KR20230124541A - Electronic control of the pH of the solution close to the electrode surface - Google Patents

Abstract

A device and method for controlling a pH or ion gradient comprising a multi-site array of electrodes and a set of feedback electrodes comprising a pH sensing element. Electrodes may include a reference electrode, a counter electrode and a working electrode. The device and method repeatedly selects the amount of current and/or voltage to be applied to each working electrode and applies the selected amount of current and/or voltage to each working electrode to determine the pH of a solution proximate to the working electrode. and measure the signal output of the sensing element. Multi-site arrays can include feedback and non-feedback electrode sets.

Description

Electronic control of the pH of the solution close to the electrode surface

Cross reference to related applications

This application claims priority to U.S. Patent Application Serial No. 16/932,096, filed on July 17, 2020, which is now U.S. Patent Application Serial No. 14/792,576, filed on July 6, 2015 (now U.S. Patent No. 10,379,080). is a continuation-in-part of U.S. Patent Application Serial No. 16/227,466, filed on December 20, 2018, a divisional application of which was granted on August 13, 2019. The entire disclosure of each of the above is incorporated herein by reference without disclaimer.

technology field

The present invention relates to a biosensor device for use in diagnostic methods for biomolecules. The invention also relates to a method and corresponding device and system for detecting the presence of air bubbles in an aqueous solution; and a glass slide for performing life science experiments, wherein at least some of the processing of data measured on the slide is performed by a computer processor located on the slide or on a peripheral component connected to the slide body or wirelessly coupled to the slide. performed by the processor. The present invention also relates to the use of electrochemical reactions, particularly redox reactions, in solutions to adjust the pH of the solution using an electric current. The present invention also relates to biological buffers and, in particular, to electrochemically active agents suitable for use in biological buffers and which can be used to facilitate pH control in biological buffers. Moreover, the present invention relates to a method for generating a pH concentration gradient near an electrode surface. The present invention provides control of biomolecular interactions in biosensors, methods of using integrated electronic systems to improve accuracy, precision and reliability in controlling pH gradients near electrode surfaces, and control of biomolecular interactions in such biosensors. It can be used in a method for controlling pH, a method for diagnosing biomolecules using a biosensor, and a method for improving such a biosensor.

Recently, there has been increased interest in predictive, preventive, and especially personalized medicine, which requires diagnostic tests with higher fidelity, eg, sensitivity and specificity. Multiplexed measurement platforms, such as protein arrays currently used in research, are one promising diagnostic technology in the near future. The sample in these tests can be a human body fluid such as blood, serum, saliva, biological cells, urine, or other biomolecules, but can also be consumables such as milk, baby food or water. In this field, there is a growing need for low-cost, multiplexed tests for biomolecules such as nucleic acids, proteins, and also small molecules. Achieving the required sensitivity and specificity in these tests is challenging. Combining these tests with integrated electronics and using complementary metal oxide semiconductor (CMOS) technology provided a solution to some of the challenges.

Two major limitations in detection assays include sensitivity and cross-reactivity. Both of these factors affect the minimum detectable concentration and thus the diagnostic error rate. Sensitivity in such assays is generally limited by the accuracy of label detection, the association factor of the probe-analyte pair (e.g. antibody-antigen pair), and the effective density of probe molecules (e.g. probe antibody) on the surface ( shown in Figure 1). Other molecules in the biological sample may also affect the minimum detectable concentration by binding to the probe molecule (eg a primary antibody) or by physically adsorbing the analyte to the surface of the test site (shown in FIG. 2 ). A detection agent (e.g., a secondary antibody) may also physisorb to the surface to increase the background signal (shown in Figure 2). Resolving cross-reactivity and background issues can take a significant amount of time in assay development of a new test and increases the cost and complexity of the overall test. Assays are typically optimized by finding the best reagents and conditions, and also by making the most specific probe molecules (eg antibodies). This results in longer development times, in some cases making tests impossible, and higher manufacturing costs. For example, typical development of an ELISA assay requires several scientists working over a year to find the right antibody as part of assay development. Cross-reactivity of the proteins may be the reason for the failure of these efforts.

A biosensor providing a multi-site testing platform was thought to provide a solution to some of the above-described limitations in assay development. US published patent applications US2011/0091870 and US2012/0115236, the contents of which are incorporated herein by reference in their entirety, can be applied to different reaction conditions to modulate the binding of a biomolecular analyte (eg protein) to a probe molecule. Such biosensors with multiple sites are described. For example, a signal detected in a biosensor having four sites may also have several components, for example four. These four terms are the concentration of the biomarker of interest, the concentration of the interfering analyte in the sample that non-specifically binds to the primary antibody (probe molecule) site and prevents binding of the biomarker, and the concentration of the interfering analyte in the sample that forms a sandwich and produces false signals. of the interfering analyte, and finally the concentration of the interfering analyte in the sample that physisorbs to the surface and produces an erroneous signal. Each term is also proportional to the binding efficiency factor, α _ij , which is a function of molecular affinity and other assay conditions, such as mass transport. By controlling the conditions at each site separately, different sites will have different efficiency factors.

Accurate and precise control of assay conditions at different sites to produce large changes in binding efficiency factors is critical to the performance of these biosensors as detection systems for biomolecular analytes of interest. In US2014/0008244 (the contents of which are incorporated herein by reference in its entirety), a CMOS, electrode array, or TFT-based setup to produce large variations in the coupling efficiency between test sites in a biosensor having an array of multiple test sites. Such biosensors and such methods that can be readily integrated with are described. In order to accurately measure a biomolecular analyte of interest, biosensors require high reliability and reproducibility. Fluctuations in the control of local pH due to repeated use of biosensors and between subsequent measurements can reduce the accuracy of determination of the biomolecule analyte of interest by such biosensors.

Additionally, pH is a factor that plays an important role in binding interactions between biomolecules, enzymatic activity, chemical transformations such as protection/deprotection of functional groups, chemical/biochemical reaction kinetics, and visualization of pH-sensitive reporter molecules. Since pH can serve as a universal switch or controller for many types of processes, precise control of pH, especially to control multiple conditions in parallel, can provide great opportunities in a variety of applications. Therefore, the adjustment of pH at each site of the multi-site array of the biosensor must be accurately controlled, and fluctuations in this pH adjustment must be corrected. Accordingly, there is a need for a biosensor in which pH can be accurately, reliably, and reproducibly controlled at each multi-site array test site.

General methods for measuring and controlling pH are known in the art. (Durst　 et al., "Hydrogen-Ion Activity," Kirk-Othmer Encyclopedia of Chemical Technology, pp. 1-15(2009)). Currently, the pH of a sample is usually changed either by exchanging the entire buffer solution to the target pH or by adding an acid or base to the solution. This process is time consuming, error prone, and in many cases significantly dilutes the sample. When the sample volume is small, or when several rounds of pH change are required during the course of an assay or reaction, current commercially available technologies cannot provide a good solution. Therefore, a technical solution enabling pH control by a flexible temporal and spatial target value and minimum dilution factor could be beneficial for several research and industrial applications.

Control of the active pH of a solution in contact with the electrode surface includes protein-protein interactions, isoelectric focusing, electrophoresis, combined pH studies of chemical and biochemical processes, DNA denaturation and regeneration, control of enzymatic processes, cell manipulation, high spatial and temporal Potentially applied as a means to accelerate or inhibit chemical reactions analytically, or in other processes involving pH as a variable. For example, US2014/0008244 describes a biosensor capable of adjusting a pH or ionic concentration gradient near an electrode in the biosensor to modulate the binding interaction of a biological sample of interest. In another example, US2014/0274760, which is incorporated herein by reference in its entirety, describes an improved biosensor with increased accuracy, reliability and reproducibility.

Attempts to control solution properties through surface-attached electrochemical agents have been described. Electrochemically triggered release of biotin from gold electrode surfaces modified through reduction of the quinone tether and subsequent lactonization has been demonstrated (Hodneland et al., "Biomolecular surfaces that release ligands under electrochemical control," J. Am. Chem. Soc. 122, pp. 4235-36 (2000)). Electrochemical control of self-assembly and release of antibodies from surfaces into solution was achieved by reduction and oxidation of n-decanethiol-benzoquinone (Artzy-Schnirman et al., Nano Lett. 8, pp. 3398-3403( 2008)). The release of protons from a 3D layer of electroactive material was demonstrated by Frasconi et al. using a material composed of gold nanoparticles and thioaniline (Frasconi et al., "Electrochemically Stimulated pH Changes: A Route To Control Chemical Reactivity ," J. Am. Chem. Soc. 132(6), pp. 2029-36(2010)). Electrochemical oxidation of the thioaniline groups produced protons that diffused from the electrode surface into the surrounding solution, altering its pH.

Electrochemical pH control in biological solutions presents significant challenges due to the complex nature of the system. Limitations include the presence of buffer components that limit pH change, limitations on the co-solvents that can be used, the presence of strong nucleophiles such as amines and thiols, and the presence of interfering electrochemically active components such as DNA bases, ascorbic acid and glutathione. is included

Quinones are one of the most extensively studied classes of electrochemically active molecules (see Thomas Finley, "Quinones," Kirk-Othmer Encyclopedia of Chemical Technology, 1-35 (2005), which is incorporated by reference in its entirety), [ Chambers, J. Q. Chem. Quinonoid Compd. 1974, Pt. 2:737-91; Chambers, J. Q. Chem. Quinonoid Compd. 1988, 2:719-57; ] see also). The hydroquinone/benzoquinone conversion was used as a model system for generating proton gradients at electrode surfaces (Cannan et al., Electrochem. Communications 2002, 4:886-92). A combination of para-hydroquinone and anthraquinone was used for the generation of an acidic pH in an organic solution as the first step in DNA synthesis, and an organic base was added to the solution to localize the acidic pH to the electrode surface (Maurer, PLOS One 2006, 1:e34). However, these systems are biologically challenged due to the reactivity of benzoquinone (a product of hydroquinone oxidation) towards nucleophiles that are often present in biological systems such as peptides, proteins and glutathione, additionally due to the insufficient solubility of unsubstituted anthraquinones in water. It cannot be adapted for use in solution (Amaro et al., Chem Res Toxicol 1996, 9(3):623-629).

Electrochemical time-of-flight measurements demonstrated that H ⁺ ions generated on the electrodes would diffuse (Slowinska et al., "An electrochemical time-of-flight technique with galvanostatic generation and potentiometric sensing," J. Electroanal. Chem. Vol. 554-555, pp. 61-69(2003);Eisen et al., "Determination of the capacitance of solid-state potentiometric sensors: An electrochemical time-of-flight method," Anal. Chem. 78(18), pp. 6356-63 (2006)). It has also been shown that the open circuit potential of the electrode surface is a function of the ion concentration in the solution, including the H ⁺ concentration in the solution, and thus the pH of the solution (Yin et al., "Study of indium tin oxide thin film for separative extended gate ISFET," Mat. Chem. Phys. 70(1), pp. 12-16 (2001)). Similarly, the redox rate of electrochemical species is also pH dependent (Quan et al., "Voltammetry of quinones in unbuffered aqueous solution: reassessing the roles of proton transfer and hydrogen bonding in the aqueous electrochemistry of quinones," J. Am Chem. Soc. 129 (42), pp. 12847-56 (2007)). To improve the accuracy of pH sensing, work has been done to improve the pH sensitivity of the electrode by the inclusion of novel pH sensitive coatings (Ge et al., "pH-sensing properties of poly(aniline) ultrathin films self-assembled on indium-tin oxide," Anal. Chem. 79(4), pp. 1401-10(2007)).

Several life science applications (proteomics, genomics, microfluidics, cell culture, etc.) use glass slides as substrates to conduct experiments. Examples of glass slides include protein microarrays, lysate arrays, DNA microarrays, and cell culture platforms. One use of protein microarrays is to analyze biological material (eg, serum) from a patient with a specific disease compared to corresponding material from healthy or control subjects. Biological materials are applied to microarrays containing many (often thousands) of human proteins. Antibodies in pathogenic substances can react (bind) to specific antigens on the microarray to identify antigens as disease-specific biomarkers. Besides protein detection, other types of detection are also possible with glass slides, such as colorimetric, chemiluminescent and fluorescence detection.

Often experiments are performed under aqueous conditions, and the substance of interest is combined with water or a water-containing liquid and placed on a slide for analysis. In many cases, the presence of air bubbles (formed of air or other gases) interferes with the experiment and adversely affects the results. An example of an adverse effect is when air bubbles dry the test solution. This can result in erroneous binding events in which a substance of interest (eg, a biomolecular analyte) fails to bind to a molecule presumed to interact with the biomolecular analyte. Another example is where air bubbles change the effective flow rate of the test solution and the flow rate is measured as part of the experiment. Therefore, it is desirable to detect air bubbles and indicate their presence so that the experimental results can be accurately interpreted.

One way to detect air bubbles is to manually check each slide under a microscope. However, microscopy is not always practical, as the field of view is typically limited to a small area of the slide and viewing the entire slide is time consuming. Additionally, using light to illuminate a slide under a microscope can sometimes have a destructive effect on the material of interest.

Electronic pH control described in US Pat. No. 10,379,080, incorporated herein by reference in its entirety, may provide a solution to some of these problems. Similar pH control schemes can be used in a variety of design formats, particularly array formats, to perform highly multiplexed independent measurements and reactions in parallel within the same sample solution. When using arrays of electrodes to locally control the microenvironment near each electrode, “crosstalk” or “bleed-over” between different sites is a concern. This problem is solved by spacing the individual sites apart or by adding a buffering reagent to the bulk solution. The former approach reduces the array density (larger device size), while the latter requires the electrochemical reaction rate to 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 methods may result in side reactions involving other components of the reaction system.

Closed-loop control is provided herein to address several of the problems described above. Several implementations of closed-loop in a high-density array of several individually position-sensitive electrodes are described herein. Disclosed herein are devices and methods for controlling a pH or ion gradient near an electrode surface in an array format. A device may contain one or more working electrode(s), pH sensing element(s) (which may be electrode(s) in some examples), counter electrode(s), and reference electrode(s). Each individual working electrode can electrochemically introduce a pH change within a small physical space close to the surface in a format on demand. Counter and reference electrodes can be shared by multiple actuating and sensing elements. The pH adjusting reagent can be electrochemically oxidized or reduced to create a pH adjusting zone covering nanometer to micrometer distances from the surface. Electrical output parameters to the working electrode can be adjusted based on feedback from the sensing element, so that faster and more precise pH control can be achieved.

Some aspects disclosed herein include array-based pH control, devices containing array-based pH control, and methods of use thereof. In some examples, the multi-electrode array contains a set of feedback-control electrodes consisting of a working electrode, a reference electrode, a counter electrode, and a sensing element. Reference and counter electrodes may be shared by multiple electrode sets. In some examples, each set contains at least independently controlled working electrodes and sensing elements. In some examples, each electrode set can be programmed for specific pH conditions with temporal variations. The number of electrodes in the array can range from one to hundreds of millions. In some examples, an array may be contained in a single or multi-layer device. Devices can have a variety of fabrication methods, including or excluding complementary metal oxide semiconductor (CMOS), thin film-transistor (TFT), and the like.

In some aspects, a device may contain an array of electrodes in a single device. The device may contain one single section of a set of feedback-control electrodes, or multiple sections of sets of feedback-control electrodes. The feedback-control electrode set may contain working, reference and counter electrodes, and sensing elements, which may be electrodes. Reference and counter electrodes may be shared by multiple electrode sets. The section(s) containing the feedback-control electrode set may be a feedback-control section. You can use this section to determine the required electrical parameters to achieve your target pH value. The target pH value can be different and/or varied over time or between different feedback-controlled electrode sets. In some examples, various pH conditions may be tested and/or provided simultaneously with an array of electrodes in a single device. The device may also contain an electrode set that does not contain a sensing element and/or is not a feedback-controlled electrode set. Section(s) containing electrode sets that are not feedback-controlled electrode sets may be non-feedback-controlled sections. Electrical parameters identified by use of the feedback-control section may be applied to the non-feedback control section. The non-feedback-control electrode set may have at least one electrode as a working electrode, optionally together with a reference electrode and a counter electrode.

In some examples, the device can be used to modify pH values for multiple rounds of reaction steps, some of which require distinct pH values to be assigned to specifically selected electrodes in the array. Non-limiting examples of reactions having multiple rounds of reaction steps include preparing library arrays of polymers such as peptides and nucleic acids. In some examples, the device may be used to continuously and/or visualize the pH of one or more area(s) one or more times.

In some embodiments, the feedback-control electrode sets are distributed throughout the substrate, each one or more of which is surrounded by a non-feedback-control electrode set. In some instances, the non-feedback-controlled electrode sets have a target pH value that varies between non-feedback-controlled electrode sets and/or over time. In some examples, electrical parameters identified by use of a feedback-controlled electrode set may be modified prior to application to a non-feedback-controlled electrode set to overcome influences (if any) from neighboring electrodes. The electrical parameters applied can be modified by averaging the effects from various pH values. In some instances, this configuration can help minimize the effect of pH control from adjacent electrode sets.

In some aspects, disclosed herein are closed-loop pH control, devices containing closed-loop pH control, and methods of use thereof. In some examples, closed-loop pH control includes a set of feedback-controlled electrodes of any of the arrays disclosed herein. In some examples, the array-based pH control contains closed-loop pH control, or a device containing closed-loop pH control. In some instances, closed-loop pH control regulates the pH electrochemically. A closed-loop pH control can contain a sensing element, a working electrode, a counter electrode, and a reference electrode. In some examples, such as but not limited to, the sensing element is used as a reference electrode when the sensing element is in a stable and/or stable pH solution. The counter electrode and reference electrode may be shared for multiple working electrodes and/or sensing elements. In some examples, an external counter electrode and/or reference electrode may be used. In some examples, patterned on-chip counter and/or reference electrodes may be used. In some examples, external and/or on-chip counter and/or reference electrodes may be used. In some examples, the counter electrode is disposed around the working electrode. Placement of a counter electrode around the working electrode can improve the precision of the pH control in some instances.

The solution may contact the device and/or closed-loop pH control. In some instances, a pH adjusting reagent is present in solution. A pH sensing element may be configured to measure an initial pH value of a solution. Using the initial pH value of the solution, the amount of current or voltage can be calculated and applied to the working electrode. When current or voltage is applied to the working electrode, electrochemical oxidation and/or reduction of the pH adjusting reagent can occur. Oxidation and/or reduction can introduce local pH changes. Local pH changes can occur through an equilibrium between the production or consumption of protons and the buffering capacity of the buffer solution. In some instances, pH changes may occur in unbuffered solutions. In some instances, the local pH change creates a pH control zone with a small vertical distance from the electrode surface (a few nm to a few mm). In some instances, the pH control zone allows pH-dependent chemical/biochemical reactions to occur. In some instances, pH-dependent reactions in solution occur only within the pH control zone. The size of the control zone may depend on the buffering capacity of the solution. In some instances, the control zone is smaller in stronger buffers compared to weaker buffers or non-buffered solutions. In some instances, the pH sensing element monitors the actual pH during pH adjustment. In some examples, when the pH sensing element monitors the actual pH, the electrical output continues through closed-loop control. Simultaneously monitoring and adjusting pH may allow faster and/or more precise control of pH.

The working electrode and sensing element can have various types of shapes and sizes. In some examples, shape and size depend on the application and/or requirements of the array format. In some examples, the working electrode and sensing element are arranged in a slide design. In some examples, the working electrode and sensing element are in contact with each other. In some examples, the sensing element is physically separated from the working electrode. In some examples, the sensing element is physically separated from the working electrode by a gap. In some examples, the gap may be between 1 nm and 100 microns. In some examples, an insulating layer is disposed between the sensing element and the working electrode. In some examples, the counter electrode at least partially surrounds the working electrode. In some examples, the counter electrode at least partially surrounds the working electrode when the working electrode is configured to be in contact with, or is in contact with, the unbuffered solution.

In some examples, the sensing element contains an electrode coated with a pH sensitive material. In some examples, the sensing element contains a semiconductor-based electrical component. In some examples, the sensing element contains a field-effect transistor. In some examples, the sensing element contains a polymeric semiconductor.

These closed-loop controls and devices containing them can be used, for example, in methods that can be integrated with CMOS, electrode array, or TFT-based biosensors, such as coupling efficiency between test sites in a biosensor having an array of multiple test sites. can make a big difference in In some examples, current applications provide methods for adjusting the pH or ion concentration near the electrode surface of an array, such as a biosensor. In some instances, arrays are used to modulate biomolecular interactions between a probe biomolecule and a biomolecule analyte of interest.

The array-based pH control and devices disclosed herein can be used in many types of applications including:

- control and/or monitoring of binding interactions between biomolecules;

- control and/or switching of enzyme activity on/off;

- control of chemistry for surface modification;

- chemical transformation of molecules;

- construction of the polymer structure through repeated steps of blocking and deblocking together with conjugation;

- control and/or monitoring of chemical reaction kinetics; and/or

- Visualization of pH sensitive reporter molecules.

According to an exemplary embodiment, one or more of aspects 1 to 52 below are provided.

Aspect 1 is a device for controlling the pH of a solution on an array of electrodes, the device comprising:

support; and

An array of electrodes comprising one or more feedback-control electrode sets, each of the one or more feedback-control electrode sets:

one or more reference electrodes;

one or more counter electrodes; and

one or more subsets comprising a pH sensing element electrically coupled to a working electrode, wherein a reference electrode and/or counter electrode are electrically coupled with at least one subset;

The device is:

a) selecting the amount of current and/or voltage to be applied to each working electrode to minimize the difference between the signal output of the sensing element and the target sensing value;

b) applying a selected amount of current and/or voltage to each working electrode to change the pH of the solution proximate the working electrode;

c) iteratively performing measuring the signal output of the sensing element.

Aspect 2 is the device of aspect 1, wherein the at least one reference electrode and the at least one counter electrode are electrically coupled with the at least one subset via a processor.

Aspect 3 is the device of any one of Aspects 1-2, wherein the support comprises a glass slide, plastic plate, silicon wafer, glass wafer, quartz wafer, flexible plastic sheet, polymer layer, paper, or mixtures thereof.

Embodiment 4 is wherein one or more of the working electrode, reference electrode, counter electrode, and/or sensing element are independently composed of metal oxides, glassy carbon, graphene, metals, conducting polymers, silver chloride, saturated calomel, and combinations thereof. The device of any one of aspects 1 to 3 comprising a material selected from the group.

Aspect 5 is wherein the at least one working electrode, reference electrode, counter electrode, and/or sensing element independently comprises a gold electrode, a silver electrode, a platinum electrode, a standard hydrogen electrode, a mercury drop electrode, or a combination thereof. It is the device of any one of aspects 1-4.

Embodiment 6 is the device of any one of Aspects 1-5, wherein the sensing element comprises a field-effect transistor, a polymeric semiconductor, a metal electrode, an inorganic electrode, an organic electrode, or a pH sensitive coating material, or a combination thereof.

Embodiment 7 is the device of embodiment 6, wherein the pH sensitive coating material comprises polyaniline, polypyrrole, iridium oxide, indium tin oxide, an ion-selective polymer, or a combination thereof.

Aspect 8 is the device of any of aspects 1-7 wherein at least one of the reference electrode(s) is also a sensing element.

Embodiment 9 is the device of any one of aspects 1 to 8, wherein the solution is buffered, unbuffered, aqueous, organic or a mixture thereof.

Embodiment 10 is the device of any one of Aspects 1 to 9, wherein the solution contains at least one redox active species selected from the group consisting of quinones, catechols, aminophenols, hydrazines, derivatives thereof, and combinations thereof. am.

Aspect 11 is the device of any one of aspects 1 to 10 wherein the selected current and/or voltage applied to the working electrode is applied galvanostatically or potentiostatically.

Aspect 12 is a device comprising or communicatively coupled to a processor, wherein selecting an amount of current and/or voltage to be applied comprises applying an algorithm using an input comprising a signal output of a sensing element. It is the device of any one of aspects 1 to 11.

Aspect 13 is the device of any of aspects 1-12, wherein the working electrode, reference electrode, counter electrode, and/or sensing element are electrically coupled to the switch-matrix module.

Aspect 14 is the device of aspect 13, wherein the switch-matrix module includes complementary metal oxide semiconductor (CMOS) and/or thin film transistors (TFT).

Aspect 15 is the device of any of aspects 1-14, wherein the feedback-control electrode set is electrically coupled to the device via a physical coupling.

Aspect 16 is the device of any of aspects 1-15, wherein at least one of the reference electrodes and/or at least one of the counter electrodes are electrically coupled to multiple subsets.

Aspect 17 is the device of any one of aspects 1 to 16, wherein the array of electrodes further comprises one or more non-feedback-controlled electrode set(s), wherein the one or more non-feedback-controlled electrode set(s) Is:

one or more reference electrodes;

one or more counter electrodes; and

comprising one or more working electrodes;

wherein a reference electrode of the non-feedback-controlled electrode set and/or a counter electrode of the non-feedback-controlled electrode set are electrically coupled with at least one working electrode of the non-feedback-controlled electrode set;

one or more non-feedback-controlling electrode set(s) do not include a pH sensing element;

here:

i. One or more all feedback-control electrode set(s) are included in one section of the device and one or more all non-feedback-control electrode set(s) are arranged in a physically separate second section of the device; or

ii. one or more feedback-control electrode set(s) interspersed between two or more non-feedback-control electrode sets;

At least one of the feedback-control electrode set(s) is electrically coupled to at least one of the non-feedback-control electrode set(s), and the coupled electrode set is coupled to 1 of the coupled feedback-control electrode set and to also apply the selected amount of current and/or voltage applied to the one or more working electrodes to one or more working electrodes of the coupled non-feedback-control electrode set.

Embodiment 18 is a method for controlling the pH of a solution,

a. Obtaining a device comprising a feedback-control electrode set, the feedback-control electrode set comprising:

one or more reference electrodes;

one or more counter electrodes; and

one or more subsets comprising a pH sensing element electrically coupled to a working electrode, wherein a reference electrode and/or counter electrode are electrically coupled with at least one subset;

b. selecting a target sensing value for the sensing element based on at least one signal output from the sensing element in a solution comprising the target pH;

c. of the following:

i. selecting the amount of current and/or voltage to be applied to each working electrode to minimize the difference between the signal output of the sensing element and the target sensing value;

ii. applying a selected amount of current and/or voltage to each working electrode to change the pH of the solution proximate the working electrode;

iii. Iteratively performing measuring the signal output of the sensing element.

Embodiment 19 is a method wherein the solution comprises water and/or one or more redox active species, and the current and/or voltage applied to the working electrode generates hydrogen ions by an electrochemical reaction of the water and/or one or more redox active species. is the method of aspect 18, wherein a is electrochemically produced and/or consumed.

Embodiment 20 is the method of any one of embodiments 18-19, wherein the solution is buffered, unbuffered, aqueous, organic or a mixture thereof.

Embodiment 21 is the method of Aspects 19-20, wherein the at least one redox-active species is selected from the group consisting of quinones, catechols, aminophenols, hydrazines, derivatives thereof, and combinations thereof.

Embodiment 22 includes one or more working electrodes, reference electrodes, counter electrodes, and/or sensing elements independently consisting of metal oxides, glassy carbon, graphene, metals, conducting polymers, silver chloride, saturated calomel, and combinations thereof. A method of any one of aspects 18 to 21 comprising a material selected from the group.

Embodiment 23 is wherein the at least one working electrode, reference electrode, counter electrode, and/or sensing element independently comprises a gold electrode, a silver electrode, a platinum electrode, a standard hydrogen electrode, a mercury drop electrode, or a combination thereof. A method of any one of aspects 18 to 22.

Embodiment 24 is the method of any one of Aspects 18-23, wherein the sensing element comprises a field-effect transistor, a polymeric semiconductor, a metal electrode, an inorganic electrode, an organic electrode, a pH sensitive coating material, or a combination thereof.

Embodiment 25 is the method of embodiment 24, wherein the pH sensitive coating material comprises polyaniline, polypyrrole, iridium oxide, indium tin oxide, an ion-selective polymer, or a combination thereof.

Aspect 26 is a method of any of aspects 18-25 comprising determining the pH of the solution by comparing the signal output of the sensing element to predetermined calibration data correlating the pH and the signal output value of the sensing element.

Aspect 27 is the method of any one of aspects 18-26 wherein the selected current and/or voltage applied to the working electrode is applied galvanostatically or potentiostatically.

Aspect 28 is the method of any one of aspects 18-27, wherein the obtained device comprises an array of two or more feedback-controlled electrode sets.

Aspect 29 is any one of aspects 18 to 28 wherein at least two of the feedback-controlled electrode sets are individually controlled by independent target pH values and/or independent time programs to apply selected amounts of current and/or voltage. One way.

Aspect 30 is the method of any one of aspects 18 to 29, wherein the feedback-control electrode set is electrically coupled to the switch-matrix module, and control of each feedback-control electrode set is performed by the switch-matrix module. .

Aspect 31 is the method of aspect 30, wherein the switch-matrix module includes complementary metal oxide semiconductor (CMOS) and/or thin film transistors (TFT).

Aspect 32 is the method of any of aspects 18-31, wherein the feedback-control electrode set is electrically coupled to the device via a physical coupling.

Aspect 33 is the method of any of aspects 18-32, wherein at least one of the reference electrodes and/or at least one of the counter electrodes are electrically coupled to multiple subsets.

Aspect 34 is the method of any one of aspects 18-33, wherein the obtained device is the device of any one of aspects 1-16.

Embodiment 35 is a method of controlling the pH of a solution,

a. Obtaining a device comprising an array of one or more feedback-control electrode set(s) and one or more non-feedback-control electrode set(s) electrically coupled to the feedback-control electrode set.

- one or more feedback-controlled electrode set(s):

one or more reference electrodes;

one or more counter electrodes; and

one or more subsets comprising a pH sensing element electrically coupled to a working electrode, wherein a reference electrode and/or counter electrode are electrically coupled with at least one subset;

The one or more non-feedback-control electrode set(s):

one or more reference electrodes;

one or more counter electrodes; and

one or more working electrodes, wherein the reference electrode of the non-feedback-control electrode set(s) and/or the counter electrode of the non-feedback-control electrode set(s) are the non-feedback-control electrode set(s) electrically coupled with at least one working electrode of the one or more non-feedback-controlling electrode set(s) not including a pH sensing element;

b. selecting a target sensing value for the sensing element based on at least one signal output from the sensing element in a solution comprising the target pH; and

c. of the following:

i. selecting an amount of current and/or voltage to be applied to each working electrode of the one or more feedback-controlled electrode set(s) to minimize the difference between the signal output of the sensing element and the target sensing value;

ii. A selected amount of current and/or voltage is applied to each working electrode of the one or more coupled feedback-control electrode sets and also to the non-feedback-control electrode set coupled to the one or more coupled feedback-control electrode sets. applied to the one or more working electrodes to change the pH of the solution proximal to the working electrode;

iii. Iteratively performing measuring the signal output of the sensing element.

Embodiment 36 is a method wherein the solution comprises water and/or one or more redox active species, and the current and/or voltage applied to the working electrode generates hydrogen ions by an electrochemical reaction of the water and/or one or more redox active species. It is the method of aspect 35 wherein a is electrochemically produced and/or consumed.

Embodiment 37 is the method of any one of embodiments 35-36, wherein the solution is buffered, unbuffered, aqueous, organic or a mixture thereof.

Embodiment 38 is the method of any one of embodiments 36 to 37, wherein the at least one redox active species is selected from the group consisting of quinones, catechols, aminophenols, hydrazines, derivatives thereof, and combinations thereof.

Embodiment 39 is characterized in that one or more of the working electrode, reference electrode, counter electrode, and/or sensing element are independently composed of metal oxides, glassy carbon, graphene, metals, conducting polymers, silver chloride, saturated calomel, and combinations thereof. A method of any one of aspects 35 to 38 comprising a material selected from the group.

Embodiment 40 is wherein the at least one working electrode, reference electrode, counter electrode, and/or sensing element independently comprises a gold electrode, a silver electrode, a platinum electrode, a standard hydrogen electrode, a mercury drop electrode, or a combination thereof. A method of any one of aspects 35 to 39.

Embodiment 41 is the method of any one of Aspects 35-40, wherein the sensing element comprises a field-effect transistor, a polymeric semiconductor, a metal electrode, an inorganic electrode, an organic electrode, a pH sensitive coating material, or a combination thereof.

Embodiment 42 is the method of embodiment 41, wherein the pH sensitive coating material comprises polyaniline, polypyrrole, iridium oxide, indium tin oxide, an ion-selective polymer, or a combination thereof.

Aspect 43 is a method of any of aspects 35 through 42 comprising determining the pH of the solution by comparing the signal output of the sensing element with predetermined calibration data correlating the pH and the signal output value of the sensing element.

Aspect 44 is the method of any one of aspects 35-43 wherein the selected current and/or voltage applied to the working electrode is applied galvanostatically or potentiostatically.

Aspect 45 is the method of any one of aspects 35-44, wherein the obtained device comprises an array of two or more feedback-controlled electrode sets.

Aspect 46 provides an independent target for at least two of the feedback-control electrode sets to apply selected amounts of current and/or voltage to the working electrode of the feedback-controlled electrode set and to the working electrode of the coupled non-feedback-control electrode set. The method of aspect 45, which is individually controlled by pH value and/or an independent time program.

Aspect 47 is the method of any one of aspects 35 to 46, wherein the feedback-control electrode set is electrically coupled to the switch-matrix module, and control of each feedback-control electrode set is performed by the switch-matrix module. .

Aspect 48 is the method of aspect 47, wherein the switch-matrix module includes complementary metal oxide semiconductor (CMOS) and/or thin film transistors (TFT).

Aspect 49 is the method of any one of aspects 35 to 48 wherein the feedback-controlled electrode set and the non-feedback-controlled electrode set are electrically coupled to the device via a physical coupling.

Embodiment 50 relates to a reference electrode and/or counter electrode of at least one of the feedback-control electrode sets to multiple working electrodes of multiple subsets of feedback-control electrode sets and/or multiple working electrodes of non-feedback-control electrode sets. The method of any one of aspects 35 to 49, wherein the method is electrically coupled.

Aspect 51 is the method of any one of aspects 35 to 50, wherein the working electrode of the coupled non-feedback-control electrode set has a similar shape and design as the working electrode of the coupled feedback-control electrode.

Aspect 52 is the method of any one of aspects 35 to 51, wherein the device obtained is the device of aspect 17.

As used herein, various terms are used only for the purpose of describing a particular implementation and are not intended to limit the implementation.

For example, as used herein, ordinal terms used to modify elements such as structures, components, operations, etc. (e.g., "first," "second," "third," etc.) It does not in itself indicate any priority or order of an element in relation to another element, but rather merely distinguishes an element from another element having the same name (but using an ordinal term).

The term “coupled” is defined as connected, not necessarily directly and not necessarily mechanically. Additionally, two items that are “coupled” may be singular to each other. For illustrative purposes, components may be coupled by physical proximity, integrated into a single structure, or formed from the same piece of material. Coupling may also include mechanical, thermal, electrical, communicative (eg, wired or wireless), or chemical coupling (eg, chemical bonds) in some contexts.

Singular terms are defined as one or more unless expressly required otherwise in this disclosure. The term “substantially” is defined as generally but not necessarily all of what is specified (and includes what is specified; e.g. substantially 90 degrees includes 90 degrees, and Substantially parallel includes parallel). As used herein, the terms “about” and “about” may be substituted for “within 10%” of that specified. Additionally, the terms “substantially” and “about” may be substituted for “within [percentage]” of that specified, and percentages may include .1, 1, or 5%; or design, manufacturing or measurement tolerances. The phrase “and/or” means and or or. For illustrative purposes, A, B, and/or C may include A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or A, B, and C A combination of In other words, “and/or” serves as an inclusive or.

The terms “comprises” (and any form of includes, such as “comprising”), “has” (and any form of has, such as “having”), “contains” (and any form of contains , such as “comprising”) and “comprises” (and any form of encompasses, such as “comprising”) is non-limiting and open-ended and may include additional steps, materials, etc. within its scope. As a result, a device that "comprises", "has", "includes" or "includes" one or more elements has those one or more elements, but is not limited to having only these one or more elements. Likewise, A method that "comprises," "has," "includes," or "comprises" one or more steps has, but is not limited to, these one or more steps.

Any aspect of any material, composition, system, method and article of manufacture may consist of, or consist essentially of (including/having/not encompassing) any of the recited steps, elements and/or features. Thus, the terms "consisting of" or "consisting essentially of" in any claim may be replaced with any open linking verb recited above, using an open linking verb to change the scope of a given claim from what it does not. Additionally, it will be appreciated that the term “where” may be used interchangeably with “here”.

Additionally, a device or system configured in a particular way is configured at least that way, but it may also be configured in a manner other than that specifically described. A feature or features of one embodiment, even if not described or illustrated, may be applied to other embodiments unless expressly prohibited by the disclosure or nature of the embodiment.

Some details related to aspects of the present disclosure have been described above and others are described below. Other implementations, advantages and features of the present disclosure will become apparent after reviewing the entire application including the following sections: BRIEF DESCRIPTION OF THE DRAWINGS, DETAILED DESCRIPTION AND CLAIMS.

Figure 1: Illustration of the steps of a typical and well-known ELISA assay: a) the sample is introduced into an immobilized primary antibody on a blocked surface, incubated, b) the sample is washed, c) a labeled secondary antibody is added . The number of labels is proportional to the concentration of the target antigen.
Figure 2: Illustration of undesirable cross-reactivity. Molecules other than the antigen of interest (diamond) may bind to the primary antibody or surface and generate false signals or prevent the antigen from forming a sandwich.
Figure 3: Illustration of multi-site sensors and components in the detected signal. The two schematics at the bottom correspond to two of the sites.
Figure 4: Illustration of the composition of the sensor test site in a multi-site sensor.
Figure 5: Schematic diagram of pH change on the electrode surface using electrochemical methods.
Figure 6: Example of pH change by an enzymatic reaction upon proximity to a protein surface using magnetic micro/nanoparticles. Micro/nano cavities help localize pH changes.
Figure 7a: Cyclic voltammetry of an indium tin oxide (ITO) electrode in PBS alone. A region where a change in pH may occur is where there is oxygen evolution above 1V relative to the Ag/AgCl reference electrode.
Figure 7b: Cyclic voltammetry study of oxidation of ascorbic acid test on ITO electrode. A current increase of approximately 0.25 V indicates the onset of oxidation at the ITO electrode.
Figure 8a: Application of 1V on the surface of ITO-PEG in phosphate buffer. The change in impedance before and after application of 1 V indicates the change or removal of PEG from the electrode.
Figure 8b: Oxidation of ascorbic acid at 0.5 V and 0.75 V on ITO-PEG surfaces. No change in impedance during ascorbic acid oxidation indicates that the PEG layer did not undergo any change.
Figure 9: Biomolecular interface layer 10 comprising immobilized fluorescent protein (eg green fluorescent protein (GFP)) spots 5 and immobilized probes 4 using polyethylene glycol (PEG) linkers 3 Illustration of a substrate (glass or plastic) 1 with an array 2 of applied electrodes.
Figure 10: Shows the change in fluorescence intensity of GFP covalently linked to PEG-coated ITO in response to changes in solution pH. The solution pH was adjusted by adding HCl to dilute phosphate buffer (pH 7.4).
Figure 11: ITO working electrode produced through current-induced oxidation of the redox active molecule 2-methyl-1,4-dihydroquinone in dilute phosphate buffer (pH = 7.4) containing 0.1 M Na ₂ SO ₄ . The pH change at the surface is plotted. After 10 seconds induction, current (50 microamperes) was applied for 30 seconds, and the solution pH dropped to 5.5, as observed by a change in GFP fluorescence intensity (FIG. 10 is a calibration curve for evaluating pH values). used as). After the current was turned off, the pH returned to neutral values within 50 seconds.
12A - 12B: Illustrates visual changes in GFP spots before, during and after pH control experiments. In FIG. 12A, the profile of fluorescence intensity across the spot is shown. In FIG. 12B, changes in GFP spot fluorescence intensity are shown before (0 sec), during (40 sec) and after (110 sec) application of current through the electrode.
Figure 13: Shows a glass slide with an ASIC chip connected to a transparent ITO electrode.
Figure 14: A block diagram of a system for bubble detection and/or pH control in accordance with an exemplary embodiment of the present invention.
15 : A top view of an exemplary electrode array according to an exemplary embodiment of the present invention.
16 and 17 : show different electrode shapes according to an exemplary embodiment of the present invention.
Figures 18-20: Each depicts slides with data processing capabilities in accordance with exemplary embodiments of the present invention.
21 : shows a graphical representation of a system for electrochemical pH generation comprising a biological buffer with an electrochemically active agent dissolved in a bulk solution above an electrode in accordance with an exemplary embodiment of the present invention, wherein the pH change is dependent on the buffering of the bulk solution. Through action, it is confined to the vicinity of the electrode surface.
22A-22C : Provides structures of hydroquinone and benzoquinone that can be used for pH generation in biological solutions according to exemplary embodiments of the present invention.
Figure 23: Demonstrates the effect of substitution of benzoquinones on the stability of proteins according to an exemplary embodiment of the present invention.
Figure 24: shows a square wave voltammetry of a substituted quinone in a buffered solution (versus Ag/AgCl) according to an exemplary embodiment of the present invention.
25A and 25B: Shows the steps for the synthesis of substituted hydroquinones and benzoquinones.
Figure 26: illustrates an open loop waveform used to maintain the pH of a solution in proximity of an electrode in accordance with an exemplary embodiment of the present invention.
Figure 27: Illustrates an example of waveform formation for pH control in accordance with an exemplary embodiment of the present invention.
Figure 28: Illustrates the response of the open circuit potential for a PANI coated surface as a function of pH according to an exemplary embodiment of the present invention, 60 mV/pH is close to the Nerstian limit.
29A-29B: In accordance with an exemplary embodiment of the present invention, FIG. 29A illustrates a controlled OCP on a sensing electrode SE using a closed loop feedback method with a single OCP V _target , and in FIG. 29B a defined top and Experimental results of OCP controlled on SE using closed-loop feedback with lower OCP V _target value are illustrated.
Figure 30: Closed look feedback method of controlling the pH of a solution as indicated by the open circuit potential voltage measured by the sensing electrode (SE) by applying a potential to the working electrode (WE) according to an exemplary embodiment of the present invention. shows the experimental results of
31 : Illustrates an open loop schematic according to an exemplary embodiment of the present invention, in which a controlled current source is used in part A, while a controlled voltage source is used in part B.
32: illustrates a closed loop single controlled current source according to an exemplary embodiment of the present invention.
33 : illustrates a closed loop dual controlled current source according to an exemplary embodiment of the present invention.
Figure 34: illustrates a closed loop single controlled current source with PANI coated sensing electrodes in accordance with an exemplary embodiment of the present invention.
35 : Illustrates a closed loop dual controlled current source with PANI coated sensing electrodes in accordance with an exemplary embodiment of the present invention.
36 : illustrates a closed loop single controlled current source with combined actuation and sensing electrodes in accordance with an exemplary embodiment of the present invention.
37 : illustrates a closed loop dual controlled current source with combined actuation and sensing electrodes in accordance with an exemplary embodiment of the present invention.
38 : illustrates a closed loop single controlled current source with combined working and sensing electrodes with a PANI coating in accordance with an exemplary embodiment of the present invention.
39 : Illustrates a closed loop dual controlled current source with combined working and sensing electrodes with a PANI coating in accordance with an exemplary embodiment of the present invention.
40 : Illustrates a closed loop single controlled potential source with PANI coated sensing electrodes in accordance with an exemplary embodiment of the present invention.
41 : Illustrates a closed loop dual controlled potential source with PANI coated sensing electrodes in accordance with an exemplary embodiment of the present invention.
42: illustrates a closed loop single controlled potential source with combined actuation and sensing electrodes, feedback controlled Phi1 and Phi2 switches for WE potential input and SE measurement output, in accordance with an exemplary embodiment of the present invention.
43 : illustrates a closed loop dual controlled potential source with combined actuation and sensing electrodes, feedback controlled Phi1 and Phi2 switches for WE potential input and SE measurement output, in accordance with an exemplary embodiment of the present invention.
Figure 44: illustrates a closed loop single controlled potential source with combined actuation and sensing electrodes with PANI coating, feedback controlled Phi1 and Phi2 switches with WE potential input and SE measurement output, in accordance with an exemplary embodiment of the present invention. It is for.
45 : Illustrates a closed loop dual controlled potential source with combined actuation and sensing electrodes with PANI coating, feedback controlled Phi1 and Phi2 switches with WE potential input and SE measurement output in accordance with an exemplary embodiment of the present invention. It is for.
46 : Illustrates a closed loop dual controlled current source with separate actuating and sensing electrodes with PANI coating, and analog controller architecture in accordance with an exemplary embodiment of the present invention.
47 : Illustrates a closed loop dual controlled current source with separate actuating and sensing electrodes with PANI coating, and digital controller architecture in accordance with an exemplary embodiment of the present invention. Also shown is a controller architecture design with analog signal processing for closed loop feedback control in Part A and digital signal processing for closed loop feedback control in Part B in accordance with an exemplary embodiment of the present invention, the architecture of which is shown in Figures 32-45. It can be applied to the specified controller schematic.
48A-48D : Illustrates a potential electrode configuration (routing not shown) for pH sensing in accordance with an exemplary embodiment of the present invention.
49 : Illustrating a multi-electrode array with feedback-control options in accordance with an exemplary embodiment of the present invention, wherein each set of electrodes with actuation, sensing, reference, and counter electrodes has a unique target pH value and temporal control scheme can be independently controlled for , and the reference and counter electrodes can be shared by multiple electrode sets.
50 : A multi-electrode array with both feedback-controlled and non-feedback-controlled sections is illustrated according to an exemplary embodiment of the present invention. The feedback-control section contains one or more sets of feedback-control electrodes consisting of a working electrode, a reference electrode, a counter electrode, and a sensing element. The reference and counter electrodes may be shared by multiple electrode sets. Each feedback-control set can target an independent pH value, the same pH value, or a combination thereof. For each target pH value, there is one or more sets of non-feedback-controlled electrodes in the non-feedback-controlled section also assigned to the same target pH value. When the shape and size of the working electrode of the non-feedback-controlled electrode set(s) is similar to that of the working electrode of the feedback-controlled electrode set(s), the electrical parameters obtained from the feedback-controlled section are It can be applied directly to the working electrode of the feedback-control section. This configuration helps achieve the same level of control with a simpler device architecture.
51 : illustrates a multi-electrode array with a set of feedback-controlled electrodes distributed across the substrate and distributed between sets of non-feedback-controlled electrodes. The feedback-control electrode sets may be distributed in a pattern and/or randomly among the non-feedback-control electrode sets. The feedback-control electrode set consists of a working electrode, a reference electrode, a counter electrode, and a sensing element. The reference and counter electrodes may be shared by multiple electrode sets. Each feedback-control electrode set can target an independent pH value, the same pH value, or a combination thereof. For each target pH value, there is one or more sets of non-feedback-control electrodes also assigned to the same target pH value (pH coupled). This configuration helps achieve more than the same level of control with a simpler device architecture. In some examples, this configuration minimizes the effect on the sensing electrode from an adjacent working electrode having a different target pH value, such as when a set of feedback-controlled electrodes is surrounded by its respective set of pH-coupled non-feedback-controlled electrodes. can do.
Figure 52: illustrates a schematic diagram of adjusting the pH of a solution via oxidation/reduction of a redox active species (quinone in this example) by closed-loop control in accordance with an exemplary embodiment of the present invention.
53 : Illustrates an example of changing the pH of a solution through oxidation/reduction of quinone in 1 mM phosphate buffer on an indium tin oxide electrode according to an exemplary embodiment of the present invention. The pH value was determined with a pre-calibrated iridium oxide sensing electrode patterned on the surface. Closed-loop control achieved the target pH value in an accurate and rapid manner.
54A-54B: FIG. 54A illustrates a pH control device setup with external counter and reference electrodes in accordance with an exemplary embodiment of the present invention. 54B illustrates a pH control device setup with counter and reference electrodes on a substrate in accordance with an exemplary embodiment of the present invention. The device may have one or more sets of individually controlled working electrodes and sensing elements.
55A-54D : Illustrates an example of a pH control electrode design according to an exemplary embodiment of the present invention. The working and sensing electrodes can have various types of shapes and sizes depending on the application (Figures a and b). The sensing electrode can be located on the same plane as the working electrode (Fig. c) or on top of the working electrode with an insulating layer in the middle (Fig. d). A counter electrode can be patterned around the working electrode, which helps to minimize diffusion effects and control the pH within a more confined physical space (Fig. e).

Closed-loop control is provided herein to solve the problems associated with electronic control of pH. Several implementations of closed-loop in high-density arrays of individually position-sensitive electrodes are described herein. Disclosed herein are devices and methods for controlling a pH or ion gradient near an electrode surface in an array format. Each individual working electrode can electrochemically introduce a pH change within a small physical space close to the surface in a format on demand. The electrical configuration may consist of one or more working electrodes, a pH sensing element such as a sensing electrode, a counter electrode, and a reference electrode. The counter electrode and reference electrode may be shared by multiple working electrodes and sensing elements. The pH adjusting reagent can be electrochemically oxidized or reduced to create a pH adjusting zone covering nano- to micro-meter distances from the surface. Electrical output parameters to the working electrode can be adjusted based on feedback from the sensing element, so that faster and more precise pH control can be achieved.

To vary the pH or ion concentration gradient in a multi-site array, a device is provided, the device comprising:

support; and

An array of electrodes comprising one or more feedback-control electrode sets, the one or more feedback-control electrode sets comprising:

one or more reference electrodes;

one or more counter electrodes; and

and one or more subsets comprising a pH sensing element electrically coupled to a working electrode, wherein a reference electrode and/or counter electrode are electrically coupled with at least one subset.

The device is configured to repeatedly perform the following:

a.) select the amount of current and/or voltage to be applied to each working electrode to minimize the difference between the signal output of the sensing element and the target sensing value;

b.) applying a selected amount of current and/or voltage to each working electrode to change the pH of the solution proximate the working electrode;

c.) Measure the signal output of the sensing element.

The device further comprises one or more sets of non-feedback-control electrodes, the one or more sets of non-feedback-control electrodes comprising:

one or more reference electrodes;

one or more counter electrodes; and

one or more working electrodes, wherein a reference electrode and/or a counter electrode are electrically coupled with at least one working electrode;

at least one set of non-feedback-controlling electrodes does not include a pH sensing element;

here:

One or more all feedback-control electrode set(s) are included in one section of the device, and one or more all non-feedback-control electrode set(s) are arranged in a physically separate second section of the device; or

one or more feedback-control electrode set(s) interspersed between two or more non-feedback-control electrode sets;

The at least one feedback-control electrode set is electrically coupled to the at least one non-feedback-control electrode set, the coupled at least one feedback-control electrode set and the at least one non-feedback-control electrode set electrically coupled to the feedback - configured to also apply the selected amount of current and/or voltage applied to the one or more working electrodes of the control electrode set to the one or more working electrodes of the coupled non-feedback-control electrode set.

A method of controlling the pH of a solution is also disclosed herein, the method comprising:

a. Obtaining a device comprising a feedback-control electrode set, the feedback-control electrode set comprising:

one or more reference electrodes;

one or more counter electrodes; and

comprising at least one subset comprising a pH sensing element electrically coupled to a working electrode, wherein a reference electrode and/or counter electrode are electrically coupled with at least one subset;

b. selecting a target sensing value for the sensing element based on at least one signal output from the sensing element in a solution comprising the target pH;

c. of the following:

i. selecting the amount of current and/or voltage to be applied to each working electrode to minimize the difference between the signal output of the sensing element and the target sensing value;

ii. applying a selected amount of current and/or voltage to each working electrode to change the pH of the solution proximate the working electrode;

iii. Iteratively performing measuring the signal output of the sensing element.

Further disclosed herein is a method of controlling the pH of a solution comprising:

a. Obtaining a device comprising an array of one or more feedback-control electrode set(s) and one or more non-feedback-control electrode set(s) electrically coupled to the feedback-control electrode set:

- one or more feedback-control electrode set(s):

one or more reference electrodes;

one or more counter electrodes; and

one or more subsets comprising a pH sensing element electrically coupled to a working electrode, wherein a reference electrode and/or counter electrode are electrically coupled with at least one subset;

wherein the one or more non-feedback-control electrode set(s) are:

one or more reference electrodes;

one or more counter electrodes; and

one or more working electrodes, wherein a reference electrode and/or a counter electrode are electrically coupled with at least one working electrode, and the one or more non-feedback-controlling electrode set does not include a pH sensing element;

b. selecting a target sensing value for the sensing element based on at least one signal output from the sensing element in a solution comprising the target pH;

c. of the following:

i. selecting an amount of current and/or voltage to be applied to each working electrode of the one or more feedback-controlled electrode set(s) to minimize the difference between the signal output of the sensing element and the target sensing value;

ii. A selected amount of current and/or voltage is applied to each working electrode of the one or more coupled feedback-control electrode sets and also to the non-feedback-control electrode set coupled to the one or more coupled feedback-control electrode sets. applied to the one or more working electrodes to change the pH of the solution proximal to the working electrode;

iii. Iteratively performing measuring the signal output of the sensing element.

The method may use the device described above.

By use of the devices described above in the methods described above, local pH or ion concentration gradients can be generated at various working electrode sites, such as biosensors or reactors for generating libraries of compounds or reactors for rapidly producing large quantities of compounds. It can be obtained in multi-site arrays. By way of non-limiting example, fluctuations in the local pH and/or ionic concentration gradient near a probe, such as a biomolecular probe, of a biomolecular interfacial layer at an electrode, particularly on a subset of a multi-site array of biosensors, can be tested from probes and biological samples. It allows control of the binding efficiency of the analyte to be split. The analyte of interest can then be detected upon binding to the probe using a detection agent such as, for example, a labeled secondary antibody. Modulation of the binding efficiency in a subset of multi-site arrays provides a method for accurate determination of these analytes of interest.

As another non-limiting example, devices and methods can be used to modify pH values for multiple rounds of reaction steps, some of which require distinct pH values assigned to specifically selected electrodes in an array. . Non-limiting examples of reactions having multiple rounds of reaction steps include the preparation of library arrays of polymers such as peptides and nucleic acids.

As another non-limiting example, the device and method can be used to visualize the pH of one or more regions continuously or more than once.

The device preferably includes a multi-site array of feedback-controlled electrode sets. Such multi-site arrays preferably include a number of different test sites, reaction sites, or other means of changing the pH at different feedback-control electrode set sites. Each site may also represent a site for performing analysis of a (biomolecule) analyte from a biological sample through detection of the (biomolecule) analyte using a (biomolecule) probe. The assay conditions at each site of each feedback-control electrode set are different signals to generate several unknowns and several equations from which the concentration of the (biomolecule) analyte can be determined to obtain an accurate measurement of the (biomolecule) analyte. can be varied to obtain a set of Each site may represent a site for carrying out a different chemical reaction requiring one or more different steps with different pH requirements. Assay conditions at each site of each feedback-control electrode set can be varied to obtain a collection of different chemical reaction products that will result in a library of chemicals, such as a protein library, a nucleic acid library, or an organic chemical library.

For visualization, testing of analyte concentration, and the like, the various unknowns in the obtained various signals each include a term proportional to the binding efficiency factor, α _ij , and the concentration of various molecules in the biological sample binding detected at the test site. Several equations with several unknowns can be expressed, for example, as

where C _an corresponds to the targeted biomolecule analyte concentration, C _j1 , C _j2 , C _j3 correspond to the total concentrations of molecules that produce different terms in the background signal, and from a set of equations the targeted biomolecule The concentration of the molecular analyte can be determined.

The number of feedback-control electrode sets, as well as the number of sites within each feedback-control electrode set, can vary as needed. Some of these analysis conditions include parameters such as, for example, temperature, shear stress and pressure. For example, the temperature of a solution in a biological sample in which a biomolecular probe and an analyte of interest interact or in which a reaction occurs can be varied using electromagnetic heat at the test site. Another important condition for an interaction or reaction between a biomolecular probe and an analyte of interest is pH or ionic concentration. The methods and devices described herein modulate this pH or ion concentration in the local environment of the site to affect, for example, the efficiency of binding in the vicinity of a biomolecule probe or the rate of reaction in the vicinity of a reactant.

As a non-limiting example of a device disclosed herein, each site in a multi-site array can include a support on which one or more electrodes and/or pH sensors are disposed. The biomolecular probe(s) or reactants may also be immobilized or bound to the surface. This immobilization of the probe or reactant to a solid surface or support helps to reduce the amount of probe or reactant required for the analytical method, and also localizes the detection/reaction region to obtain accurate measurements and/or to different reaction sites. Reduces bleed-over of Probes and/or reactants may be attached to supports such as silicon, glass, metals and semiconductor materials and/or solid surfaces of electrodes (shown in FIGS. 3, 4 and 52).

Probes and/or reactants may be attached or immobilized on the support and/or electrode(s) within the biomolecule interfacial layer (shown in FIG. 4 ). The biomolecule layer may comprise a layer of an immobilized polymer, preferably silane immobilized polyethylene glycol (PEG). Surface-immobilized polyethylene glycol (PEG) can be used to prevent non-specific adsorption of biomolecular analytes and/or reactants onto surfaces. At least a portion of the surface-anchored PEG may include terminal functional groups capable of conjugation, such as N-hydroxysuccinimide (NHS) esters, maleimides, alkynes, azides, streptavidin or biotin. Probes and/or reactants may be immobilized by conjugation with surface-immobilized PEG. It is important that the method used to change the pH does not damage, for example, the covalent attachment of PEG to the surface of the solid support, or the linker conjugating the probe or reactant to PEG (shown in Figure 5). Methods of adjusting pH or ion concentrations as described herein can protect these surface chemistries while affecting pH/ion concentration changes in the environment of probes and/or reactants.

Suitable biomolecular probes can be carbohydrates, proteins, glycoproteins, glycoconjugates, nucleic acids, cells or ligands with specific affinity for the analyte of interest. Such probes can be, for example, antibodies, antibody fragments, peptides, oligonucleotides, DNA oligonucleotides, RNA oligonucleotides, lipids, lectins that bind glycoproteins and glycolipids on the cell surface, sugars, agonists, or antagonists. In a specific example, the biomolecule probe is, for example, a protein antibody that interacts with an antigen present in a biological sample, wherein the antigen is the biomolecule analyte of interest.

In the assay methods described herein, the analyte of interest in a biological sample is, for example, a protein, such as an antigen or enzyme or peptide, a whole cell, a cell membrane component, a nucleic acid, such as DNA or RNA, or a DNA oligonucleotide, or an RNA oligonucleotide. can be

Suitable reactants can be, but are not limited to, carbohydrates, proteins, glycoproteins, glycoconjugates, nucleic acids, nucleotides, amino acids, or other organic chemical precursors upon which further reactants can react to produce the desired product or intermediate. Such reactants may be, but are not limited to, peptides, oligonucleotides, DNA oligonucleotides, RNA oligonucleotides, lipids, or sugars.

A biosensor comprising a device provided herein may be, for example, a protein, such as an antigen or enzyme or peptide, a whole cell, a cell membrane component, a biological sample, which may be a nucleic acid, such as DNA or RNA, or a DNA oligonucleotide, or an RNA oligonucleotide. It can be used in an analytical method to determine a biomolecule of interest in an analyte.

In this method, local pH or ion concentration gradients can be obtained at various test sites of a multi-site array biosensor. At the working electrode, especially in the vicinity of the (biomolecular) probe in the biomolecular interfacial layer over a subset of the multi-site array of biosensors, fluctuations in the local pH and/or ionic concentration gradients can be detected from the (biomolecular) probes and the biological sample to be tested. It allows control of the binding efficiency of analytes. The analyte of interest can then be detected upon binding to the (biomolecule) probe using a detection agent such as, for example, a labeled secondary antibody. Modulation of the binding efficiency in a subset of multi-site arrays provides a method for accurate determination of these analytes of interest.

A reaction vessel comprising a device provided herein can be, for example, a protein, such as a peptide, or a nucleic acid, such as DNA or RNA, a DNA oligonucleotide, an RNA oligonucleotide, or a protein and nucleic acid conjugate, or an organic compound, such as a polymer or a drug. It can be used in an analytical method to generate a library of compounds with

In this method, local pH or ion concentration gradients can be obtained at various sites of a multi-site array. At the working electrode, especially in the vicinity of a reactant bound to a (biomolecular) probe or surface of a biomolecular interfacial layer on a subset of a multisite array, fluctuations in the local pH and/or ion concentration gradient from the (biomolecular) probe and the biological sample It allows control of the binding efficiency of the analyte to be tested or control of the reaction rate of the reactants. In some instances, the analyte of interest may then be detected upon binding to the (biomolecule) probe, using a detection agent such as, for example, a labeled secondary antibody. Modulation of the binding efficiency in a subset of multi-site arrays provides a method for accurate determination of these analytes of interest. In some instances, the product of interest resulting from a reaction promoted near the working electrode may be released from the surface by further reaction.

Each of the electrodes is suitable for the intended purpose of the device, such as a metal oxide, glassy carbon, graphene, metal, gold, silver, platinum, conducting polymer, silver chloride, standard hydrogen, mercury drops, saturated calomel, or combinations thereof. It can be any electrode. For biosensors, the electrodes may include indium tin oxide (ITO), gold, silver electrodes, or combinations thereof. In a preferred embodiment, the electrode in the biosensor in the methods described herein is an indium tin oxide (ITO) electrode.

The pH sensing element can be any element suitable for sensing pH, such as field-effect transistors, polymer semiconductors, metal electrodes, inorganic electrodes, organic electrodes, and polyaniline, polypyrrole, iridium oxide, indium tin oxide and ion- It may be formed by a combination of one or more electrical components comprising a pH sensitive coating material comprising a selective polymer. In some instances, the pH at each site is determined by the fluorescence intensity of a pH-sensitive fluorescent protein.

The solution in contact with the working electrode may be any solution suitable for the intended purpose of the device, eg buffered, unbuffered, aqueous, organic solution, or mixtures thereof. In some instances, the solution contains one or more redox active species. The redox active species can be any species suitable for the intended purpose of the device, such as quinones, catechols, aminophenols, hydrazines, derivatives thereof, and combinations thereof. Solutions may contain water, buffer inhibitors, probes, targets, reactants, suspended particles, reporting molecules, enzymes, substrates, cells, viruses, solvents, co-solvents, catalysts, and the like. In some examples, the solution is a biological sample. In some examples, the solution contains a pH sensitive fluorescent protein, such as green fluorescent protein (GFP). In some examples, the solvent or cosolvent is water, acetonitrile, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), and/or N,N-dimethyl acetamide (DMAc). In some instances, the solution contains a buffer, such as a phosphate buffer. In some examples, the solution contains an electrolyte, such as a salt, such as sodium sulfate, sodium chloride, or potassium chloride. In some examples, the amount of water in the solution is between 0.0001% and 100% by weight, such as 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 , 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.9% by weight, or any range or percentage therebetween. can be

The support can be any support suitable for the intended purpose of the device, for example a material that can have patterned electrodes. The surface can be a material such as a glass slide, plastic plate, silicon wafer, glass wafer, quartz wafer, flexible plastic sheet, polymer layer, paper, or mixtures thereof. The support may contain on or therein one or more electrodes, sensors, electromagnets, interfacial layers with one or more immobilized detection agents, probes, reactants, enzymes, cells, catalysts, cells, viruses, substrates, reporting molecules, etc. can In some instances, the surface has a pH sensitive fluorescent protein, such as green fluorescent protein (GFP), immobilized thereon. In some examples, the support is coated with PEG. The surface and what is contained on or within it can be site-to-site to facilitate detection of different targets or production of different products.

A biomolecular analyte may be detected using any suitable detection method. Known methods for detecting such analytes include luminescence, fluorescence, colorimetric methods, electrochemical methods, impedance measurements, or magnetic induction measurements. In these various methods, an analyte binds to an immobilized biomolecule probe and a detection agent is introduced, such as a secondary labeled probe that specifically binds to the analyte bound to the immobilized probe. This detecting agent or secondary labeled probe can generate a detectable signal such as, for example, luminescence or fluorescence.

In analytical methods such as biomolecular probe detection and chemical reactions, the pH of the solution surrounding the biomolecule probe or reactant is known to affect the reaction rate or to a significant extent the binding/activity between the probe and analyte. Concentrations of other ions can also greatly affect binding/activity/reaction. Devices and methods for adjusting pH and/or ion concentration in the vicinity of a probe or reactant in proximity of a working electrode are provided herein. Adjustment of the pH near the working electrode also affects non-specific interactions of the analyte and reactant with respect to molecules other than the probe or intended reactant, and interactions of the probe, analyte, or reactant with other molecules in solution. can affect However, adjustment of pH or ion concentration can be controlled so as not to impair any surface chemistry, such as immobilization of biomolecular probes or reactants to a solid support at the site of a multisite array. Methods of adjusting the pH or ion concentration as described herein can protect these surface chemistries while affecting pH/ion concentration changes in the environment of the probe or reactant.

Surface chemistry compatibility can be considered when the methods described herein are practiced. Changes in pH are caused by changes in the concentration of hydrogen ions or hydroxyl ions. As shown in Figure 5, the various chemical reactions occurring at the electrode-liquid, electrode-crosslinker, crosslinker-protein, and protein-protein interfaces also begin to interfere with pH changes occurring near the solid surface, causing the probe or reactants can be reached. They can act as a diffusion barrier for ions and can interfere with pH changes around the probe, analyte, and reactant. These methods of controlling pH or ion concentrations described herein help maximize changes in hydrogen or hydroxyl ion concentrations to overcome any diffusion barriers imposed by surface chemistry.

Another important aspect is the buffering capacity of the solution in contact with the solid interface. The buffering effect can be sufficiently large that the pH change at the interface never reaches the immobilized probe or reactant away from it. The distance may vary based on the interfacial layer deposited on top of the solid interface. Such an interfacial layer may have a thickness of 300 nm or less, preferably 1-150 nm, even more preferably 5-100 nm. Thus, the distance between the solid interface and the probes or reactants in the interfacial layer may be in the range of 0.1-300 nm. The use of a buffer inhibitor in solution or on a surface that extends the pH change on the electrode interface to reach the interacting probe-analyte pair or reactant can serve to control the pH or ion concentration in the vicinity of the probe and/or reactant. .

The following is an example of how to control the pH/ion concentration at the solid-liquid interface. These include: 1) Electrochemical generation of ions at the electrode surface by adding electrochemically active species to a solution which upon electrochemical oxidation/reduction produces the ion of interest (eg H ⁺ , Mg ⁺ , OH ^- ) ; 2) bringing the enzyme close to the site of interest releasing these ions of interest from the enzyme substrate reacting with the enzyme; 3) Incorporation of buffer inhibitors, for example by mixing polymers that selectively reduce the rate of diffusion of ions (e.g., phosphates) in solution. US Patent No. 7,948,015 describes the use of such inhibitors for applications where measurement of small local pH changes is of interest (eg, in DNA sequencing). However, in methods of locally adjusting the pH, similar inhibitors can be used to extend the local pH change further away from the electrode-liquid interface; and 4) redistribution of ions already present near the electrode surface due to electrostatic forces.

In one embodiment of the method of adjusting the pH or ion concentration as described herein, an electrochemically active agent is added to a solution at a site of a multi-site array, said site comprising a probe, detection agent or reactant and comprising an electrochemically active agent It has an interfacial layer that oxidizes or reduces. The electrochemically active agent may be added at a concentration of 1 nM to 100 mM, preferably at a concentration of 10 nM to 10 mM, more preferably at a concentration of 100 nM to 5 mM. Electrochemically active agents can be electro-oxidized or electro-reduced at electrode potentials ranging from -2V to +2V (relative to the Ag/AgCl reference electrode). Preferably the electrode potential is in the range of -1V to +1V, even more preferably the electrode potential is in the range of -0.5V to +0.5V. The voltage required to drive the redox reaction can be used as a real-time feedback method to monitor the pH produced at the electrode surface.

Devices provided herein may include such an array of multiple sites in solution to adjust the pH at each test site. In some instances, it is used to determine the presence and concentration of a biomolecular analyte of interest in a biological sample or to facilitate multi-step chemical reactions. In this use, the device may be contacted with a solution comprising a phosphate buffer, preferably a dilute phosphate buffer (preferably having a concentration of 0.1 mM to 100 mM). In a preferred embodiment, the pH of the dilute phosphate buffer is between 5 and 8, preferably between 7 and 8, more preferably between 7 and 7.5.

Adjustment of pH or ion concentration on a device described herein by an electrochemical reaction at one or more working electrodes can be performed in a galvanostatic or potentiostatic mode. Additionally, electrical pulses of any type may be applied to the electrodes of the device in a method for adjusting the pH. These pulses may be in the form of annealing pulses and may vary by pulse frequency, pulse width and pulse shape. In the annealing pulse, a voltage sufficient to change the pH is applied so that non-covalently bound molecules are removed from the device. This annealing pulse eliminates or reduces the need to clean the substrate after initial contact with the sample or reactant solution to remove non-covalently bound materials. Another advantage is that an annealing pulse can be more efficient in removing these non-covalently bound materials from the device than simple cleaning. Preferred pulse widths for pH control range from 1 nanosecond to 60 minutes.

The solution may further contain one or more electrolytes, such as sodium sulfate, for example, or any other suitable strong electrolyte. Preferably, the electrolyte is sodium sulfate, sodium or potassium chloride, sodium or potassium bromide, sodium or potassium iodide, sodium or potassium perchlorate, sodium or potassium nitrate, tetraalkylammonium bromide and tetraalkylammonium selected from iodides. Buffer-inhibitors may also be used in solution. Suitable buffer inhibitors include poly(allylamine hydrochloride), poly(diallyldimethyl ammonium chloride), poly(vinylpyrrolidone), poly(ethylenimine), poly(vinylamine), poly(4-vinylpyridine) and tris( 2-carboxyethyl)phosphine hydrochloride. When used in a method for adjusting the pH as described in U.S. Patent Application Serial No. 13/543,300, the aqueous solution is preferably acetonitrile, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), N,N-dimethyl acetic acid. Also included are water-miscible organic co-solvents selected from the group consisting of amides (DMAc), and mixtures thereof.

Suitable electrochemically active agents include dopamine hydrochloride, ascorbic acid, phenol and derivatives, benzoquinone and derivatives such as 2,5-dihydroxy-1,4-benzoquinone, 2,3,5,6-tetrahydride hydroxy-1,4-benzoquinone and 2,6-dichloroquinone-4-chloroimide; Naphthoquinone and derivatives such as hydroxy-1,4-naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone, and potassium 1,4-naphthoquinone-2-sulfo Nate; and 9,10-anthraquinone and derivatives such as sodium anthraquinone-2-carboxylate, potassium 9,10-anthraquinone-2,6-disulfonate. Preferably the concentration of the electrochemically active agent in the aqueous solution is between 1 nM and 100 mM.

In another embodiment of the method of adjusting pH or ion concentration in a solution, the enzyme is immobilized in an interfacial layer or magnetic particle. The interfacial layer or magnetic particle may also have one or more immobilized biomolecular probes or reactants therein or thereon. An enzyme substrate is then added to the solution at a site of the multi-site array, which site has an interfacial layer, and enzymatically oxidizes the enzyme substrate.

Enzymes suitable for immobilization in the interfacial layer or on magnetic micro- or nano-particles include, for example, oxidase, urease, or dehydrogenase. Such immobilized oxidases can be, for example, glucose oxidases where the enzyme substrate is glucose (shown in Figure 6). The amount of immobilized enzyme and added enzyme substrate can be varied at different sites of the multi-site array to provide a pH or ion concentration gradient in the multi-site array.

Alternatively, the enzyme is added to the solution at the site of the multi-site array, rather than being immobilized on a solid surface as in the above methods immobilized on an interfacial layer or on magnetic micro- or nano-particles. Through electrolysis, the enzyme undergoes a redox reaction at the electrode surface and perturbs the local pH.

In each of these embodiments, the pH or ionic concentration can be further adjusted by adding a buffer inhibitor to the solution. Such addition of a buffer inhibitor helps diffuse a given ion of interest into the location of a probe, reactant, detector, or the addition inhibits an interaction between such a given ion and a buffer salt. Alternatively, in the methods of adjusting pH or ionic concentration described herein, a buffer inhibitor is added to a solution at a site in the absence of an electrochemical active agent or immobilized enzyme. In this embodiment, the buffer inhibitor is added to the solution and either facilitates the diffusion of H ⁺ ions or OH ^- ions generated at the working electrode of the site, or prevents interactions between H ⁺ ions or OH ^- ions and the buffer salt. restrain

Suitable buffer inhibitors include poly(allylamine hydrochloride), poly(diallyldimethyl ammonium chloride), poly(vinylpyrrolidone), poly(ethylenimine), poly(vinylamine), poly(4-vinylpyridine) and tris. soluble polymers selected from (2-carboxyethyl)phosphine hydrochloride. The amount of buffer inhibitor added can be varied at different sites in each subset of the multi-site array to provide a pH or ion concentration gradient in the multi-site array.

When a method of controlling pH in a multi-site array of sites is used in a biosensor or reactor, accuracy, reliability and reproducibility of pH control at each site are important. However, pH control at each site may vary depending on subsequent use. In order to accurately determine the amount of an analyte of interest in a sample or to accurately prepare reaction conditions using the devices and methods described above, the pH at each site must be accurately adjusted or controlled. The devices provided herein enable accurate determination and control of pH at each site.

When an electrode (the working electrode) causes a pH adjustment at a specific site of the multi-site array, the pH sensor enables sensing of the pH at the electrode. In one example, the fluorescence intensity of a fluorescent protein is used by a sensor. Fluorescence intensity can change due to pH adjustment. A change in fluorescence intensity of a fluorescent protein can be proportional to a change in pH (eg, when there is a linear relationship between pH and fluorescence intensity). Thus, as also shown in FIG. 10, the pH value at each position at any time point can be easily obtained by correlating the fluorescence intensity of a fluorescent protein with pH. An accurate calibration of the correlation between pH and fluorescence intensity can be performed before or during use of the device. Preferably, the fluorescent protein is selected from green fluorescent protein, yellow fluorescent protein, and cyan fluorescent protein. More preferably, the fluorescent protein is immobilized green fluorescent protein (GFP). In an alternative embodiment, a pH-sensitive dye may be used in place of a pH-sensitive fluorescent protein. In another alternative embodiment, pH sensitive binding proteins may be used.

In a multi-site array of sites, the pH sensor can cover areas on the surface that are also covered by electrodes and areas not covered by electrodes. The electrode covered by the pH sensor is the working electrode or counter electrode. Preferably, the pH sensors are applied on the surface in discrete spots, each spot overlapping only one area and the area not covered by the electrode as shown in FIG. 12A. When the pH is not controlled by the electrode, the presence of the pH sensor on the area not covered by the electrode allows determination of the pH sensor output. This pH sensor output can be used as a standard and control in determining whether the pH sensor output returns to its original strength after discontinuation of pH control by the electrode. Thus, a pH sensor that is not located on or near the electrode can be used as an internal reference for signal normalization.

If calibration is performed while using the device, one or more sites in the multi-site array may be dedicated to calibrating the pH sensor output for pH correlation. When the pH is no longer regulated by the electrode (working electrode) at this site, the pH sensor output can return to its strength before current is applied through the electrode.

The device includes at least one counter electrode and at least one working electrode. In the device, one or more electrodes may be arranged in a multi-site array, and each site of the multi-site array may include a working electrode and/or a counter electrode. The electrode may be any electrode suitable for the intended purpose of the device, for example an indium tin oxide (ITO), gold, or silver electrode. In a preferred embodiment, the electrode in the device is an indium tin oxide (ITO) electrode. In an alternative embodiment, the working electrode is an indium tin oxide electrode and the counter electrode(s) are selected from indium oxide electrodes, gold electrodes, platinum electrodes, silver electrodes, and carbon electrodes.

The electrodes in the device can be used to adjust the pH or as sensing electrodes or both. In a device, or a biosensor using a device, one or more electrodes may be connected to an electronic board via pogo-pins, to a chip on a foil via a z-axis adhesive, or to a chip on a substrate. An electronic board or chip may be powered by a printed battery, a small battery coupled to the board, magnetically coupled power transfer using a coil on the board, or rf-coupled power transfer using a coil on the board.

The following description is an example of specific embodiments that can be modified within the scope of the description as understood from general knowledge. 9 shows a side view of a part of a device comprising a substrate 1, for example glass or plastic. One or more electrodes 2 are covered on a substrate 1 which is also covered with a biomolecular interfacial layer 10 . The biomolecular interfacial layer (10) includes immobilized PEG (3), an immobilized probe (4) and an immobilized pH-sensitive fluorescent protein (5) in the form of a green fluorescent protein spot. GFP spot 5 overlaps electrode 2 and the area not covered by the electrode. Electrodes 2 and GFP spots 5 are arranged in a multi-site array to provide multiple test sites on the device.

The position of the luminescent signal generated by the luminescent molecule can be controlled by directly controlling the position of the luminescent molecule itself. This includes, for example, immobilizing the luminescent molecule. However, by including the ability to control the pH of a solution near the electrode with the pH-sensitive luminescent molecule, the location of the luminescent signal produced by the free-floating luminescent molecule can also be controlled.

14 depicts an exemplary system 100 for controlling pH in accordance with an exemplary embodiment of the present invention. In the example shown in FIG. 14 , the system 100 includes a slide 30 comprising an area 11 in which a test solution containing a substance of interest is disposed, a control unit 12 and a power source 14 . Slide 30 may be formed of any electrically insulating material. For example, glass will typically be used for this purpose and to act as the substrate on which the region 11, control unit 12 and power source 14 are formed. The glass may be formed of, for example, silicon dioxide (SiO2), possibly with additives. Alternatively, other types of silicate glass may be used.

Region 11 contains an array of electrodes used to control pH. In an exemplary embodiment, at least a portion of the electrode in region 11 is used to detect the pH level of the test solution. (For example, the aforementioned U.S. Patent Application Serial No. 13/543,300 describes the use of an electrode for pH control in a biosensor, and the control can be performed using the electrodes discussed herein.)

15 is a top view of an exemplary electrode array, wherein sets of column electrodes X01-X12 are arranged at regular spaced distances from each other. The set of row electrodes Y01 to Y09 are also arranged at regularly spaced distances and separated from the column electrodes X01 to X12, for example by a glass intervening layer. Each electrode includes one or more contact pads 31, 32 for use in pH control. The shape of the pad is variable and in an exemplary embodiment substantially rectangular. 16 shows a close-up view of an exemplary square-shaped pad. 17 shows an alternative embodiment in which the pads form an interdigitated structure, and thus are frame-shaped.

In the example illustrated in FIG. 14 , the control unit 12 is electrically connected to the electrode array 11 and controls the array 11 to perform pH adjustment. Control unit 12 may be, for example, a microprocessor or an application specific integrated circuit (ASIC). In an exemplary embodiment, the control unit 12 is located on an electronic circuit board removably connected to the slide 30 using, for example, pogo pins. The control unit 12 may be located within a packaged chip coupled directly to the rigid glass substrate using, for example, a chip-on-glass process. In an alternative exemplary embodiment, slide 30 is formed from a flexible foil-type substrate, and control unit 12 is configured to form a chip-on-foil in a manner similar to the way chips are incorporated in certain liquid crystal displays. It is adhered to the slide 30 using z-axis adhesive. Control unit 12 may include, for example, a non-transitory computer readable storage medium containing program code implementing the pH adjustment techniques described herein. In addition to pH adjustment, control unit 12 may control the electrodes to perform other types of sensing or to control other sensing structures, such as bubble detection, or other sensing or control known in the art of biosensors.

In an exemplary embodiment, control unit 12 transmits control signals that cause input pulses to be applied at specific electrodes. Capacitance and/or pH value may be measured in control unit 12 in some instances based on the response of the electrode to the input pulse. Measurement of capacitance is known in the field of touch screen displays using measurements of self-capacitance (eg, single electrode) or mutual capacitance (eg, between two electrodes). In some examples, to also support bubble detection, control unit 12 has a larger capacitance detection range than typical control units that measure capacitance in life science experiments. An input pulse for pH control may be applied using a control signal. Control signals for pH adjustment can be initiated by the control unit 12 according to a predefined program sequence designed for pH adjustment, for example. Alternatively, the control signal for pH adjustment is initiated externally, for example in response to a command from the data processing unit 50 . In one example, control unit 12 is a hardware and/or software component that performs preliminary signal processing on the measured capacitance value and/or pH value, including converting the measurement from analog to digital format and/or filtering the measurement. includes In one example, the processed measurements are then output to data acquisition unit 40 as raw data.

Power source 14 provides power to control unit 12 and electrode array 11 . For example, in an exemplary embodiment, power source 14 is a battery, such as a coin-cell or printed battery. In one exemplary embodiment, the slide 30 is designed for single use and is disposable, such that the battery provides a small energy capacity sufficient for a single measurement, for example, and the battery can be permanently attached to the slide and , for example bonded or adhered to a glass surface. In an exemplary embodiment in which slide 30 is reusable, the battery may be rechargeable or user replaceable. Other forms of power delivery may alternatively be used. In one exemplary embodiment, electrical power is wirelessly transferred via magnetic coupling between an external power supply (eg, data acquisition unit 40) and one or more resonant coils in the slide. As an alternative to magnetic coupling, but also using wireless power transfer, an external power supply can be coupled to the resonant coil using a radio frequency (RF) signal. In yet another exemplary embodiment, slide 30 receives power through a wired connection to data acquisition unit 40 .

In one example, data acquisition unit 40 is a device that communicates with slide 30 to receive the measured capacitance and/or pH values from control unit 12 in the form of raw data. For example, in one example, data acquisition unit 40 includes a wired communication interface 20 to a corresponding interface on slide 30 . In one exemplary embodiment, raw data are output in parallel from control unit 12 . For example, in an exemplary embodiment, control unit 12 includes multiple output channels, and data from a single row or single column is output for the corresponding channel. In this embodiment, interface 20 converts, for example, parallel data into a format suitable for transmission to data processing unit 50 . Conversion may involve parallel-to-serial conversion using a Universal Asynchronous Receiver/Transmitter (UART) or other conventional data conversion device. In an alternative embodiment, interface 20 wirelessly communicates with slide 30 using, for example, an RF signal.

In the exemplary embodiment, data processing unit 50 receives raw data from output interface 22 of data acquisition unit 40, for example from the transmitter portion of a UART. Output interface 22 may be a wired serial interface, such as a Universal Serial Bus (USB) interface. Alternatively, the output interface 22 may be a wireless interface, for example a Bluetooth or WiFi interface. In one example, the interface is a Bluetooth low energy (LE) interface. The data processing unit 50 may be a general-purpose computer, for example in the form of a desktop, laptop or tablet, and includes, for example, a processor and memory for storing instructions for further processing the raw data. For example, in an exemplary embodiment, further processing normalizes the raw data to a predefined scale and uses the normalized data for display on display device 60 to produce an output image, such as a two- or three-dimensional graph. including creating When the data processing unit 50 is a laptop or tablet, the display device 60 may be integrated into the housing of the data processing unit 50 as a single unit. The display device 60 may alternatively be externally connected, for example the data processing unit 50 is a desktop. The output images can be combined to form a video showing changes in data over time. In one embodiment, an output image representing the measured capacitance value and/or pH value is displayed along with additional output images corresponding to other measured data. For example, the output image and the additional output image may be displayed on different parts of the same display screen or may be overlaid (superimposed) on the same part of the display screen.

In an exemplary embodiment, data processing unit 50 is also configured to issue commands to control unit 12 for pH adjustment. For example, when the processor of data processing unit 50 determines that the pH level of the test solution should be adjusted, a command may be automatically generated. Alternatively or additionally, the command may be user initiated.

According to an exemplary embodiment, slide 30 may include a layered structure in which one or more electrode layers are positioned on top of a glass substrate. A layered structure may be formed, for example, using lamination techniques in which two or more layers are formed separately and then laminated together, for example using adhesives or bonds. Alternatively, the layered structure may be monolithically formed as a single unit using techniques known in the art of semiconductor device fabrication. The layered structure may include one or more passivation layers formed, for example, of SiO ₂ (also referred to as an oxide). However, the composition and size of the passivation layer can vary, for example from atomic layer SiO ₂ to several micrometers SiO ₂ , and various techniques such as low pressure chemical vapor deposition (LPCVD) or plasma-enhanced chemical vapor deposition (PECVD) can be used. It will be understood that it can be formed using Silicon nitride (Si3N4) is another exemplary passivation material. When the layered structure is formed using lamination, the passivation layer may be formed as a laminated thin film.

One way to perform pH control is to separate the pads of adjacent electrodes to form a channel to collect the test solution. The channels bring the test solution into contact with the electrodes, allowing the pH level of the solution to be adjusted by passing an electrical current between adjacent electrodes. According to an exemplary embodiment, the electrode may be formed of any suitable conductive material, but is preferably indium tin oxide (ITO), as ITO is transparent and relatively colorless, making it suitable for experiments involving optical measurements. am. This makes the entire measurement area 11 transparent, as shown in FIG. 13 . An oxide layer may be used as a passivation layer to cover the electrode. When pH control is implemented using a channel, in an exemplary embodiment, the oxide layer does not completely fill the channel, but instead allows a lateral portion of the electrode to remain exposed and in contact with the test solution.

The pH measurement result may be output for display on the display device 60 . According to an exemplary embodiment, raw data values are displayed in the form of a two-dimensional table. Each table entry corresponds to the measured pH value obtained from the corresponding electrode pad. The raw data can be displayed as a three-dimensional graph, eg a 3-D mesh where the x and y values correspond to the electrode positions and the z values correspond to the measured pH values. To facilitate visual perception, in an exemplary embodiment, the graph may be color coded, for example using a gradient scheme, eg a gray scale scheme, or a heatmap where the color gradually changes with pH change. In another embodiment, color coding is used to indicate pH position on a two-dimensional graph where pH values are displayed using color changes. Alternatively or additionally to the display of the raw data, according to an exemplary embodiment, the data processing unit 60 processes the raw data by normalizing it to a predefined scale. The graphs described above can be displayed alone or with additional values from other parameters under test, such as capacitance values and flow rates. In one embodiment, the additional values are displayed on the same graph, eg using a different color scheme, and superimposed on the pH values.

Advantageously, the graphical display of the pH value allows the user to quickly determine the pH as a specific location and to perform appropriate corrective actions in response to the pH. A user may, for example, decide to discard values associated with the location of a particular pH value while retaining those additional values not associated with the location of the particular pH value (corresponding to one or more parameters determined empirically). Alternatively, the user may decide that the entire data set should be discarded due to the abundance of pH values outside the desired range, thus rendering additional values entirely unreliable.

According to an exemplary embodiment, the pH value is superimposed on additional measurement data, and the additional data is stored in relation to layout data representing the physical configuration of the measurement area. Layout data is an electronic file in the form of an image (e.g., a scanned image of the measurement area) or text (e.g., a configuration file for a microarray spotter used to fabricate an array, or a GenePix array list (GenePix Array List; GAL) file). The additional measurement data may also be an image or text (eg, measured capacitance values stored in a GAL file, or measured capacitance values presented in gray scale in a scanned image of the measurement area).

According to an exemplary embodiment in which the pH values are superimposed, a composite display can be created wherein the display shows a graphical representation of the array with the pH values superimposed on additional measured values at corresponding locations of the array. Overlapping can be presented as text-on-text, text-on-image, or image-on-image. The text-on-text example displays the pH value in half of the array positions and additional measurement data in the other half. The text-on-image example uses a heat map to display pH, while displaying additional measurement data as numerical values on the heat map. An example of an image-on-image displays pH using a heat map, while displaying additional measurement data using a 3-D mesh. Superimposed data may be stored in the electronic layout file prior to or in conjunction with the superimposed display.

According to an exemplary embodiment, a processor on the slide or on an external computer is configured to automatically invalidate additional measurement data in response to pH values outside a desired range (eg, by replacing measurement values with null values). . For example, a processor on a slide can detect the pH of some location, output an indication where the pH is located to an external computer, and then perform an invalidation based on the indicated location. This eliminates the need for the user to manually review the pH values to determine whether to retain additional measurement data or products generated by reactions within these locations.

According to exemplary embodiments, electrodes may be used to perform functions other than pH detection and pH control. For example, the electrodes can be used for bubble detection. In some instances, electrodes may be used for temperature control. In another example, capacitance measurements can be used to estimate the dielectric constant of a solution, which is then correlated with the rate of cell growth, the rate of production of a reaction product, or the rate at which a substance of interest binds to a biomolecule. In some instances, the function of an electrode may change between one function and another.

An exemplary embodiment in which electrodes are arranged in two layers is described. However, the number of layers may be more or less. In practice, a single layer may be sufficient for both pH control and pH measurement. Additionally, not all electrode layers need be used for pH control or pH measurement. Instead, additional electrode layers can be used for other purposes depending on the use of the electrodes in a conventional biosensor or reactor.

An exemplary embodiment of the present invention relates to a glass slide, wherein at least part of the measurement data processing is performed on the slide itself or on a peripheral device connected to the slide body, rather than on an external computer responsible for displaying the processed data. Such slides are referred to herein as instrument-on-glass. 18-20 each depict an exemplary embodiment of an on-glass device.

18 shows a slide 400 according to an exemplary embodiment of the present invention. Slide 400 includes a pH measurement/adjustment area 405 , a power source 410 and processing circuitry 420 . Region 405 may be formed of TFT (for switch) together with ITO (for electrode). Alternatively, region 405 may be formed using ITO alone or combining ITO with other metals. Power source 410 may be similar to power source 14 of FIG. 14 and may be powered by a battery or passive power source using magnetic or RF coupling, for example.

Processing circuit 420 may be similar to control unit 12 of FIG. 14 and may perform preliminary signal processing. In addition, processing circuitry 420 may perform some of the functions previously described with respect to data processing unit 50 (eg, normalizing or scaling pH values or controlling pH adjustment). Processing circuitry 420 may include a processor (eg, one or more CMOS chips) that processes the raw data obtained from region 405 . Processing circuitry 420 may further include memory to store instructions or data used by the processor to process the raw data. Processing circuitry 420 may be configured to arrange the raw data into a format suitable for output to an external computer, or to perform preliminary data analysis (eg, pH detection and invalidation of data associated with a particular pH). The processor may control the sensing operation of region 405 (e.g., driving and reading data from the array), perform data compression, and perform wired or wireless transmission of pre-processed data to an external computer. . Post-processing and output of data for display can be performed in an external computer.

19 shows a slide 500 according to an exemplary embodiment of the present invention. Components 505, 510 and 520 may be similar to and perform the same functions as components 405, 410 and 420 of FIG. 18, respectively. However, instead of being located on the body of the slide 500, the power source 510 and processing circuitry 520 are connected externally to a peripheral circuit board 515 that fits into the hardware interface of the slide 500, for example.

20 shows a slide 600 according to an exemplary embodiment of the present invention. Components 605, 610 and 620 may be similar to and perform the same functions as components 405, 410 and 420 of FIG. 18, respectively. In the embodiment of FIG. 20 , power supply 610 and processing circuitry are connected externally similar to FIG. 19 . However, circuit board 615 includes a serial port connector for transfer of data and power between board 615 and an external computer. Specifically, a serial port can be used to transfer measurement data to an external computer, supply power to operate measurement area 605 or recharge power source 610 .

Exemplary embodiments of the present invention perform any of the methods described herein, alone or in combination, to execute code provided on a non-transitory hardware computer-readable medium, including, for example, any conventional memory device. Any conventional processing circuitry and device or combination thereof, such as a personal computer (PC) or other workstation processor, to output any one or more of the graphical user interfaces described, for example, centrally. It relates to one or more processors that may be implemented using a processing unit (CPU). The memory device may include any conventional permanent and/or temporary memory circuitry or combination thereof, including, but not limited to, random access memory (RAM), read only memory (ROM), compact disk (CD), These include digital versatile disks (DVDs), flash memory, and magnetic tape.

Exemplary embodiments of the present invention relate to a non-transitory hardware computer-readable medium, for example described above, having stored thereon instructions executable by a processor to perform any one or more of the methods described herein. .

Exemplary embodiments of the present invention relate to methods, eg, hardware components or machines, for transmitting instructions executable by a processor to perform any one or more of the methods described herein.

Exemplary embodiments of the present invention relate to one or more of the devices and methods described above, eg, computer-implemented methods, alone or in combination.

Exemplary embodiments of the present invention provide a device and method for using electronics to control the pH of a solution proximate to an electrode, and a device for implementing the method in an integrated electronic system. Preferably, the device and method of the present invention can control the pH of the solution surrounding the electrode within a distance of about 1 cm. Devices and methods using electronic devices to control the pH of a solution may also include detecting the pH of a solution by devices and methods described above and elsewhere herein. In some instances, both pH detection and pH adjustment are performed by the same device and/or method.

According to an exemplary embodiment, a method of changing the pH of a solution by electronic control provides an electrical input to the solution using three or more electrodes and a pH sensing element to electrochemically produce and/or consume hydrogen ions in the solution. includes providing The production and/or consumption of hydrogen ions is accomplished by an electrochemical reaction of one or more redox active species in solution. Preferably, the at least one redox active species is selected from: quinones, catechols, aminophenol hydrazines, and derivatives thereof. More preferably, the at least one redox active species is a quinone (see Thomas Finley, “Quinones,” Kirk-Othmer Encyclopedia of Chemical Technology, 1-35 (2005)). Even more preferably, the at least one redox active species is selected from: hydroquinone, benzoquinone, naphthoquinone naphthoquinone. Most preferably, the at least one redox active species is a quinone derivative further defined below.

Preferably, the three or more electrodes include a counter electrode, a working electrode and a reference electrode (RE). In some exemplary embodiments, the sensing element may be a sensing electrode. In certain exemplary embodiments of the present invention, the sensing electrode may also function as the working electrode. In certain exemplary embodiments of the present invention, the three or more electrodes are each independently made of metal oxide, gold, glassy carbon, graphene, silver, platinum, silver chloride, standard hydrogen, mercury drops, or saturated calomel. In certain exemplary embodiments of the invention, the solution is buffered, unbuffered, aqueous, organic or a mixture thereof. In certain exemplary embodiments of the present invention, the amount of electrical input is provided by providing the amount of electrical current. In certain exemplary embodiments of the present invention, the electrical source waveform is selected based on a given amount of electrical input. Preferably, the electric source waveform is a galvanostatic waveform or a potentiostatic waveform. More preferably, the electrical source waveform is selected from a predetermined map that maps electrical inputs to respective solution pH values.

According to an exemplary embodiment of the present invention, a method of controlling the pH of a solution using three or more electrodes is provided to control the pH of a solution and/or the pH of a solution while not applying an electrical input between the two or more electrodes. obtaining an open circuit potential (OCP) of , selecting an electrical input based on the pH and/or OCP, and applying the electrical input to the solution between two or more electrodes to change the pH of the solution. In certain exemplary embodiments of the present invention, OCP is obtained by measuring the OCP of two or more electrodes in solution, or calculated from a known or measured initial pH. In certain exemplary embodiments of the present invention, the method also includes determining the pH of a solution based on the OCP of two or more electrodes in the solution. In certain exemplary embodiments of the present invention, the method also includes selecting an electric source waveform based on an electrical input quantity, and the electrical input quantity is provided in accordance with the selected electric source waveform.

In certain exemplary embodiments of the present invention, the method further comprises determining the pH of the solution based on the measured OCP of two or more electrodes in the solution. In certain exemplary embodiments of the present invention, the electrical input is selected based on the determined pH.

According to an exemplary embodiment of the present invention, a method of monitoring the pH of a solution using a sensing element, a reference electrode, a counter electrode, and a working electrode includes selecting a target pH value and/or open circuit potential (OCP), and selecting a working electrode. characterizing the pH and/or OCP of the solution between the reference and sensing electrodes while no electrical input is applied to the solution, and repeatedly performing the following steps: pH and/or OCP of the solution and a target pH value; /or select the amount of electrical input to be applied to the working electrode to minimize the difference between the OCPs; An electrical input is applied to the working electrode to adjust the pH and/or OCP of the solution.

In certain exemplary embodiments of the present invention, the target pH value and/or OCP is a fixed value, and in other exemplary embodiments, the target pH value and/or OCP is a variable value. In certain exemplary embodiments of the present invention, the target pH value and/or OCP is a range having upper and lower limits or a single value. Preferably, the target OCP is selected based on the target pH value. Preferably, the target pH value is user defined. In some instances, the pH changes over time and/or is different for different working electrodes. In certain exemplary embodiments of the present invention, the sensing electrode may also function as the working electrode. In certain exemplary embodiments of the present invention, the sensing and working electrodes are separate electrodes. Preferably, the distance between the sensing electrode and the working electrode is 0 cm to 1 cm. Electrical input may be provided as electrical current or electrical potential. Preferably, the electrical input is provided by applying an electrical potential to the working electrode. More preferably, the electrical potential is provided according to the electrical source waveform. The electric source waveform is a constant current waveform or a potentiostatic waveform. Even more preferably, the electrical source waveforms are selected from predetermined maps that map electrical inputs to respective solution pH values. Preferably, the sensing element is a sensing electrode coated with a pH sensitive coating, and the OCP of the sensing electrode and the pH sensitive coating is governed by the H ⁺ ion concentration. In certain exemplary embodiments of the present invention, the pH sensitive coating is an organic material. In another exemplary embodiment of the present invention, the pH sensitive coating is an inorganic material. Preferably, the pH sensitive coating is made of a material selected from the group consisting of polyaniline, polypyrrole, and iridium oxide.

Exemplary embodiments involve monitoring and/or characterizing electrochemical parameters and using the detected signal as a feedback control to generate a desired pH waveform for a specified period of time. All of the methods described involve the use of a redox couple, which upon oxidation releases H ⁺ ions to lower the pH and upon reduction consumes H ⁺ ions to increase the pH, based on the equation:

An example of a redox reaction is the reaction of quinone derivatives. There are several different quinone derivatives, each with specific oxidative and reductive peaks, and the rate of the redox reaction has been shown to be dependent on the pH of the solution. Regarding the reference electrode, the electrochemical properties of the electrode, including electron transfer coefficient and open circuit potential (OCP), are also properties that may be considered. OCP has previously been demonstrated to be dependent on the pH of the solution.

According to an exemplary embodiment of the present invention, a device for controlling the pH of a solution includes a controller, three or more electrodes, and a solution containing at least one redox active species. In some embodiments, a device may be configured to measure the pH of a solution and/or OCP between two or more electrodes in the solution, generate measured OCP data, and transmit the measured pH and/or OCP data to a controller. there is. In some embodiments, a device may be configured to measure the pH of a solution by measuring the fluorescence of a pH-sensitive fluorescent molecule or molecules. In some embodiments, the device may be configured to measure the pH of a solution by use of a pH sensing electrode. In some embodiments, the device can be configured to measure the pH of a solution by measuring the pH by any method known in the art. The controller may be configured to repeatedly perform the following steps: a difference between a target pH value and/or OCP data and measured pH and/or OCP data or between a target output of the pH sensing element and a measured output of the pH sensing element. A current amount or an electric source waveform is selected based on the difference in , an electric current or an electric potential is applied to one or more of the two or more electrodes according to the electric potential waveform to apply the selected amount of current to the solution, and the OCP's or Sends a request to the device for another measurement and/or output of the pH sensing element.

Preferably, the one or more redox active species in the solution generate and/or consume hydrogen ions through an electrochemical reaction induced by an electrical current or electrical potential applied to the solution. Preferably, the one or more redox active species is selected from: quinones, catechols, aminophenols, hydrazines, and derivatives thereof. More preferably, the at least one redox active species is a quinone. (See Thomas Finley, "Quinones," Kirk-Othmer Encyclopedia of Chemical Technology, 1-35 (2005)). Even more preferably, the quinone is selected from: hydroquinone, benzoquinone, naphthoquinone, and derivatives thereof. Most preferably, the at least one redox active species is a quinone derivative. In certain exemplary embodiments of the present invention, the solution is buffered, unbuffered aqueous, organic or a mixture thereof. In certain exemplary embodiments of the present invention, the electric source waveform is a galvanostatic waveform or a potentiostatic waveform. Preferably, the electrical source waveforms are selected from predetermined maps that map electrical inputs to respective solution pH values. In certain exemplary embodiments of the invention, the three or more electrodes are made of metal oxides, glassy carbon, graphene, gold, silver, or platinum. Preferably, the three or more electrodes include a reference electrode, a working electrode and a counter electrode, and the sensing element is a sensing electrode. Each of the three or more electrodes is each made of a metal oxide, gold, glassy carbon, graphene, silver, platinum, silver chloride, standard hydrogen, mercury drops, or saturated calomel. In certain exemplary embodiments of the present invention, the sensing electrode may also function as the working electrode. Preferably, the sensing electrode is coated with a pH sensitive coating, and the OCP of the sensing electrode and the pH sensitive coating are governed by the H ⁺ ion concentration. In certain exemplary embodiments of the present invention, the pH sensitive coating is an organic or inorganic material. Preferably, the pH sensitive coating is made of a material selected from the group consisting of polyaniline, polypyrrole, and iridium oxide.

Also provided are quinone derivatives that can be added to solutions and are suitable for electrochemical pH adjustment in biological buffers, electrochemically active compositions comprising quinone derivatives, methods of making the derivatives and/or compositions, and uses thereof.

According to an exemplary embodiment, the electrochemically active composition comprises a quinone derivative, the reactivity between the nucleophile and the quinone derivative is reduced compared to the reactivity between the nucleophile and the unsubstituted quinone from which the quinone derivative is derived, and the composition is It is configured such that the pH is controlled electrochemically through a quinone derivative. More preferably, the reactivity between the nucleophile and the quinone derivative is reduced by at least 50% compared to the reactivity between the nucleophile and the unsubstituted quinone from which the quinone derivative is derived. Preferably, the quinone derivative is defined by a formula selected from the group consisting of:

In Formulas I to XII above, each R group is H; C _n H _2n+1 ; Cl; F; I; Br; OM; NO ₂ ; 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 ; SO ₃ H; SO ₃ M; NH ₂ ; NHC _n H _2n+1 ; N(C _n H _2n+1 ) ₂ ; NHC _n H _2n OH; NHC _n H _{2 n} NH ₂ ; N(C _n H _2n OH) ₂ ; N(C _n H _2n NH ₂ ) ₂ ; NHCOC _n 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 NH ₂ ; 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 ; 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 N(C _n H _2n+1 )C _n H _2n ; C _n H _2n OH; C _n H _2n 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 SO ₃ H; C _n H _{2 n} SO ₃ M; C _n H _2n NH ₂ ; 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 NH ₂ ; C _n H _2n N(C _n H _2n OH) ₂ ; C _n H _2n N(C _n H _2n NH ₂ ) 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 NH ₂ ; 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; at least one of the R groups is not hydrogen; M is any metal cation or NH ₄ ⁺ ; n is an integer from 1 to 10 ⁹ ; y is an integer from 1 to 10 ⁹ .

According to an exemplary embodiment, all R groups of the quinone derivative are different from each other. According to an exemplary embodiment, at least two of the R groups of the quinone derivative are the same. Preferably, the composition is an aqueous solution. According to exemplary embodiments, the composition may comprise a group consisting of aqueous buffers, organic solvents, electrolytes, buffer salts, bioreagents, biomolecules, surfactants, preservatives, cryoprotectants, one or more reactants, catalysts, enzymes, co-factors, and the like. and combinations thereof. Preferably the at least one nucleophile is selected from the group consisting of amines, thiols, amino acids, peptides, proteins, and combinations thereof. According to an exemplary embodiment, the reactivity between the nucleophile and the quinone derivative is due to (i) increased steric hindrance of the nucleophile binding site by one or more of the R groups; (ii) the reactivity between the nucleophile and the unsubstituted quinone from which the quinone derivative is derived is reduced due to removal of the nucleophile binding site by covalent bond between the nucleophile binding site and one of the R groups; Preferably the reactivity between the nucleophile and the quinone derivative is reduced due to both (i) and (ii) compared to the reactivity between the nucleophile and the unsubstituted quinone from which the quinone derivative is derived.

Also provided are methods of modifying quinones having one or more R groups by substituting a substituent for one or more of the R groups to provide quinone derivatives, wherein the quinone derivatives have reduced reactivity with nucleophiles compared to the reactivity between quinone and nucleophiles. become; Substituents are H; C _n H _2n+1 ; Cl; F; I; Br; OM; NO ₂ ; 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 ; SO ₃ H; SO ₃ M; NH ₂ ; NHC _n H _2n+1 ; N(C _n H _2n+1 ) ₂ ; NHC _n H _2n OH; NHC _n H _{2 n} NH ₂ ; N(C _n H _2n OH) ₂ ; N(C _n H _2n NH ₂ ) ₂ ; NHCOC _n 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 NH ₂ ; 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 ; 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 N(C _n H _2n+1 )C _n H _2n ; C _n H _2n OH; C _n H _2n 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 SO ₃ H; C _n H _{2 n} SO ₃ M; C _n H _2n NH ₂ ; 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 NH ₂ ; C _n H _2n N(C _n H _2n OH) ₂ ; C _n H _2n N(C _n H _2n NH ₂ ) 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 NH ₂ ; 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; M is any metal cation or NH ₄ ⁺ ; n is an integer from 1 to 10 ⁹ ; y is an integer from 1 to 10 ⁹ . According to exemplary embodiments, one or more R groups are substituted with polar groups. Preferably, polar groups have atoms containing lone pairs of electrons. Preferably, the polar groups are capable of forming hydrogen bonds with water. Preferably, the polar groups contain at least one of oxygen, nitrogen and sulfur atoms. More preferably, the polar group is selected from the group consisting of OH, CH ₂ OH, OCH ₃ , COOH, SO ₃ H, NH ₂ , NH ₃ Cl, ONa, sugars, amino acids, and peptides.

(i) a halide substitution step of reacting a starting material with a hydrogen halide in the presence of acetic acid and an aldehyde; (ii) reaction of the material produced by step (i) with the nucleophile of structure RX; (iii) reaction of the material produced by step (ii) with an oxidizing agent; and (iv) reacting the material produced by step (iii) with a reducing agent, wherein R is H; C _n H _2n+1 ; Cl; F; I; Br; OM; NO ₂ ; 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 ; SO ₃ H; SO ₃ M; NH ₂ ; NHC _n H _2n+1 ; N(C _n H _2n+1 ) ₂ ; NHC _n H _2n OH; NHC _n H _{2 n} NH ₂ ; N(C _n H _2n OH) ₂ ; N(C _n H _2n NH ₂ ) ₂ ; NHCOC _n 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 NH ₂ ; 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 ; 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 N(C _n H _2n+1 )C _n H _2n ; C _n H _2n OH; C _n H _2n 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 SO ₃ H; C _n H _{2 n} SO ₃ M; C _n H _2n NH ₂ ; 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 NH ₂ ; C _n H _2n N(C _n H _2n OH) ₂ ; C _n H _2n N(C _n H _2n NH ₂ ) 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 NH ₂ ; 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; M is any metal cation or NH ₄ ⁺ ; n is an integer from 1 to 10 ⁹ ; y is an integer from 1 to 10 ⁹ ; X is OH, NH ₂ , NHR, SH, O ^- , or S ^- .

According to an exemplary embodiment, the starting material is dialkoxybenzene and the result of the halide substitution step is ortho-quinone, para-quinone, or a combination thereof. Preferably, the number of halide groups per molecule of ortho-quinone or para-quinone is 1, 2, 3 or 4. According to an exemplary embodiment, the starting material is dialkoxynaphthalene and the result of the halide substitution step is ortho-naphthoquinone, para-naphthoquinone, or a combination thereof. Preferably, the number of halide groups per molecule of ortho-naphthoquinone or para-naphthoquinone is 1 or 2. Preferably, the hydrogen halide is selected from the group consisting of HCl, HBr, HI, and combinations thereof. Preferably, the oxidizing agent is selected from the group consisting of cerium ammonium nitrate, iodine, hydrogen peroxide, ultra-expensive iodine, iodobenzene diacetate, bromine compounds, and combinations thereof. Preferably, the reducing agent is selected from the group consisting of sodium borohydride, potassium borohydride, sodium hydrosulfite, trichlorosilane, and combinations thereof.

providing a biosensor comprising a support in a solution, the support comprising one or more electrodes and a biomolecular interface layer having one or more probes immobilized thereon, and the solution comprising a quinone derivative; adding a biomolecular analyte to the solution; The quinone derivative is electrochemically reacted using one or more electrodes to produce an amount of H ⁺ ions and/or an amount of OH ^- ions, and the pH of the solution in the vicinity of the one or more electrodes is proportional to the amount of H ⁺ ions produced. controlled by the amount and/or amount of OH ^- ions; Methods comprising collecting a signal from the biosensor and reducing the reactivity between the nucleophile and the quinone derivative compared to the reactivity between the nucleophile and the unsubstituted quinone from which the quinone derivative is derived are also provided.

Preferably, the pH of the solution before electrochemically reacting the quinone derivative using one or more electrodes is 1 to 14. Preferably, the pH of the solution after electrochemically reacting the quinone derivative using one or more electrodes is 1 to 14. Preferably, the solution contains one or more nucleophiles. Preferably, the one or more nucleophiles are selected from the group consisting of amines, thiols, amino acids, peptides, proteins, and combinations thereof. According to an exemplary embodiment, the solution contains the reduced quinone derivative, and electrochemically reacting the quinone derivative causes an electrochemical oxidation reaction of the reduced quinone derivative to make the pH of the solution more acidic. Preferably, the concentration of the reduced quinone derivative is 0 to 1M. According to an exemplary embodiment, the solution contains an oxidized quinone derivative, and electrochemically reacting the quinone derivative causes an electrochemical reduction reaction of the oxidized quinone derivative to make the pH of the solution more basic. Preferably, the concentration of the oxidized quinone derivative is 0 to 1M. Preferably, the solution contains one or more buffer components provided at a concentration of 0 to 1M. Preferably, the one or more buffer components are selected from the group consisting of organic solvents, electrolytes, bioreagents, biomolecules, surfactants, and combinations thereof. According to an exemplary embodiment, the method further comprises measuring the pH of the solution. Preferably, the pH is continuously measured. Preferably, the quinone derivative reacts electrochemically by providing a quantity of electrical current. Preferably, the pH is measured prior to providing the amount of electrical current. Preferably, the amount of electric current is selected based on the measured pH.

Quinones are defined herein as unsubstituted quinones. For example, chemical structures I-XII represent quinones when all R groups are hydrogen. A quinone derivative is defined herein as a compound structurally similar to quinone except that at least one hydrogen has been replaced by a substituent. When more than one hydrogen is replaced by a substituent, the type of substituent may be independently selected, but need not be unique. Compared to their unsubstituted counterparts, the quinone derivatives of the present invention have reduced reactivity with nucleophiles, and the pH of biological buffers containing quinone derivatives can be controlled electrochemically.

Measuring the pH of a solution and/or the open circuit potential (OCP) of two or more electrodes in a solution while no current is applied between the two or more electrodes, and selecting the amount of current based on the measured pH and/or OCP and providing a selected amount of current to the solution to change the pH of the solution by at least one electrochemically generating or consuming hydrogen ions. , and methods of controlling the pH of a solution using a pH sensing element, which can be a sensing electrode, are also provided. Preferably, the generation and/or consumption of hydrogen ions is achieved by electrochemical reaction of one or more redox active species in solution. Preferably, the at least one redox active species is selected from the group consisting of quinones, catechols, aminophenols, hydrazines, derivatives thereof, and combinations thereof. Preferably, the at least one redox active species is a quinone selected from: hydroquinone, benzoquinone, naphthaquinone, derivatives thereof, and combinations thereof. Preferably, the three or more electrodes include a counter electrode, a working electrode and a reference electrode, and preferably a pH sensing electrode. According to an exemplary embodiment, the sensing electrode is configured to also function as a working electrode. Preferably, the three or more electrodes are each independently a material selected from the group consisting of metal oxides, gold, glassy carbon, graphene, silver, platinum, silver chloride, standard hydrogen, drops of mercury, saturated calomel, and combinations thereof. is manufactured with Preferably, the solution is buffered, unbuffered, aqueous, organic or a mixture thereof. Preferably, the method further comprises determining the pH of the solution based on the measured OCP of the two or more electrodes in the solution. Preferably, selecting the electrical waveform is based on the determined pH. Advantageously, the method further comprises selecting an electrical waveform and providing the electrical waveform to the solution. The electric waveform is a galvanostatic waveform or a potentiostatic waveform. Preferably, the electrical waveforms are selected from predetermined maps that map respective amounts of current to respective electrical waveforms.

a support comprising a pH sensing element, a counter electrode, a reference electrode, and a working electrode; A biosensor system comprising an electrochemically active agent is also provided, wherein the biosensor is configured to control a change in the redox state of the electrochemically active agent, and the biosensor is configured to repeatedly perform the following: pH and/or OCP of a solution. select an amount of current applied to the working electrode to minimize the difference between the pH value and/or the target pH value and/or OCP; applying a selected amount of current to the working electrode to adjust the pH and/or OCP of the solution; Measure the pH and/or OCP of the solution. Preferably, the solution is an aqueous solution. Preferably, the pH sensing element is a sensing electrode. According to an exemplary embodiment, the sensing electrode can also function as a working electrode. According to an exemplary embodiment, the sensing and working electrodes are separate electrodes, and the distance between the sensing and working electrodes is between 0 cm and 1 cm. Preferably, the amount of current applied to the working electrode is provided by applying an electrical waveform. The electric waveform is a galvanostatic waveform or a potentiostatic waveform. Preferably, the electrical waveforms are selected from predetermined maps that map respective amounts of current to respective electrical waveforms. Preferably, the sensing electrode is coated with a pH sensitive coating. pH sensitive coatings are organic or inorganic materials. Preferably, the pH sensitive coating is made of a material selected from the group consisting of polyaniline, polypyrrole, iridium oxide, and combinations thereof.

selecting a target pH value and/or an open circuit potential (OCP) based on the target pH value for the solution; determining the pH and/or characterizing the OCP of the solution using a sensing element between the reference and sensing electrodes while no current is applied to the working electrode; Also provided are methods of monitoring the pH of a solution using a sensing element, a counter electrode, a reference electrode, and a working electrode comprising repeatedly performing the OCP and/or pH of the solution and the target OCP and/or selecting the amount of current applied to the working electrode to minimize the difference between pH; applying a selected amount of current to the working electrode to adjust the OCP and/or pH of the solution; Measure the OCP and/or pH of the solution. Preferably, the solution is an aqueous solution. Preferably, the target pH value is set by including an electrochemical delta-sigma-regulator. The target pH value may change over time and/or may differ between different working electrodes. More preferably, the output-signal of the electrochemical delta-sigma-regulator is digitally filtered to obtain a digital representation of the charge required to produce the target pH value. Preferably, the target pH value and/or OCP is a range having upper and lower limits. More preferably, the target pH value is a single target value. According to an exemplary embodiment, the sensing electrode can also function as a working electrode. According to an exemplary embodiment, the sensing and working electrodes are separate electrodes, and the distance between the sensing and working electrodes is between 0 cm and 1 cm. Preferably, the amount of current applied to the working electrode is provided by applying an electrical waveform. The electric waveform is a galvanostatic waveform or a potentiostatic waveform. Preferably, the electrical waveforms are selected from predetermined maps that map respective amounts of current to respective electrical waveforms. Preferably, the sensing electrode is coated with a pH sensitive coating. pH sensitive coatings are organic or inorganic materials. Preferably, the pH sensitive coating is made of a material selected from the group consisting of polyaniline, polypyrrole, iridium oxide, and combinations thereof.

controller; three or more electrodes; pH sensing element; and a solution containing at least one redox active species, a device for controlling the pH of a solution is also provided, the device being configured to repeatedly perform the following: pH between two or more electrodes in a solution and / or measuring an open circuit potential (OCP) to generate measured pH and / or OCP data; using the controller, selecting an amount of current or electrical potential waveform based on a difference between the target pH value and/or OCP data and the measured pH and/or OCP data; Using a controller, a selected amount of current or selected electrical potential waveform is applied to the solution through one or more of the three or more electrodes. Preferably, the solution is an aqueous solution. Preferably, the one or more redox active species generate or consume hydrogen ions through an electrochemical reaction induced by an electrical current or electrical potential applied to the solution. Preferably, the at least one redox active species is selected from: quinones, catechols, aminophenol hydrazines, derivatives thereof, and combinations thereof. Preferably, the at least one redox active species is a quinone selected from: hydroquinone, benzoquinone, naphthaquinone, derivatives thereof, and combinations thereof. Preferably, the solution is buffered or unbuffered aqueous, organic or a mixture thereof. Preferably, the electric potential waveform is a galvanostatic waveform or a potentiostatic waveform. More preferably, the electric potential waveform is selected from a predetermined map mapping each amount of current to each electric potential waveform. Preferably, the three or more electrodes include a reference electrode, a working electrode, and a counter electrode, and the pH sensing element is a sensing electrode. The three or more electrodes are made of materials independently selected from the group consisting of metal oxides, gold, glassy carbon, graphene, silver, platinum, silver chloride, standard hydrogen, drops of mercury, saturated calomel, and combinations thereof. According to an exemplary embodiment, the sensing electrode can also function as a working electrode. Preferably, the sensing electrode is coated with a pH sensitive coating. pH sensitive coatings are organic or inorganic materials. Preferably, the pH sensitive coating is made of a material selected from the group consisting of polyaniline, polypyrrole, iridium oxide, and combinations thereof.

A diagram of a system with an electrochemically active agent in solution, in accordance with an exemplary embodiment of the present invention, is presented in FIG. 21 . In the previously described system, the electrochemically active agent was attached to the electrode surface rather than being in solution. As shown in FIG. 21 , the system can provide an anode electrode and electrochemically active agent in solution. Applying current to the electrode induces the electrochemical active agent to undergo an electrochemical redox reaction, making the pH of the solution near the electrode more acidic. Having the electrochemically active agent in solution rather than adhering to the electrode surface has several advantages. For example, when the amount of electrochemically active agent is not limited by the density of the surface layer, the capacity of the device can be increased with a more significant change to the surrounding environment; Diffusion from the bulk solution may provide fresh electrochemically active agents to the electrode surface, enabling cycling capability; A universal electrochemistry can be applied to all types of electrodes, so it will not interfere with the adhesion of other surface chemistries, such as antifouling reagents or biomolecules. Additionally, to use quinones as electrochemically active agents for pH generation in biological solutions, the structure of quinones can be modified to meet the requirements for use in biological solutions. To be useful for pH control in biological buffers, a molecule must meet the following requirements: release or consumption of protons through an electrochemical reaction upon electron stimulation, sufficient water solubility, reduction and oxidation potentials to prevent water hydrolysis or other redox in solution. Must be lower than the potential of the active species, stability in solution in the absence of electronic excitation (i.e., no self-oxidation/reduction), low reactivity towards nucleophiles, biological samples (eg: proteins, peptides, cells, DNA, and enzymes) compatibility with.

22A-22C depict quinone derivatives that can be used for pH adjustment in aqueous solutions according to exemplary embodiments of the present invention.

The above description is illustrative and not intended to be limiting. A person skilled in the art can understand from the above description that the present invention may be embodied in various forms, and that the various embodiments may be embodied singly or in combination. Thus, while embodiments of the present invention have been described in connection with specific examples thereof, the true scope of the embodiments and/or methods of the present invention lies, as other variations will become apparent to those skilled in the art upon a study of the drawings, detailed description and appendices. It shouldn't be so limited. Additionally, steps illustrated in the flowcharts may be omitted and/or the order of certain steps may be changed, and in certain instances several illustrated steps may be performed automatically.

The following are examples that illustrate specific methods without being limiting in any way. Examples may be modified within the scope of the detailed description as understood from common knowledge.

examples

Example 1 - Electrochemical generation of H+ or OH- ions at electrode surfaces .

Electrode material used: The electrode material was indium tin oxide. It is a semiconductor electrode surface with a very large potential range in aqueous solution.

Electro-oxidation of species to produce H ⁺ ions . Oxidation of ascorbic acid at the electrode surface produced H ⁺ ions and changed the electrode surface pH to a more acidic state:

AH ₂ → A + 2H ⁺ + 2e ^-

Here, AH ₂ is ascorbic acid (C6H6O6) (shown in FIG. 5). The oxidizing electrode potential was less than 0.5V for the indium tin oxide material compared to the Ag/AgCl reference electrode (shown in FIG. 7). This potential was lower than that required for the oxygen evolution reaction in aqueous solution. Higher electrode potentials (eg > 1V for ITO electrodes in phosphate buffer only) can damage the PEG layer (shown in Figure 8). Ascorbic acid also served as a sacrificial species to prevent electrochemical degradation of the surface chemistry.

OH ^- electro-reduction of species to produce ions . Reduction of benzoquinone (C6H4O2) to hydroquinone (C6H6O2) can produce OH ^- ions at -0.1V:

BQ + 2e ^- + 2H ₂ O → HQ + 2OH ^-

This reduction reaction increased the pH at the electrode interface.

In the example above, the amount of H ⁺ or OH ^- ions produced depends on the concentration of the species present in the solution (in the nM-mM range), the applied potential (-2V to +2V), and the type of waveform (pulls at different frequencies and duty cycles). spar, constant, sawtooth, sine, square), and diffusion of the species (which may vary due to additives in solution). These parameters can be optimized to obtain a different pH at each working electrode present in the multi-site array.

Example 2 - pH change using an enzymatic reaction

Enzymes such as oxidase, urease or dehydrogenase are known to consume or produce hydrogen during reactions. for example:

ß-d-glucose + O ₂ → d-glucose-δ-lactone + H ₂ O ₂

d-glucose-δ-lactone + H ₂ O → d-gluconate + H ⁺

Oxidation of glucose in the presence of glucose oxidase can generate H ⁺ ions that are used to change the pH near the protein of interest.

Example 3 - Co-immobilization of Enzymes with Biomolecular Probes in a Biomolecular Interfacial Layer

Enzymes bring proteins, reactants, catalysts, etc., into close proximity when co-immobilized on a surface, so that the H ⁺ produced by the enzymatic reaction can lead to protein binding (e.g. antigen-antibody binding and non-specific binding), reaction will cause localized pH changes that can affect rates and the like.

Example 4 - Attachment of Enzymes to Magnetic Micro/Nanoparticles

Proteins, reactants, catalysts, etc. are attached to the micro/nanocavities of the solid surface on the electromagnet. Enzymes are separately attached to magnetic micro/nanoparticles in solution. Controlling the electromagnet built/placed underneath controls the local pH value. The enzymatic reaction is triggered by introducing the corresponding enzyme substrate (shown in Figure 6). Alternatively, electrochemically active enzymes are used. pH changes are localized in the cavity, and protein interactions, reaction rates, etc. are regulated.

Example 5 - Electrochemical modulation of pH monitored by fluorescence intensity by green fluorescent protein (GFP)

Electrode material used: The electrode material was indium tin oxide. The fluorescent protein used is GFP immobilized on a glass substrate containing an array of electrodes. GFP is applied in spots, each spot covering an area overlapping with one electrode and an area not overlapping with an electrode.

The pH change at the surface of the ITO working electrode is via current-induced oxidation of the redox active molecule 2-methyl-1,4-dihydroquinone in dilute phosphate buffer containing 0.1 M Na ₂ SO ₄ (pH = 7.4). is created After 10 seconds of induction, a current (50 microamps) was applied for 30 seconds and the solution pH lowered to 5.5 as observed by a change in GFP fluorescence intensity. 10 is used as a calibration curve for evaluating pH values. After the current was turned off, the pH recovered to a neutral value within 50 seconds (shown in FIGS. 11 and 12B).

Example 6 - Prevention of Reaction with Nucleophiles

Para-benzoquinone and ortho-benzoquinone (compounds 2, 4, 6 and 9 in FIGS. 22A-22C) are susceptible to nucleophilic attack at the ring double bond (position 2 in structure 2 of FIGS. 22A-22C). 3, 5 and 6, positions 3, 4, 5 and 6 in structure 4 of FIGS. 22A-22C, and positions 2 and 3 in structure 6 of FIGS. 22A-22C). Introduction of substituents at some or all of these positions can alleviate the problem of nucleophilic attack. For example, 1,4-benzoquinone (structure 2 in FIGS. 22A-22C, where R1, R2, R3 and R4 are H) undergoes a Michael addition reaction with amino groups of proteins ([Loomis et al. al., Phytochemistry, 5, 423, (1966)] and US6753312 B2). On the other hand, 2,5-disubstituted 1,4-benzoquinones (structure 2 in Figure 21, where R1 and R3 are H and R2 and R4 are groups other than H) are susceptible to Michael addition reactions in the presence of proteins. and is more suitable for use as a biological buffer. 23 demonstrates the effect of substitution on protein stability in the presence of benzoquinone. The fluorescence intensity of green fluorescent protein (GFP) was measured after incubation for 30 minutes with three different benzoquinones in phosphate buffered saline (benzoquinone concentration was 0.5 mM). Differences in fluorescence intensity indicate the different effects of different substitutions in benzoquinone on the stability of GFP. The fluorescence intensity of GFP is an indicator of its structural integrity. Loss of fluorescence intensity usually indicates loss of its tertiary structure (protein denaturation) (Yin D. X., Zhu L., Schimke R. T. Anal. Biochem. 1996, 235:195-201). As shown in Figure 23, GFP retains 100% of its fluorescence intensity after incubation with disubstituted benzoquinones for 30 minutes, whereas incubation with unsubstituted and monosubstituted benzoquinones, respectively It causes 25% and 7% loss in fluorescence intensity.

Example 7 - Soluble adjustment

The water solubility of aromatic compounds can be improved by introducing charged groups or atoms with lone pairs of electrons capable of participating in hydrogen bonding. Such groups are for example - OH, - CH ₂ OH, - OCH ₃ , - COOH, - SO ₃ H, - NH ₂ , - NH ₃ Cl, - ONa. Sugars, amino acids and peptides can also improve the water solubility of quinones. Synthetic macromolecules such as polyethylene glycol may also be used as substituents.

Example 8 - Adjustment of redox range

The reduction/oxidation potential of quinones can be tailored to the needs of specific applications. The redox potential can be pushed towards higher voltages by introducing electron-donating groups (such as alkyl, hydroxyl, alkoxy, methoxymethyl, morpholinomethyl, amino and chloro substituents). Conversely, electron-withdrawing groups (such as nitro, cyano, carboxylic acid, or carboxylic acid ester groups) will push the redox potential toward lower voltages.

In an exemplary embodiment, the mechanism of oxidation of hydroquinone in aqueous solution involves two steps: transfer of electrons and transfer of protons. The introduction of electron-withdrawing or electron-donating substituents covers the first step of the process. The oxidation potential of hydroquinone can also be lowered by introducing a substituent capable of forming an intramolecular hydrogen bond with the hydroxyl group of hydroquinone. These hydrogen bonds weaken the bond between the hydrogen and oxygen of the hydroxyl group, lowering the overall energy barrier to the oxidation reaction. Examples of such R groups are CH ₂ OH, CH ₂ OCH ₃ , morpholinomethyl, and COOCH ₃ .

Having the ability to select molecules that undergo electrochemical transformation through the same mechanism but at different potentials can accommodate different pH conditions and avoid undesirable electrochemical reactions involving other redox active components in the system. For example, in applications involving DNA synthesis, it is desirable to keep the voltage below the reduction potential of pyrimidine bases, nucleosides, and nucleotides (1 V relative to NHE in an aqueous solution pH ~8) (Steenken, S J Am Chem Soc, 1992, 114: 4701-09).

Autooxidation of hydroquinone is another issue to be considered. In an exemplary embodiment, to avoid oxidation, a quinone derivative with a sufficiently high electrochemical oxidation potential that is resistant to spontaneous chemical oxidation by molecular oxygen is used.

Conversely, benzoquinones with sufficiently low reduction potentials should be selected for systems where reducing agents such as mercaptoethanol, glutathione and dithiothreitol are present in solution. Examples of such applications are DNA synthesis, electrophoresis, and immunoassays.

The redox potential of quinones is affected by the pH of the solution. It is easier to oxidize hydroquinone at a more basic pH, while a more acidic pH will require a higher oxidation potential. Thus, it affects the stability of hydroquinone against auto-oxidation by oxygen in air. Thus, when working at basic pH, the use of quinones at higher oxidation potentials will improve the stability of the electrochemical system. Figure 24 shows that there are different oxidation potentials for different substituents in quinones (Figure 24, A-D), so oxidation potentials can be tuned by changing substituents in quinones.

Example 9 - Synthesis of Substituted Quinones

Scheme 1, shown in Figures 25A and 25B, shows the synthesis of substituted quinones according to an exemplary embodiment of the present invention.

Example 10 - 2,5-Dimethyl-1,4-hydroquinone

Sodium dithionate (18.7 g, 107.3 mmol, 7.3 equiv) was dissolved in 20 mL H ₂ O and loaded into a separatory funnel. Next, a solution of benzoquinone (2 g, 14.7 mmol, 1 equiv) in 75 mL diethyl ether was added. The biphasic mixture was vigorously stirred for 30 minutes and the organic layer turned from orange to pale yellow. The organic phase was washed with brine, dried over MgSO ₄ and concentrated to give a white solid (1.69 g, 83%).

Example 11 - 1,4-bis(bromomethyl)-2,5-dimethoxybenzene

Paraformaldehyde (Aldrich, 4.27 g, 144.75 mmol) and HBr/AcOH (Fluka, 33%, 30 mL) were mixed with 1,4-dimethoxy in glacial acetic acid (Fisher, 50 mL). To a stirred solution of benzene (Aldrich, 10.00 g, 72.37 mmol) was added slowly. The mixture was stirred at 50 °C for 1 hour, cooled to room temperature and hydrolyzed in water (200 mL). A white solid was obtained by filtration, suspended in CHCl ₃ (50 mL) and refluxed for 10 min. After cooling to room temperature, the white solid was collected again by filtration and washed with water (15.75 g, 67%). An NMR spectrum was obtained experimentally to confirm the chemical structure of the resulting compound and its purity. The NMR results were as follows: ¹ H NMR (300 MHz, CDCl ₃ ) δ: 6.88 (s, 2H), 4.54 (s, 4H), 3.87 (s, 6H) ppm.

Example 12 - 1,4-dimethoxy-2,5-dimethoxymethylbenzene

A dry round bottom flask was charged with 2,5-dibromomethyl-1,4-dimethoxy-benzene (3 g, 9.26 mmol, 1.0 equiv), anhydrous K ₂ CO ₃ (25.6 g, 185 mmol, 20 equiv), and dried Charged with methanol (200 mL). The reaction mixture was heated to reflux for 30 min, then cooled to ambient temperature, filtered and concentrated to a crude white solid. The solid was resuspended in water, extracted with ethyl acetate, dried over MgSO ₄ and concentrated. The residue was recrystallized from hexane as a pale yellow powder (1.2 g, 57%). Retention factor (Rf) (50% EtOAc/Hexanes) = 0.65.

Example 13 - 2,5-dimethoxymethyl-1,4-benzoquinone

A solution of 1,4-dimethoxy-2,5-dimethoxymethylbenzene (1.2 g, 5.24 mmol, 1.0 equiv) in acetonitrile (0.1 M, 52 mL) was dissolved in cerium ammonium nitrate (5.8 mL) in water (8 mL). g, 10.6 mmol, 2.02 equiv). The reaction mixture was stirred under argon at ambient temperature for 30 min, then diluted with water and extracted with dichloromethane. The combined organic layers were washed with water, dried over Na ₂ SO ₄ and concentrated to an orange solid. The crude mixture was purified by column chromatography on deactivated silica gel (0-5% ethyl acetate/hexanes) to give yellow crystals (300 mg, 67%). Rf (50% EtOAc/Hexanes) = 0.7; ¹ H NMR (300 MHz, CDCl ₃ ) δ: ppm; UV-Vis = 268 nm.

Example 14 - 2,5-dimethoxymethyl-1,4-hydroquinone

Benzoquinone (200 mg, 1.02 mmol, 1.0 equiv) obtained according to the reaction of Example 13 in 2.5 mL EtOAc was treated with a solution of sodium dithionate (1.3 g, 7.44 mmol, 7.3 equiv) in 2 mL H ₂ O. . The biphasic mixture was intensively mixed for 30 minutes and the solution turned from light to pale yellow. The mixture was diluted with water, extracted with ethyl acetate, dried over MgSO ₄ and concentrated to a white powder (96 mg, 48%).

Example 15 - 1,4-dimethoxy-2,5-dihydroxymethylbenzene

A dry round bottom flask was charged with 2,5-dibromomethyl-1,4-dimethoxy-benzene (5 g, 15.4 mmol, 1.0 equiv) and NaOH (77 mL of 1.0 M solution, 77 mmol, 5.0 equiv), 12 mL H ₂ O, and 38 mL THF. The reaction mixture was sealed and heated to 80° C. for 6 hours. After cooling, the reaction mixture was concentrated by rotary evaporation to give a crude solid which was recrystallized in hexanes as a white powder (3 g, 60%). Rf (80% EtOAc/Hexanes) = 0.2.

Example 16 - 2,5-dimethoxymethyl-1,4-benzoquinone

A solution of 1,4-dimethoxy-2,5-dihydroxymethylbenzene (1.0 g, 5.04 mmol, 1.0 equiv) in acetonitrile (0.2 M, 25 mL) was dissolved in cerium ammonium in water (33 mL) at 0 °C. It was treated with a solution of nitrate (5.5 g, 10.1 mmol, 2.0 equiv). The reaction mixture was stirred under argon at ambient temperature for 30 min and then extracted with EtOAc. The combined organic layers were dried over Na ₂ SO ₄ and concentrated to an orange-red solid. The crude mixture was purified by column chromatography on deactivated neutral alumina (0-100% ethyl acetate/hexanes) to give yellow crystals (300 mg, 67%). Rf (80% EtOAc/Hexanes) = 0.4; ¹ H NMR (DMSO, 500 MHz): 6.6 (s, 2H), 5.3 (s, 2H), 4.3 (s, 4H), ppm; ¹³ C NMR (DMSO, 125 MHz): 187.4, 149.1, 129.8, 57.0 ppm; UV-Vis = 260 nm.

Example 17 - 2,5-dihydroxymethyl-1,4-hydroquinone

Benzoquinone (60 mg, 0.36 mmol, 1.0 equiv) obtained according to the reaction of Example 16 in 0.6 mL EtOAc was treated with a solution of sodium dithionate (453 mg, 2.6 mmol, 7.3 equiv) in 0.7 mL H ₂ O. . The biphasic mixture was vigorously stirred for 30 minutes and the solution turned from light to pale yellow. The mixture was diluted with water, extracted with ethyl acetate, dried over MgSO ₄ and concentrated to a crude solid which was purified by column chromatography over deactivated neutral alumina under Ar to give a white powder (26 mg, 30%) was obtained. UV-Vis = 297 nm.

Example 18 - Open Loop Method

According to an exemplary embodiment, a method termed open loop pH control involves using an electric current or electric potential buildup to maintain a desired pH of a solution in proximity of an electrode. The method uses an understanding of the electrochemical components of the system, the main components being the reduction/oxidation properties of the quinone, the starting pH of the solution, the electron transfer coefficient of the electrode material, the redox molecule concentration, the salt concentration, and the buffer composition and is the concentration All of these components can affect how the electrochemical reaction changes the pH near the electrode. By incorporating this understanding and experimental data, a series of models can be used to define waveforms to change pH as needed.

26 shows results from open loop waveform experiments designed to hold the pH of a solution at pH 6.5 over 15 minutes in close proximity to the electrodes. A stepped trace extending approximately between current values 3.12e ^-6 and 3.50e ^-6 correlates with the current applied to the system (right axis). The approximately straight trace correlates with the pH observed close to the electrode surface (left axis) as measured by analyzing the pH-dependent fluorescence of the green fluorescent protein bound to the surface.

27 shows four different examples of waveforms (A-E) that can be used to form and control the pH of a solution. The black line is the current/potential driven input plotted over time, and the gray line is the pH change produced in solution near the electrode surface.

If the relevant electrochemical components of the system remain fixed, these waveforms can be used to reproducibly generate the same pH change profile of a solution on demand without additional complexity. The method can be implemented using a variety of electrode systems, and schematic diagrams of exemplary setups are shown in FIGS. 31 , A-B. The method requires at least two electrodes 815 and 820. Initially, the open circuit potential of the solution near electrode 700 can be measured by applying zero current between the two electrodes 815, 820. In this state, one electrode acts as a sensing electrode (SE) 815 and the second acts as a reference electrode (RE) 820 . Once the starting OCP is known, a current 800 (FIG. 31 A) or electrical potential 804 (FIG. 31 B) is applied to the electrodes 815, 820 based on the desired pH change. While current or potential is applied, one electrode 815 serves as a working electrode WE, and the second electrode 820 serves as a counter electrode CE. This is the simplest case in that the condition of the system must remain fixed, but to improve accuracy, a continuous feedback method of the system state is desirable.

Example 19 - Closed Loop Method

According to another exemplary embodiment, a method termed closed loop pH control uses open circuit potential as a feedback measurement to control current or potential.

29A illustrates OCP controlled on sensing electrode SE using a closed loop feedback method with a single OCP V _target . In one setup (shown in FIG. 32 ), the system uses a current source (shown in FIG. 32 ) to increase the H ⁺ concentration in solution 700 until V _in 830 detected at SE 817 reaches a single OCP V _target value. 800) is driven to apply current. Current source 800 is then shut off using switch 802 and the diffusion of H ⁺ ions away from SE 817 reduces the H ₊ concentration in solution 700 . Similarly, the system can be set to induce a decrease in H ⁺ concentration until an OCP V _target value is reached, then diffusion of H ⁺ ions towards the SE increases the H ⁺ concentration (not shown). In another setup (shown in FIG. 33 ), positive (800) and negative (801) current sources are used so that the system does not have to rely on diffusion to facilitate pH changes. This system can actively change pH in both positive and negative directions. In this setup, the system is driven to apply positive current from current source 800 to increase the H ⁺ concentration until V _in 830 reaches a single OCP V _target value, using transfer switch 803. Thus, a negative current is applied by connecting the negative current source 801 to the working electrode (WE) 816, and the setting is reversed to reduce the H ⁺ concentration. In this way, the system does not rely on passive diffusion, but actively monitors and adjusts the pH by electronic control.

29B shows experimental data of OCP between SE 817 and RE 822, which changes as current is applied to WE 816. In this setup (shown in FIG. 32 ), upper and lower targets are preset via controller 900 for OCP values on SE 817 . When the potential is below the lower limit, the feedback activates the current driven pH change, and when the potential is above the upper limit, the switch 802 turns off the current. The data represent an ascending OCP until the upper target is achieved. Feedback then turns off WE 816, which causes a drop in OCP as the buffer from the bulk begins to restore the local pH of solution 700. When OCP reaches the lower limit target, switch 802 is used to resume current to increase OCP again. This feedback mechanism may allow a defined pH to be maintained for solution 700 close to WE 816 . In another setup (shown in FIG. 33 ), a positive current is applied to actively increase OCP until an upper limit is reached, then a negative current is applied to actively decrease OCP until a lower limit is reached. To be applied in reverse, the feedback can be activated to continuously change the OCP. In this way, the system does not rely on passive diffusion, but actively monitors and adjusts the pH by electronic control.

Additionally, FIG. 30 shows experimental results of the open circuit potential voltage measured on the sensing electrode adjacent to the energized working electrode. The working electrode is in closed loop feedback with the sensing electrode. The open circuit potential (OCP) of the working electrode as detected by the sensing electrode is similar to the pH of the solution near the working electrode. The feedback was set to apply current to the WE only when the sensing electrode open circuit potential was less than 0.2V. The feedback then borders between 0.19V and 0.2V (target OCP), turning the potential to the working electrode ON below 0.19 and OFF above 0.2V. The shift at time 560 s is an interruption caused by pipetting the bulk solution onto the working electrode. This perturbs the pH gradient created by the current applied to the working electrode, causing the pH of the solution to immediately return to that of the bulk solution. After pipetting stops, the closed loop feedback system is restored to maintain the target OCP thereafter.

32-47 show a schematic diagram of a closed loop feedback setup, wherein the OCP between a sensing electrode (SE) 816 and a reference electrode (RE) 822 is one or more switches 802-803, 806-810 is provided as an input to the controller 900 that regulates the current source(s) 800-801 or the potential source(s) 804-805 via In this way, the system is triggered on and off to increase or decrease the electrochemical production/consumption of H ⁺ ions, thereby increasing or decreasing the rate of diffusion of buffer ions from the bulk solution. A specific pH value defined by a target OCP (V _target ) between SE 816 and RE 822 can be achieved.

Example 20 - Use of a pH Sensitive Coating

To further improve the closed loop pH control method, improved pH sensitivity may be included by adding a pH sensitive coating 840 on the actuation and/or sensing electrodes 815, 816, 817. An example of such a coating is PANI, which has been shown to have exceptional pH sensitivity. PANI contains charged groups that interact with hydrogen ions and change the conductivity of the polymer. The response of PANI as a function of pH is close to the Nerstian limit of pH detection (59 mV/pH). The change in the open circuit potential of PANI is highly selective for H ⁺ ions, unlike just the bare electrode surface, which is sensitive to other ions in solution other than H ⁺ . 28 shows the response of open circuit potential (OCP) as a function of pH, the surface of the electrode being coated with a pH sensitive PANI coating. The response of OCP as a function of pH is plotted with the slope of the line being approximately 60 mV/pH close to the Nerstian limit.

Example 21 - Device design

31 shows an exemplary schematic diagram of a device design using an open loop method. In part A a controlled current source is used, whereas in part B a controlled voltage source is used.

A variety of designs can be used with closed loop methods. 32, 34, 36, 38, 40, 42 and 44 show various designs for the closed loop method with a single controlled current source 800, and FIGS. 33, 35, 37, 39, 41, 43 and 45- 47 shows an alternative design using dual controlled current sources 800, 801. Closed loop methods can also be implemented in designs with PANI coated 840 sensing electrodes 817 (FIGS. 34, 35, 38-41 and 44-47). The WE and SE can also be combined with a single electrode 815 that can function as both a sensing and actuating electrode (FIGS. 36-39). The current 800-801 and voltage sources 804-805 are connected and disconnected using the various switches 802-803 and 806-810. 32, 34, 36 and 38 show a simple switch 802 for connecting and disconnecting the current source 800. 40 shows a similar switch 806 for connecting and disconnecting the voltage source 804. 33 , 44 , 46 and 48 show a transfer switch 803 for switching between a positive current source 800 and a negative current source 801 . 42 shows a similar transfer switch for switching between a positive voltage source 804 and a negative voltage source 805. 42-45, switches 808-810 operate with clock phases Phi1 and Phi2. Switches 808 and 810 operate with clock phase Phi1 and switch 809 with Phi2. In each of the above examples, it is the various switches controlled by the controller 900 based on feedback to enable an additional level of control over the WE potential input and SE measurement output.

46 schematically illustrates a system architecture with a closed loop controller. This system controls the pH to a single target value V _target (907). The same architecture can be applied to electrochemical cells with systems that use electrical voltage instead of electrical current to regulate pH. In an exemplary embodiment, the controller operates as follows. The input voltage (V _in ) 830 is sampled (901), compared to the target voltage V _target (907) (902), the difference is amplified (903) and processed by a loop filter (904). It can also be part of a loop filter and can also be realized in a different way, for example as a switched-capacitor amplifier or a switched-capacitor loop filter. One example for loop filter 904 is a PID-controller with a proportional part (P), an integral part (I) and a derivative part (D). The output signal of loop filter 904 is compared to a fixed threshold by comparator 905. The output of comparator 905 can be positive or negative. It is equivalent to digital display. This digital signal is then stored in a clock module 906, such as a flip-flop or latch. In an exemplary embodiment, the frequency of clock 908 is much greater than the frequency determined by the reciprocal of the diffusion time constant of H ⁺ ions. The output signal of the flip-flop then determines whether a positive or negative integrated current is applied to WE 816. This equation can also be applied to a system with only one current source 800 or a system with voltage sources 804 and 805 instead of current sources 800 and 801 . The system operates as an "electrochemical delta-sigma-regulator", wherein the quantization error of the integrated current source is "shaped" by the transfer function of the electrochemical cell and the transfer function of the electronic loop filter 904, and the clock CLK 908 It is distributed over a wide frequency spectrum determined by frequency. The 1-bit-output of the comparator may be filtered by digital filter 909 as shown in FIG. This creates a digital indication of what the system must apply to the electrochemical cell to maintain the pH at the target value.

There is flexibility in controller architecture design in that analog controllers, digital controllers, or controllers using both analog and digital signal processing may be used. Figure 47 shows the second controller architecture, the main difference from Figure 46 is the way the target value for pH is set. In FIG. 46 , the target pH value is set by analog voltage V _target 907 . This V _target 907 can be created by a digital to analog converter (DAC). In FIG. 47 , the input voltage V _in 830 is digitized by an analog-to-digital converter (ADC) 911 and then compared to a digital target value 910 (912). As mentioned above, electrons are clocked at a frequency 908 much higher than the reciprocal of the diffusion time constant of H ⁺ ions.

The controller in FIG. 47 can also be used to “measure” the electrical current or voltage needed to set a specific target pH value using a closed loop method. This information can be used to characterize the system and derive stimuli for the open loop system. One example where this can be very useful is an array-structure of several sites where one site is used to "measure" the correct value using a closed loop, and then these values are then plotted against several other "mirrored" sites. Applied according to open-loop.

48A-48D show examples of exemplary electrode configurations for pH sensing (routing not shown). Examples of working and sensing electrode geometries include: FIG. 48A sensing electrode positioned adjacent to the working electrode, FIG. 48B sensing electrode positioned within the working electrode, FIG. 48C multiple sensing electrodes to a single working electrode, and 48D Interdigitated actuation and sensing electrodes. Inclusion of a PANI surface is beneficial to improve the accuracy of closed loop pH control. There are several different methods for incorporating a sensing electrode that monitors pH. For example, as yet another alternative, the working and sensing electrodes can also be the same as one by switching between active and passive/measuring phases.

Example 22 - pH control

Reversible electrochemical oxidation/reduction of pH adjusting reagents such as quinone derivatives, hydrazine derivatives, or water has been demonstrated for rapid pH changes in localized areas (Fomina et al., Lab Chip 16, pp. 2236-2244 (2016)) . The pH control limits may depend on the pKa and oxidation/reduction potential of specific pH control reagents, and their concentrations. On-demand pH control by oxidation of 2,5-dimethyl-1,4-hydroquinone and reduction of 2,5-dimethyl-1,4-benzoquinone was tested on an indium-tin oxide electrode in 1 mM phosphate buffer. 53 shows that when an anode current is applied to the electrode, proton production overcomes the buffer capacity and the pH of the solution becomes more acidic and vice versa.

Example 23 - Multi-electrode array design

49 shows a non-limiting example of a multi-electrode array containing feedback-control electrodes. Connection electrodes and contact pads that may be needed in some instances to connect to the control unit are not shown for clarity. This approach is particularly useful for performing multiple rounds of reaction or visualization steps. Each feedback-control set in the feedback-control section can target an independent pH value, the same pH value, or a combination thereof.

50 shows a non-limiting example of a control scheme for a single device containing a feedback-controlled section and a non-feedback-controlled section. This approach is particularly useful for performing multiple rounds of reaction or visualization steps. Each feedback-control set in the feedback-control section can target an independent pH value, the same pH value, or a combination thereof. For each target pH value, there may be one or more sets of non-feedback-controlled electrodes in the non-feedback-controlled section assigned to the same target pH value. When the shape and size of the working electrode of the non-feedback-controlled electrode set(s) is similar to that of the working electrode(s) of the feedback-controlled electrode set, the electrical parameters obtained from the feedback-controlled section are - Can be applied directly to the working electrode of the control section.

51 shows a non-limiting example of a feedback-control electrode set distributed among non-feedback-control electrode sets. Each feedback-control electrode set can target an independent pH value, the same pH value, or a combination thereof. For each target pH value, there is one or more sets of non-feedback-control electrodes also assigned to the same target pH value (pH coupled). This configuration can help achieve the same level of control or a greater level of control with a simpler device architecture. In some examples, this configuration minimizes the effect on the sensing electrode from an adjacent working electrode having a different target pH value, such as when a set of feedback-control electrodes is surrounded by its respective set of pH-coupled non-feedback-control electrodes. can do. Non-feedback-control electrode sets assigned to the same target pH value of the feedback-control electrode set may be positioned adjacent to and/or not adjacent to each feedback-control electrode set assigned the same target pH value. The feedback-control electrode sets may be pattern-distributed and/or randomly-distributed with respect to each non-feedback-control electrode set assigned the same target pH value. In some examples, the feedback-control electrode set is distributed randomly or in a pattern designed to provide a uniform solution pH across the working electrode. In some examples, the feedback-control electrode sets are distributed in a pattern designed to provide maximum versatility in providing a unique solution pH and/or a unique series of pH changes at each working electrode. In some examples, the feedback-control electrode sets are distributed in a pattern designed to provide maximum feedback or minimize feedback from the collection of sensing elements.

As described in further detail in the paragraphs below, in a non-limiting example, a multi-electrode array design may include a pH coupled electrode that is pH coupled via direct electrical coupling between the working electrodes of a set of pH coupled electrodes. contains a set; In another non-limiting example, a multi-electrode array design includes a set of pH coupled electrodes that are pH coupled via each working electrode of the set of pH coupled electrodes receiving the same electrical parameters from the processor; In another non-limiting example, a multi-electrode array design is a pH coupled electrode that receives different electrical parameters from a processor but is pH coupled via each working electrode of a set of pH coupled electrodes configured to produce the same target pH value. contains a set

Direct electrical coupling may be electrical coupling that bypasses or does not include a processor in the direct electrical coupling path. In some examples, direct electrical coupling is achieved through coupling through electrical wires. When the working electrode of a set of pH coupled electrodes is directly electrically coupled, in some instances, the feedback-controlled working electrode obtains an electrical parameter from a processor, and the feedback-controlled electrode converts the electrical parameter to a direct electrical coupling. through the non-feedback-controlled actuation electrode. Preferably, the shape and size of the working electrode of the non-feedback-control electrode set(s) is similar to the shape and size of the working electrode(s) of the feedback-control electrode set. In this way, the working electrode of the pH-coupled electrode set can receive the same electrical parameters and in the same way can transform the solution surrounding the working electrode of the pH-coupled electrode set.

When each working electrode of the pH-coupled electrode set receives the same electrical parameters from the processor, the working electrode of the pH-coupled electrode set modifies the solution surrounding the working electrode of the pH-coupled electrode set in the same way. To ensure this, the working electrodes of the pH coupled electrode set preferably have the same size and/or shape. In some examples, the reassignment of pH coupling of the non-feedback-controlled electrode set(s) to the feedback-controlled electrode set(s) can be readily changed when the electrical parameters are received from the processor.

When each working electrode of the pH-coupled electrode set receives electrical parameters from the processor to generate the same target pH value, the working electrodes of the pH-coupled electrode set may have different sizes and/or shapes. In these examples, the processor (1) provides an electrical parameter to the working electrode(s) of the pH coupled feedback-controlled electrode set to obtain a solution surrounding the working electrode(s) of the pH coupled feedback-controlled electrode set. (2) providing working electrode(s) of a set of pH-coupled non-feedback-controlled electrodes having different sizes and/or shapes for the modified electrical parameter, The pH of the solution surrounding the working electrode(s) of the coupled non-feedback-control electrode can be modified to the same target pH value. In some examples, the modified electrical parameter is the electrical parameter required for the working electrode(s) of the pH coupled feedback-controlled electrode set and the electrical parameter required for the working electrode(s) of the pH coupled non-feedback-controlled electrode set. Modified according to predetermined calibration data correlated with electrical parameters, both yield the same target pH value. In some examples, the reassignment of pH coupling of the non-feedback-controlled electrode set(s) to the feedback-controlled electrode set(s) can be readily changed when the electrical parameters are received from the processor.

In some examples, pH coupling may change over time during the same use or between uses of a device that includes a set of pH coupled electrodes. Since the device can provide a unique set of pH conditions at each individual working electrode to produce a unique product at each working electrode, this means that devices incorporating these pH-coupled electrodes can produce more than one product at the same time. , for example, can be advantageous when used to generate a library of products. This may also be advantageous in reducing the number of sensing electrodes and thus the amount of feedback from the collection of sensing elements to be processed that would otherwise be required. This may be advantageous when the same device, which further contains a set of pH coupled electrodes, is used for several different purposes between uses. In some examples, the change in pH coupling is controlled through a network of switches and/or through a processor that individually controls each working electrode. In some examples, pH coupling and/or changes in pH coupling are user-defined, such as through selection of pH coupling through a graphical user interface.

Example 24 - Closed-Loop Control and Design

52 shows a non-limiting example of a schematic diagram of how closed-loop pH control can work with surface-patterned electrodes.

54A-54B are non-limiting examples of closed-loop pH control with external counter and reference electrodes (FIG. 54A) and closed-loop pH control with surface patterned on-chip counter and reference electrodes. An example (FIG. 54B) is shown.

55A-54D show non-limiting examples of designs of sensing elements and working electrodes and in some examples other components. 55A and 55B show a slide design that can be used in a variety of applications. In some instances, the sensing element must be physically separated from the working electrode to avoid crosstalk or shorting. If the working electrode and the sensing element are to be in the same plane, a small gap of 1 nm to 100 micrometers between the working electrode and the sensing element can be used (FIG. 55C). Another possible design option is to build a multi-layer stack: a thin insulating layer can be placed between the sensing element and the working electrode (Fig. 55d). The counter electrode can also be patterned around the working electrode, which can minimize diffusion effects and can help control the pH in a more restrictive shaped pH control zone, especially in non-buffered solutions ( Figure 55e).

Claims (20)

A device for controlling the pH of a solution on an array of electrodes,
support; and
An array of electrodes comprising one or more feedback-control electrode set(s), each of the one or more feedback-control electrode set(s):
one or more reference electrode(s);
one or more counter electrode(s); and
one or more subset(s) comprising a pH sensing element electrically coupled to the working electrode;
here:
the reference electrode of the one or more reference electrode(s) and/or the counter electrode of the one or more counter electrode(s) are electrically coupled with at least one of the one or more subset(s);
The device is:
a) selecting the respective amount of current and/or voltage to be applied to at least one of the one or more working electrode(s) to minimize the difference between the signal output of the coupled sensing element(s) and the target sensing value; do;
b) applying a selected respective amount of current and/or voltage to at least one of the one or more working electrode(s) to change the pH of the solution proximate each working electrode(s);
c) a device configured to repeatedly measure the signal output of the coupled sensing element(s). The device of claim 1 , wherein at least one of the reference electrode(s) is also a sensing element. The device of claim 1 , wherein the solution contains at least one redox active species selected from the group consisting of quinones, catechols, aminophenols, hydrazines, derivatives thereof, and combinations thereof. The method of claim 1 , wherein the device comprises or is communicatively coupled to a processor, wherein selecting the amount of current and/or voltage to be applied is performed using an input comprising a signal output of the sensing element(s) to be applied to an algorithm. A device comprising executing a. The method of claim 1 , wherein at least one of the working electrode(s), at least one of the reference electrode(s), at least one of the counter electrode(s), and/or at least one of the sensing element(s) is a switch. - a device that is electrically coupled to the matrix module. The device of claim 1 , wherein at least one of the reference electrode(s) and/or at least one of the counter electrode(s) are electrically coupled to at least two of the subset. The method of claim 1 , wherein the array of electrodes further comprises one or more non-feedback-controlled electrode set(s), each of the one or more non-feedback-controlled electrode set(s):
one or more reference electrode(s);
one or more counter electrode(s); and
comprises one or more working electrode(s);
here:
Each one or more reference electrode(s) of each non-feedback-control electrode set and/or each one or more counter electrode(s) of each non-feedback-control electrode set is electrically coupled with at least one of the working electrode(s) of the control electrode set;
Each set of one or more non-feedback-controlled electrode(s) does not include a pH sensing element;
(i) all feedback-control electrode set(s) are included in one section of the device and all non-feedback-control electrode set(s) are arranged in a physically separate second section of the device, or (ii) one or more of the feedback-control electrode set(s) are interspersed between two or more of the non-feedback-control electrode set(s);
At least one of the feedback-control electrode set(s) is electrically coupled to at least one of the non-feedback-control electrode set(s), and at least one of the coupled feedback-control electrode set(s) is A selected amount of current and/or voltage applied to one or more working electrode(s) of at least one coupled feedback-control electrode set is applied to each of the at least one coupled non-feedback-control electrode set(s) A device configured to also be applied to one or more of the working electrode(s) of the device. A method for controlling the pH of a solution using a device comprising a set of feedback-controlled electrodes,
A feedback-control electrode set includes (1) one or more reference electrode(s), (2) one or more counter electrode(s), and (3) one or more subset(s), and includes one or more sub-electrode(s). Each of the set(s) includes a pH sensing element electrically coupled to a working electrode, and the reference electrode of one or more reference electrode(s) and/or the counter electrode of one or more counter electrode(s) are a subset ( s) is electrically coupled with at least one of the methods,
b. selecting a target sensing value for the sensing element based on one or more signal(s) output from the sensing element in a solution having a target pH;
c. of the following:
i. selecting respective amounts of current and/or voltage to be applied to at least one of the working electrode(s) of the feedback-controlled electrode set to minimize the difference between the signal output of the sensing element and the target sensing value;
ii. applying a selected respective amount of current and/or voltage to at least one of the working electrode(s) of the feedback-control electrode set to change the pH of the solution proximate to each working electrode of the feedback-control electrode set;
iii. A method comprising repeatedly measuring a signal output of a sensing element. 9. The method of claim 8, wherein the solution comprises water and/or one or more redox active species(s), and the applied current and/or voltage is applied to the electrical current and/or one or more redox active species(s). wherein hydrogen ions are electrochemically produced and/or consumed by a chemical reaction. 10. The method of claim 9, wherein the at least one redox active species is selected from the group consisting of quinones, catechols, aminophenols, hydrazines, derivatives thereof, and combinations thereof. 9. The method of claim 8, comprising determining the pH of the solution by comparing the signal output of the sensing element to predetermined calibration data correlating the pH and the signal output value of the sensing element. 9. The method of claim 8, wherein the device used comprises an array of two or more feedback-controlled electrode sets. 13. The method of claim 12, wherein at least two of the feedback-controlled electrode sets are each individually controlled by independent target pH values and/or independent time programs to apply selected amounts of current and/or voltage. 9. The method of claim 8, wherein a set of feedback-control electrodes is electrically coupled to the switch-matrix module, and control of each set of feedback-control electrodes is performed by the switch-matrix module. 9. The method of claim 8, wherein at least one of the reference electrode(s) and/or at least one of the counter electrode(s) are electrically coupled to two or more of the subsets. 9. The method of claim 8, wherein the device used comprises an array of one or more feedback-controlled electrode set(s) and one or more non-feedback-controlled electrode set(s), and the non-feedback-controlled electrode set(s) ) is electrically coupled to one or more of the feedback-control electrode set(s), and each of the one or more non-feedback-control electrode set(s):
one or more reference electrode(s);
one or more counter electrode(s); and
comprises one or more working electrode(s);
here:
Each one or more reference electrode(s) of each non-feedback-control electrode set and/or each one or more counter electrode(s) of each non-feedback-control electrode set is electrically coupled with at least one of the one or more working electrode(s) of the control electrode set;
each of the one or more non-feedback-controlled electrode set(s) does not include a pH sensing element;
Applying the selected respective amount of current and/or voltage applies the selected amount of current and/or voltage to one or more of the working electrode(s) of the coupled feedback-controlled electrode set, and the current and/or voltage is applied to one or more of the working electrode(s) of the coupled non-feedback-control electrode set(s) so that the working electrode of the coupled non-feedback-control electrode set(s) ( s) changing the pH of the solution in the vicinity of the 17. The method of claim 16, wherein the device used comprises an array of two or more coupled feedback-control electrode sets. 18. The method of claim 17 wherein at least two of the coupled feedback-control electrode set(s) transmit selected amounts of current and/or voltage to each one or more working electrode(s) of the coupled feedback-control electrode set(s) and wherein each is individually controlled by independent target pH values and/or independent time programs for application to each one or more working electrode(s) of the coupled non-feedback-controlled electrode set. 17. The method of claim 16, wherein the reference electrode and/or counter electrode of one or more feedback-controlled electrode sets is a subset of two or more feedback-controlled electrode sets and/or the working electrode of two or more non-feedback-controlled electrode sets. electrically coupled to 17. The method of claim 16, wherein one or more working electrode(s) of the coupled non-feedback-control electrode set has a similar shape and design as the one or more working electrode(s) of the coupled feedback-control electrode set. way of being.

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