 proximate to the sensing area SAi. In some embodiments, the current or voltage is applied to the working electrode 106 in a constant current or constant potential manner. In Figure 6 In the embodiment shown in FIG. 1, the electronic controller 104 applies a current to the working electrode 106 via a current source 126 coupled between the working electrode 106 and the counter electrode 108. The current source 126 supplies a positive current to increase the hydrogen ion concentration and decrease the pH proximate to the sensing area of the first ISFET 112. The current source 126 also supplies a negative current to decrease the hydrogen ion concentration and increase the pH proximate to the sensing area of the first ISFET 112. In Figure 6 In the embodiment shown in FIG. 1, the current source 126 is separate from the electronic controller 104, and the electronic controller 104 is configured to generate an analog or digital signal that sets the output current of the current source 126. In other embodiments, the current source 126 is included in the electronic controller 104. In still other embodiments, the electronic controller 104 applies a voltage between the counter electrode 108 and the working electrode 106 via an external or internal voltage source.

[0045] The electronic controller 104 sets the current or voltage to be applied to the working electrode 106 to reduce the difference between the differential voltage and a target voltage. In some embodiments, the electronic controller 104 determines the target voltage based on the reference voltage measured by the reference electrode 110. For example, the target voltage can be substantially equal to the reference voltage from the reference electrode 110. In other embodiments, the target voltage is fixed at a predetermined value, or dynamically set based on other parameters of the closed loop system 100.

[0046] In closed loop system 100, the output of first ISFET 112 is used to sense pH, and electronic controller 104 actively adjusts the current or voltage to raise or lower the pH to a desired level. The current or voltage can be adjusted to a higher value in response to a difference between a target pH value and an actually measured pH or a disturbance in the solution to cause the pH to quickly reach the desired value. The current or voltage can also be adjusted by a small amount to maintain a constant pH over a long period of time.

[0047] Figure 9 is a flowchart of one example of a closed loop method 300 for controlling pH. At block 302, the sensing area SAl of working electrode 106, counter electrode 108, reference electrode 110, and first ISFET 112 is immersed in active solution 116. At block 304, the sensing area SA2 of second ISFET 114 is immersed in reference solution 118. At block 306, a first amount of current is applied to working electrode 106. For example, electronic controller 104 generates a signal and sends the signal to current source 126, which causes current source 126 to apply the first amount of current to working electrode 106. At block 308, a differential voltage between first ISFET 112 and second ISFET 114 is determined (e.g., by electronic controller 104). In some embodiments, the differential voltage between first ISFET 112 and second ISFET 114 is the differential voltage between drain terminal Dl of first ISFET 112 and drain terminal D2 of second ISFET 114. At block 310, a difference between the differential voltage and a target voltage is determined (e.g., by electronic controller 104). At block 312, a second amount of current is set to reduce the difference between the differential voltage and the target voltage. As a first example, when the differential voltage is less than the target voltage, electronic controller 104 sets the second amount of current to be higher than the first amount of current. As a second example, when the differential voltage is greater than the target voltage, electronic controller 104 sets the second amount of current to be lower than the first amount of current. At block 314, the second amount of current is applied to working electrode 106. For example, electronic controller 104 generates a signal and sends the signal to current source 126, which causes current source 126 to apply the second amount of current to working electrode 106. In some embodiments, method 300 returns to block 308 after block 314 to continuously control pH. For example, electronic controller 104 determines a new (or updated) differential voltage between first ISFET 112 and second ISFET 114 resulting from the second amount of current applied to working electrode 106.

[0048] In some embodiments, feedback electrode set 102 is one of a plurality of feedback electrode sets arranged in an array. Figure 10 is one example of an array of feedback electrode sets. In some embodiments, the array is arranged in a different manner than Figure 10The configuration shown in FIG. 1 includes fewer or additional components. For example, in practice, the array can include additional components such as connection electrodes, contact pads, etc. These additional components are not shown here for ease of illustration. In some embodiments, multiple feedback electrode groups share the counter electrode 108 and / or the reference electrode 110.

[0049] In some embodiments, the array further includes non-feedback electrode groups. Figure 11 FIG. 2 is an example of an array including feedback electrode groups and non-feedback electrode groups. Each of the non-feedback electrode groups includes a working electrode 106. In some embodiments, each of the non-feedback electrode groups further includes a counter electrode 108 and a reference electrode 110. The feedback electrode groups are used to identify electrical parameters for achieving one or more target pH values for each round by a feedback control scheme. The identified electrical parameters are applied to the non-feedback electrode groups. For example, each feedback electrode group targets an independent pH value. For each target pH value, there is one or more non-feedback electrode groups that are also assigned the same pH target. Because the working electrodes 106 are similar in shape and size, the electrical parameters obtained from the feedback electrode groups can be directly applied to the working electrodes 106 in the non-feedback electrode groups. Such a control scheme can be used, for example, to perform multiple rounds of reaction steps or visualizations, such as making a library array of polymers including peptides and nucleic acids.

[0050] In some embodiments, the feedback electrode groups and the non-feedback electrode groups are disposed in different sections of the array. For example, in FIG. 2, the feedback electrode groups are disposed in a feedback control section and the non-feedback electrode groups are disposed in a non-feedback control section. Figure 11 In other embodiments, such as shown in FIG. 3, the feedback electrode groups are interspersed throughout the array, surrounded by non-feedback electrode groups. Figure 12 In some embodiments, the feedback electrode groups are interspersed throughout the array, surrounded by non-feedback electrode groups. In such embodiments, the electrical parameters are selected to overcome the effects from neighboring electrodes, if any, by averaging the effects from various pH values. Figure 12

[0051] In some embodiments, the active solution includes a pH modulating reagent and an analyte labeled with a pH sensitive tag, and a pH modulating region is formed near the surface of the working electrode as a result of the pH control process as described herein. In some embodiments, the pH sensitive tag of the analyte generates a signal in the pH modulating region. The presence and intensity of the signal can vary in response to the pH value in the pH modulating region. In some embodiments, the method further includes detecting the signal generated by the pH sensitive tag in the pH modulating region.

[0052] In particular embodiments, the present disclosure provides an assay method, comprising:

[0053] ​(a) immersing a feedback electrode set of a closed loop system as described herein in a solution comprising a pH modulating reagent and an analyte labeled with a pH sensitive tag;

[0054] (b) performing a method by an electronic controller, the method comprising:

[0055] (b1) measuring an output sense value from the first sense terminal;

[0056] (b2) determining a difference between the output sense value and a target sense value; and

[0057] (b3) applying an amount of current or voltage to the working electrode to reduce the difference between the output sense value and the target sense value;

[0058] whereby the method (b) forms a pH modulating region near the surface of the working electrode;

[0059] wherein the pH sensitive tag of the analyte generates a signal in the pH modulating region; and

[0060] (c) detecting the signal generated by the pH sensitive tag in the pH modulating region.

[0061] A pH modulating reagent refers to a compound or composition that undergoes a chemical reaction in response to an electrical potential or current in a solution, thereby causing a change in the pH of the solution. The chemical reaction can be a redox reaction in which the redox state of the pH modulating reagent is changed. Electrochemical oxidation and / or reduction of the pH modulating reagent via electrical stimulation can introduce a local pH change through the balance between the production or consumption of protons and the buffer capacity of the buffer solution. This can generate a pH modulating region that is very short in vertical distance from the surface of the electrode (e.g., a few nanometers to a few microns). In some embodiments, the pH modulating reagent can include a material that can enable proton-coupled electron transfer. Suitable pH modulating reagents include, but are not limited to, quinone derivatives, aminophenol derivatives, aniline derivatives, benzidine derivatives, hydrazine derivatives, phenol-Ru(2,2'-bipyridine)3 2+ and combinations thereof. Suitable pH modulating reagents can also include other known compounds with pH responsive moieties not exemplified above.

[0062] In some embodiments, the pH modulating reagent is a quinone derivative of any one of formulas (I)-(XII)

[0063] (I), (II),

[0064] (III), (IV),

[0065] (V), (VI),

[0066] (VII), (VIII),

[0067] (IX), (X),

[0068] (XI) (XII),

[0069] X = C or N X = C or N

[0070] wherein R1, R2, R3, R4, R5, R6, R7and R8are each independently selected from:

[0071] H; C n H 2n+1 ; Cl; F; I, Br, OM, N02, OH, OC n H 2n+1 , OC n H 2n OH, O(C n H 2n O) y H, O(C n H 2n O) y C n H 2n+1 , O(C n H 2n O) y COOH; O(C n H 2n O) y COOM; COOH; COOM; COOC n H 2n+1 ; CONHC n H 2n+1 ; CON(C n H 2n+1 )2; SO3H; SO3M; NH2; NHC n H 2n+1 ; N(C n H 2n+1 )2; NHC n H 2n OH; NHC n H 2n NH2; N(C n H 2n OH)2; N(C n H 2n NH2)2; NHCOCn H 2n+1 ; NC n H 2n COC n H 2n+1 ; NC n H 2n COC n H 2n OH; NC n H 2n COC n H 2n NH2; NHC n H 2n COC n H 2n SH; SH; SC n H 2n+1 ; SC n H 2n OH; S(C n H 2n O) y H; S(C n H 2n O) y C n H 2n+1 ; S(C n H 2n O) y COOH; S(C n H 2n O) y COOM; OC n H 2n SH; O(C n H 2n O) y C n H 2n SH; O(C n H 2n O) y C n H 2n SC n H 2n+1 ; C n H 2n OC n H 2n+1 ; C n H 2n SC n H 2n+1 ; C n H 2n NHC n H 2n+1 ; C n H 2n OH; C n H2n OC n H 2n+1 ; C n H 2n OC n H 2n OH; C n H 2n O(C n H 2n O) y COOH; C n H 2n O(C n H 2n O) y COOM; C n H 2n COOH; C n H 2n COOM; C n H 2n COOC n H 2n+1 ; C n H 2n CONHC n H 2n+1 ; C n H 2n CONH(C n H 2n+1 )2; C n H 2n SO3H; C n H 2n SO3M; C n H 2n NH2; C n H 2n N(C n H 2n+1 )2; C n H 2n NHC n H 2n OH; C n H 2n NHC n H 2n NH2; C n H 2n N(C n H 2n OH)2; C n H 2n N(C n H 2n NH2)2; C n H 2n NHCOC n H 2n+1 ; C n H2n NHC n H 2n COC n H 2n OH; C n H 2n NHC n H 2n COC n H 2n NH2;C n H 2n NHC n H 2n COC n H 2n SH;C n H 2n SH;C n H 2n SC n H 2n+1 C n H 2n SC n H 2n OH; C n H 2n S(C n H 2n O) y H;C n H 2n S(C n H 2n O) y C n H 2n+1 C n H 2n S(C n H 2n O) y C n H 2n COOH; C n H 2n S(C n H 2n O) y C n H 2n COOM; sugar; peptide; and amino acids,

[0072] in

[0073] M can be any metal cation or NH4. + ,

[0074] n is 1 to 10 9 an integer, and

[0075] y is 1 to 10 9 Integers.

[0076] In some implementations, R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from C. n H 2n+1 C n H 2n OH; C n H 2n OC n H 2n OH; C n H 2n O(C n H 2n O) y COOH; C n H 2n O(C n H 2n O) y COOM; C n H 2n COOH; C n H 2n COOM; C n H 2n COOC n H 2n+1 C n H 2n CONHC n H 2n+1 C n H 2n CONH(C n H 2n+1 )2; C n H 2n SO3H; C n H 2n SO3M; C n H 2n NH2;C n H 2n NHC n H 2n+1 C n H 2n N(C n H 2n+1 )2; C n H 2n NHC n H 2n OH; C n H 2n NHC n H 2n NH2;C n H 2n N(C n H 2n OH)2;C n H2n N(C n H 2n NH2)2; C n H 2n NHCOC n H 2n+1 ; C n H 2n NHC n H 2n COC n H 2n OH; C n H 2n NHC n H 2n COC n H 2n NH2; C n H 2n NHC n H 2n COC n H 2n SH; C n H 2n SH; C n H 2n SC n H 2n OH; C n H 2n S(C n H 2n O) y H; C n H 2n S(C n H 2n O) y C n H 2n+1 ; C n H 2n S(C n H 2n O) y C n H 2n COOH; and C n H 2n S(C n H 2n O) y C n H 2n COOM.

[0077] Suitable quinone derivatives can contain various functional groups to modulate their solubility, biocompatibility, and electrochemical properties. Other examples of suitable quinone derivatives include those described in US9766197, US9874538, US9910008, US10011549, US10041905, US20170010238, and WO2017005587 (PCT / EP2016 / 065252), the entire contents of which are incorporated herein by reference.

[0078] The analyte can be a synthetic or natural molecule, a biological sample, a chemical complex, or a combination thereof. In some embodiments, the analyte is a synthetic or natural small molecule (e.g., a molecular weight of 500 Da or less), a polymer, or a biological molecule such as a protein, a polypeptide, DNA, RNA, and a lipid. In some embodiments, the analyte is a biological sample such as a fixed tissue, a cell such as a fixed cell and a live cell, an extracellular vesicle, or a combination thereof. In some embodiments, the analyte is a chemical complex such as a metal chelate or a complex of biological molecules (e.g., a protein-protein complex, a protein-ligand complex, a protein-antibody complex, or a protein-DNA complex). The complex of biological molecules can have two, three, four, five, six, seven, or eight biological molecules as components, and the complex can be formed by affinity binding and / or conjugation between the individual components.

[0079] The analyte can be labeled with the pH-sensitive tag by chemical bonding or attachment. As used herein, the term “chemical bonding” or “chemical attachment” refers to the formation of a chemical bond between two substances. The chemical bond can be an ionic bond, a covalent bond, a dipole-dipole interaction, or a hydrogen bond. In some embodiments, the analyte is a biological molecule chemically attached to the pH-sensitive tag. In some embodiments, the analyte includes a protein or a peptide chemically attached to the pH-sensitive tag. For example, the analyte can be a protein or a peptide attached to the pH-sensitive tag via a covalent bond.

[0080] The pH-sensitive tags disclosed herein include any reagent that produces a detectable signal, particularly an optical signal, directly or indirectly in response to a change in pH. Suitable pH-sensitive tags include, but are not limited to, fluorescent dyes, fluorescent proteins, enzymes, and combinations thereof. The analyte can be labeled with the pH-sensitive tag using methods known in the art. In some embodiments, the labeling method can include the use of known labeling detection agents, such as antibodies, DNA, RNA, aptamers, peptides, lipids, and small molecules. The labeling can include the direct attachment of the pH-sensitive tag to the analyte. Alternatively, the analyte can be indirectly labeled. For example, the labeling can include the attachment of the pH-sensitive tag to a detection agent that is attached to the analyte. In some embodiments, the analyte is a biomolecule that is directly or indirectly labeled with the pH-sensitive tag. In some embodiments, the labeling method can include chemical modification by functional groups, such as methoxy- or ethoxy-, acetoxy- and trichlorosilane, primary or secondary amines, NHS esters, maleimides, azides, or thiols.

[0081] In some embodiments, the pH-sensitive tag is a pH-sensitive fluorescent dye. Suitable fluorescent dyes include, but are not limited to, pHrodo, Protonex, Oregon Green, LysoSensor Green, pHAb, fluorescein, FAM, rhodamine B derivatives, and SNARF.

[0082] Suitable fluorescent proteins include, but are not limited to, green fluorescent protein, yellow fluorescent protein, and cyan fluorescent protein. In some embodiments, the fluorescent protein is green fluorescent protein (GFP) and mutants thereof (eGFP).

[0083] Suitable enzymes that can be used as pH-sensitive tags include, but are not limited to, horseradish peroxidase (HRP), glucose oxidase, and alkaline phosphatase.

[0084] The solution can be buffered or unbuffered. A buffered solution refers to an aqueous or organic solution whose pH can be maintained at a nearly constant level and does not interfere with the operation of the imaging instrument. In some embodiments, the buffered solution is an aqueous solution, such as a phosphate buffer, a citrate buffer, an acetate buffer, or other buffers used for biological applications. In some embodiments, the buffer is a solution in which the biological function of a biological sample can be detected or monitored. For example, the buffered solution can be a culture medium used for cell culture. In some embodiments, the analyte is contained in a sample of a human body fluid (such as blood, serum, saliva, urine, or sweat), a consumable (such as milk, food, or drinking water), an industrial liquid, or a natural resource (such as lake water, river water, or sea water). The sample can be added to the buffered solution prior to performing a method as described herein.

[0085] In some embodiments, the optical signal generated by the pH-sensitive tag is a colorimetric signal, such as a color change, a chemiluminescent signal, such as chemiluminescent emission, or a fluorescent signal, such as fluorescent emission. The generation and intensity of the optical signal can depend on the amount of pH-sensitive tag detected. In some embodiments, the optical signal is fluorescent emission from the pH-sensitive tag in response to the pH modulation, which can be detected by a fluorescence microscope known in the art. In particular embodiments, the optical signal can also refer to the difference between the optical output (e.g., fluorescent intensity) detected at various stages of the method. For example, the optical signal can refer to the change in fluorescent intensity detected before and after applying an electrical potential or current to the electrode as disclosed herein.

[0086] The intensity of the detection signal (e.g., the magnitude of fluorescent emission) generated by the pH-sensitive tag of the labeled analyte in the pH-modulated region can be correlated to the presence and amount of the analyte in the solution. For example, a standard curve can be calculated using the signal obtained from a standard solution with a known concentration of the analyte, which can then be used to determine the concentration of the analyte in an unknown sample. The method can further comprise quantifying the concentration of the analyte in the solution using the intensity of the detected signal.

[0087] In some embodiments, the surface of the working electrode can comprise a capture agent. The capture agent can be coated onto the surface or chemically attached to the surface. The capture agent can be a small molecule, a biomolecule (e.g., a protein, an antibody, a peptide, or a nucleic acid), or a complex of biomolecules. The analyte can be coupled to the capture agent through affinity binding or chemical attachment. The analyte is thus immobilized near the surface of the working electrode.

[0088] The methods described herein can be used to detect an analyte in various chemical and biological assays, such as immunoassays and other types of assays based on protein-protein interactions. In particular embodiments, an enzyme-linked immunosorbent assay (ELISA) can be performed by the method. For example, to detect a target protein in a solution, an antibody can be attached to the surface of the working electrode as a capture agent (e.g., through ssDNA spotting on the surface and subsequent antibody-oligonucleotide conjugation). The solution includes a pH-modulating reagent (e.g., 2,5-dimethyl-1,4-hydroquinone) and a pH-modulated region is formed as described herein. The target protein is immobilized near the surface of the working electrode by binding to the capture agent. A detection antibody and a pH-sensitive tag (e.g., a fluorescent dye) then bind to the target protein directly or indirectly to form a protein complex as the analyte, which can be detected by measuring the signal generated by the tag (e.g., fluorescent imaging).

[0089] In one example of an ELISA, spots of capture antibodies are prepared on the surface of a working electrode incubated with a buffer solution containing a target protein, a detection antibody labeled with biotin, streptavidin labeled with pHAb, and 2,5-dimethyl-l,4-hydroquinone and 2,5-dimethylbenzoquinone as pH modulating reagents. pH modulation and control are performed according to the methods described herein, and fluorescent imaging is performed to detect the signal generated by the labeled target protein.

[0090] Various aspects of the present disclosure can employ any one or more of the following example configurations.

[0091] EEE(1) A closed loop system for controlling pH, which in some embodiments includes a feedback electrode set and an electronic controller. The feedback electrode set includes a working electrode, a counter electrode, a reference electrode, a first ion-sensitive field effect transistor (ISFET), and a second ISFET. The working electrode, the counter electrode, and the reference electrode are submersible in an active solution. The first ISFET includes a first sensing terminal submersible in the active solution. The second ISFET includes a second sensing terminal submersible in a reference solution. The electronic controller is coupled to the feedback electrode set. The electronic controller is configured to apply a first amount of current or voltage to the working electrode. The electronic controller is further configured to determine a differential voltage between the first ISFET and the second ISFET. The electronic controller is further configured to determine a difference between the differential voltage and a target voltage. The electronic controller is further configured to set a second amount of current or voltage to reduce the difference between the differential voltage and the target voltage. The electronic controller is further configured to apply the second amount of current or voltage to the working electrode.

[0092] EEE(2) The closed loop system of EEE(1), wherein the electronic controller is coupled to a first drain terminal of the first ISFET and to a second drain terminal of the second ISFET, and wherein to determine the differential voltage between the first ISFET and the second ISFET, the electronic controller is further configured to determine the differential voltage between the first drain terminal and the second drain terminal.

[0093] EEE(3) The closed loop system of EEE(1) or EEE(2), wherein a first source terminal of the first ISFET is coupled to a second source terminal of the second ISFET, and wherein the first source terminal and the second source terminal are biased using at least one selected from a current source, a reference terminal with a pseudo-differential configuration, and a load impedance.

[0094] EEE(4) The closed loop system of EEE(2), wherein the first drain terminal is coupled to a first load, and wherein the second drain terminal is coupled to a second load.

[0095] EEE(5) The closed loop system of EEE(4), wherein the first load comprises at least one selected from a fixed current source, a p-channel metal oxide semiconductor field effect transistor (MOSFET), a cascaded p-channel MOSFET, a resistor, and a resonant circuit.

[0096] EEE(6) The closed loop system of any one of EEE(l) to EEE(5), wherein the active solution is physically separated from the reference solution.

[0097] EEE(7) The closed loop system of any one of EEE(l) to EEE(5), wherein the active solution is isolated from the reference solution.

[0098] EEE(8) The closed loop system of any one of EEE(l) to EEE(7), wherein the electronic controller is further configured to measure a reference voltage from a reference electrode and determine a target voltage based at least in part on the reference voltage.

[0099] EEE(9) The closed loop system of any one of EEE(l) to EEE(8), wherein the working electrode and the counter electrode are coupled to a current or voltage source, and wherein the electronic controller is further configured to apply a first amount of current or voltage and a second amount of current or voltage via the current or voltage source.

[0100] EEE(10) The closed loop system of any one of EEE(l) to EEE(9), wherein the feedback electrode set is one of a plurality of feedback electrode sets arranged in an array.

[0101] EEE(11) The closed loop system of EEE(10), further comprising a plurality of non-feedback electrode sets arranged in an array, wherein each of the plurality of non-feedback electrode sets comprises a working electrode and a counter electrode.

[0102] EEE(12) The closed loop system of EEE(l), wherein the plurality of feedback electrode sets and the plurality of non-feedback electrode sets are disposed in different sections of the array.

[0103] EEE(13) The closed loop method of EEE(l), wherein the working electrode is a first working electrode, wherein the closed loop system further comprises a non-feedback electrode set comprising a second working electrode that is submersible in the active solution, and wherein the electronic controller is further configured to apply a second amount of current or voltage to the second working electrode.

[0104] EEE(14) A closed loop method for controlling pH comprising submerging a working electrode, a counter electrode, a reference electrode, and a first sensing terminal of a first ion-sensitive field effect transistor (ISFET) in an active solution. The closed loop method further comprises submerging a second sensing terminal of a second ISFET in a reference solution. The working electrode, the counter electrode, the reference electrode, the first ISFET, and the second ISFET are included in a feedback electrode set. The closed loop method further comprises applying a first amount of current or voltage to the working electrode with an electronic controller. The closed loop method further comprises determining a differential voltage between the first ISFET and the second ISFET with the electronic controller. The closed loop method further comprises determining a difference between the differential voltage and a target voltage with the electronic controller. The closed loop method further comprises setting a second amount of current or voltage to reduce the difference between the differential voltage and the target voltage with the electronic controller. The closed loop method further comprises applying the second amount of current or voltage to the working electrode with the electronic controller.

[0105] EEE(15) The closed loop method of EEE(14), wherein determining the differential voltage between the first ISFET and the second ISFET with the electronic controller comprises determining a differential voltage between a first drain terminal of the first ISFET and a second drain terminal of the second ISFET.

[0106] EEE(16) The closed loop method of EEE(14) or EEE(15), further comprising biasing a first source terminal of the first ISFET and a second source terminal of the second ISFET with at least one selected from a current source, a reference terminal having a pseudo-differential configuration, and a load impedance.

[0107] EEE(17) The closed loop method of any one of EEE(14) to EEE(16), further comprising: measuring a reference voltage from the reference electrode with the electronic controller; and determining the target voltage based at least in part on the reference voltage with the electronic controller.

[0108] EEE(18) The closed loop method of any one of EEE(14) to EEE(17), wherein the working electrode is a first working electrode, and wherein the closed loop method further comprises: submerging a second working electrode of a non-feedback electrode set in the active solution; and applying a second amount of current or voltage to the second working electrode with the electronic controller.

[0109] EEE(19) The closed loop method of any one of EEE(14) to EEE(18), wherein the active solution comprises a pH modulating reagent and an analyte labeled with a pH-sensitive tag, wherein a pH modulated region is formed near a surface of the working electrode, wherein the pH-sensitive tag of the analyte generates a signal in the pH modulated region, and wherein the closed loop method further comprises detecting the signal generated by the pH-sensitive tag in the pH modulated region.

[0110] EEE (20) The closed loop method of EEE (19), wherein the surface of the working electrode comprises a capture agent, and wherein the closed loop method further comprises coupling an analyte to the capture agent.

[0111] Thereby, the present disclosure provides, inter alia, closed loop systems and methods for controlling pH. Various features and advantages will be set forth in the following claims.

[0112] In the foregoing specification, specific embodiments have been described. However, one of ordinary skill in the art appreciates that various modifications and changes can be made without departing from the scope of the claims set forth hereinbelow. Accordingly, the specification and figures are to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of the present disclosure.

[0113] The benefits, advantages, solutions to problems, and any one or more elements of any benefit, advantage or solution are not to be construed as critical, required, or essential features or elements of any or all the claims. The application is defined solely by the appended claims including any amendments made during the pendency of this application and all equivalents of the claims as

[0114] Furthermore, relational terms such as first and second, top and bottom, and the like can be used herein solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms "comprises," "comprising," "has," "having," "includes," "including," "contains," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises, has, includes, or contains a list of elements does not include only those elements but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by "comprises... a," "has... a," "includes... a," or "contains... a" does not, without more constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises, has, includes, or contains that element. The terms "a" and "an" are defined as one or more unless explicitly stated otherwise herein. The terms "substantially," "approximately," "about," or any other version thereof, are defined as being close to as understood by one of ordinary skill in the art, and in one non-limiting embodiment the term is defined to be within 10% of the value stated in a claim, in another embodiment within 5%, in another embodiment within 1%, and in another embodiment within 0.5%. The term "coupled" as used herein is defined as connected, although not necessarily directly and not necessarily mechanically. A device or structure that is "configured" in a certain way is configured in at least that way, but can also be configured in ways not listed.

[0115] The Abstract is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or the meaning of the claims. In addition, in the foregoing Detailed Description, for purposes of convenience and candor, the disclosure can be in terms of one embodiment or implementation, or it may CLAIM

Claims

1. A closed loop system for controlling pH, the closed loop system comprising: a feedback electrode set comprising: a working electrode immersed in an active solution, a counter electrode immersed in the active solution, a reference electrode immersed in the active solution, a first ion-sensitive field effect transistor having a first sensing terminal immersed in the active solution, and a second ion-sensitive field effect transistor having a second sensing terminal immersed in a reference solution; and an electronic controller coupled to the feedback electrode set and configured to: apply a first amount of current or voltage to the working electrode, determine a differential voltage between the first ion-sensitive field effect transistor and the second ion-sensitive field effect transistor, determine a difference between the differential voltage and a target voltage, set a second amount of current or voltage to reduce the difference between the differential voltage and the target voltage, and apply the second amount of current or voltage to the working electrode, wherein the active solution is physically separated from the reference solution.

2. The closed loop system of claim 1, wherein the electronic controller is coupled to a first drain terminal of the first ion-sensitive field effect transistor and to a second drain terminal of the second ion-sensitive field effect transistor, and wherein, To determine the differential voltage between the first ion-sensitive field effect transistor and the second ion-sensitive field effect transistor, the electronic controller is further configured to determine the differential voltage between the first drain terminal and the second drain terminal.

3. The closed loop system of claim 2, wherein the first source terminal of the first ion-sensitive field effect transistor is coupled to the second source terminal of the second ion-sensitive field effect transistor, and wherein the first source terminal and the second source terminal are biased using at least one selected from a current source, a reference terminal having a pseudo-differential configuration, and a load impedance.

4. The closed loop system of claim 2, wherein the first drain terminal is coupled to a first load, and wherein the second drain terminal is coupled to a second load.

5. The closed loop system of claim 4, wherein the first load comprises at least one selected from a fixed current source, a p-channel metal-oxide-semiconductor field effect transistor, a cascode p-channel metal-oxide-semiconductor field effect transistor, a resistor, and a resonant circuit.

6. The closed loop system of claim 1, wherein the electronic controller is further configured to: measure a reference voltage from the reference electrode, and determine the target voltage based at least in part on the reference voltage.

7. The closed loop system of claim 1, wherein the working electrode and the counter electrode are coupled to a current or voltage source, and wherein the electronic controller is further configured to apply the first amount of current or voltage and the second amount of current or voltage via the current or voltage source.

8. The closed loop system of claim 1, wherein the feedback electrode set is one of a plurality of feedback electrode sets arranged in an array.

9. The closed loop system of claim 8, further comprising a plurality of non-feedback electrode sets arranged in the array, wherein each of the plurality of non-feedback electrode sets comprises a working electrode and a counter electrode.

10. The closed loop system of claim 9, wherein the plurality of feedback electrode sets and the plurality of non-feedback electrode sets are disposed in different sections of the array.

11. The closed loop system of claim 1, wherein the working electrode is a first working electrode, wherein the closed loop system further comprises a non-feedback electrode set comprising a second working electrode immersed in the active solution, and wherein the electronic controller is further configured to apply a second amount of current or voltage to the second working electrode.

12. A closed loop method for controlling pH, the closed loop method comprising: immersing a working electrode, a counter electrode, a reference electrode, and a first sensing terminal of a first ion-sensitive field effect transistor in an active solution; immersing a second sensing terminal of a second ion-sensitive field effect transistor in a reference solution, wherein the working electrode, the counter electrode, the reference electrode, the first ion-sensitive field effect transistor, and the second ion-sensitive field effect transistor are included in a feedback electrode set; applying, with an electronic controller, a first amount of current or voltage to the working electrode; determining, with the electronic controller, a differential voltage between the first ion-sensitive field effect transistor and the second ion-sensitive field effect transistor; determining, with the electronic controller, a difference between the differential voltage and a target voltage; setting, with the electronic controller, a second amount of current or voltage to reduce the difference between the differential voltage and the target voltage; and applying, with the electronic controller, the second amount of current or voltage to the working electrode, wherein the active solution is physically separated from the reference solution.

13. The closed loop method of claim 12, wherein determining, with the electronic controller, the differential voltage between the first ion-sensitive field effect transistor and the second ion-sensitive field effect transistor comprises determining a differential voltage between a first drain terminal of the first ion-sensitive field effect transistor and a second drain terminal of the second ion-sensitive field effect transistor.

14. The closed loop method of claim 13, further comprising biasing a first source terminal of the first ion-sensitive field effect transistor and a second source terminal of the second ion-sensitive field effect transistor with at least one selected from a current source, a reference terminal having a pseudo-differential configuration, and a load impedance.

15. The closed loop method of claim 12, further comprising: measuring, with the electronic controller, a reference voltage from the reference electrode; and determining, with the electronic controller, the target voltage based at least in part on the reference voltage.

16. The closed loop method of claim 12, wherein the working electrode is a first working electrode, and wherein the closed loop method further comprises: immersing a second working electrode of a non-feedback electrode set in the active solution; and applying, with the electronic controller, a second amount of current or voltage to the second working electrode.

17. The closed loop method of claim 12, wherein the active solution comprises a pH modulating reagent and an analyte labeled with a pH-sensitive tag, wherein a pH modulating region is formed near a surface of the working electrode, wherein the pH-sensitive tag of the analyte generates a signal in the pH modulating region, and the signal generated by the pH-sensitive tag in the pH modulating region is detected.

18. The closed loop method of claim 17, wherein the surface of the working electrode comprises a capture agent, and the analyte is coupled to the capture agent. ​ ​ ​

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