Measuring ionic strength using closed loop electrochemical ph adjustment

Abstract

The present invention relates to the measurement of ionic strength using closed loop electrochemical pH regulation. A method for monitoring changes in ionic strength in a sample solution by means of a closed loop device is provided, which provides for continuous electrochemical pH regulation cycles between predetermined pH values. In particular, changes in ionic strength can be induced by chemical reactions and can eventually change the electrical control parameters of the closed loop device. By measuring these electrical control parameters, the extent and progress of the respective chemical reactions can be monitored.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a method of monitoring the progress of a chemical reaction in a solution by a closed loop device capable of electrochemical pH regulation. BACKGROUND

[0002] pH plays an important role in intermolecular interactions, chemical modifications, enzyme activity, chemical / biochemical reaction kinetics, and visualization of pH sensitive reporter molecules. As pH can be used as a universal switch or controller for various types of processes, monitoring pH over time helps to ensure that experimental conditions remain within effective and expected ranges throughout the experiment. On the other hand, pH can be a good indicator of the extent and progress of a reaction of interest, as biological or chemical reactions often induce ion concentration changes in the sample solution. At the beginning of this process, such changes are still compensated by the buffering capacity of the buffer solution and the pH is maintained for a while until the ion concentration changes exceed the buffering capacity.

[0003] Typically, quantitative polymerase chain reaction (qPCR) uses optical characterization to determine the progress of the PCR reaction, where the fluorescence signal of an intercalating dye is proportional to the amount of DNA in the sample. As the PCR progresses, the amount of DNA is monitored by fluorescence, and by analyzing the exponential change in signal during the cycles, the original concentration of DNA can be quantitatively determined. pH sensing can also be used to monitor and quantify the PCR progress, as the incorporation of nucleotides during PCR releases protons as a byproduct. Thus, the pH change can be related to the amount of DNA, and monitoring the pH change during the exponential phase of DNA replication can be used to quantify the original DNA concentration.

[0004] For example, it has been reported that direct measurement of pH change as a method for detecting and quantifying the progress of DNA amplification in qPCR. This method works well in a weakened buffer that allows the pH change induced by the reaction under study (DNA amplification). In this case, a diluted buffer solution is used to improve the measurement sensitivity. However, any pH change complicates the direct correlation with the original DNA, as enzyme efficiency is also bound to some extent by the pH of the solution. As a diluted buffer solution is used, there is a risk that enzyme efficiency can be sacrificed. Thus, monitoring the pH of the solution would require deconvolution of this contribution to the DNA quantification calculation. The same problem remains for optical systems, where the pH change of the sample is not captured by the optical reader.

[0005] Accordingly, there remains a need for a method of monitoring a chemical reaction with high sensitivity in a constant pH environment that measures changes in ionic concentration at early stages of the chemical reaction, preserves the functionality of any pH-sensitive components of the reaction (e.g., enzymes), avoids the need to deconvolute as the reaction proceeds, and more desirably provides faster detection and improved stability for biosensors and bioreactors. SUMMARY

[0006] In one aspect, the present disclosure provides a method for monitoring a chemical reaction, comprising

[0007] (a) initiating the chemical reaction in a buffered solution equipped with a closed loop device, wherein

[0008] the buffered solution comprises a pH adjusting agent;

[0009] the closed loop device comprises an electronic controller, a working electrode, a counter electrode, a reference electrode, and a sensing element, wherein the working electrode, the counter electrode, the reference electrode, and the sensing element are immersed in the buffered solution; and

[0010] the chemical reaction causes a change in ionic strength of the buffered solution;

[0011] (b) applying a current or voltage to the working electrode, whereby the pH adjusting agent causes a change in pH in a region adjacent to a surface of the working electrode;

[0012] (c) detecting the change in ionic strength of the buffered solution with the closed loop device.

[0013] Other aspects, features, and embodiments will become apparent by consideration of the detailed description and accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 A schematic showing the use of closed loop control to adjust solution pH through oxidation / reduction of a redox active species, in this example a quinone.

[0015] Figure 2 An example showing the change in solution pH through oxidation / reduction of a quinone on an indium tin oxide electrode in 1 mM phosphate buffer. The pH is determined by a pre-calibrated iridium oxide sensing electrode patterned on the surface, and the closed loop control achieves the target pH value in a precise and fast manner. In a representative method, the adjustment of pH in the vicinity of the working electrode surface is achieved, while the pH of the bulk solution is kept constant by a strong buffer.

[0016] Figure 3 An ionic concentration change caused by a biological or chemical reaction and sensing assembly is shown.

[0017] Figure 4 An example of a pH adjustment cycle performed between two pH values (top) and the corresponding required voltage of the working electrode (bottom) is shown.

[0018] Figure 5 An example of the change in electrical parameters for pH adjustment at different buffer capacities is shown. To achieve a pH change of 2 pH units (shown as the voltage output of the sensing element depicted in the orange line), a higher working electrode voltage is required as the buffer strength of the solution increases. At the high buffer capacity (1x PBS) in this example, the target pH change is not fully achieved even when the working electrode voltage reaches the limit of the hardware (1 V). The target pH change can be selected depending on the buffer, the pH adjuster, and the chemical reaction being studied.

[0019] Figure 6 A representative pH adjustment-based monitoring of ionic strength using a closed-loop device is shown. Because a pH adjustment between two values is performed, the actual pH adjustment trajectory is monitored over time (top). The current value required to achieve the target pH value is continuously monitored (middle). The actual pH change can be captured at a later time point than the method (bottom). In the representative method, the parameters of the closed-loop device required to achieve the target pH value (such as time and current / voltage) change over time in response to the change in ionic strength of the solution as the reaction proceeds. DETAILED DESCRIPTION

[0020] Before any embodiments are explained in detail, it is to be understood that the disclosure is not intended to be limited to the details of construction and the arrangements of components set forth in the following description or illustrated in the following drawings. Embodiments can be capable of other configurations and capable of being practiced or being carried out in various ways.

[0021] The present disclosure provides a method for monitoring chemical reactions in a solution by a closed-loop device that measures changes in ionic strength in the solution. Advantageously, the present method can be used to monitor chemical and biological reactions with high sensitivity at an early stage while keeping the pH of the solution constant.

[0022] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” and the like are open-ended transitional phrases, terms, or words that encompass the items following the transitional phrase, term, or word and any equivalents thereof as well as additional items. The singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Whether or not explicitly stated, in instances where the term “comprising” is used, the present disclosure also contemplates “comprising the embodiments or elements presented herein,” “consisting of the embodiments or elements presented herein,” and “consisting essentially of the embodiments or elements presented herein” other embodiments.

[0023] Any numerical range recited herein is intended to include all sub-ranges of the same entire range. For example, a range of "1% to 50%" is intended to include all sub-ranges, e.g., 2% to 40%, 10% to 30%, or 1% to 3%, etc. in the specified range, and any other subrange between the stated "lowest value and the stated "highest value." These are only specific examples of the intended scope, and all possible sub-ranges between the stated lowest value and the stated highest value are intended to be explicitly stated.

[0024] The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context, (e.g., it includes the degree of error associated with the measurement of the particular quantity). The modifier "about" should also be considered in its normal sense as permitting a range of values. For example, "about 2 to about 4" is also disclosing the range "2 to 4". The term "about" can mean plus or minus 10% of the indicated number. For example, "about 10%" can mean a range of 9% to 11%, and "about 1" can mean 0.9-1.1. Other meanings of "about" can be apparent from the context, such as rounding off, so for example "about 1" can also mean 0.5 to 1.4.

[0025] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described herein. Organic Chemistry , Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry , 5thEd., John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations: A Guide to Functional Group Comprehensive Organic Transformations Transformations, 2ndEd., VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of the Organic Some Modern Methods of Organic Synthesis Chemist, 3rdEd., Cambridge University Press, Cambridge, 1987; each of which is incorporated herein by reference in its entirety.

[0026] In one aspect, the present disclosure provides a method for monitoring a chemical reaction, comprising:

[0027] (a) initiating a chemical reaction in a buffered solution equipped with a closed loop device, wherein

[0028] The buffer solution contains a pH adjuster;

[0029] The closed-loop device includes an electronic controller, a working electrode, a counter electrode, a reference electrode, and a sensing element, wherein the working electrode, counter electrode, reference electrode, and sensing element are immersed in a buffer solution; and

[0030] Chemical reactions cause changes in the ionic strength of buffer solutions;

[0031] (b) Applying a current or voltage to the working electrode, thereby causing the pH adjuster to cause a change in pH value in a region adjacent to the surface of the working electrode;

[0032] (c) Use a closed-loop device to detect changes in the ionic strength of the buffer solution.

[0033] Chemical reactions described herein involve the production of positively charged and / or negatively charged ions in solution, such as H+. + Na + K + OH - and Cl - Any chemical or biological process that causes a change in the ion concentration or ionic strength of a solution. In some embodiments, the chemical reaction produces hydrogen ions (H+). + ) or hydroxide ions (OH-) - In a particular implementation, the chemical reaction produces H₂. + .

[0034] A buffer solution is an aqueous or organic solution whose pH value can be maintained at a nearly constant level without interfering with the operation of the chemical reaction under study or the closed-loop device. In some embodiments, the buffer solution is an aqueous solution containing a buffering agent. Suitable buffers include, but are not limited to, phosphates, acetates, {[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine (Bicine), tris(hydroxymethyl)aminomethane (Tris), N-tris(hydroxymethyl)methylglycine (Tricine), 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), {[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), dimethylarsonic acid (dimethylarsonic acid salt), sodium citrate saline (SSC), 2-(N-morpholino)ethanesulfonic acid (MES), and other buffers known to be used in biological applications. In some implementations, the buffer solution is a solution in which biological events such as protein binding, DNA replication, enzymatic hydrolysis, or enzymatic synthesis can be detected or monitored.

[0035] The buffering capacity of the buffer solution is related to the concentration of the buffering agent. The buffering agent can be present at a concentration of at least 1 mM, at least 5 mM, at least 10 mM, at least 50 mM, at least 100 mM, or at least 500 mM. In some embodiments, the concentration of the buffering agent is at least 5 mM. In some embodiments, the concentration of the buffering agent is from about 5 mM to about 500 mM, such as from about 5 mM to about 250 mM, from about 10 mM to about 250 mM, or from about 50 mM to about 250 mM.

[0036] In some embodiments, chemical reactions involved in known biological processes, such as cell signaling, metabolism, and cell growth and reproduction, can be studied. The chemical reactions can include biological samples or samples derived from biological samples. The biological samples can be bodily fluids, fixed tissues, cells such as fixed cells and live cells, extracellular vesicles, and surface patterned biomolecules such as proteins, DNA, RNA, and peptides from animals, or combinations thereof. In some embodiments, the chemical reactions include biomolecules such as DNA, RNA, or peptides derived from animals or cells.

[0037] In some embodiments, the chemical reactions are DNA amplification reactions. In some embodiments, the chemical reactions are quantitative polymerase chain reactions (qPCR). Sample preparation and qPCR experiments can be performed using known techniques. For example, during a PCR reaction in solution, hydrogen ions (H + ) are released as nucleotides are incorporated into growing nucleic acid chains, thus changing the ionic strength of the solution, which can be detected and quantified by the present method.

[0038] The buffer solution absorbs excess H + or OH - to maintain the pH. The present method can actively increase the H + or OH - near the surface of the electrode, thereby changing the pH near the electrode. As the reaction proceeds, the buffering capacity of the solution can become weaker as the buffering agent is consumed, which can eventually change the pH of the solution. In some embodiments, the present method can detect changes in ionic strength in the solution before the change in pH is measurable.

[0039] Advantageously, the present method can be used to detect and quantify the extent of a corresponding chemical reaction (e.g., qPCR) by measuring changes in the ionic strength of the buffer solution while keeping the pH of the solution nearly constant. In particular embodiments, the present method measures changes in the ionic strength of the buffer solution while the buffer solution maintains a constant pH. Thus, the present method can be used to monitor chemical or biological processes without interfering with the function of any pH-sensitive components (e.g., enzymes) involved in the process.

[0040] A pH adjusting agent refers to a compound or composition that undergoes a chemical reaction in response to an electrical potential or current in a solution, thereby causing a change in the pH of the solution. The chemical reaction can be a redox reaction in which the redox state of the pH adjusting agent changes. Electrochemical oxidation and / or reduction of the pH adjusting agent induced by electrical stimulation can introduce local pH changes through the balance between the production or consumption of protons and the buffering capacity of the buffer solution. This can create a pH adjusting zone with a very short vertical distance from the electrode surface, e.g., a few nanometers to a few microns. In some embodiments, the pH adjusting agent can comprise a material that can undergo a proton-coupled electron transfer. Suitable pH adjusting agents include, but are not limited to, quinone derivatives, aminophenol derivatives, aniline derivatives, benzidine derivatives, hydrazine derivatives, phenol-Ru(2,2'-bipyridine)3 2+ and combinations thereof. Suitable pH adjusting agents can also include other known compounds with pH-responsive moieties not exemplified above.

[0041] In some embodiments, the pH adjusting agent is a quinone derivative of any one of formulas (I)-(XII)

[0042]

[0043] wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from: H; C n H 2n+1 ; Cl; F; I, Br, OM, NO2, OH, OC n H 2n+1 , OC n H 2n OH, O(C n H 2n O) y H, O(C n H 2n O) y C n H 2n+1 , O(C n H 2n O) y COOH; O(C n H 2n O) y COOM; COOH; COOM; COOC n H 2n+1 ; CONHC n H 2n+1 ; CON(C n H 2n+1 )2; SO3H; SO3M; NH2; NHC n H 2n+1 ; N(C n H2n+1 )2; NHC n H 2n OH; NHC n H 2n NH2; N(C n H 2n OH)2; N(C n H 2n NH2)2; 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 NH2; NHC n H 2n COC n H 2n SH; SH; SC n H 2n+1 ; SC n H 2n OH; S(C n H 2n O) y H; S(C n H 2n O) y C n H 2n+1 ; S(C n H 2n O) y COOH; S(C n H 2n O) y COOM; OC n H 2n SH; O(C n H 2n O) y C n H 2n SH; O(C n H 2n O) y C n H 2n SC n H 2n+1 ; C n H 2n OC n H 2n+1 ; C nH 2n SC n H 2n+1 ; C n H 2n NH n C 2n+1 ; 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 SO3H; C n H 2n SO3M; C n H 2n NH2; C n H 2n N(C n H 2n+1 )2; C n H 2n NHC n H 2n OH; C n H 2n NHC n H 2n NH2; C n H 2n N(C n H 2nOH)2; C n H 2n N(C n H 2n NH2)2; C n H 2n NHCOC n H 2n+1 ; C n H 2n NHC n H 2n COC n H 2n OH; C n H 2n NHC n H 2n COC n H 2n NH2; C n H 2n NHC n H 2n COC n H 2n SH; C n H 2n SH; C n H 2n SC n H 2n+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; sugars; phthaleins; and amino acids,

[0044] wherein

[0045] M is any metal cation or NH4 + ,

[0046] n is an integer from 1 to 10 9 , and

[0047] y is an integer from 1 to 10 9 .

[0048] In some embodiments, R1, R2, R3, R4, R5, R6, R7, and R8are each independently selected from the group consisting of: C n H 2n OH; C n H 2n OC n H 2n OH; C n H 2n O(C n H 2n O) y COOH; C n H 2n O(C n H 2n O) y COOM; C n H 2n COOH; C n H 2n COOM; C n H 2n COOC n H 2n+1 ; C n H 2n CONHC n H 2n+1 ; C n H 2n CONH(C n H 2n+1 )2; C n H 2n SO3H; C n H 2n SO3M; C n H 2n NH2; C n H 2n NHC n H 2n+1 ; C n H 2n N(C n H 2n+1 )2; C n H 2n NHC n H 2n OH; C n H2n NHC n H 2n NH2; C n H 2n N(C n H 2n OH)2; C n H 2n N(C n H 2n NH2)2; C n H 2n NHCOC n H 2n+1 ; C n H 2n NC n H 2n COC n H 2n OH; C n H 2n NC n H 2n COC n H 2n NH2; C n H 2n NC n H 2n COC n H 2n SH; C n H 2n SH; C n H 2n SC n H 2n OH; C n H 2n S(C n H 2n O) y OH; C n H 2n S(C n H 2n O) y H; C n H 2n S(C n H 2n O) y C n H 2n+1 ; C n H 2n S(C n H 2n O) y C n H 2n COOH; and C n H 2n S(C n H2n O) y C n H 2n COOM. In some embodiments, the pH adjusting agent is a quinone derivative of Formula (I).

[0049] Suitable quinone derivatives can contain various functional groups to modulate their solubility, biocompatibility, and electrochemical properties. Further examples of suitable quinone derivatives include those described in US 9,766,197, US 9,874,538, US 9,910,008, US 10,011,549, US 10,041,905, US 2017 / 0010238, and WO 2017 / 005587, the entire contents of which are incorporated herein by reference.

[0050] The closed-loop control device can include a set of electrodes, e.g., working electrodes, sensing elements, a counter electrode, and a reference electrode. The reference electrode provides a stable potential reference for the measurements. In some implementations, the sensing elements are used as the reference electrode when they have good stability and are placed in a stable pH solution. Further, in some implementations, the counter electrode and the reference electrode are shared for multiple working electrodes and sensing elements. In some implementations, external counter and reference electrodes are used. In other implementations, surface-patterned on-chip counter and reference electrodes are used.

[0051] In some embodiments, the working electrodes, counter electrode, reference electrode, and sensing elements are immersed in a buffered solution.

[0052] The working electrodes and sensing elements can have various shapes and sizes. In some implementations, the sensing elements are used as the working electrodes. In some implementations, the sensing elements and working electrodes are different electrodes. The sensing elements can need to be physically separated from the working electrodes to avoid cross-talk or short-circuiting. In some implementations, the sensing elements are positioned in the same plane as the working electrodes with a small gap in between to provide the physical separation. The gap between the sensing elements and working electrodes can be, for example, 1 nanometer to 100 micrometers. In other implementations, the sensing elements are placed on top of the working electrodes with an insulating layer in between to provide the physical separation.

[0053] In some implementations, the counter electrode is patterned around the working electrodes, which minimizes the diffusion effects and helps to control the pH in a more well-defined pH modulation zone shape.

[0054] The sensing element can include an ion-sensitive field effect transistor (ISFET) or a metal oxide electrode, such as a thallium oxide electrode, an aluminum oxide electrode, a tin oxide electrode, a zinc oxide electrode, a ruthenium oxide electrode, a titanium dioxide electrode, or an iridium oxide electrode. An ISFET is a specific type of chemically sensitive field effect transistor (chemFET) that has sensitivity to ion concentrations in a solution. An ISFET is similar to a metal oxide semiconductor field effect transistor (MOSFET) and has a source (S), a drain (D), and a body (or bulk) connection. However, an ISFET has an ion-sensitive region immersed in a solution and a separate reference electrode, without a metal gate electrode. An ISFET can be configured to be sensitive to ions, such as hydrogen ions, and thus to the pH of a solution.

[0055] Suitable sensing elements also include electrodes coated with a pH-sensitive coating. Such a coating can comprise, for example, an organic or inorganic material. In some embodiments, the pH-sensitive coating comprises a material selected from the group consisting of polyaniline, polypyrrole, polyaminoanthracene, polycarbazole, polybisphenol A, polyethyleneimine, poly(p-phenylenediamine), poly-1,5-diaminonaphthalene, titanium nitride, thallium oxide, aluminum oxide, tin oxide, zinc oxide, ruthenium oxide, and iridium oxide.

[0056] The closed-loop device can include, for example, multiple ISFETs used in a differential manner in order to read and control ion concentrations and pH in a closed-loop system. The electrodes are composed of a material comprising, for example, a metal oxide, glassy carbon, graphene, a metal, gold, silver, platinum, a conductive polymer, silver chloride, normal hydrogen, a mercury drop, saturated calomel, or a combination thereof. In some implementations, the electrodes are patterned on a carrier comprising, for example, a glass slide, a plastic plate, a silicon wafer, a glass wafer, a quartz wafer, a flexible plastic sheet, a polymer layer, paper, or a combination thereof.

[0057] The closed-loop device can use open-circuit potential (OCP) as a feedback measurement to control current or potential. Suitable electrical control units include, but are not limited to, electronics with current / voltage source outputs and sense inputs, and software to control electrical parameters. Suitable materials, device designs, electrical component configurations, and control methods for the closed-loop device include those described in US 10,379,080 and US Application No. 16 / 931,727 (“Closed-loop pH control with differential sensor,” filed July 17, 2020), the entire contents of which are incorporated herein by reference.

[0058] The pH adjustment of the present method can be performed by applying a current or voltage to the electrode. The potential or current used for pH adjustment herein can be defined by a waveform that can be adjusted based on a closed loop control scheme to vary the size of the pH adjustment zone. For example, the size of the pH adjustment zone adjacent to the surface of the electrode can be controlled by adjusting parameters of the waveform.

[0059] The present method can include the following steps for detecting changes in ionic strength of the buffer solution using a closed loop device:

[0060] (c1) controlling the current or voltage applied to the working electrode to adjust the pH value in the zone between a first target pH value and a second target pH value;

[0061] (c2) measuring one or more parameters of the closed loop device; and

[0062] (c3) quantifying changes in ionic strength of the buffer solution based on the one or more parameters.

[0063] The first and second target pH values can be selected based on the pH adjuster, the type of chemical reaction being studied, the buffer, and the ionic strength of the buffer solution. The difference between the first target pH value and the second target pH value can be about 0.5 pH units to about 3.0 pH units, for example, the difference is about 1.0 pH units, about 1.5 pH units, about 2.0 pH units, or about 2.5 pH units. For example, the first and second target pH values can be about 6.0 and about 8.5, about 6.0 and about 8.0, about 6.0 and about 7.5, about 6.0 and about 7.0, about 6.5 and about 8.5, about 6.5 and about 8.0, about 6.5 and about 7.5, about 7.0 and about 8.5, or about 7.0 and about 8.0. In some embodiments, the first and second target pH values are separated by 1.5 pH units or 2.0 pH units. In some embodiments, the first and second target pH values are about 6.0 and about 8.0, or about 6.0 and about 7.5.

[0064] The biological / chemical reaction can cause changes in the concentration of ions and ionic strength of the solution, while the pH of the solution is nearly constant due to the strong buffering capacity of the bulk solution. The local pH adjustment provided by the closed loop device can be used as an additional perturbation to the buffer solution. At the same time, the ionic strength of the solution can affect the ability of the closed loop device to cycle between the target pH values. As the ionic strength of the buffer solution changes, the electrical parameters of the closed loop device need to be adjusted accordingly in order to maintain the pH adjustment between the two predetermined pH values. The rate of the pH adjustment reaction (e.g., oxidation / reduction of quinone derivatives) can also change in response to changes in the ionic strength in the bulk solution, which can be detected by the closed loop device as a change in parameters such as rise time, decay time, or slope.

[0065] Thus, changes in the closed loop device parameters in response to changes in the ionic strength in the solution can be monitored to detect the extent and progress of the biological / chemical reaction and / or to assess the experimental conditions, even before changes in the pH of the solution can be detected by conventional methods.

[0066] Suitable parameters of the closed loop device can include: current or voltage applied to the working electrode, first target pH value, second target pH value, pulse duration at the first target pH value (tsl), pause duration at the second target pH value (ts2), pH rise time (tl), pH decay time (t2); rise peak current (cl), steady current (c2), decay peak current (c3), current rise time (t3), and current decay time (t4).

[0067] In some embodiments, the first target pH value, the second target pH value, the pulse duration at the first target pH value (tsl), and the pause duration at the second target pH value (ts2) can be set to predetermined values. The closed loop algorithm continuously controls the current or voltage that needs to be applied to the working electrode based on the difference between the current pH value and the target pH value. The pH adjustment is performed by repeating a cycle consisting of the pulse duration at the first target pH value (tsl) and the pause duration at the second target pH value (ts2). In some embodiments, one or more parameters selected from the group consisting of pH rise time (tl), pH decay time (t2), rise peak current (cl), steady current (c2), decay peak current (c3), current rise time (t3), and current decay time (t4) are measured by the closed loop device during each pH adjustment cycle.

[0068] In some embodiments, the method comprises the steps of:

[0069] (A) iteratively performing the following operations for a first target pH:

[0070] measuring a signal output of the sensing element;

[0071] selecting an amount of current to be applied to the working electrode to minimize a difference between the signal output of the sensing element and a first target sensing value;

[0072] applying the specifically selected amount of current to the working electrode to change the pH of the local solution in the vicinity of the working electrode;

[0073] determining, with the electronic controller, a voltage required for the amount of current, a time for the current to reach a steady value;

[0074] determining, with the electronic controller, a time for the pH to reach the first target pH; and

[0075] (B) iteratively performing the following operations for a second target pH:

[0076] measuring the signal output of the sensing element;

[0077] selecting an amount of current to be applied to the working electrode to minimize the difference between the signal output of the sensing element and the second target sensing value;

[0078] applying the specifically selected amount of current to the working electrode to change the pH of the local solution in the vicinity of the working electrode;

[0079] determining with the electronic controller the voltage required for the amount of current, the time for the current to reach a steady value;

[0080] determining with the electronic controller the time for the pH to reach the second target pH.

[0081] Representative methods of monitoring chemical reactions

[0082] Methods have been developed to monitor changes in ionic concentration of a buffer solution by measuring electrical parameters of a closed loop device during local electrochemical pH adjustment. Figure 1 A schematic of electrochemical pH adjustment using a surface patterned electrode is shown. The electrical configuration consists of a working electrode, a pH sensing element, a counter electrode and a reference electrode. A pH adjuster that generates or consumes protons during the reaction is added to the sample solution. The pH sensing element measures the initial pH value from which the amount of current or voltage to be applied to the working electrode is calculated. When the current or voltage is applied to the working electrode, the electrochemical oxidation and / or reduction of the pH adjuster introduces a local pH change through the balance between the generation or consumption of protons and the buffering capacity of the buffer solution. This local pH change creates a pH adjusted zone with a very short vertical distance to the electrode surface, from a few nanometers to a few millimeters. Since the size of the pH adjusted zone depends on the balance between the amount of protons changed and the buffering capacity, the buffer strength of the solution is an important control factor. Monitoring the actual pH during the adjustment with the pH sensing element allows continuous changes in the electrical output by closed loop control, which enables faster and more accurate pH control. In closed loop control, the amount of current / voltage to be applied to the working electrode to generate the target pH change depends on the ionic strength and the buffering capacity of the solution. Advantageously, changes in the ionic strength of the solution can be measured by the present method while keeping the pH of the solution constant.

[0083] Reversible electrochemical oxidation / reduction of pH adjusters such as quinone derivatives, hydrazine derivatives or water has been demonstrated for fast pH changes in local regions. The pH adjustment limit depends on the pKa and the oxidation / reduction potential of the specific pH adjuster, as well as their concentration. Figure 2Multi-step pH adjustment by oxidation of 2,5-dimethyl-l,4-hydroquinone on an indium tin oxide electrode and reduction of 2,5-dimethyl-l,4-benzoquinone on an indium tin oxide electrode in 1 mM phosphate buffer is demonstrated. When an anodic current is applied to the electrode, the proton production overcomes the buffer capacity and the pH of the solution becomes more acidic, and vice versa.

[0084] Electrochemical pH adjustment with closed loop control involves a set of electrodes: a working electrode, a sensing element, a counter electrode, and a reference electrode. These electrodes can be in any form, including external electrodes, device integrated / surface patterned electrodes, etc. Multiple sets of electrodes can be used to improve reliability, uniformity, and speed. For the sensing element, an electrode coated with a pH sensitive material can be the common configuration. However, a semiconductor based electrical component, such as a field effect transistor or a polymer semiconductor, can also be used. In particular, an ion-sensitive field effect transistor (ISFET) is a specific type of chemically sensitive field effect transistor (chemFET) that has sensitivity to ion concentrations in solution. The counter electrode and the reference electrode can be shared for multiple working electrodes and sensing elements. Since the role of the reference electrode is to provide a stable potential reference for the measurement, if the sensing electrode has good stability and is placed in a stable pH solution, it is possible to choose to use the sensing electrode as the reference electrode.

[0085] Figure 3 Biology / chemistry reactions can introduce ion concentration changes as the reaction proceeds. At least one set of working electrodes and sensing elements are located in the same sample solution. Electrochemical pH adjustment with closed loop control can continuously adjust the pH between at least two predetermined pH values with high precision and high speed. Figure 4 An example of a pH adjustment cycle between two pH values is shown. Typically, the pH only changes within a small pH adjustment region without affecting the bulk pH value (top figure). A small voltage is applied to the working electrode to achieve the specified pH change (bottom figure). Depending on the difference between the actual pH of the pH adjustment region and the set pH value, the current level determined by the closed loop algorithm changes. The current is applied to the working electrode and the voltage generated on the working electrode is monitored (bottom figure). Figure 4 Figure 4 , bottom figure). Depending on the difference between the actual pH of the pH adjustment region and the set pH value, the current level determined by the closed loop algorithm changes. The current is applied to the working electrode and the voltage generated on the working electrode is monitored (bottom figure). Figure 4

[0086] Figure 5 An example of how the electrical parameters change as the buffer capacity and ionic strength of the solution increase from 0.1x PBS (10x diluted buffer) to 1x PBS (undiluted) is shown. Higher voltages are needed to achieve the same level of pH change in solutions with higher buffer capacity and ionic strength. Figure 6 ​​A more detailed example of the parameters of the closed-loop device is illustrated, which can be measured in this method to monitor changes in ionic strength. For pH adjustment, at least four input parameters can be monitored: a first pH setpoint, a second pH setpoint, the duration of the pH adjustment pulse (ts1), and the pause duration (ts2). Based on pH monitoring, the pH rise time (t1) and pH decay time (t2) can be monitored. Based on electrical monitoring, the peak current at rise (c1), the steady current (c2), the peak current at decay (c3), the current rise time (t3), and the current decay time (t4) can be monitored. These parameters are non-limiting examples of this method and can be varied for specific experimental designs. Variations in these parameter values ​​can be used to quantify the extent and progress of reactions of interest.

[0087] In one exemplary embodiment, this method is used to monitor DNA replication via qPCR. For example... Figure 6 As shown, pH regulation is controlled by a closed-loop device, which cycles repeatedly between target pH 6 and pH 7.5 (top figure). The DNA amplification reaction occurring in the sample solution causes changes in the ionic strength of the bulk solution, while the pH is maintained at a constant level by a strong buffer. However, as the ionic concentration of the buffer solution changes, the required electrical parameters of the closed-loop device change accordingly to maintain pH regulation between the two target pH values ​​(e.g., reducing the magnitude of c1 and c3, middle figure), which is detected by the closed-loop device. As the reaction proceeds further, the pH can be recorded due to H... + The actual pH change of the solution caused by the release of [the substance] (see figure below). Notably, this method can detect changes in ionic strength caused by the reaction at an earlier time point than directly measuring pH changes in the solution (see figures above and below). Therefore, this method offers the following advantages: maintaining a stable pH environment in the buffer solution to allow enzymes (e.g., DNA polymerase) to continue functioning, while simultaneously detecting changes in the solution's buffering capacity during the PCR reaction to quantify proton concentration changes, eliminating the need for deconvolution.

[0088] Various features, advantages and embodiments are set forth in the appended claims.

Claims

1. A method for monitoring chemical reactions, comprising: (a) The chemical reaction is initiated in a buffer solution equipped with a closed-loop device, wherein The buffer solution contains a pH adjuster; The closed-loop device includes an electronic controller, a working electrode, a counter electrode, a reference electrode, and a sensing element, wherein the working electrode, the counter electrode, the reference electrode, and the sensing element are immersed in the buffer solution, and wherein the sensing element is placed on top of the working electrode, with an insulating layer between them to provide physical separation. and The chemical reaction causes a change in the ionic strength of the buffer solution; (b) Applying a current or voltage to the working electrode, thereby causing the pH adjuster to induce a continuous pH adjustment cycle between at least two target pH values ​​in a region adjacent to the surface of the working electrode and the sensing element; (c) Detecting the change in ionic strength of the buffer solution using the closed-loop device, wherein the detection includes: (c1) Controlling the current or voltage applied to the working electrode to adjust the pH value in the region between the at least two target pH values; (c2) Analyze the current or voltage applied to the working electrode and the signal output from the sensing element; and (c3) Quantify the change in ionic strength of the buffer solution based on the current or voltage applied to the working electrode and the signal output from the sensing element; and (d) Repeat steps (b) and (c) at least once.

2. The method according to claim 1, wherein the detection further comprises analyzing one or more other parameters of the closed-loop device, the one or more other parameters being selected from: a first target pH value, a second target pH value, a pulse duration (ts1) at the first target pH value, a pause duration (ts2) at the second target pH value, a pH rise time (t1), a pH decay time (t2); a rising peak current (c1), a steady current (c2), a decaying peak current (c3), a current rise time (t3), and a current decay time (t4).

3. The method according to claim 1, further comprising: (e) Determine the progress of the chemical reaction.

4. The method according to claim 1, wherein the sensing element comprises: Ion-sensitive field-effect transistor (ISFET); The metal oxide electrode is selected from thallium oxide electrode, aluminum oxide electrode, tin oxide electrode, zinc oxide electrode, ruthenium oxide electrode, titanium dioxide electrode and iridium oxide electrode; or An electrode coated with a pH-sensitive coating, the pH-sensitive coating comprising materials selected from: polyaniline, polypyrrole, polyaminoanthracene, polycarbazole, polybisphenol A, polyethyleneimine, poly(p-phenylenediamine), poly-1,5-diaminonaphthalene, titanium nitride, thallium oxide, aluminum oxide, tin oxide, zinc oxide, ruthenium oxide, and iridium oxide.

5. The method of claim 1, wherein the bulk of the buffer solution, except for the region adjacent to the surface of the working electrode, maintains a constant pH value.

6. The method of claim 1, wherein the buffer solution comprises a buffer selected from the group consisting of phosphates, TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, dimethylarsine, SSC, and MES.

7. The method of claim 6, wherein the concentration of the buffer in the buffer solution is at least 1 mM.

8. The method according to claim 1, wherein the chemical reaction produces H + or OH - .

9. The method according to claim 1, wherein the chemical reaction is a DNA amplification reaction.

10. The method according to claim 1, wherein the chemical reaction is a quantitative polymerase chain reaction (qPCR).

11. The method according to claim 1, wherein the pH adjuster is a quinone derivative, an aminophenol derivative, an aniline derivative, a benzidine derivative, a hydrazine derivative, or a combination thereof.

12. The method according to claim 1, wherein the pH adjuster is a quinone derivative of any one of formulas (I)-(XII). R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from: H; C n H 2n+1 ;Cl;F;I, Br, OM, NO2, OH, OC n H 2n+1 OC n H 2n OH, O(C) n H 2n O) y H, O(C) n H 2n O) y C n H 2n+1 O(C) n H 2n O) y COOH;O(C n H 2n O) y COOM; COOH; COOM; COOC n H 2n+1 CONHC n H 2n+1 CON(C) n H 2n+1 )2;SO3H;SO3M;NH2;NHC n H 2n+1 ;N(C n H 2n+1 )2; NHC n H 2n OH; NHC n H 2n NH2; N(C) n H 2n OH)2;N(C n H 2n NH2)2;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 NH2; NHC n H 2n COC n H 2n SH; SH; SC n H 2n+1 ;SC n H 2n OH;S(C n H 2n O) y H;S(C n H 2n O) y C n H 2n+1 ;S(C n H 2n O) y COOH;S(C n H 2n O) y COOM;OC n H 2n SH;O(C n H 2n O) y C n H 2n SH;O(C n H 2n O) y C n H 2n SC n H 2n+1 ;C n H 2n OC n H 2n+1 ;C n H 2n SC n H 2n+1 ;C n H 2n NHC n H 2n+1 ;C n H 2n OH;C n 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 CHUHC n H 2n+1 C n H 2n CONH(C) n H 2n+1 )2;C n H 2n SO3H; C n H 2n SO3M;C n H 2n NH2; C n H 2n N(C n H 2n+1 )2;C n H 2n NHC n H 2n OH; C n H 2n NHC n H 2n NH2; C n H 2n N(C n H 2n OH)2; C n H 2n N(C n H 2n NH2)2;C n H 2n NHCOC n H 2n+1 C n H 2n NHC n H 2n COC n H 2n OH; C n H 2n NHC n H 2n COC n H 2n NH2; C n H 2n NHC n H 2n COC n H 2n SH;C n H 2n SH;C n H 2n SC n H 2n+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; phthalic acid; and amino acids in M can be any metal cation or NH4. + , n is 1 to 10 9 integers, and y is 1 to 10 9 Integers.

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