WO2021025998A1

WO2021025998A1 - Systems and methods for liquid-liquid electrochemical sensing

Info

Publication number: WO2021025998A1
Application number: PCT/US2020/044500
Authority: WO
Inventors: Hyejin Moon; Ali Farzbod
Original assignee: The Board Of Regents Of The University Of Texas System
Priority date: 2019-08-02
Filing date: 2020-07-31
Publication date: 2021-02-11

Abstract

The present disclosure provides methods and systems for performing liquid-liquid electrochemical sensing. The method includes the steps of depositing a droplet of an extractant liquid on a surface, the extractant liquid comprising a selective element; depositing a droplet of a sample liquid on the surface, the sample liquid being immiscible with the extractant liquid and containing target molecules that are to be sensed; bringing the two droplets together to form a liquid-liquid interface at which the droplets contact each other; enabling the target molecule to attach to the selective element at the liquid-liquid interface and transfer into the extractant liquid; separating the two droplets from each other; and evaluating at least one electrical parameter of the extractant liquid both before and after contact with the sample liquid.

Description

SYSTEMS AND METHODS FOR LIQUID-LIQUID ELECTROCHEMICAL SENSING

Related Application

This application claims the benefit of U.S. Provisional Application Ser. No. 62/882,322, filed August 2, 2019, which is incorporated herein by reference in its entirety.

Field

The present disclosure relates to systems and methods for performing liquid- liquid electrochemical sensing.

Background

Biosensing is often performed using a sensor that may include digital microfluidics (DMFs). In a typical scenario, selective elements, such as antibodies, are attached to a surface of the sensor using a particular protocol. Next, a liquid sample, which may or may not contain a target molecule, is then deposited on the surface so that the target molecules, if present, can attach to the selective elements. One or more electrical parameters associated with the surface can then be measured to determine whether or not the target molecules are present in the sample and/or to determine the concentration of the target molecules in the sample.

While the above-described sensing method is viable, it always requires attachment of the selective elements to the surface of the sensor. As this attachment adds additional steps and difficulty to the sensing process, it can be appreciated that it would be desirable to be able to perform sensing without such attachment. Summary

The present disclosure provides systems and methods for performing liquid- liquid electrochemical sensing.

In one aspect, the present disclosure provides a method for performing liquid- liquid electrochemical sensing. The method includes the steps of depositing a droplet of an extractant liquid on a surface, the extractant liquid comprising a selective element; depositing a droplet of a sample liquid on the surface, the sample liquid being immiscible with the extractant liquid and containing target molecules that are to be sensed; bringing the two droplets together to form a liquid-liquid interface at which the droplets contact each other; enabling the target molecule to attach to the selective element at the liquid-liquid interface and transfer into the extractant liquid; separating the two droplets from each other; and evaluating at least one electrical parameter of the extractant liquid both before and after contact with the sample liquid.

In another aspect, the present disclosure provides a method for detecting a target molecule in a sample liquid.

In another aspect, the present disclosure provides a method for quantitatively assessing a target molecule in a sample liquid.

Brief Description of the Drawings

The present disclosure may be better understood with reference to the following figures. Matching reference numerals designate corresponding parts throughout the figures, which are not necessarily drawn to scale.

Figs. 1A-1C are schematic diagrams that illustrate an embodiment of liquid- liquid electrochemical sensing in accordance with the present disclosure.

Fig. 2 is a drawing of the chemical formula of 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide.

Fig. 3A is a schematic view of an interdigitated electrode (IDE) integrated on an electrowetting-on-dielectric (EWOD) electrode to be used in experiments used to evaluate use of an EWOD device for electrochemical sensing.

Fig. 3B is an image of the actual IDE used in the experiments.

Fig. 3C is an image of the sensing platform used in the experiments.

Fig. 4 is a diagram of an equivalent circuit for the initial electrical impedance spectroscopy (E IS) data.

Fig. 5 is a Bode plot that shows curve fitting of the initial EIS data with the equivalent circuit model.

Fig. 6 is a Nyquist plot that shows curve fitting of the initial EIS data with equivalent circuit model. Fig. 7 is a diagram of an equivalent circuit for the secondary EIS data.

Fig. 8 is a Bode plot that shows curve fitting of the secondary EIS data with the equivalent circuit model.

Fig. 9 is a graph that plots EIS data for a pure ionic liquid as a selective medium in liquid-liquid extraction (LLE). Fig. 10 is a graph that plots EIS data for initial values.

Fig. 11 is a graph that plots impedance differences between various sample liquids having different KCI concentrations. Detailed Description

Definitions

For convenience, before further description of the present invention, certain terms used in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances.

The articles “a,” “an” and “the” are used to refer to one or to more than one (i.e. , to at least one) of the grammatical object of the article.

The terms “comprise”, “comprising”, “including”, “containing”, “characterized by”, and grammatical equivalents thereof are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only.”

As used herein, the term “about” or “approximately” usually means within 20%, more preferably within 10%, and most preferably still within 5% of a given value or range.

An “ionic liquid” is a salt, formed by the association of a cation and of an anion, in the liquid state at a temperature generally less than 100 °C, advantageously at a temperature less than or equal to the ambient temperature.

The “selective element” used herein is capable of binding or capturing a target molecule.

The term “hydrophobic”, as used herein, refers to substances that lack an affinity for water; tending to repel and not absorb water as well as not dissolve in or mix with water.

As used herein, the term “specifically binds” can refer to non-covalent physical association of a first and a second moiety wherein the association between the first and second moieties is at least 2 times as strong, at least 5 times as strong as, at least 10 times as strong as, at least 50 times as strong as, at least 100 times as strong as, or stronger than the association of either moiety with most or all other moieties present in the environment in which binding occurs. Binding of two or more entities may be considered specific if the equilibrium dissociation constant, is 10 ³ M or less, 10 ⁴ M or less, 10 ⁵ M or less, 10 ⁶ M or less, 10 ⁷ M or less, 10 ⁸M or less, 10 ⁹ M or less, 10 ¹⁰ M or less, 10 ¹¹ M or less, or 10 ¹² M or less under the conditions employed. Examples of specific binding interactions include primer- polynucleotide interaction, aptamer-aptamer target interactions, antibody-antigen interactions, avidin-biotin interactions, ligand-receptor interactions, metal-chelate interactions, hybridization between complementary nucleic acids, etc.

Reference throughout this specification to “one embodiment”, “an embodiment”, “another embodiment”, “some embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” “in another embodiment”, or “in some embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

As described above, it would be desirable to be able to perform sensing, such as biosensing, without having to attach selective elements to a surface of the sensor. Disclosed herein are electrochemical sensors and sensing methods that do not require such attachment.

In the following disclosure, various specific embodiments are described. It is to be understood that those embodiments are example implementations of the disclosed inventions and that alternative embodiments are possible. Such alternative embodiments include hybrid embodiments that include features from different disclosed embodiments. All such embodiments are intended to fall within the scope of this disclosure.

In one aspect, the present disclosure provides a method for performing liquid- liquid electrochemical sensing. Sensing is achieved utilizing the interface between two immiscible liquids. An extractant liquid (solution), such as an ionic liquid, is provided that contains selective elements to which target molecules can attach. A sample liquid (solution) that is immiscible with the extractant liquid and that may or may not contain the target molecules is then brought into contact with the extractant liquid. When the two liquids are brought into contact, a liquid-liquid interface is formed between them. The formation of the liquid-liquid interface enables the target molecules, if present, to attach to the selective elements contained in the extractant fluid and cross over into the extractant fluid. After a period of time, the sample liquid is separated from the extractant liquid and one or more electrical parameters of the extractant fluid measured before and after contact with the sample liquid can be evaluated as a means of determining whether or not the target molecule is present and, if so, in what concentration. In some embodiments, the above process can be performed using a sensor that incorporates digital microfluidics (DMFs) to facilitate contact and separation of the two immiscible liquids.

In one embodiment, sensing can be achieved without having to attach selective elements to a surface of a sensor by instead placing an extractant liquid containing the selective elements in contact with a sample liquid that may contain the target molecules. The target molecules in the sample liquid can attach to the selective elements in the extractant liquid at the interface between the two liquids and the target molecules can then cross over into the extractant liquid such that the target molecules are selectively transferred to the extractant liquid. When this occurs, one or more electrical parameters of the extractant liquid are changed and this change can be correlated to a concentration of the target molecules in the extractant liquid and, therefore, a concentration of the target molecules in the sample liquid.

Figs. 1A-1C schematically illustrate an embodiment of the present disclosure. In Fig. 1A, the extractant liquid is deposited on the sensing electrode and an EIS is acquired for determination of the initial values. In Fig. 1B, the sample liquid is deposited on the electrode and the electrode is then activated to drive the sample liquid into contact with the extractant liquid, thereby forming a liquid-liquid interface between the two immiscible liquids. In Fig. 1 C, the sample liquid is pulled away from the extractant liquid and another EIS acquired to determine the secondary values. The initial values can then be compared with the secondary values to determine the concentration of the target molecules (potassium ions) in the extractant liquid and, therefore, the sample liquid.

In some embodiments, the target molecule is one selected from the group consisting of nucleic acids, proteins, peptides, antibodies, enzymes, small molecules, oligo or polysaccharides, mixtures thereof; and the like. In another embodiment, the target molecule is an ion. In one embodiment, the target molecule is potassium cation. In one embodiment, the selective element specifically binds to the target molecule.

In some embodiments, a droplet of a sample liquid is derived from a sample selected from the group consisting of whole blood, lymphatic fluids, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluids, amniotic fluids, seminal fluids, vaginal excretions, serous fluids, synovial fluids, pericardial fluids, peritoneal fluids, pleural fluids, transudates, exudates, cystic fluids, bile, urine, gastric fluids, intestinal fluids, fecal samples, fluidized tissues, fluidized organisms, biological swabs and biological washes.

In one embodiment, the selective element is an element selected from the group consisting of ionophores, antigens, antibodies, enzyme, peptides, proteins, nucleic acids, nucleic acid or peptide aptamers, ligands, receptors, and the like. In another embodiment, the selective element is one element selected from the group consisting of antibodies, enzymes, and ionophores. In another embodiment, the selective element is ionophores.

In one embodiment, the extractant liquid comprises an ionic liquid. In one embodiment, the ionic liquid includes at least one cation selected from the group consisting of imidazolium, pyridinium, pyrrolidinium, guanidinium, the cation being unsubstituted or substituted with at least one group selected from the group consisting of aryl and C1 to C4 alkyl, the aryl group and the C1 to C4 alkyl group being unsubstituted or substituted with at least one group selected from the group consisting of halogen, C1 to C4 alkyl, hydroxyl group and amino.

In another embodiment, the ionic liquid includes at least one cation selected from the group consisting of an imidazolium cation, a C1 to C4 alkyl imidazolium cation, a pyridinium cation, a C1 to C4 alkyl pyridinium cation, a pyrrolidinium cation, and a C1 to C4 alkyl pyrrolidinium cation. In one embodiment, the ionic liquid includes at least one cation selected from the group consisting of a C1 to C4 alkyl imidazolium cation and a C1 to C4 alkyl pyrrolidinium cation.

In another embodiment, the ionic liquid includes at least one anion selected from the group consisting of a halide anion, a nitrate anion, a nitrite anion, a tetrafluoroborate anion, a hexafluorophosphate anion, a polyfluoroalkane sulphonate anion, a bis(trifluoromethylsulfonyl)imide anion, an alkyl sulphate anion, an alkane sulphonate anion, an acetate anion, and an anion of a fluoroalkane acid. In one embodiment, the ionic liquid includes at least one anion selected from the group consisting of a tetrafluoroborate anion, a hexafluorophosphate anion, and a bis(trifluoromethylsulfonyl)imide anion. In another embodiment, the ionic liquid includes a bis(trifluoromethylsulfonyl)imide anion.

In another embodiment, the ionic liquid includes at least one anion selected from the group consisting of a C1-C6 alkyl sulphate anion and a C1-C6 alkane sulphonate anion. In one embodiment, the ionic liquid includes at least one anion selected from the group consisting of a methyl sulphate anion, an ethyl sulphate anion, a butyl sulphate anion, a methanesulphonate anion, an ethanesulphonate anion and a butanesulphonate anion.

In one embodiment, the ionic liquid is 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide. In another embodiment, the ionic liquid is a hydrophobic ionic liquid. In one embodiment, the hydrophobic ionic liquid has a water solubility between 0.001% and 25% (mol). In another embodiment, the water solubility is between 0.01% and 10%.

In another embodiment, the water solubility is between 0.1% and 5%. In another embodiment, the water solubility is between 0.5% and 2%.

In some embodiments, the hydrophobic ionic liquid includes at least one anion selected from the group consisting of hexafluorophosphate, tetrafluoroborate, bis (trifluoromethylsulfonyl)imide, nonaflate, bis(tosyl) imide, and trifluoromethanesulfonate (CFOS). In some embodiments, the hydrophobic ionic liquid includes at least one cation selected from the group consisting of an imidazolium cation, a C1 to C4 alkyl imidazolium cation, a pyridinium cation, a C1 to C4 alkyl pyridinium cation, a pyrrolidinium cation, and a C1 to C4 alkyl pyrrolidinium cation.

In one embodiment, the ratio of the ionic liquid to the selective element is between about 1000:1 and about 10:1 by weight. In one embodiment, the ratio of the ionic liquid to the selective element is between about 100:1 and about 20:1. In one embodiment, the ratio of the ionic liquid to the selective element is between about 70:1 and about 30:1. In some embodiments, the ratio of the ionic liquid to the selective element by weight is about 1000:1, 500:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, or 10:1.

In another embodiment, the extractant liquid comprises an ionic liquid and potassium ionophore. In one embodiment, the ratio of the ionic liquid to potassium ionophore is between about 500:1 and about 10:1 by weight. In one embodiment, the ratio of the ionic liquid to potassium ionophore is between about 80:1 and about 20:1. In one embodiment, the ratio of the ionic liquid to potassium ionophore is between about 70:1 and about 30:1. In some embodiments, the ratio of the ionic liquid to potassium ionophore by weight is about 500:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, or 10:1. In one embodiment, the ionic liquid is 1 -ethyl-3- methylimidazolium bis(trifluoromethylsulfonyl)imide.

In one embodiment, bringing the two droplets together comprises moving one or more of the droplets using a liquid-liquid electrochemical sensor that incorporates digital microfluidics.

In one embodiment, the liquid-liquid electrochemical sensor incorporates an electrowetting-on-dielectric (EWOD) device.

In one embodiment, evaluating at least one electrical parameter comprises evaluating an electrical parameter indicating a property selected from the group consisting of amperometric, potentiometric conductometric, and impedance property. In another embodiment, evaluating at least one electrical parameter comprises evaluating an electrical impedance of the extractant liquid. In one embodiment, evaluating the electrical impedance comprises performing electrical impedance spectroscopy on the extractant liquid.

Examples

The examples disclosed herein demonstrated that a liquid-liquid electrochemical sensor is compatible with DMF devices. For the first time, a modified ionic liquid has been used as a selective medium for the impedimetric measurement instead of a hydrogel or polymeric membrane, as is commonly used. Unlike any other sensor in use today, the liquid-liquid electrochemical sensor discolsed herein does not require selective elements (enzymes, antibodies, ionophores, etc.) to be immobilized on the surface of an electrode. Instead, the selective elements are mixed with the ionic liquid, which results in less inactivation due to a more controlled environment. This characteristic makes the fabrication of the sensors, such as biosensors, much easier and provides more variability. It is worth noting that biosensors are widely used in medical diagnostics, biological research, environmental protection, and food analysis in which one of the significant hurdles is immobilizing the selective element. The introduced sensor is the perfect match for DMF devices, such as EWODs, in which liquids (~650 nl_) are manipulated as droplets by applying electrical potential on a series of dielectric-coated electrodes. This integration of the sensor and a DMF device paves the path to low-cost home- use sensors in which ease of use, automation, and minimal consumption of reagents are crucial.

Example 1. Methods and Materials

A gold (Au, 1000 A)/Chromium (Cr, 100 A) coated glass wafer was used to form the integrated device. Metal layers (Au/Cr) were used for the EWOD electrodes as well as the interdigitated electrodes (IDE). S1813 (MICROPOSIT) was used as the photoresist for patterning and mask layer in different stages of chip fabrication. SU-82005 (Micro-Chem) was used as the dielectric layer. Teflon AF1600S (Du Pont) powder dissolved in Fluorinert FC-40(Sigma-Aldrich) solution was used as the hydrophobic layer on top of the dielectric layer.

1-Ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, a hydrophobic ionic liquid, was been purchased from lolitec. This ionic liquid has a high electrochemical window that makes it compatible with non-faradaic electrochemical sensors. Table 1 presents the physicochemical properties of the ionic liquid and Fig. 2 shows the chemical formula.

Table 1: Physicochemical properties of1-Ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide.

Potassium ionophore was dissolved in tetrahydrofuran (THF) and then mixed with the ionic liquid. It is worth noting that the usage of TFIF as an organic solvent is a standard method for fabrication of ion-selective membranes (ISM). Later, the mixture was placed in an oven to evaporate the THF. A dehydration process is standard for ionic liquids due to their high absorption of water. Bath sonication can also be used for enhancing the mixing of ionophore and ionic liquids during the mixing process.

While studies have been performed regarding the usage of ionic liquid in the structure of ion-selective membranes in the construction of Cu(ll) ion-selective electrodes with solid contacts, in these types of sensors, the ionic liquid typically replaces the plasticizer in the structure of the ISMs. Because a gel-phase membrane layer is desired, polyvinyl chloride (PVC) has been used for forming the structure. In this study, however, 98% ionic liquid and 2% potassium ionophore were used as the liquid phase to perform the liquid-liquid extraction (LLE).

The choice of weight percentage for the potassium ionophores in the extractant liquid was based on their standard weight percentage in potassium ISMs. Ionophores are organic molecules that are insoluble in aqueous solutions, which make them a perfect match as the selection element for LLE-based electrochemical sensing.

An interdigitated electrode arrays (IDEA) was used to measure impedance changes in the non-Faradaic measurements. The Reference 300 EIS instrument from Gamry Instruments was used to acquire the EIS data and that data was fit using Echem Analyst also from Gamry Instruments. Different molarities of KCI solution (1 pM-1 M) were prepared by serial dilution of stock solution (1 M KCI) to acquire a calibration curve. The stock KCI solution was prepared by dissolving potassium chloride powder (Sigma-Aldrich) in deionized water.

Example 2. Design and test sensors

Experiments were performed as part of a study to evaluate this new sensing protocol. In the study, a liquid-liquid electrochemical sensor was integrated into an electrowetting-on-dielectric (EWOD) device. Such devices are capable of changing the contact angle of a polarizable liquid droplet when it is placed on a dielectric- coated electrode and an electric potential is applied across the droplet. This change in contact angle can be used to drive the liquid droplet across the surface of the electrode and, therefore, drive the droplet into and out of contact with another liquid droplet. Accordingly, a droplet of a sample liquid can be driven into and then out of contact with an extractant liquid. The goal of the study was to design and test a sensor that has compatibility with EWOD DMFs. Integrating electrochemical sensors into an EWOD has two main challenges: (1) complete removal of the droplet containing the analyses from the sensing surface and (2) biochemical regeneration of the biorecognition element immobilized on sensor electrode after each round of measurement.

The second problem was eliminated for the experimental sensor by using an LLE method in which the selective elements are entrapped in a desired ionic liquid. Experiments were performed to study the effect of the ratio between the actuating electrode and sensor electrode (i.e. , the hydrophilic-to-hydrophobic ratio) on movability of the droplets for the purpose of enhancing droplet removal. With knowledge that the signal derived from the sensor electrode is dependent on the sensor area, 33% was chosen as the ratio to achieve movability in the proposed microfluidic device while having maximum area possible.

Although the liquid-liquid sensing on EWOD DMF was not actually performed in the study, electrowetting was used to form and control the interfaces between the ionic liquid and the sample liquids. Fig. 3 shows the IDE used in the experiments.

In recent studies, it has been demonstrated that the change in the contact angle of the droplet can change the resistor-capacitor (RC) behavior of the ionic liquid. Therefore, in the experimental sensor, the impedance of electrochemical cell (i.e., extractant liquid) was measured. Because this impedance can be affected by the contact angle of the droplet on the IDE, hydrophilic opening and the EWOD electrodes were used to control the contact angle and time of the liquid-liquid interface formation.

Development of an equivalent circuit for an electrochemical cell is important for collecting quantitative information on parameter variations. Flowever, these models can vary in different systems. An ideal circuit can deviate from values measured in the experiment, which typically can be corrected by considering phenomena like ionic chemical/physical adsorption, diffusional impedance, and incomplete polarization.

In the experiments, two equivalent circuits were developed for the proposed IDE sensor because, during LLE, potassium ions are transferred, which changes the electrical behavior of the extractant liquid. Fig. 4 shows the equivalent circuit model for the initial EIS. As shown in this figure, the electrochemical cell of the IDEs is represented by an electric double-layer capacitance (CPEdi), resistance at the double-later interface (Rint), bulk capacitance (CPEbuik), bulk resistance (Rbuik), and the solution resistance (R_s).

The curve fitting of the EIS response of the electrochemical cell to the response of equivalent circuit model was performed using the Gamry data analyzer software, which has an agreement factor of 2 x 10⁶ Figs. 5 and 6 are graphs that show good agreement between the response of modeled equivalent circuit (Fig. 4) and the experimental data in Bode and Nyquist plots for the initial response of the modified ionic liquid.

Transfering potassium ions can introduce diffusion resistance after EDL in the bulk layer, which can be represented by a Warburg element in the equivalent circuit model, while entrapment of ions within the electrode array can be presented by a capacitor, as shown in Fig. 7. Fig. 8 shows the good agreement between the response of modeled equivalent circuit (Fig. 7) and the experimental data with the Bode plot for the secondary response of the extractant liquid.

In impedance spectroscopy of an electrochemical cell, high-frequency impedance represents the resistance of the liquid because the impedance of the capacitance approaches zero at high frequencies. Therefore, impedance differences obtained at high frequencies were used to avoid the complexity of capacitance behavior at lower frequencies.

Although the equivalent circuit model identified above was developed to investigate the EIS data of electrochemical cell and not for use of the calculated values (through fitting the response of the equivalent model and EIS data), a more comprehensive analysis can be performed to identify changes of the extractant liquid before and after the sample interaction. Therefore, high-frequency impedances were used in the study for calculating the calibration graphs.

Example 3. Determine the selectivity of 1-Ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)

This example shows that the ionic liquid itself does not have selectivity towards potassium ions. To determine the selectivity of 1 -Ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl), six tests with different KCI molarities (i.e. , sample liquids) and pure ionic liquids (i.e., extractant liquids) were conducted. Fig. 9 shows that the ionic liquid itself does not have selectivity towards potassium ions. To confirm the repeatability of extractant liquid (i.e., the ionic liquid mixed with ionophores), the EIS of seven different samples were acquired and compared. As shown in Fig. 10 and Table 2, the standard deviation was below 0.5% at high frequencies, illustrating the consistency in the initial EIS values for each test.

Table 2: Standard deviations of initial impedance values for the modified ionic liquid.

Example 4. Measure KCI in a solution

In the study, an ionic liquid mixed with potassium ionophore was used as the extractant liquid and different molarities of KCI solutions were used as sample liquids. Electrical impedance spectroscopy (EIS) was used to obtain impedance measurements of the extractant liquid both before and after the sample liquid interaction. A normalized number, which is the high-frequency impedance difference of the extractant liquid before and after sample interaction, was used for calibrating the sensor. In the experiments, a single droplet (3 pi) of ionic liquid containing potassium ionophores (the extractant liquid) was dispensed over an interdigitated electrode integrated into an EWOD electrode. Initial values of electrical parameters for the extractant liquid were obtained by acquiring electrical impedance spectra (EIS) and fitting the spectra to an equivalent circuit model. Then, a sample droplet of KCI solution containing potassium ions was deposited next to extractant liquid to create a liquid-liquid interface. Due to the functionality of ionophores, the ions transfer to the extractant liquid. Finally, after removing the sample liquid from the extractant liquid, EIS was performed a second time to identify secondary values of components of the equivalent circuit of the extractant liquid. The difference between the initial and secondary values of electrical elements can then be correlated to the concentration of targeted molecule in the sample solution.

Finally, after dispensing the KCI solutions and acquiring secondary impedance values, it was found that the impedance difference is lower for the samples having higher concentrations of potassium (Fig. 11). This can be explained by the intrinsic behavior ionophores, which act as ion carriers in an organic medium and the fact that they form complexes in the presence of targeted ions when in contact with sample liquids.

It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.

The term "substantially" is meant to permit deviations from the descriptive term that don't negatively impact the intended purpose. Descriptive terms are implicitly understood to be modified by the word substantially, even if the term is not explicitly modified by the word substantially.

It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations ( e.g ., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range. The term “about” can include traditional rounding according to significant figures of numerical values. In addition, the phrase “about ‘x’ to ‘y’” includes “about ‘x’ to about ‘y’”·

Claims

CLAIMS What is claimed is:

1. A method for performing liquid-liquid electrochemical sensing, the method comprising: depositing a droplet of an extractant liquid on a surface, the extractant liquid comprising a selective element; depositing a droplet of a sample liquid on the surface, the sample liquid being immiscible with the extractant liquid and containing a target molecule that is to be sensed; bringing the two droplets together to form a liquid-liquid interface at which the droplets contact each other; enabling the target molecule to attach to the selective element at the liquid- liquid interface and transfer into the extractant liquid; separating the two droplets from each other; and evaluating at least one electrical parameter of the extractant liquid both before and after contact with the sample liquid.

2. The method of claim 1 , wherein the extractant liquid is an ionic liquid.

3. The method of claim 2, wherein the ionic liquid is a hydrophobic ionic liquid.

4. The method of claim 2, wherein the ionic liquid is 1 -ethyl-3- methylimidazolium bis(trifluoromethylsulfonyl)imide.

5. The method of any one of the proceeding claims, wherein the selective element is one element selected from the group consisting of antibodies, enzymes, and ionophores.

6. The method of any one of the proceeding claims, wherein bringing the two droplets together comprises moving one or more of the droplets using a liquid- liquid electrochemical sensor that incorporates digital microfluidics.

7. The method of claim 6, wherein the liquid-liquid electrochemical sensor incorporates an electrowetting-on-dielectric (EWOD) device.

8. The method of any one of the proceeding claims, wherein evaluating at least one electrical parameter comprises evaluating an electrical impedance of the extractant liquid.

9. The method of claim 8, wherein evaluating the electrical impedance comprises performing electrical impedance spectroscopy on the extractant liquid.

10. A method for detecting a target molecule in a sample liquid, the method comprising: depositing a droplet of an extractant liquid on a surface, the extractant liquid comprising a selective element; depositing a droplet of the sample liquid on the surface, the sample liquid being immiscible with the extractant liquid and containing the target molecule that is to be sensed; bringing the two droplets together to form a liquid-liquid interface at which the droplets contact each other; enabling the target molecule to attach to the selective element at the liquid- liquid interface and transfer into the extractant liquid; separating the two droplets from each other; and evaluating at least one electrical parameter of the extractant liquid both before and after contact with the sample liquid, thereby determining the presence of the target molecule in the sample liquid.

11. The method of claim 10, wherein the extractant liquid comprises an ionic liquid.

12. The method of claim 11, wherein the ionic liquid is a hydrophobic ionic liquid.

13. The method of claim 11, wherein the ionic liquid comprises an anion of bis(trifluoromethylsulfonyl)imide.

14. The method of any one of claims 11-13, wherein the ionic liquid comprises a C1 to C4 alkyl imidazolium cation.

15. The method of claim 11, wherein the ionic liquid is 1 -Ethyl-3- methylimidazolium bis(trifluoromethylsulfonyl)imide.

16. The method of any one of claims 10-15, wherein the selective element is one element selected from the group consisting of antibodies, enzymes, and ionophores.

17. The method of any one of claims 10-16, wherein bringing the two droplets together comprises moving one or more of the droplets using a liquid-liquid electrochemical sensor that incorporates digital microfluidics.

18. The method of claim 17, wherein the liquid-liquid electrochemical sensor incorporates an electrowetting-on-dielectric (EWOD) device.

19. The method of any one of claims 10-18, wherein evaluating at least one electrical parameter comprises evaluating an electrical impedance of the extractant liquid.

20. The method of claim 19, wherein evaluating the electrical impedance comprises performing electrical impedance spectroscopy on the extractant liquid.

21. A method for quantitatively assessing a target molecule in a sample liquid, the method comprising: depositing a droplet of an extractant liquid on a surface, the extractant liquid comprising a selective element; depositing a droplet of the sample liquid on the surface, the sample liquid being immiscible with the extractant liquid and containing the target molecule that are to be sensed; bringing the two droplets together to form a liquid-liquid interface at which the droplets contact each other; enabling the target molecule to attach to the selective element at the liquid- liquid interface and transfer into the extractant liquid; separating the two droplets from each other; and evaluating at least one electrical parameter of the extractant liquid both before and after contact with the sample liquid; thereby quantitively assessing the target molecule in the sample liquid.

22. The method of claim 21 , wherein the extractant liquid comprises an ionic liquid.

23. The method of claim 22, wherein the ionic liquid is a hydrophobic ionic liquid.

24. The method of claim 22, wherein the ionic liquid comprises an anion of bis(trifluoromethylsulfonyl)imide.

25. The method of any one of claims 22-24, wherein the ionic liquid comprises a C1 to C4 alkyl imidazolium cation.

26. The method of claim 22, wherein the ionic liquid is 1 -Ethyl-3- methylimidazolium bis(trifluoromethylsulfonyl)imide.

27. The method of any one of claims 21-26, wherein the selective elements include one or more of antibodies, enzymes, and ionophores.

28. The method of any one of claims 21-27, wherein bringing the two droplets together comprises moving one or more of the droplets using a liquid-liquid electrochemical sensor that incorporates digital microfluidics.

29. The method of claim 28, wherein the liquid-liquid electrochemical sensor incorporates an electrowetting-on-dielectric (EWOD) device.

30. The method of any one of claims 21-29, wherein evaluating at least one electrical parameter comprises evaluating an electrical impedance of the extractant liquid.

31. The method of claim 30, wherein evaluating the electrical impedance comprises performing electrical impedance spectroscopy on the extractant liquid.