WO2026019784A2 - Hydrocarbon enhanced ionophore-based ion-selective optodes - Google Patents

Abstract

Hydrocarbon enhanced ionophore-based ion-selective optodes (ISOs) and methods of their use are provided. The ISOs exhibit improved ultrasensitive ion sensing due to the addition of ultralow-polarity hydrocarbons to the sensing phase using specific ratios of sensing phase components. The ISOs can be operated under an exhaustive or non-exhaustive extraction response mode.

Description

HYDROCARBON ENHANCED IONOPHORE-BASED

ION-SELECTIVE OPTODES

BACKGROUND OF THE INVENTION

Technical Field.

The invention generally relates to the use of hydrocarbon to improve ionophorebased ion-selective optodes (ISOs) and methods of their use. In particular, the invention relates to ISOs that exhibit enhanced responses due to the addition of ultralow-polarity hydrocarbons to the sensing phase.

Description of Related Art

Ionophore-based sensors are one of the most powerful technologies for the detection of ionic species such as electrolytes. Ionophore-based ion- selective electrodes (ISEs) offer highly specific ion measurements in complicated samples like untreated whole blood and have been dominating the blood electrolyte analyzer market for decades. Ionophore-based ion-selective optodes (ISOs) feature similar specificities and antifouling properties but do not rely on electrochemical cells. Instead, ISOs employ a dye as an indirect optical reporter for the ion-ionophore binding events. In both ISEs and ISOs, the ionophore is dissolved in a water-immiscible sensor phase for specific ion binding. This water-immiscible matrix or is integral and crucial to these biphasic sensors and forms the sensing phase of the ISO.

Ions have different Gibbs free energies of partition in different sensor matrices. The dielectric constant of a matrix affects ion pairing formation and ion-ionophore binding. Some water-immiscible matrices with electronegative atoms have coordinating abilities that favor cation binding. These factors affect the specificity, sensitivity, and response range of ionophore-based ion sensors. For ISOs, the role of the sensor matrix is even more complicated because the acid-base property, spectrophotometric property, and solubility of the dye varies in different media. Moreover, the viscosity of the water-immiscible matrix is a determining factor for the resistance of ISEs and response time of ISOs.

Given the important and multifaceted role of the water-immiscible sensor matrix in ISEs and ISOs, a wide variety of matrices have been examined. The most classical matrix is plasticized polyvinyl chloride (PVC) for both ISEs and ISOs. The most common plasticizers are dioctyl sebacate (DOS) and o-nitrophenyl octyl ether (NPOE) with dielectric constants of 4.2 and 21, respectively, representing the most apolar and polar examples. Other ester type plasticizers based on sebacates, adipates, and phthalates have also been used. The PVC polymer may be replaced by other hydrophobic polymers such as polyurethane, polyacrylate, and silicone rubber. Self-plasticized polymers have been employed as sensor matrices to eliminate the need for a small-molecule plasticizer that usually leaks over time. Examples of plasticizer-free matrices include polysiloxanes, polyacrylates, and polymethacylates with a glass transition temperature above room temperature. Recently, lipids such as aliphatic alcohols and triglyceride esters of fatty acids have been used to fabricate ISOs. Adsorption-based ISEs and ISOs have also been reported, in which the hydrophobic environment is provided by the sensing chemicals themselves or other hydrophobic co-adsorbates. Although the dielectric constant of an exact matrix is often not specified, these sensor constituents have a dielectric constant of at least 4.

Ultralow-polarity matrix of ISEs based on fluorous membranes have been reported. Perfluorocarbons are known to be the least polar and polarizable condensed phase. Due to dramatically enhanced ion pair formation constants and minimum coordinating and solvating ability for solvate interference ions, unprecedented selectivity was obtained for these ISEs. Relatively few ion sensors using fluorous membranes have been reported probably due to the need for synthesis of perfluorinated ionophores and ion exchangers and no ISOs based on fluorous phases have been reported, probably due to the rarity of perfluorinated dyes that can serve as the optical reporter in the sensor phase.

It would be advantageous to have available ISOs having matrices with low dielectric constants, e.g. less than 4, especially if made from readily available components.

SUMMARY OF THE INVENTION

Other features and advantages of the present invention will be set forth in the description of invention that follows, and in part will be apparent from the description or may be learned by practice of the invention. The invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof.

Hydrocarbons have poor solubility toward sensing chemicals used in ionophorebased ISOs. Therefore, hydrocarbons are not suitable solvents to create ISOs. Solvents that can dissolve sensing chemicals have highly electronegative atoms such as oxygen, nitrogen, sulfur, and chlorine. Correspondingly, their polarities are high due to these electronegative atoms. Dielectric constants of hydrocarbons typically range from 1.8 to 4 (often 1.8-2.5), which is lower than the most solvents used in ISOs. For example, DOS, a commonly used low-polarity plasticize, has a dielectric constant of 4.1. Mixing a hydrocarbon with a solvent represents a new way to reduce the overall polarity of the sensor matrix while maintaining the solubility of the sensing chemicals in the sensor matrix. The reduced polarity of the sensor matrix is found to enhance the response of ISOs under both exhaustive mode and non-exhaustive mode. In other words, depending on the components of the optode (i.e. how it is configured and/or formulated) , it can operate under either i) an exhaustive response mode or ii) a non-exhaustive response mode. Therefore, mixing a hydrocarbon and a solvent is a new method to enhance the performance of ISOs. In some aspects, this method is applied to ultrasensitive exhaustive ISOs with well-defined detection ranges.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Response of Ca²⁺ ISOs comprising hydrocarbons with different polarities (dielectric constants, E ). Low-polarity hydrocarbons enhance the response compared to high-polarity liquids when mixed with the DOS-based stock oil containing the sensing chemicals. 3-pL diluent is used with 1-pL aqueous sample/standard solution. The diluent is 0.2 M HEPES/Tris buffer at pH 7.4.

Figure 2A and B. Response of the Ca²⁺ ISO to Na⁺. When the sensing oil does not contain hexadecane, it requires more ionophore to ensure the exhaustive response. As a result, the response to Na⁺ is larger (A) compared to that obtained from a hexadecane-based sensing oil containing much less ionophore (B). The red number is the hue value of the oil. A larger hue indicates more deprotonation of Chi, which is more response.

Figure 3. Responses of K+ ISOs comprising liquids of different polarities. The sensing chemicals are dissolved in DOS to create a stock oil. The stock oil is mixed with pure DOS, squalane, hexadecane, or mineral oil to create the final sensing oil. Squalane, hexadecane, and mineral oil have dielectric constants of about 2 while DOS has a dielectric constant of about 4. Larger responses that are close to the theoretical response of the exhaustive ISO are observed when a low-polarity (low dielectric constant) liquid is used. The more reddish color indicates the larger response since the deprotonated dye is reddish in these oil formulations.

Figure 4A and B. The effect of hydrocarbon on the response of Ca²⁺ ISOs using new methylene blue as a cationic optical reporter. The oil phase contains NaTFPB and calcium ionophore II. The theoretical response range under exhaustive response mode is designed to be 1.0-2.0 mM. The inclusion of squalane improves the response since more cationic dye is expelled from the oil phase to the aqueous phase. A, without squalane; B, with squalane. Figure 5A and B. Fluorescence response of a non-exhaustive K⁺ ISO. The extraction of K⁺ into the sensing oil deprotonates the chromoionophore III (Chill) and the deprotonated dye has negligible fluorescence compared to the highly fluorescent protonated form. The inclusion of hexadecane in the sensing oil enhances the response. A, without hexadecane; B, with hexadecane.

DETAILED DESCRIPTION

Disclosed herein are optical ion sensors with matrices comprising at least one ultralow-polarity hydrocarbon present in the matrix (“sensing matrix”, “sensing phase”). "Ultralow-polarity hydrocarbons" refers to hydrocarbon compounds with extremely low polarity, meaning they exhibit minimal charge separation within their molecules. Ultralow- polarity hydrocarbons are composed primarily of carbon and hydrogen atoms, which have very similar electronegativities. The small electronegativity difference between carbon and hydrogen results in nearly equal sharing of electrons in the covalent bonds, leading to a lack of significant polarity in the molecule. Electronegativity is the ability of an atom to attract electrons in a chemical bond. Carbon and hydrogen have very similar electronegativity values (2.55 and 2.20 respectively). When the electronegativity difference between two bonded atoms is small (generally less than 0.4), the bond is considered nonpolar, meaning the electrons are shared relatively equally. The hydrocarbons employed in the present disclosure are nonpolar. In some aspects, the hydrocarbons employed in the present disclosure are symmetrical molecules so that even if there are polar bonds within the molecule, their effects can cancel each other out due to the symmetrical arrangement of atoms.

“Exhaustive mode" as used herein and in the context of optodes (optical sensors) refers to a measurement approach where the sensor consumes a significant portion or all of the analyte being measured during the detection process. This is in contrast to traditional optodes, which are designed to measure the analyte concentration without significantly altering it.

Also as used herein, in optode sensors, "non-exhaustive mode" refers to a measurement approach where the analyte (the substance being measured) is not significantly depleted from the sample during the sensing process. This contrasts with "exhaustive mode," where the sensor extracts a substantial portion of the analyte. In non-exhaustive mode, the sensor response reaches an equilibrium with the analyte concentration in the sample, allowing for a measurement of the analyte's activity or concentration without significantly altering the sample's composition.

In some aspects, the ultralow-polarity hydrocarbon is an acyclic, saturated, branched or unbranched hydrocarbon (an alkane) having the generic formula of CnH2n+2, where n represents the number of carbon atoms. In some aspects, n is from 5 to 30, inclusive. In some aspects, n is 7 to 25 inclusive, i.e. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25. In further aspects, n is from 10 to 20 inclusive, i.e., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. Examples of suitable alkanes include but are not limited to: decane (C10H22), undecane (C11H24), dodecane (C12H26), tridecane (C13H28), tetradecane (C14H30), pentadecane (C15H32), hexadecane (C16H34), heptadecane (C17H36), octadecane (CisELs), nonadecane (C19H40), eicosane (C20H42), 2-methylnonane, 3-ethyloctane, 2,2,4- trimethylpentane, 3-methylundecane, 2,6-dimethyldecane, 4-ethyltetradecane, 3,7- dimethyloctadecane, and 2,6,10,15,19,23-hexamethyltetracosane (squalane).

In other aspects, the ultralow-polarity hydrocarbon is an acyclic, branched or unbranched unsaturated hydrocarbon with at least one double bond (an alkene) having the generic formula CnH2n where n is 5-30, inclusive. In some aspects, n is 7 to 25 inclusive, i.e. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25. In further aspects, n is from 10 to 20 inclusive, i.e., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. Examples of suitable alkenes include but are not limited to: 1 -pentene, 2-pentene, 1 -hexene, 2-hexene, 1 -heptene, 1 -octene, 1 -nonene, and 1 -decene, 1 -undecene, 1 -dodecene, 1- tridecene, 1 -tetradecene, 1 -pentadecene, 1 -hexadecene, 1 -heptadecene, 1 -octadecene, 1- nonadecene, 1-eicosene, squalene, 3-methyl-l -butene, 2-methyl-l -pentene, 2-methyl-2- pentene, 3-methyl-l -pentene, 4-methyl-l -pentene, 3-ethyl-l -pentene, 2,3-dimethyl-2-butene, 2-ethyl-l -butene, 4-methyl-l -hexene, 5-methyl-l -heptene, and 6-methyl-l -octene, isoprene (2-methyl-l, 3-butadiene), 1,3-pentadiene, 1,4-pentadiene, 2,4-hexadiene, 2,4-heptadiene, and 2,4-octadiene, 1,3-decadiene, 1,3-dodecadiene, 6-methyl- 1 -tridecene, 7-methyl-l - tetradecene, 8-methyl-l -pentadecene, 9-methyl-l -hexadecene, 10-methyl-l -heptadecene, 11 -methyl- 1 -octadecene, 12-methyl-l -nonadecene, 13-methyl- 1-eicosene, and squalene.

In further aspects, the ultralow-polarity hydrocarbon is an aromatic hydrocarbon (an arene), characterized by the presence of a benzene ring. Examples of suitable aromatic hydrocarbons include but are not limited to: benzene, toluene, ethylbenzene, propylbenzene, isopropylbenzene, butylbenzene, sec -butylbenzene, tert-butylbenzene, pentylbenzene, hexylbenzene, heptylbenzene, octylbenzene, nonylbenzene, decylbenzene, o-xylene, m- xylene, p-xylene, 1,2-diethylbenzene, 1,3-diethylbenzene, 1,4-diethylbenzene, l-methyl-2- ethylbenzene, l-methyl-3 -ethylbenzene, l-methyl-4-ethylbenzene, 3-methylpropylbenzene, mesitylene, hemimellitene, pseudocumene, prehnitene, isodurene, durene, pentamethylbenzene, and hexamethylbenzene.

In further aspects, the ultralow-polarity hydrocarbon is a cyclic, saturated or unsaturated hydrocarbon with the number of carbons ranging from 5 to 30, inclusive. Examples of cyclic hydrocarbons include but are not limited to cyclopentane, cyclopentene,

1.3-cyclopentadiene, methylcyclopentane, dimethylcyclopentane, cyclohexane, cyclohexene,

1.4-cyclohexadiene, methylcyclohexane, dimethylcyclohexane, cycloheptane, cycloheptene, cyclooctane, cyclooctene, 1,3-cyclooctadiene, 1,5-cyclooctadiene, cyclononane, cyclodecane, cycloundecane, cyclododecane, cyclotridecane, cyclotetradecane, cyclopentadecane, cyclohexadecane, cycloheptadecane, cyclooctadecane, cyclononadecane, cycloeicosane, cycloheneicosane, cyclodocosane, cyclotricosane, cyclotetracosane, cyclopentacosane, cyclohexacosane, cycloheptacosane, cyclooctacosane, cyclononacosane, cy dotriacontane .

In further aspects, the ultralow-polarity hydrocarbon is an oil or polymer. Examples include mineral oil, white mineral oil, light mineral oil, heavy mineral oil, liquid paraffin, petroleum jelly oil fraction, vaseline oil, hydrotreated paraffinic oil, hydrotreated naphthenic oil, polydecene, hydrogenated polydecene, polyisobutene, polybutene, isododecane, isohexadecane, isoeicosane, neopentane oils, alkylcyclohexane oil, cyclohexane oil, cyclododecane oil, methylcyclohexane oil, decalin oil, tetralin oil, 1 -methylnaphthalene oil, 2-methylnaphthalene oil, alkylnaphthalene oils, nonylbenzene, dodecylbenzene, tridecylbenzene, C9-C12 aromatic solvent oil, aromatic hydrocarbon solvent oil, solvent naphtha (aromatic), high-flash aromatic oil, light aliphatic solvent oil, naphthenic base oil, synthetic hydrocarbon fluid (SHF), synthetic isoalkane oil, branched paraffinic oil, hydrocarbon cosmetic emollient oils, branched isoparaffin fluid, isoalkylcyclohexane, saturated isoprenoid hydrocarbon oils, C13-C18 isoparaffinic oils, C20-C30 branched alkanes, low-aromatic content transformer oil, hydrophobic GC-grade hydrocarbon oil, hydrocarbon immersion oil, synthetic diesel-range paraffinic fluid, white cosmetic hydrocarbon oil, branched C24H50 synthetic base oil, unsaturated polyterpene oil, and linear alpha-olefin oil (e.g., 1-decene oligomers).

Thus, in some aspects, the ultralow-polarity hydrocarbon is saturated, unsaturated, or aromatic, but saturated hydrocarbons which are branched or unbranched are preferred.

In some aspects, the matrix comprises a mixture of hydrocarbons comprising at least one ultralow-polarity hydrocarbon. Examples of suitable mixtures include but are not limited to: mineral oil, liquid paraffin, and naphthalic oil.

Suitable ultralow-polarity hydrocarbons have a boiling point of at least about 60 °C to about 400 °C, such as at least about 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200,

205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 245, 250, 255, 260, 265, 270, 275, 280,

285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 345, 350, 355, 360,

365, 370, 375, 380, 385, 390, 395, or 400 °C, including all whole and decimal integers between these values. In some aspects, the boiling point is at least about 200 °C. In further aspects, the boiling point is at least about 250 °C. Hydrocarbons with higher boiling points are less volatile and easy to store without concerns of evaporation.

Suitable ultralow-polarity hydrocarbons have a melting point ranging from at most about -30 °C to about 150 °C, inclusive, e.g., at most about -30, -20, -10, 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 °C. In some aspects, the melting point is 150 °C or less, or 90 °C or less, or even 30 °C or less. Hydrocarbons with melting points of below room temperature (~ 25 °C) are liquid at room temperature and therefore preferred. However, even if hydrocarbons are solid at room temperature, the overall optode matrix can still be liquid at room temperature after the solid hydrocarbons are mixed with other components including a solvent.

In some aspects, the hydrocarbons that are employed are not pentane, hexane, heptane, octane, petroleum ether, squalene, cyclohexane, cyclooctane, benzene, toluene or a xylene.

In some aspects, the sensor matrix further comprises a solvent, e.g., typically a known solvent which is suitable for use in an optical ion sensor. Examples include plasticizers and oils that have at least one electronegative atom such as oxygen, nitrogen, and/or sulfur. Exemplary plasticizers include but are not limited to: dioctyl sebacate, 2- nitrophenyl octyl ether, polyethylene glycol, epoxidized soybean oil, isosorbide ester, isosorbide diester, diethyl succinate, dimethyl glutarate, dimethyl adipate, succinic acid, dimethyl, glyceryl oleate, glyceryl linoleate, glyceryl palmitate, diethyl adipate, sorbitan distearate, glyceryl stearate, sucrose distearate, rape seed methyl ester, diethylhexyl adipate, sorbitan tristearate, diisopropyl adipate, succinate, dibutyl sebacate, diethyl phthalate, dibutyl phthalate, triethyl citrate, tributyl citrate, triacetin, acetylated mono glycerides, phthalate esters, tri(ethylene glycol) bis(2- ethylhexanoate), tri(ethylene glycol) bis(n- octanoate), tetra(ethylene glycol) bis(2- ethylhexanoate), tetra(ethylene glycol) dihexanoate, di(propylene glycol) bis(2- ethylhexanoate), tri(propylene glycol) bis(2-ethylhexanoate), and tri(propylene glycol) dihexanoate, castor oil, and the like. Other examples of liquids suitable for use are described in International Patent Application No. PCT/ US2006 /047486 and in International Patent Application No. PCT /US2008 /072604, each of which is incorporated herein by reference. Exemplary oils that are used include but are not limited to Olive oil, sunflower oil, canola oil, soybean oil, com oil, peanut oil, flaxseed oil, sesame oil, avocado oil, almond oil, castor oil, coconut oil, palm oil, jojoba oil, oleic acid, linoleic acid, linolenic acid, palmitic acid, stearic acid, lauric acid, myristic acid, caprylic acid, capric acid, cetyl alcohol, stearyl alcohol, oleyl alcohol, glycerol, 1,2-hexanediol, 1,3-butanediol, propylene glycol, myristyl myristate, cetyl palmitate, isopropyl myristate, isopropyl palmitate, cetearyl ethylhexanoate, caprylic/capric triglyceride, neopentyl glycol diheptanoate, diisopropyl adipate, pentaerythrityl tetraisostearate, ethylhexyl palmitate, octyldodecyl myristate, trimethylolpropane trioleate, and C12-C15 alkyl benzoate.

This solvent and the ultralow-polarity hydrocarbon are fully or partially miscible. The volume ratio of the solvent to ultralow-polarity hydrocarbon ranges from 10:1 to 1:10 but is preferred to be 4:1 to 1:4, and even more preferred to be 2:1 to 1:2.

The sensor further comprises sensing chemicals that are suitable for use in optical ion sensors. They include an optical reporter that is a pH indicator or a permanently charged dye, an ionophore for specific ion binding (e.g., which specifically or at least selectively bind to an ion of interest), and an ion exchanger.

Examples of suitable pH indicators include but are not limited to: Chromoionophores (pH indicators) such as one or more of: Nile blue, chromoionophore I (9-(diethylamino)-5- (octadecanoylimino)-5H- benzo [a] phenoxazine) designated ETH5249; chromoionophore II (9-dimethylamino-5-[4- (16-butyl-2,14-dioxo-3,15 ioxaeicosyl)phenylimino]benzo[a]phenoxazine) designated ETH2439 and having light absorbance peaks at 520 nm and 660 nm and a fluorescent emission peak at 660 nm; chromionophore III (9-(diethylamino)-5-[(2-octyldecyl)imino] benzo [a]phenoxazine), designated ETH 5350 and having light absorbance peaks at 500 nm and 650 nm and fluorescent emission peaks at 570 nm and 670 nm; chromoionophore IV (5- octadecanoyloxy-2- (4-nitrophenylazo)phenol), designated ETH2412; chromoionophore V (9-(diethylamino)- 5-(2-naphthoylimino)-5H-benzo[a]phenoxazine); chromoionophore VI (4,5- dibromofluorescein octadecyl ester) designated ETH7075; chromoionophore XI (fluorescein octadecyl ester) designated ETH7061; [ll-[(l-butylphenyl)oxy]-l l- oxoundecyl-4- { [9-(dimethylamino)-5H-benzo [a]phenoxazine-5-ylidene] -amino } - benzenate] }; 2,4,5,7-tetraiodofluorescein octadecyl ester; and combinations thereof;

Examples of suitable permanently charged dyes (positively charged) include but are not limited to: Rhodamine B, rhodamine 6G, rhodamine 123, rhodamine 110, rhodamine 101, sulforhodamine B, tetramethylrhodamine (TMR), carboxytetramethylrhodamine (TAMRA), lissamine rhodamine B, methylene blue, new methylene blue, methylene green, thionine, azure A, azure B, azure C, toluidine blue O, toluidine blue, thioflavin T, thioflavin S, crystal violet, methyl violet 10B, basic fuchsin, pararosaniline, rosaniline, magenta, gentian violet, victoria blue B, victoria blue R, brilliant cresyl blue, Nile blue A, Nile blue sulfate, malachite green, brilliant green, aniline blue, brilliant cresyl violet, safranin O, safranin T, acridine orange, acridine yellow, acriflavine, 9-aminoacridine, proflavine, celestine blue, cresyl violet, methyl green, BODIPY -based pyridinium dyes, styryl-9M, 4’,6- diamidino-2-phenylindole (DAPI), SYTO 9, SYTOX Green, SYTO 13, SYTO 21, TO- PRO-1, TO-PRO-3, YO-PRO-1, YOYO-1, TOTO-1, TOTO-3, DiSC₃(5), DiOC₂(3), JC-1 monomer (cationic form), cyanine 3 (Cy3), cyanine 5 (Cy5), Cy7, indocyanine green, 2-[4- (dimethylamino) styryl] - 1 -methylpyridinium iodide, 4-dimethylaminostyryl-N- methylpyridinium, 4-dimethylaminostyryl-N-methylquinolinium, hemicyanine dyes such as JC-9 and Styryl- 11, ethidium bromide, propidium iodide, and mitotracker red CMXRos.

Examples of suitable permanently charged dyes (negatively charged) include but are not limited to: Coomassie Brilliant Blue G-250, Coomassie Brilliant Blue R-250, Amido Black 10B, Acid Black 1, Acid Orange 7, Acid Red 1, Acid Red 14, Acid Red 87, Acid Blue 25, Acid Blue 40, Acid Blue 113, Acid Blue 185, Acid Green 3 (Fast Green FCF), Acid Violet 17, Acid Yellow 9, Alizarin Red S, Alizarin Blue S, Bromophenol Blue, Bromocresol Green, Bromocresol Purple, Bromothymol Blue, Congo Red, Crocein Scarlet 7B, Eosin Y, Eosin B, Eriochrome Black T, Eriochrome Blue SE, Eriochrome Cyanine R, Evans Blue, Fluorescein, Fluorescein sodium salt, Fluorescein isothiocyanate (FITC), HABA (4^Z - hydroxyazobenzene-2-carboxylic acid), Lissamine Green B, Lissamine Rhodamine sulfonyl derivatives, Methyl Orange, Methyl Red sodium salt, Methyl Yellow, Metanil Yellow, Naphthol Blue Black (Acid Black 1), Naphthol Yellow S, Orange G, Ponceau S, Ponceau 4R, Ponceau SX, Reactive Blue 2, Reactive Red 120, Remazol Brilliant Blue R, Rose Bengal, Sirius Red F3B, Sudan IV, Tartrazine, Thymol Blue, Xylenol Orange, and Trypan Blue.

Examples of analytes that are extracted from the samples include but are not limited to: protons, K⁺, Na⁺, Li⁺, Ca²⁺, Mg²⁺, Pb²⁺, Cd²⁺, Cu²⁺, Cr²⁺, Ag⁺, Hg²⁺, Zn²⁺, CP, SO₄ ²’, CO₃ ²’, nitrate, nitrite, phosphate, phosphate monobasic, phosphate dibasic, creatinine, lactate, drugs that are ionic at the applied pH, i.e., the pH of the aqueous sample, and radioactive ions such as Sr²⁺.

Examples of suitable ionophores for binding specific ions include but are not limited to: potassium ionophore I (valinomycin), potassium ionophore II (BB15C5, Bis[(benzo-15- crown-5)-4^z -ylmethyl] pimelate), potassium ionophore III (2-Dodecyl-2-methyl-l,3- propanediyl bis[N-[5^z -nitro(benzo-15-crown-5)-4^z -yl] carbamate], BME 44), sodium ionophore IV (2,3:l l,12-Didecalino-16-crown-5, 2,6,13,16,19- Pentaoxapentacyclo[18.4.4.4^{7 12}.0¹’²⁰.0^{7 12}]dotriacontane, DD-16-C-5), sodium ionophore VI (Bis[(12-crown-4)methyl] dodecylmethylmalonate, Dodecylmethylmalonic acid bis[(12- crown-4)methyl ester]), sodium ionophore X (4-tert-Butylcalix[4]arene-tetraacetic acid tetraethyl ester), sodium ionophore III (ETH 2120, N,N,N^Z ,N^Z -Tetracyclohexyl- 1,2- phenylenedioxy diacetamide), sodium ionophore I (ETH 227, N,N^Z ,N^Z ' -Triheptyl- N,N^Z ,N^Z ' -trimethyl-4,4^z ,4^Z ' -propylidynetris(3-oxabutyramide)), calcium ionophore II (N,N,N^Z ,N^Z -Tetra[cyclohexyl]diglycolic acid diamide, N,N,N^Z ,N^Z - Tetracyclohexyl-3-oxapentanediamide, ETH 129), calcium ionophore III (Calcium Ionophore A23187, Antibiotic A 23187, Calimycin), calcium ionophore IV (N,N- Dicyclohexyl-N^z ,N^Z -dioctadecyl-3-oxapentanediamide, N,N-Dicyclohexyl-N^z ,N^Z - dioctadecyl-diglycolic diamide, ETH 5234), calcium ionophore I ((-)-(R,R)-N,N^z -Bis-[ 11 - (ethoxycarbonyl)undecyl]-N,N^z ,4,5-tetramethyl-3,6-dioxaoctane-diamide, Diethyl N,N^Z - [(4R,5R)-4,5-dimethyl-l,8-dioxo-3,6-dioxaoctamethylene]bis(12- methylaminododecanoate), ETH 1001), calcium ionophore V (10,19- Bis [(octadecylcarbamoyl)methoxy acetyl] - 1 ,4,7 , 13 , 16-pentaoxa- 10,19- diazacycloheneicosane, K23E1), magnesium ionophore I (ETH 1117, Magnesium-ligand, N,N^Z -Diheptyl-N,N^Z -dimethyl- 1,4-butanediamide), magnesium ionophore III (ETH 4030, N,N^Z ' -Octamethylene-bis(N^z -heptyl-N^z -methylmalonamide)), magnesium ionophore IV (ETH 7025, N,N^Z ,N^{Z z} -Tris[3-(heptylmethylamino)-3-oxopropionyl]- 8,8^Z -iminodioctylamine), magnesium ionophore VII (4,13-[Bis(N- adamantylcarbamoyl)acetyl]-l,7,10,16,tetraoxa-4,13-diazacyclooctadecane, K22B5), magnesium ionophore VI (l,3,5-Tris[10-(l-adamantyl)-7,9-dioxo-6,10- diazaundecyl]benzene, ETH 5506), lithium ionophore IV (5-Butyl-5-ethyl-N,N,N,N^z - tetracyclohexyl-3,7-dioxaazelaic diamide, ETH 2137, N,N,N,N-Tetracyclohexyl(2-butyl-2- ethyltrimethylenedioxy)diacetamide), lithium ionophore VI (6,6-Dibenzyl- 1,4,8- 11- tetraoxacyclotetradecane, 6,6-Dibenzyl-14-crown-4), lithium ionophore VIII (N,N,N^Z ,N^Z ,N^ZZ ,N^ZZ -Hexacyclohexyl-4,4^Z ,4^ZZ -propylidynetris(3-oxabutyramide)), lead ionophore IV (tert-Butylcalix[4]arene-tetrakis(N,N-dimethylthioacetamide)), nitrate ionophore VI (9-Hexadecyl-l,7,l l,17-tetraoxa-2,6,12,16-tetraazacycloeicosane), chloride ionophore IV (4,5-Bis-[N^z -(butyl)thioureido]-2,7-di-tert-butyl-9,9-dimethylxanthene), and other commercially available chemicals such as monensin (Coban and Rumensin), lasalocid (Avatec and Bovatec), salinomycin (Bio-cox and Sacox), narasin (Monteban and Maxiban), maduramicin (Cygro), semduramicin (Aviax), laidlomycin propionate (Cattlyst), A23187 (Calbiochem).

Examples of suitable ion exchangers include but are not limited to: sodium tetrakis [3 ,5-bis(trifluoromethyl)phenyl]borate, potas sium tetrakis [3,5- bis(trifluoromethyl)phenyl]borate, potassium tetrakis(4-chlorophenyl)borate, sodium tetrakis(4-chlorophenyl)borate, tridodecylmethylammonium chloride, tetradodecylammonium chloride, and tetradodecylammonium nitrate.

The use of ultralow-polarity hydrocarbon(s) in the matrix typically and advantageously reduces the required amount of ionophore to its theoretical amount with negligible excess. Thus, in preferred sensors, the ratio of the ionophore to the ion exchanger is lower than typical values due to the use of ultralow-polarity hydrocarbon. If the analyte has a charge of z, the ion exchanger has a charge of 1, and the ionophore- analyte binding has a stoichiometry of s:l, the minimum required ionophore to ion exchanger ratio to fully extract the analyte ion is s x 1/z = s/z (relative to 1 ion exchanger), wherein z is the charge of the analyte ion. Usually the actual amount of ionophore is 2-6 times as much as this theoretical value. However, because of the presence of the ultralow-polarity hydrocarbon, in some aspects, the required ionophore is reduced to only 100% to 200% of the theoretical value. In further aspects, it is only 100-130% of the theoretical value or even only 100-110% of the theoretical value.

In some aspects, the molar ratio of e.g. the permanently charged dye to the ion exchanger is 1 : 10 to 2: 1 and the molar ratio of the ion exchanger to the ionophore is 1 : 1 to 1:50, including all ratios in between these ranges.

In some aspects, the disclosed optodes are liquid sensors at the operating temperature. The liquid sensor can be in contact with the sample in a liquid channel such as microfluidic channels and millifluidic channels for ion sensing. The fluidics and operation are described in International Patent Application No. PCT/US23/65882 and in US Patent 11724260 (16/841,215), each of which is incorporated herein by reference.

However, in other aspects, the liquid optode can be dispersed in an aqueous phase as nanoparticles or microparticles. The liquid optode particles may further contain a hydrophobic polymer to enhance its mechanical stability. Examples of the hydrophobic polymer includes but are not limited to polytetrafluoroethylene (PTFE), poly vinylidene fluoride (PVDF), polypropylene (PP), polyethylene (PE), low-density polyethylene (LDPE), high-density polyethylene (HDPE), polystyrene (PS), poly(methyl methacrylate) (PMMA), polycarbonate (PC), poly(4-methyl-l -pentene) (PMP), polyether ether ketone (PEEK), polyimide (PI), polysiloxane, polyvinyl chloride (PVC), polyvinyl acetate (PVAc), polybutylene, polyisobutylene, polyisoprene, polychlorotrifluoroethylene (PCTFE), perfluoroalkoxy alkane (PFA), fluorinated ethylene propylene (FEP), Teflon AF, polyacrylonitrile (PAN, in hydrophobically modified forms), poly(ethylene-co- tetrafluoroethylene) (ETFE), and silicone rubber. Polyvinyl chloride, polyurethane, polyacrylate, polysiloxanes, and polymethacylates are preferred in some embodiments. The weight ratio of the polymer and the liquid (hydrocarbon and solvent) ranges from 1:10 to 10:1. The liquid optode particles may also be stabilized by one or more surfactants. Examples of surfactants include but are not limited to sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), didodecyldimethylammonium bromide (DDAB), dodecyltrimethylammonium bromide (DTAB), sodium dodecylbenzenesulfonate (SDBS), dioctyl sodium sulfo succinate (AOT), lauryl sulfate, sodium laurate, sodium oleate, stearic acid, linoleic acid, sodium cholate, sodium taurocholate, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), Brij 35, Brij 58, Tween 20, Tween 40, Tween 60, Tween 80, Triton X-100, Triton X-114, Span 20, Span 40, Span 60, Span 80, Pluronic F68, Pluronic F127, Pluronic P123, Poloxamer 188, Poloxamer 407, lecithin, egg phosphatidylcholine, soy phosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), lysophosphatidylcholine (LPC), CHAPS, sodium deoxycholate, sodium taurodeoxycholate, cetrimonium tosylate, octadecylamine, hexadecylamine, oleylamine, laurylamine, poly(ethylene-alt-maleic anhydride), polyethyleneimine (PEI), poly(acrylic acid), carboxymethyl cellulose (CMC), gum arabic, sodium caseinate, gelatin, gelatin methacryloyl (GelMA), and sodium alginate. The weight ratio of the surfactant/stabilizer to the liquid (hydrocarbon and solvent) is 1:1000 to 1:1, and is preferred to be 1:100 to 1:10. There are numerous publications on particle-based ion-selective optodes. However, hydrocarbons have not been used to enhance the optode response.

In other aspects, the liquid optode can contain polymers to create. The polymer, solvent, hydrocarbon, and sensing components can be dissolved in an organic solvent. The the cocktail can be cast, coated, or printed onto a glass or plastic substrate. After the solvent evaporation, a membrane is formed and can be used to detect analytes in aqueous samples. Examples of the hydrophobic polymer includes but are not limited to polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polypropylene (PP), polyethylene (PE), low-density polyethylene (LDPE), high-density polyethylene (HDPE), polystyrene (PS), poly(methyl methacrylate) (PMMA), polycarbonate (PC), poly(4-methyl- 1-pentene) (PMP), polyether ether ketone (PEEK), polyimide (PI), polysiloxane, polyvinyl chloride (PVC), polyvinyl acetate (PVAc), polybutylene, polyisobutylene, polyisoprene, polychlorotrifluoroethylene (PCTFE), perfluoroalkoxy alkane (PFA), fluorinated ethylene propylene (FEP), Teflon AF, polyacrylonitrile (PAN), poly(ethylene-co-tetrafluoroethylene) (ETFE), and silicone rubber. There are numerous publications on membrane-based ion- selective optodes. However, hydrocarbons have not been used to enhance the optode response.

In other aspects, the liquid optode can be formed on a membrane. The liquid with sensing components can be adsorbed onto a membrane with hydrophobic domains. This membrane can be used to detect analytes in aqueous samples. Examples of the membrane include but are not limited to polytetrafluoroethylene (PTFE) membranes, polyvinylidene fluoride (PVDF) membranes, polypropylene (PP) membranes, polyethylene (PE) membranes, polydimethylsiloxane (PDMS) membranes, poly(ethylene-co- tetrafluoroethylene) (ETFE) membranes, poly (ether ether ketone) (PEEK) membranes, poly(4-methyl-l -pentene) (PMP) membranes, polysulfone (PSf) membranes, polyetherimide (PEI) membranes, polycarbonate membranes, polystyrene membranes, silicone rubber membranes, cellulose acetate membranes, polyacrylonitrile membranes, Teflon AF membranes, Nafion membranes, polyimide membranes, and cellulose membranes.

In some aspects, the present formulations ensure an exhaustive response mode. A feature of an exhaustive response mode is that ionic analytes in an aqueous samples are mostly extracted into the sensing phase. This is in contrast to the equilibrium response mode, in which most analyte ions are not extracted into the sensing phase. There is no specific criterion on how much extraction is considered exhaustive, but it should be over 50% through the entire response range. Typically, at least about 50 to about 90% or more of the analyte ions are extracted into the sensing phase in the exhaustive response mode, e.g. at least about 50, 55, 60, 65, 70, 75, 80, 85, or 90%, or even up to about 95, 96, 97, 98 or 99% of the analyte ions are extracted. The exhaustive mode requires enough ion exchanger and ionophore in the ISO to extract most analyte ions.

For traditional optical ion sensors, there is no precisely defined lower and upper limit of detection. In contrast, in some aspects, the present disclosure provides a formulation strategy to obtain a precisely defined response range that matches the levels of ionic analytes in biological samples. This response range of the sensor is defined by a lower limit of detection and a upper limit of detection that are precisely controlled by the ISO formulation, i.e. the sensing chemicals and their ratios are purposefully selected to obtain the desirable response range. In particular, the ratio of the number of moles of dye to the number of moles of ion exchanger is specifically formulated to define the lower and upper limit of detection to make the sensor slope conveniently steep. Under the exhaustive response mode, the ratios of chemicals described herein define precise response ranges with a precise lower limit of detection (not zero and not an unspecific value) and upper limit of detection. Previous technologies using the exhaustive response mode have a detection limit of zero or an undefined value, which does not match the need for many real- sample tests. For example, electrolyte ions (e.g. calcium, potassium, sodium, magnesium, chloride, and phosphate ions) in biological samples like blood and plasma are never zero. Instead, their ranges are very narrow (e.g., a total calcium concentration of about 1.0 to 3.0 mM, potassium concentration of 2 to 8 mM). Therefore, a detection limit of zero is not useful and wastes a portion of the signal, ultimately reducing the accuracy and precision of the test for the clinically relevant concentration range. In the present matrix formulations, the amount of dye is less (lower) than that of the ion exchanger by a specific amount. This difference in the number of moles precisely determines the lower limit of detection. Also, the total amount of ion exchanger defines the upper limit of detection since no more analyte ions can be extracted when the ion exchanger is used up.

This is achieved by a reduction in the amount of dye relative to the ion exchanger, compared to conventional ISOs. When the ion exchanger is monovalent and the dye is monovalent or neutral, the present sensors extract (y-x /z) moles of analyte where the amount of the dye is x moles, the amount of the ion exchanger is y moles, and z is the magnitude of the charge of the analyte that is detected. Although the sample has approximately (y-x /z) moles of the analyte and most of these analyte ions are extracted into the ISO, the response of the ISO is nearly the same as that in analyte-free blank samples. Therefore, (y-x /z) moles is the approximate lower limit of the detection for this sensor. If the dye is divalent, y-x /z becomes y-2x /z. The upper limit of detection of the sensor is approximately y/z moles. Therefore, according to the required detection range of the real- world samples, the number of moles of the ion exchanger and the ionophore can be calculated. A large portion of the sensor response occurs over the biologically relevant concentration range instead of irrelevant concentrations that are too low or too high. Notably, the actual detection limit may slightly vary from the theoretical detection limit due to the presence of interference species or other factors. The deviation from the theoretical detection limit is usually less than ± 50% of the detection limit and most often less than ± 20% of the detection limit. The deviation is more preferred to be less than ± 10% of the detection limit. For example, if the theoretical upper limit of detection is 3 mmol for an analyte, the actual detection limit usually falls in the range of 2.7 to 3.3 mmol (± 10% deviation) but may have larger deviations.

From another way of calculation, the upper end of the detection range (upper limit of detection: m mole) is defined by the amount of ion exchanger in the sensor and the lower end of the detection range (lower limit of detection: n mole) is defined by the difference between the amount of the ion exchanger and the amount of the dye. In order to obtain the detection range of n to m moles of analyte, the optode needs to contain (m x z) moles of ion exchanger to maintain an upper limit of detection of “m mole” and contains ((m-n) x z) moles of optical reporter to maintain a lower limit of detection of “n mole”. Wherein z is the absolute value of the charge of the analyte, the ion exchanger has a charge of one, and the optical reporter has a charge of zero or one. Notably, the actually amounts of ion exchanger and dye in the sensing system may deviate from the theoretically calculated amounts due to the presence of interference ions and other factors. The deviation is usually less than ± 50% of the calculated amount and most often less than ± 20% of the calculated amount. The deviation is more preferred to be less than ± 10%.

However, the sensors disclosed herein are not limited to operation under the exhaustive mode. In some aspects, the sensors operate under a non-exhaustive mode, in which a significant amount of analytes stay in the sample phase. The use of hydrocarbons can still improve the response of these ion- selective optodes.

METHODS

During use, the ISO is in contact with an aqueous sample under conditions which allow mass transfer of at least one analyte of interest between the two phases (from the aqueous sample and into the sensing phase) and binding of the analyte to an ionophore in the sensing phase. Binding produces a response, which is converted to a detectable signal, usually an electrical signal.

When the optical reporter is a pH indicator, the optical property (e.g., color, absorbance, fluorescence, etc.) of the sensing phase changes due to the protonation or deprotonation of the pH indicator and generates a detectable signal. A variety of pH indicators are available for use, with hydrophobic pH indicators being preferred. Exemplary pH indicators that are employed include but are not limited to: Nile blue, chromoionophore I (9-(diethylamino)-5-(octadecanoylimino)-5Hbenzo[a]phenoxazine) designated ETH5249; chromoionophore II (9-dimethylamino-5-[4- (16-butyl-2,14- dioxo-3,15 ioxaeicosyl)phenylimino]benzo[a]phenoxazine) designated ETH2439; chromionophore III (9- (diethylamino)-5-[(2-octyldecyl)imino] benzo [a] phenoxazine), designated ETH 5350; chromoionophore IV (5-octadecanoyloxy-2- (4-nitrophenylazo)phenol), designated ETH2412; chromoionophore V (9- (diethylamino)- 5-(2-naphthoylimino)-5H- benzo[a]phenoxazine); chromoionophore VI (4,5 -dibro mofluorescein octadecyl ester) designated ETH7075; chromoionophore XI (fluorescein octadecyl ester) designated ETH7061; [ 11 - [( 1 -butylphenyl)oxy ] - 11 - oxoundecyl-4- { [9-(dimethylamino)- 5Hbenzo[ a]phenoxazine-5-ylidene]-amino}-benzenate] }; 2,4,5,7-tetraiodofluorescein octadecyl ester; and combinations thereof. When the optical reporter is a dye that is charged in both the ISO matrix and the sample, the sensor response is based on ion exchange of the analyte with the ionic dye. For example, a cationic analyte in the sample can be extracted into the sensor phase with a cationic dye as the optical reporter. The extraction of the analyte leads to expulsion of the cationic dye from the sensor phase to the sample phase. Thus, the optical properties of both the sensor phase and the sample phase will change. Typically, the color intensity, characteristic absorbance, and the fluorescence of the oil phase reduces due to the loss of the dye and these optical signals of the aqueous phase increase due to gaining the dye. Similarly, an anionic dye can be used in an ISO for anionic analytes. The anionic analyte is extracted into the sensor phase to replace the anionic dye. Examples of dyes that are used in the present ISOs include but are not limited to: cationic dyes such as rhodamine B, rhodamine 6G, rhodamine 123, rhodamine 110, rhodamine 101, sulforhodamine B, tetramethylrhodamine (TMR), carboxytetramethylrhodamine (TAMRA), lissamine rhodamine B, methylene blue, new methylene blue, methylene green, thionine, azure A, azure B, azure C, toluidine blue O, toluidine blue, thioflavin T, thioflavin S, crystal violet, methyl violet 10B, basic fuchsin, pararosaniline, rosaniline, magenta, gentian violet, victoria blue B, victoria blue R, brilliant cresyl blue, Nile blue A, Nile blue sulfate, malachite green, brilliant green, aniline blue, brilliant cresyl violet, safranin O, safranin T, acridine orange, acridine yellow, acriflavine, 9-aminoacridine, proflavine, celestine blue, cresyl violet, methyl green, BODIPY-based pyridinium dyes, styryl-9M, 4’,6-diamidino-2-phenylindole (DAPI, under acidic conditions), SYTO 9, SYTOX Green, SYTO 13, SYTO 21, TO-PRO-1, TO-PRO-3, YO-PRO-1, YOYO-1, TOTO-1, TOTO-3, DiSC₃(5), DiOC₂(3), JC-1 monomer (cationic form), cyanine 3 (Cy3), cyanine 5 (Cy5), Cy7, indocyanine green (cationic in some environments), 2- [4-(dimethylamino)styryl]-l -methylpyridinium iodide, 4- dimethylaminostyryl-N-methylpyridinium, 4-dimethylaminostyryl-N-methylquinolinium, hemicyanine dyes such as JC-9 and Styryl- 11, ethidium bromide, propidium iodide, and mitotracker red CMXRos as well as anionic dyes such as coomassie Brilliant Blue G-250, Coomassie Brilliant Blue R-250, Amido Black 10B, Acid Black 1, Acid Orange 7, Acid Red 1, Acid Red 14, Acid Red 87, Acid Blue 25, Acid Blue 40, Acid Blue 113, Acid Blue 185, Acid Green 3 (Fast Green FCF), Acid Violet 17, Acid Yellow 9, Alizarin Red S, Alizarin Blue S, Bromophenol Blue, Bromocresol Green, Bromocresol Purple, Bromothymol Blue, Congo Red, Crocein Scarlet 7B, Eosin Y, Eosin B, Eriochrome Black T, Eriochrome Blue SE, Eriochrome Cyanine R, Evans Blue, Fluorescein, Fluorescein sodium salt, Fluorescein isothiocyanate (FITC), HABA (4^Z -hydroxyazobenzene-2-carboxylic acid), Lissamine Green B, Lissamine Rhodamine sulfonyl derivatives, Methyl Orange, Methyl Red sodium salt, Methyl Yellow, Metanil Yellow, Naphthol Blue Black (Acid Black 1), Naphthol Yellow S, Orange G, Ponceau S, Ponceau 4R, Ponceau SX, Reactive Blue 2, Reactive Red 120, Remazol Brilliant Blue R, Rose Bengal, Sirius Red F3B, Sudan IV, Tartrazine, Thymol Blue, Xylenol Orange, and Trypan Blue.

A wide variety of samples are assessed using the devices and methods disclosed herein. The samples that are assessed using the method are generally aqueous liquid samples. In some aspects, the samples are physiological samples, e.g. samples taken from an organism such as an animal or plant. In some aspects, the samples are obtained from a mammal such as a human. However, veterinary applications of this technology are not excluded, i.e., samples from nonhuman animals may also be assessed. Any liquid sample that can be mixed with the sensing phase in a manner that permits extraction of analytes into the sensing phase, can be assessed. The types of samples that are analyzed using the disclosed ISOs include but are not limited to: biological fluid (e.g. bodily fluid) samples such as blood, blood products including blood fractions, plasma, serum, platelets and the like, urine, tears, saliva, sweat, lymphatic fluid, cerebrospinal fluid, stomach fluid, exhalations and the like and are either intracellular extracellular fluids; environmental fluids or environmental samples (e.g., for detecting pollutants and other ions) in air, water (e.g. tap water; ocean, lake pond and stream water; rain water; storm runoff; etc.), and soil extractions (e.g. from agricultural samples, lake or ocean beds, mining sites, etc.; samples from industrial processes, e.g. industrial control samples for monitoring ion concentrations in various industrial processes; research samples generated while studying e.g., ion transport and binding in biological and chemical systems; and food samples such as dairy products and beverages or extracts of solid foods. Any liquid sample that contains ionic species, or that is suspected of containing ionic species, can be anlayzed using the ISOs disclosed herein. In addition, dry or gaseous samples or scrapings can be analyzed if they are first dissolved or extracted using an aqueous liquid, and samples taken e.g., by swabs can be analyzed by soaking the swab in a dilution buffer. If desired or necessary, the sample may be diluted with a sample buffer for any of a variety of reasons, e.g. to dilute the sample, alter the pH of the sample to ensure protonation or deprotonation of the analyte, etc. Examples of commonly used buffers include but are not limited to: saline; various aqueous based buffers such as phosphate (e.g., dihydrogen phosphate), HEPEs, MOPS, MES, BES, MOPSO, ACES, TAPS, Bicine, acetic acid with sodium acetate, ammonium hydroxide with ammonium chloride, citric acid with sodium citrate, carbonic acid with bicarbonate ion, KH2PO4 with K2HPO4, Tris buffers (e.g., Tris- HCI (Tris hydrochloride), TrisEDTA (TE), Tris-buffered saline (TBS), Tris-acetate-EDTA (TAE), and Tris-borate-EDTA (TBE), Bis-Tirs buffers, and mixtures of buffer components such as HEPES-Tris buffer.

In general, the volume of a sample that is taken up for analysis generally ranges from about 0.1 - 100 |lL, such as from about 0.1, 0.5, 1.0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 |lL, or more, including all decimal fractions in between to 0.1 |lL, e.g. from about 0.1, 0.2, 0.3... to about 99.8, 99.9 or 100|lL Lower amounts of sample may also be used, e.g., from about 0.1 to about 1.0 pl, including all decimal fractions in between to the nearest 0.1 pl. Higher amounts of sample may also be used, e.g., from about 0.1 to 10 mL, including from about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ml, including all decimal fractions in between to 0.1 decimal point, e.g. about 0.1, 0.2, 0.3... to 1.0, 1.1, 1.2... to about 9.8, 9.9 or 10.0 ml.

When a dilution buffer is used, after a sample is loaded into preloaded dilution buffer, the total volume of dilution buffer plus sample generally ranges from about 0.2 - 200 p.L, such as from about 0.2, 0.3, 0.4... 199.8. 199.5 or 200.2 pL, including all decimal fractions in between to 0.1 decimal points, as described elsewhere herein.

DEVICES, SYSTEMS AND KITS

In some aspects, devices as described herein comprises a hollow tube or container, generally with two open ends. A first open end is configured to receive a sample and a second open end is configured to be attached to a suction device. The application of suction to the second end (to a volume of the tube) causes a liquid to be drawn into the tube via the first end if/when the first open end is submerged into a source of liquid. Alternatively, capillary action may be the force that drives ingress of liquids into the tube.

The tube or channel can be made of any plastic, glass, quartz, ceramic, or rubber materials that are transparent or translucent, so that color changes or other optical property changes can be detected through the material of which the tube is made. The tube can be of any shape or size to match the volume of the sensing matrix and sample. In some aspects, the tube is a pipette tube, a capillary tube or a microfabricated channel.

In some aspects, the tube of the device is preloaded e.g., by applying suction to the second open end so that the liquid sensor is taken up into the tube, and the liquid source is switched to dilution buffer and dilution buffer is taken up into the tube. Thus, as provided to the user, the tube is generally preloaded with an aliquot (segment or segments) of sensing matrix with a total volume of a few pL. Thus, when the tube is a pipette tip, the pipette tip directly contacts the sample. A tip or the tips of the tube may be sealed and the seal removed prior to use. However, other aspects are also contemplated, e.g., the user is provided with a clean (empty) tube and instructions regarding how to load the sensing matrix and sample are provided with the device.

Examples of tubes that are suitable for use in the device include but are not limited to: a pipette tip, a capillary tube, or any device with a hollow channel, e.g., a microfabricated channel. The volume of the tube is generally in the range of from about 10 pL to about 10 mL. Multibore tubing can also be used to hold different sensing oils in different lumens of the tube. One end of multiple lumens is connected to the device of suction and another end of multiple lumens can be exposed to the sample so that the sample is introduced into multiple lumens. Generally, the tubes are designed to be disposable, although washable, resusable tubes are not excluded. In this case, a washing and/or rinsing solution may also be provided to the user, e.g., in a kit, together with the tubes and one or more other items described below.

Systems comprising the device disclosed herein are also provided. The systems comprise at least a tube as described above and a source of suction. Examples of suitable sources of suction include but are not limited to: a stepper motor-based device such as an electronic pipette or syringe pump. The source of suction may also control the mixing of two phases to facilitate extraction of the analyte from the sample to the sensing oil.

Further items for inclusion in a system, which may be part of a “kit” that is provided to the user or may use one or more components otherwise available to the user, include but are not limited to: a means for detecting a detectable signal (e.g., a color change) in the sensing matrix of the device and a means of analyzing the signal, or a means for detecting absorbance and/or fluorescence, etc. For example, a LED-photodiode pair can be used to detect absorbance when they are aligned in a straight line and detect fluorescence when they are aligned perpendicularly. Examples of other light sources include but are not limited to Deuterium lamps, tungsten-halogen lamps, xenon arc lamps, laser diodes, mercury vapor lamps, hydrogen lamps, quartz halogen lamps, compact xenon flash lamps, tungsten filament lamps, white light sources (combinations of broad-spectrum lamps), supercontinuum lasers, LED arrays, and pulsed xenon lamps. Examples of other light detectors include but are not limited to silicon photodiodes, indium gallium arsenide (InGaAs) photodiodes, germanium photodiodes, avalanche photodiodes (APDs), photomultiplier tubes (PMTs), silicon photomultipliers (SiPMs), charge-coupled devices (CCDs), complementary metal-oxide-semiconductor (CMOS) sensors, scientific CMOS (sCMOS) detectors, microchannel plates (MCPs), thermopile detectors, bolometers, pyroelectric detectors, photoconductive detectors, phototransistors, photovoltaic cells, quantum dot photodetectors, superconducting nanowire single-photon detectors (SNSPDs), multi-channel plate PMTs, quadrant photodiodes, position-sensitive detectors (PSDs), and photonic crystal-based detectors.

Detecting a color change refers to detecting a change in color of the sensing matrix, for example, by comparing the color before and after mixing with the sample and/or comparing the color to standards, threshold values and/or ranges of analyte concentration, etc. Detection may be accomplished in comparison to standards such as sensing matrices comprising known amounts of an analyte of interest, sensing matrices without any analyte present (blanks), and other standards that will occur to those of skill in the art. In some aspects, it may be sufficient to simply detect a change and describe a sample as positive or negative for the presence of an analyte of interest. In some aspects, a color chart (either digital or a “hard copy” on paper) with each color indicating an analyte concentration is provided to allow users to correlate the color or other change that correlates with the amount of analyte present in the sensing matrix. In this case, the color change may be detected “by eye” by the user. In other aspects, it is preferable to analyze a color change in detail, e.g., to detect a quantity, amount or level of change, and or to detect the hue, intensity or shade of color, and to interpret/analyze the change and/or the degree of change. This analysis generally involves “capturing” a representation of the sensing oil after mixing (and optionally also before mixing), such as obtaining a digital image (photograph) of the matrix phase segment of the tube which is then compared to one or more corresponding standards. It is noted that standards may be predetermined and provided to the user (e.g., via a computer- based medium in a reader device described below) or may be established by the user following instructions provided with the device. Digital representations are obtained, e.g., with a smart phone camera, a digital camera, etc. Alternatively, a digital device (e.g., a small digital camera) to capture the representation may be specifically designed for inclusion in e.g., a kit comprising the device described herein.

In some aspects described in more detail below, the means of interpreting a digital representation comprises a computer program such as a mobile “app”. Color analyzing apps are known and may be downloaded from the internet to a device such as a portable smart phone, i-pad, personal computer, laptop, etc., and access to the digital representation is also provided on the device. Commercially available apps that fulfil this function include but are not limited to: apps such as Color Mate, Color Grab, ColorSnap® Visualizer, Google Lens, Palette Cam, Color Converter, Adobe Capture, Color Viewfinder, Pantone Studio, Coolers, TECHKON ColorCatcher, etc. Alternatively, an app or other type of program may be designed specifically for use in analyzing the results (color changes) obtained by practicing the methods disclosed herein. Further, a device for capturing and analyzing color changes may be designed specifically for use in the methods disclosed herein, e.g., a camera with a built-in or preloaded or preprogrammed analysis system.

Output from the analysis means is provided to the user and/or to suitable medical professionals by any of a variety of methods. For example, a visual output may be provided to a screen and may include a numerical read-out, a graph, etc. which shows the measurement just taken in comparison to a standard or standards and/or in comparison to previous results and/or in comparison to goals, etc. A range indicator may be included, e.g., “high”, “low”, “normal”, etc. or a numeric output (e.g. moles, nanomoles, etc.). The output may be in black and white or in color.

The app or computer program may be set to automatically transfer the results of the analysis to a suitable medical professional for analysis by a human or Al analyst, e.g., by a smartphone. Feedback may be provided to the user, such as instructions to increase or decrease a dose of medication, contact the medical provider, or “good job; continue with present dosing”, etc. Further, reminders to perform the assay may be built-in as may recognition that an analysis has been completed.

The means for detecting and/or analyzing and/or transferring such data may be referred to as a reader device. Examples of reader devices include, but are not limited to, personal electronic devices such as cell phones, smart phones, personal digital assistants (PDAs), tablet computers, laptop computers, media players, and other such devices. In particular embodiments, a reader device or a component thereof (e.g., image capture device 499) may be a mobile electronic device. A reader device may be a single device or, alternatively, a reader device may include two or more devices communicatively coupled. Therefore, in some embodiments, a “reader device” may include two or more electronic devices, and operations described and attributed herein to a reader device may be performed collectively by the two or more electronic devices.

In some embodiments, a reader device can include both a personal electronic device and an image capture device such as a camera. In various embodiments, the image capture device may be configured to communicate data to a mobile electronic device such as a smartphone or a cell phone, or to another type of electronic device. The image capture device and the personal electronic device may each be configured to perform some of the reader device functions described herein. For example, an image capture device may include one or more of a processor, an optical sensor, a memory, and a communications module (e.g., a transmitter, transceiver, or other type of communications device) coupled by circuitry. Optionally, image capture device may include a power source (e.g., a rechargeable battery or a replaceable battery).

The reader device may comprise or use an imaging application that includes one or more algorithms for color analysis, calculation of representative values for analytes, tracking of representative values over time, analysis of a user's medication, and/or other functions. For example, the imaging application may include an algorithm configured to analyze the effect of a user's medication based on user inputs (e.g., times and dosages at which a medication was taken) and the determined concentrations of an analyte of interest at specific time points. Optionally, the imaging application may track the effect of the medication as a function of dosage and/or time or suggest modifications in the dosage of the medication based on the analysis. The reader device may be configured to access a look-up table from program data or a database that stores one or more of a pre-determined pattern, reference images, calibration data, and/or ranges for some or all of the analytes of interest. The reader device may then determine or calculate a representative value for an analyte based on the image color data and corresponding detection ranges.

The concentrations/representative values, captured image, image data, and/or other relevant data (e.g., time, date, identity of analyte, etc.) may be stored in non-volatile memory as program data or imaging data. The reader device may track the concentrations/representative values over time, recording them in a table or other format that can be displayed or communicated to the user. Optionally, the reader device may display the captured image and/or determined representative value on a display, communicate the results to the user or to another device/system, and/or generate and communicate a message, notification, alert, instructions, or a representative value (e.g., a target analyte concentration) to a user of the reader device in a visual, audio, and/or tactile (e.g., vibratory) format. Optionally, the reader device may alert the user of a possible device malfunction, or that the device is approaching or has reached or exceeded the end of its recommended duration of use.

In some embodiments, the reader device may transmit a message, notification, alert, instructions, or a representative value (e.g., a target analyte concentration) to a medical service provider or caretaker. The reader device may be communicatively coupled to one or more computing devices or systems via a wireless connection or network. The reader device may exchange data with one or more of a personal computer, a network, a medical device, a first computing system, a first database, a second computing system, and/or a second database. In some examples, the first computing system/database is a medical provider or health monitoring computing system/database and may be operated or accessible by a first medical provider, such as a primary care physician of the user. The second computing system/database may be operated by a caretaker or a second medical provider such as a doctor's office, hospital, emergency medical service, or subscription-based service that notifies a medical provider of a potential emergency. Alternatively, the second computing system/database may be a computing system/database of a manufacturer that can be read by reader device. The computing system of the manufacturer may analyze and/or track data received from the reader device to assess device performance.

In some embodiments, an analyte sensor may be read by a user without the use of a reader device. For example, the user may determine an approximate analyte concentration by viewing the color changes within the tube without the aid of a reader device. Optionally, the user may be provided with a visual aid such as a chart, color key, or the like. The user may compare the response(s) of the analysis region(s) to the chart to determine an approximate analyte concentration. Alternatively, the user may interpret the response(s) of the analysis region(s) without the use of a visual aid. For example, after a period of time, the user may have sufficient experience with the use of the sensor to correlate the visible color change to an approximate analyte concentration.

In addition, although the reader device is typically used to capture images of the sensor, one or more of the other functions described herein as being performed by the reader device may instead be performed by another device or system, such as a computer, database, medical device, etc., and vice versa. For example, the reader device may capture an image of the sensor and transmit the image data to a computing system for analysis. Alternatively, image analysis functions may be divided among the reader device and another device or computing system. For example, the reader device may be configured to determine a representative value for a target analyte and the computing system may be configured to track the representative values over time and/or to generate and send instructions to the reader device to adjust one or more operational parameters.

Calculating a representative value may include comparing the representative value to one or more reference values. Some reference values may be pre-determined such as color changes corresponding to specific concentrations of an analyte.

An imaging application is one example of an application suitable for use with the present analyte monitoring system. As used herein, the term “imaging application” refers to a program that directs a processor to perform various tasks related to analyte monitoring (e.g., image analysis, calibration, tracking of data, etc.). Imaging applications and operations thereof may vary among embodiments. Optionally, an imaging application may include, or may be provided with, reference data such as reference tables/values, reference images, and/or other relevant data. Some imaging applications may be developed or configured for use with a particular type of reader device (e.g., a smartphone or tablet computer) and/or operating system (e.g., Google Android, Apple iOS, Nokia Symbian, RIM BlackBerry OS, Samsung Bada, Microsoft Windows Phone, Hewlett-Packard webOS, Linux operating system). Again, these examples are provided merely by way of illustration, and imaging applications may be configured/adapted/developed for use with many other types of reader devices (e.g., tablet computer, personal digital assistant, camera) and/or operating systems. Some imaging applications may be “cross -platform” applications developed or configured for use with multiple types of reader devices/operating systems. In some embodiments, a reader device may an iPhone or an iPad.

In some embodiments, an imaging application may be pre-installed on the reader device (e.g., by the reader device manufacturer). In other embodiments, the application may be provided in a physical medium, such as an optical disc (e.g., a CD, a DVD), a data storage disk (e.g., a ZIP disk), a flash memory device (e.g., a USB flash drive, a memory card), and the like. Alternatively, the application may be downloaded/electronically transmitted to the reader device or associated computer system (e.g., the user's personal computer) over a network (e.g., the Internet). The application may be made available for download from a computer system or database of a third party (e.g., a manufacturer of the service, a manufacturer of the reader device, a medical service provider, a software developer, a software distributor, or a web-based application store, such as the Apple App Store). In some embodiments, the imaging application may be a web-based application that resides on a server of a third party and is accessible by the reader device via the Internet (e.g., as a web application). In one example, a portion of the web-based imaging application may be downloaded to the reader device and may reside on the reader device thereafter. Alternatively, a portion of the imaging application may be downloaded to the reader device each time the reader device accesses/uses the imaging application.

Various operations, sequential orders in which operations are performed, and the distribution of operations among the reader device and other devices/computing systems may vary among embodiments. For example, in some embodiments, one or more of the operations may be performed locally by the reader device and others may be performed remotely by one or more third-party computer systems. A third-party computer system can be a computer system, website, database, server (e.g., a network server, a cloud server), or other digital distribution platform of a third party such as a manufacturer, a medical services provider, and/or an imaging application developer. Again, many variations and modifications to the illustrated processes and user interface displays will be readily understood by persons with ordinary skill in the art in light of the present disclosure, which encompasses all such variations and modifications.

In various embodiments, one or more of the user interface displays may be included in the device or system, and they may comprise additional user-selectable features (e.g., virtual buttons or keys, links, etc.) configured to provide control over, or access to, various options/displays of the imaging application.

The reader device may be calibrated based at least in part on the reference measurement(s). The calibration process may be performed by the reader device, a third - party computing system, and/or both. In some embodiments, the reader device and/or third- party computing system may track reference measurement inputs as part of the calibration process. The reader device may track reference measurement inputs and/or associated data over a period of days, weeks, months, or years. In some embodiments, the reader device may periodically transmit the reference measurement inputs and/or associated data to a third- party computing device. This may allow the reader device to store a smaller volume of tracking data in local storage. In some embodiments, tracking data may be accessed/downloaded by the reader device from the third-party computing system (e.g., analyte sensor manufacturer, cloud network, etc.) in response to a request from the user for such data.

The reader device may report one or more data trends to the user. For example, the reader device may report data trends to the user as a function of time (e.g., over a day, week, month, year, etc.) in the form of a dashboard, chart, table, or other format.

Provided herein are methods of detecting (measuring, analyzing, assaying, etc.) the amount (level, concentration, moles, etc.) of analyte in a sample.

In some aspects, the methods comprise a step of obtaining a sample that is suitable for analysis. Exemplary samples are listed elsewhere herein and include samples from the body of an animal such as a mammal, or samples of interest such as water, samples from plants, etc.

The step of obtaining a sample is conducted by any suitable means, depending on the nature of the sample and the setting for using the devices and methods. For example, if the sample is a blood sample and the analysis is being conducted in the home or other nonlaboratory setting by a patient, blood may be obtained e.g., by a finger prick conducted by the patient. Similarly, the patient may obtain saliva samples using a swab which is then placed in a buffered solution from which an aliquot is taken. Saliva samples may also be collected in a collection tube without dilution. If the sample is urine, a small amount may be obtained in a container and diluted, or not, for analysis. Other techniques for sample procurance by nonprofessional subjects are known and any technique that is suitable for the type of sample may be used. Depending on the nature of the sample, the sample may or may not be diluted prior to introducing it into the tube of the device, e.g., using a suitable amount of diluent such as an aqueous buffer, generally a physiologically comparable buffer, examples of which include water, saline, phosphate buffer, etc. and others as described elsewhere herein for the dilution buffer. Such buffers may be provided to the patient along with the device, e.g., as part of a kit. If the methods are used in a professional setting, then other options are available for obtaining a sample, such as using needles to pierce the skin, a blood vessel, an internal organ, etc. to retrieve a sample of interest. If the samples are not physiological, and method that provides a suitable amount of sample may be used, e.g., a pipette, syringe, etc.

To practice the methods, the user (patient, subject, etc.) aspirates or draws up a drop or aliquot of sample into the tube via the suction source, the tube having been preloaded with sensing matrix. Generally, the suction source is preprogrammed or “preset” to transfer a given amount of sample. The sensing oil and the diluted or undiluted sample need to be in contact so that the analyte can be at least partially or preferably fully extracted from the sample into the sensing matrix. The analyte enters the sensing phase selectively in comparison with other components of the sample fluid. The two phases may be mixed by a process such as vortexing, spinning, shaking, vibrating, stirring, or via an oscillating pressure source (e.g., vacuum or suction) that moves these two liquid phases back and forth. Generally, a stepper-motor-powered vacuum (suction) source, which is usually supplied with the device, is used. Examples of the stepper motor include but are not limited to syringe pumps and electronic pipettes.

Typically, the suction source is also preprogrammed or “preset” to cause the contents of the tube to be automatically drawn up and then mixed or agitated by rapidly “pulling” and “pushing” the contents back and forth until the phases are thoroughly mixed. During mixing, the two phases intermingle and analytes of interest come into contact with the sensing molecules in the sensing oil and are captured, i.e., they are bound to the sensing molecules and thus stay in the sensing oil. In other words, mass transfer of the analyte between the sensing oil and the dilution buffer occurs via a mixing protocol. The analyte in the aqueous samples is fully or partially extracted into the oil phase during the mixing process.

The mixing process includes multiple, alternate pushing and pulling cycles to move liquids in the tube in one first direction and then in the opposite second direction. Pushing steps move or propel liquids in the tube toward the first (proximal) end of the tube i.e., away from the motor, but without expelling liquid from the proximal tip of the tube. Pulling steps pull liquids in the tube toward the second (distal) end of the tube, i.e., toward the motor. The liquids move over the same travel distance in both directions at either equal or unequal speeds. In an exemplary protocol based on unequal speeds, in a first set of e.g., 10 cycles of pulls-pushes, the pulling speed is about 1 to 15 times higher than the pushing speed, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 times higher. After this first set, the oil segment is at the proximal end of the tube (further from the stepper motor). Then the speed of pushing and pulling is switched for another (second) set of e.g., 10 cycles of mixing in which the pushing speed is greater e.g., about 1 to 15 times higher than the pulling speed e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 times higher. During this second set, the position of the oil is gradually switched (changed, shifted) so as to be at the distal end of the tube (closer to the stepper motor) at the end of the second set. Alternating the relative positions of the sensing oil and the aqueous sample may enhance the mass transfer between the two immiscible phases and reduces the response time of the sensor. This position alternating process controlled by an unequal moving speed may be repeated multiple times to reach an equilibrium response, for example, from about 1-100 times, such as about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times or more. The travel distance in the push and pull steps is preferably greater than the length of the total liquid in the tube to achieve the shortest response time but is shorter than (less than) the total length of the tube. For example, when about 10 pl of sensing oil and about 10 pl of aqueous phase are present in a pipette tip using Format 1, the length of the segment of sensing oil is about 3-5 mm and the length of the segment of aqueous phase is about 3-5 mm, and the travel distance in the push and pull steps is preferably greater than the combined distance, i.e., greater than about 6-10 mm. The length of the liquid depends on the exact position of the liquid in the tube and the diameter and/or shape of the tube.

While the mixing protocol is typically preprogrammed, in some aspects, the user may perform the mixing protocol by following instructions provided e.g., in a kit. For example, the instructions may instruct the user to “agitate the contents of the tube up and down for a minimum of 25 strokes” or “for 20 seconds”, for example.

The optical signal of the sensing phase is detected by, for example, a color detector or absorbance detector or fluorescence detector that is located at the site of use of the device. Alternatively, a representation of the tube after mixing is obtained and provided to a computer-based analysis program such as an app that is employed by the user. In this aspect, the color change of the oil segment is typically recorded by a camera. Thus, after mixing, the user obtains a digital image (e.g., takes a photograph) of at least the oil segment of the tube. A digital image of the entire tube may be obtained since only the sensing oil changes color. Then, the digital image is loaded, transferred to, or otherwise made accessible to the app or other program which analyzes the color changes and provides an output to the user and/or optionally to at least one suitable medical professional. In some aspects, a camera associated with a smart-phone or i- pad is used. In other aspects, a separate digital or monochrome camera is used.

In some aspects, the sensing oil is brought in contact with the sample in droplet microfluidics (segmented microfluidics) as described in US Patent 11724260 (16/841,215), the complete contents of which is herein incorporated by reference in entirety. The difference is that a specific type of mixture of at least two liquids must be used in the new technology, in which one of the liquid is a hydrocarbon that does not dissolve sensing chemicals well due to the lack of functional groups forming hydrogen bonds but reduces the dielectric constant of the mixture, and another liquid is the true solvent that has functional groups containing at least one of these atoms: oxygen, nitrogen, sulfur, phosphorus, chlorine. Examples of solvents are summarized above. Plasticizers and oils (except hydrocarbons) are commonly used solvents.

In some aspects, the digital representation is sent (uploaded) e.g. to a website for analysis and processing. Remote exchange, processing, monitoring, storing, etc. of data is well known. Data are obtained, analyzed, transformed and output is provided to a user e.g., as described in United States patent applications 20220192609, 20190197858, 20130303869 and 20190361436, the complete contents of each of which is hereby incorporated by reference in entirety, i.e., via Internet of Things (loT) connections. The color parameter can be hue or any other color or fluorescence parameter, e.g., fluorescence intensity and peak wavelength. Typically, the detectable change is converted into an electronic signal which is correlated with the concentration of the analyte.

The necessary ranges of detection of a sensing phase typically encompass (bracket or include) those of e.g. a naturally occurring ion, especially in biological samples. For example, typical ranges in blood or serum include: sodium is 135 to 145 milliequivalents per liter (mEq/L); potassium is 3.5 to 5.2 milliequivalents per liter (mEq/L); calcium is 8.5 to 10.2 milligrams per deciliter (mg/dL), or 2.15 to 2.55 millimoles per liter (mmol/L); chloride is 96 to 106 milliequivalents per liter (mEq/L); magnesium is 1.7 to 2.2 milligrams per deciliter (mg/dL); and bicarbonate is 23 and 29 milliequivalents per liter (mEq/L) or 22 to 29 millimoles per liter (mmol/L); phosphate is 2.5-4.5 mg/dL (milligrams per deciliter) or 0.8- 1.4 mmol/L (millimoles per liter) for adults, 4.5-6.5 mg/dL and 1.45-2.1 mmol/L for children, and 4.3-9.3 mg/dL and 1.4-3 mmol/L for newborns. Those of skill in the art are well aware of the conversion of these values to moles to provide a suitable target range to ensure an exhaustive extraction response mode using the formulas disclosed herein.

Generally the operating temperature for conducting an analysis ranges from about 0 to about 80 °C, with a preferred range being from about 15 to about 50 °C.

The formulations disclosed herein are advantageously used in any type of ISO device. Preferably, two configurations of liquid ISOs are encompassed. They are, for example, based on i) pressure-driven droplet microfluidics in microchannels or ii) stepper motor-driven push-pull microfluidics in millichannels as discussed above. Lor the push-pull microfluidics method, an electronic pipette controls the mixing of the sensing phase and an aqueous sample in a micropipette tip by programmed pushes and pulls of the liquids. This platform only needs a few microliters of sample for the testing. The recent development of integrated optical detectors further enhances the usability of this platform.

Eurther description of ISOs is given in issued United States patent 8765060 and published international patent W02023205630, the complete contents of each of which is hereby incorporated by reference in entirety.

While the invention has been described in terms of its several exemplary embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Representative illustrative methods and materials are herein described; methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual dates of public availability and may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as support for the recitation in the claims of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitations, such as "wherein [a particular feature or element] is absent", or "except for [a particular feature or element]", or "wherein [a particular feature or element] is not present (included, etc.)...".

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended, nor should they be interpreted to, limit the scope of the invention.

EXAMPLES

EXAMPLE 1.

Figure 1 shows the response of the Ca²⁺ ISOs based on solvents of different polarities. Sensing chemicals including chromoionophore I (Chi), NaTFPB, and calcium ionophore II (Ca-II) are first dissolved in DOS at a concentration of 1 mM, 2 mM, and 3.5 mM. Then this DOS stock solution is mixed with squalane, hexadecane, dioctyl sebacate (DOS), and dibutyl phthalate (DBP) at a volume ratio of 1.5:1. If the ISO operates under exhaustive response mode, the dynamic range is supposed to span from 1.5 to 3.0 mM Ca²⁺ in 1.0 pF of sample based on the concept of ultrasensitive exhaustive ISOs with precisely defined lower and upper limits of detection. The amount of Ca²⁺ is 1.5 nmol to 3.0 nmol. Since the upper limit of detection is y/z as described above and z = 2, y =6 nmol (y is the amount of ion exchanger, TFPB, and z is the charge of the analyte). The lower limit of detection is (y-x)/z mol as described above. Since y = 6.0 nmol, z = 2, and the lower limit of detection is 1.5 nmol, so x is calculated to be 3 nmol (x is the amount of dye needed). 3 ph of sensing oil in the DOS stock solution with 1 mM Chi and 2 mM NaTFPB will contain 3 nmol Chi and 6 nmol TFPB. When the calcium concentration is 1.5 mM, the color of the optode should be similar to that of the optode exposed to buffer (blue, photo not shown) because most Chi molecules are fully protonated and the protonated Chi is known to be blue. Indeed, the color of the sensing oil is blue for 1.5 mM Ca²⁺ If the extraction of Ca²⁺ is highly effective (so-called exhaustive mode), 3.0 mM Ca²⁺ will make the sensing oil pink. Extraction of 3.0 mM Ca²⁺ into the oil will displace 6 nmol Na⁺ from NaTFPB so all TFPB needs to serve as the counterion of Ca²⁺. Since there is no extra TFPB available, Chi can only stay deprotonated due to the electroneutrality condition. Deprotonated Chi is pinkish/reddish in these formulations. If the extraction is not effective (i.e., only a limited portion of Ca²⁺ is extracted from the sample), there will be extra TFPB available to maintain protonation of some Chi molecules. For example, if only 70% of the 3 mM Ca²⁺ is extracted (2.1 mM, 2.1 nmol), only 4.2 nmol TFPB will be needed to serve as the counterion of the extracted Ca²⁺. Then there is 1.8 nmol TFPB available to maintain protonation of Chi, so approximately 1.8 nmol out of 3 nmol Chi will be protonated. The mixture of the protonated and deprotonated Chi has a color between blue and pink and looks purplish. As can be seen, the inclusion of hexadecane or squalane in the sensing oil ensures the theoretical response (pink for 3 mM Ca²⁺) by maintaining the exhaustive response mode. The response for 3 mM Ca²⁺ becomes obviously smaller (less pink) as the solvent polarity increases (1.9 of squalane ~ 2.1 of hexadecane < 4.1 of DOS < 6.4 of DBP). Even though the secondary solvent (DOS is the primary solvent used to dissolve all sensing chemicals) is only 40 N/N% of the total solvent mixture, its effect on the ISO response is remarkable. When the percentage of squalane or hexadecane in the DOS-hexadecane mixture decreases, the response becomes smaller because of the less effective Ca²⁺ extraction (data not shown). An even high percentage of squalane or hexadecane can further ensure the highly effective extraction of Ca²⁺ (the exhaustive mode) and ensure the theoretical response (data not shown). The improved extraction and response caused by the low-polarity hydrocarbon is a generic phenomenon for various optical reporters and ionophores. Notably, the minimum amount of ionophore required to extract 3 mM Ca²⁺ in 1 pL sample is 9 nmol since each calcium can bind to 3 ionophore. The actually used ionophore is only 10.5 nmol, which is only 17% more than the theoretical amount. In most ISOs, the ionophore is much more than its theoretical amount (typically at least 2 times more. For example, 6 times more ionophore is used to obtain exhaustive response in this publication: Anal. Chem. 2023, 95, 33, 12557- 12564). The current invention substantially reduces the required ionophore while maintaining highly effective extraction of the analyte ions because of the use of the ultralow- polarity hydrocarbon. Hydrocarbons reduce the dielectric constant of the overall sensor matrix and presumably increase the binding constant between the ionophore and the analyte ion. Hydrocarbons may enhance the response of ISOs via other mechanisms. This invention is not associated with a specific mechanism. Notably, it is understandable that the volume of the oil, sample, and diluent can be adjusted and volumes used here are examples.

Using more ionophores is possible to ensure highly effective extraction of the analyte ions and achieve similar exhaustive responses. However, the use of more ionophores actually increases the response of interference ions as well. Although the ionophore binds to the target ions most, it also binds other ions. For example, Ca-II binds to sodium ions to some extent. As shown in Figure 2, the use of more ionophores leads to more interference from Na⁺, which is undesirable.

EXAMPLE 2.

Figure 3 shows the hydrocarbon-enhanced response of an exhaustive potassium ion sensors using potassium ionophore I. The target K⁺ concentration for clinical applications could be 3-6 mM. For 1 pL sample, it is 3 to 6 nmol K⁺. Given the charge of 1 for K⁺, the required TFPB for the 6-mM upper limit of detection is 6 nmol / 1 = 6 nmol. The amount of chromoionophore I (Chi) is ((m-n) x z mole = 3 nmol because m=6 nmol (upper limit of detection), n=3 nmol (lower limit of detection), and z = 1. Therefore, the DOS-based stock oil needs to contain 1 mM Chi and 2 mM NaTFPB if 3 pL of the stock oil is used. The theoretical amount of ionophore to ensure full extraction of 6 mM K⁺ is 6 nmol since the binding stoichiometry of potassium ionophore I (valinomycin) to K⁺ is 1:1. In this example, we used 7.5 nmol potassium ionophore I, which is 2.5 mM potassium ionophore I for a volume of 3 pL stock oil. This amount of ionophore is only 25% more than the theoretical amount. As shown in Figure 3, when the DOS stock oil is mixed with low-polarity hydrocarbons such as squalane, hexadecane, and mineral oil, the response is obviously larger than DOS. The pinkish/reddish color means more deprotonation of Chi and larger response.

EXAMPLE 3.

The pH indicator can be replaced by a cationic dye to indicate the cation extraction process. As shown in Figure 4, when a cationic dye named new methylene blue is used as the optical reporter and the oil phase also contains NaTFPB and Ca-II as the sensing chemicals, the extraction of Ca²⁺ from the aqueous sample to the oil phase causes expulsion of the cationic dye from the oil phase to the sample phase. A decrease in the dye concentration in the oil phase or an increase in the dye concentration in the aqueous phase can be used to indicate the concentration of Ca²⁺ in the aqueous sample. We have formulated the oil to make the response exhaustive with well-defined lower and upper limits of detection (1.0-2.0 mM Ca²⁺ in this example). 4.5 pL of the DOS-based stock oil is mixed with 3 pL of pure DOS or squalane. As can be seen, inclusion of squalane in the oil phase significantly increases the sensitivity (more dye is displaced from the oil phase).

Notably, when a cationic dye is used as the optical reporter, the dye does not have to be first dissolved in the sensor phase. Instead, the dye can be dissolved in the aqueous diluent or be deposited onto the liquid channel as solid. When the sample like blood is aspirated and interacts with the sensing oil, the dye will still partition between the oil and aqueous phases based on the amount of the analyte ions in the sample. In other words, after the two-phase interaction, the amount of dye in the oil phase will be less when the analyte ions in the sample are more and vice versa regardless of where the dye is added in the beginning of the assay.

EXAMPLE 4.

Other than the exhaustive ISOs, we further tested the effect of hexadecane on the response of ISOs in non-exhaustive mode. For ISOs under non-exhaustive mode, a large portion of the analyte (usually more than 50%, but oftentimes more than 80%) stays in the sample phase during the sensing process. The stock oil used here is DOS containing 10 pM chromoionophore III (Chill), 10 pM NaTFPB, and 20 pM K-I (potassium ionophore I). As shown in Figure 5, the dynamic range of the ISO is 0 to 10'² M, which obviously indicates a non-exhaustive response because the amount of TFPB (10‘⁵ M) is far less than the amount of K⁺ in the sample (e.g., 10'³ or 10'² M) and only a very small portion of K⁺ is possible to be extracted into the sensor phase. When 3 ph of this stock oil is mixed with 3 pL pure DOS or hexadecane, the sensor response to 3 pL of pH 7.4 buffer containing 0 - 10'² M KC1 is very different. As shown in Figure 5, the inclusion of hexadecane, again, enhances the sensitivity of the ISO as evidenced by the enhanced fluorescence decrease. The fluorescence decreases because deprotonated Chill has negligible fluorescence compared to its protonated form.

Hexadecane may be preferred in some applications because it has a melting point and boiling point of 18 and 287 °C, respectively. The DOS-hexadecane mixture is liquid at room temperature and also does not evaporate during storage and use. Lighter hydrocarbons have a lower boiling point and therefore are more volatile. Heavier hydrocarbons are more difficult to handle because of the solid state at room temperature although it does not indicate that solid hydrocarbons cannot be used. Actually solid hydrocarbons can be mixed with the solvent to create mixtures that are liquid under the operating temperature. Hexadecane also has a low viscosity of 3.5 mPa- s at 20 °C, which is much lower than that of DOS (20 mPa- s) and therefore increases ion mobilities and accelerates the optode response. However, many hydrocarbons other than hexadecane can be used to enhance the ISO response.

While the invention has been described in terms of its several exemplary embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

Claims

CLAIMS We claim:

1. An ionophore-based ion selective optode, comprising: a sensing matrix comprising at least one ultralow-polarity hydrocarbon that is: an acyclic, saturated, branched or unbranched alkane having a generic formula CnH2n+2 where n ranges from 3 to 30, inclusive; an acyclic, branched or unbranched unsaturated alkene having the generic formula CnH2n where n ranges from 3-30, inclusive; a cyclic, saturated or unsaturated hydrocarbon with the number of carbons ranging from 5 to 30, inclusive; an aromatic hydrocarbon; or a mixture of one or more of these; a solvent that is fully or partially miscible with the ultralow-polarity hydrocarbon; and an optical reporter that is a pH indicator or a charged dye; an ionophore for specific analyte binding and, an ion exchanger.

2. The ionophore-based ion selective optode of claim 1, wherein the at least one ultralow- polarity hydrocarbon is an acyclic, saturated, branched or unbranched alkane; an acyclic, branched or unbranched unsaturated alkene; a cyclic, saturated or unsaturated hydrocarbon; an aromatic hydrocarbon; or a mixture of one or more of these.

3. The ionophore-based ion selective optode of claim 2, wherein the acyclic, saturated, branched or unbranched alkane is squalane.

4. The ionophore-based ion selective optode of claim 2, wherein the acyclic, saturated, branched or unbranched alkane is hexadecane.

5. The ionophore-based ion selective optode of claim 2, wherein the acyclic, saturated, branched or unbranched alkane is mineral oil.

6. The ionophore-based ion selective optode of claim 1, wherein the at least one ultralow- polarity hydrocarbon has a boiling point of at least 60 °C, or at least 150 °C or at least 200

7. The ionophore-based ion selective optode of claim 1, wherein the solvent has at least one electronegative atom.

8. The ionophore-based ion selective optode of claim 7, wherein the at least one electronegative atom is oxygen, nitrogen, sulfur, or chlorine.

9. The ionophore-based ion selective optode of claim 7, wherein the solvent is a plasticizer.

10. The ionophore-based ion selective optode of claim 7, wherein the solvent is a liquid lipid.

11. The ionophore-based ion selective optode of claim 1, wherein a volume ratio of the solvent to the ultralow-polarity hydrocarbon ranges from 10:1 to 1:10, inclusive; or from 4:1 to 1:4, inclusive; or from 2:1 to 1:2, inclusive.

12. The ionophore-based ion selective optode of claim 1, wherein the optical reporter is a hydrophobic pH indicator containing a Nile blue structure.

13. The ionophore-based ion selective optode of claim 1, wherein the optical reporter is a charged dye selected from the group consisting of a rhodamine, a phenothiazine, a benzothiazole, and a cyanine.

14. The ionophore-based ion selective optode of claim 1, wherein the ionophore-based ion selective optode is configured and/or formulated to operate i) under an exhaustive response mode; or ii) under a non-exhaustive response mode.

15. A method for detecting an analyte in an aqueous sample, comprising: mixing the aqueous sample with the sensing matrix of the ionophore-based ion selective optode of claim 1 under conditions sufficient to permit mass transfer of the analyte into the sensing matrix, binding of the analyte to the ionophore in the sensing matrix and a change in a detectable signal of the optical reporter, and detecting the detectable signal.

16. The method of claim 15 wherein the aqueous sample is a biological fluid or an environmental sample.

17. The method of claim 15, wherein the analyte is extracted into the sensing matrix selectively in comparison with other components of the aqueous sample.

18. The method of claim 15, wherein the detectable signal is color, absorbance, or fluorescence.

19. The method of claim 15, wherein the ionophore-based ion selective optode operates under i) an exhaustive response mode; or ii) a non-exhaustive response mode.

20. The method of claim 19, wherein an upper limit of detection (m moles) of the ionophorebased ion selective optode is defined by an amount of ion exchanger in the sensor and a lower limit of detection (n moles) is defined by a difference between the amount of the ion exchanger and an amount of dye.

21. The method of claim 20, wherein the ionophore-based ion selective optode contains (m x z) moles of ion exchanger to maintain the upper limit of detection and ((m-n) x z) moles of optical reporter to maintain the lower limit of detection, wherein z is the absolute value of a charge of the analyte, the ion exchanger has a charge of one, and the optical reporter has a charge of zero or one.