Detection of a target ion via ionophore-based ion-selective sensing using surface enhanced raman spectroscopy (SERS)

Abstract

Disclosed herein are assays, methods, and devices for detecting a target ion via ionophore-based ion-selective sensing using Surface Enhanced Raman Spectroscopy (SERS). For example, disclosed herein are assays for detection of a target ion via SERS, the assays comprising: an ionophore, a nanostructured metal, an ion exchanger, and an indicator, wherein the ionophore comprises a binding portion configured to bind with the target ion, wherein the nanostructured metal is configured to enhance a Raman signal of at least a portion of the indicator, wherein the Raman signal changes when the target ion is bound to the ionophore. Also disclosed herein are methods, for example, comprising contacting any of the assays described herein with a liquid sample; subsequently collecting a surface enhanced Raman signal from the liquid sample and the assay; and processing the surface enhanced Raman signal to determine a property of the liquid sample.

Description

[0001] DETECTION OF A TARGET ION VIA IONOPHORE-BASED ION- SELECTIVE SENSING USING SURFACE ENHANCED RAMAN SPECTROSCOPY (SERS) CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 562,804 filed March 8, 2024, which is hereby incorporated herein by reference in its entirety. BACKGROUND Ionophore-based ion-selective sensing has been explored through techniques such as electronic absorption (UV / Vis), emission spectroscopy (e.g., fluorescence), and ion-selective electrode (ISE) methods. However, the inherent limitations of these conventional approaches have presented substantial challenges when deploying ionophore-based ion-selective sensors for practical applications. Improved ionophore-based ion-selective sensing compositions, devices, and methods are needed. The compositions, methods, and devices discussed herein addresses these and other needs. SUMMARY In accordance with the purposes of the disclosed compositions, methods, and devices as embodied and broadly described herein, the disclosed subject matter relates to assays, methods, and devices for detecting a target ion via ionophore-based ion-selective sensing using Surface Enhanced Raman Spectroscopy (SERS). For example, disclosed herein are assays for detection of a target ion via Surfaced Enhanced Raman Spectroscopy (SERS), the assays comprising: an ionophore, a nanostructured metal, an ion exchanger, and an indicator, such as a chromoionophore, wherein the ionophore comprises a binding portion configured to bind with the target ion, wherein the nanostructured metal is configured to enhance a Raman signal of at least a portion of the indicator, wherein the Raman signal changes when the target ion is bound to the ionophore. In some examples, least a portion of the indicator changes orientation in the presence of the target ion (e.g., when the target ion is bound to the ionophore), and said change in orientation results in a detectable change in the Raman signal. In some examples, the indicator undergoes a change in protonation and orientation when the target ion is bound to the ionophore, which results in a detectable change in the Raman signal. In some examples, the ion exchanger comprises tetrakis[3,5- bis(trifluoromethyl)phenyl]borate (TFPB). In some examples, the ion exchanger comprises sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), potassium tetrakis[3,5- bis(trifluoromethyl)phenyl]borate (KTFPB), or a combination thereof. In some examples, the indicator is a chromoionophore. In some examples, the chromoionophore comprises chromoionophore I, chromoionophore III, or a combination thereof. In some examples, the ionophore selectively binds with the target ion. In some examples, the ionophore is selected in view of the target ion. In some examples, the ionophore comprises a calcium ionophore, a sodium ionophore, a potassium ionophore, a magnesium ionophore, a lithium ionophore, a copper(II) ionophore, a lead ionophore, an ammonium ionophore, a chloride ionophore, a carbonate ionophore, a nitrate ionophore, or a combination thereof. In some examples, the ionophore comprises calcium ionophore I, calcium ionophore II, calcium ionophore IV, calcium ionophore V, sodium ionophore III, sodium ionophore IV, sodium ionophore VI, sodium ionophore X, potassium ionophore I , potassium ionophore III, magnesium ionophore I, magnesium ionophore III, magnesium ionophore IV, magnesium ionophore VII, lithium ionophore VI, lithium ionophore VIII, copper(II) ionophore I, lead ionophore IV, ammonium ionophore I, chloride ionophore II, chloride ionophore III, chloride ionophore IV, carbonate ionophore VII, nitrate Ionophore VI, or a combination thereof. In some examples, the ionophore comprises a calcium ionophore, a sodium ionophore, a potassium ionophore, or a combination thereof. In some examples, the ionophore comprises calcium ionophore I, sodium ionophore X, potassium ionophore I , or a combination thereof. In some examples, the ionophore comprises calcium ionophore I, sodium ionophore X, or a combination thereof. In some examples, the target ion comprises a cation, an anion, a polyion, a zwitterion, or a combination thereof. In some examples, the target ion comprises a cation, an anion, or a combination thereof. In some examples, the target ion comprises a sodium ion, a calcium ion, a potassium ion, a magnesium ion, a lithium ion, an ammonium ion, a chloride ion, a carbonate ion, a nitrate ion, a copper(II) ion, a lead ion, or a combination thereof. In some examples, the target ion comprises an anion. In some examples, the target ion comprises a chloride ion, a carbonate ion, a nitrate ion, or a combination thereof. In some examples, the target ion comprises a cation. In some examples, the target ion comprises a sodium ion, a calcium ion, a potassium ion, a magnesium ion, a lithium ion, an ammonium ion, a copper(II) ion, a lead ion, or a combination thereof. In some examples, the target ion comprises a metal ion, such as an alkali metal ion, an alkaline earth metal ion, a transition metal ion, a heavy metal ion, or a combination thereof. In some examples, the target ion comprises an alkali metal ion, an alkaline earth metal ion, or a combination thereof. In some examples, the target ion comprises a sodium ion, a calcium ion, a potassium ion, a magnesium ion, a lithium ion, or a combination thereof. In some examples, the target ion comprises a sodium ion, a calcium ion, a potassium ion, or a combination thereof. In some examples, the target ion comprises a sodium ion, a calcium ion, or a combination thereof. In some examples, the target ion comprises a transition metal ion, a heavy metal ion, or a combination thereof. In some examples, the target ion comprises a copper(II) ion, a lead ion, or a combination thereof. In some examples, the nanostructured metal comprises a metal selected from the group consisting of Ru, Rh, Pd, Os, Ir, Pt, Au, Ag, Cu, Al, and combinations thereof. In some examples, the nanostructured metal comprises a metal selected from the group consisting of Pt, Au, Ag, Cu, Al, and combination thereof. In some examples, the nanostructured metal comprises a metal selected from the group consisting of Au, Ag, and combinations thereof. In some examples, the nanostructured metal comprises Ag. In some examples, the nanostructured metal comprises a plurality of metal particles. In some examples, the metal particles have an isotropic shape or an anisotropic shape. In some examples, the metal particles have an average particle size of from 5 nanometers (nm) to 1 micrometer (micron, µm). In some examples, the nanostructured metal comprises a metal modified with a nanostructure. In some examples, the nanostructured metal comprises a film or layer modified with a nanostructure, such as a film or layer modified with a plurality of metal particles. In some examples, the nanostructured metal comprises a two-dimensional material modified with a plurality of metal particles. In some examples, the two-dimensional material comprises a two-dimensional transition metal carbide, a two-dimensional transition metal nitride, a two-dimensional transition metal carbonitride, or a combination thereof. In some examples, nanostructured metal comprises a two-dimensional transition metal carbide, a two- dimensional transition metal nitride, and / or a two-dimensional transition metal carbonitride modified with a plurality of metal particles. In some examples, the nanostructured metal comprises a two-dimensional material modified with a plurality of metal particles; the two- dimensional material comprising a two-dimensional transition metal carbide, a two-dimensional transition metal nitride, a two-dimensional transition metal carbonitride, or a combination thereof; and the plurality of metal particles comprising Au, Ag, or a combination thereof. In some examples, the ionophore, the nanostructured metal, the ion-exchanger, the indicator, or a combination thereof are at least partially dispersed in a solvent. In some examples, the ionophore, the nanostructured metal, the ion-exchanger, the indicator, or a combination thereof are disposed on a substrate. In some examples, the ionophore, the ion-exchanger, the indicator, or a combination thereof is attached to a surface of the nanostructured metal, such that the assay comprises a modified nanostructured metal comprising the nanostructured metal having the ionophore, the ion-exchanger, the indicator, or a combination thereof attached to the surface thereof. In some examples, the modified nanostructured metal is at least partially dispersed in a solvent. In some examples, the modified nanostructured metal is disposed on a substrate. Also disclosed herein are methods of making any of the assays described herein. Also disclosed herein are methods for detecting a target ion via ionophore-based ion- selective sensing using Surface Enhanced Raman Spectroscopy (SERS). In some examples, method comprises: contacting any of the assays described herein with a liquid sample; subsequently collecting a surface enhanced Raman signal from the liquid sample and the assay; and processing the surface enhanced Raman signal to determine a property of the liquid sample. In some examples, the liquid sample comprises a bodily fluid. In some examples, the liquid sample comprises an environmental sample. In some examples, the method is performed in vitro. In some examples, the method is performed in vivo. In some examples, the property of the liquid sample comprises the presence of the target ion in the liquid sample, the concentration of the target ion in the liquid sample, the identity of the target ion, or a combination thereof. Also disclosed herein are methods of use of any of the assays described herein or any of the methods described herein. In some examples, the assay or method is used for biological research, medical diagnostics, environmental analysis, pharmaceutical research, food and / or beverage industry, quality control, or a combination thereof. In some examples, the assay or method is used for bioimaging. In some examples, the assay or method is used for Intracellular ion sensing, extracellular ion sensing, or a combination thereof. Also disclosed herein are devices comprising: a receptacle configured to at least partially contain any of the assays disclosed herein; an excitation source; a detector; and a computing device; wherein the receptacle is further configured to position the assay such that the assay is in optical communication with the excitation source and the detector; and wherein the computing device is configured to receive and process an electromagnetic signal from the detector; wherein, when the device is assembled together with a liquid sample, then: the receptacle is configured to at least partially contain the assay in contact with the liquid sample and position the assay in contact with the liquid sample such that the assay and the liquid sample are in optical communication with the excitation source and the detector; the excitation source is configured to apply an excitation signal to the liquid sample and the assay; the detector is configured to collect a surface enhanced Raman signal from the liquid sample and the assay; and the computing device is configured to process the surface enhanced Raman signal to determine a property of the liquid sample. In some examples, the excitation source and / or the detector comprise a Raman spectrometer. In some examples, the device is further configured to output the property of the liquid sample and / or a feedback signal based on the property of the liquid sample. In some examples, the feedback signal comprises haptic feedback, auditory feedback, visual feedback, or a combination thereof. In some examples, the property of the liquid sample comprises the presence of the target ion in the liquid sample, the concentration of the target ion in the liquid sample, the identity of the target ion, or a combination thereof. In some examples, the device comprises a microfluidic device. Additional advantages of the disclosed compositions, devices, and methods will be set forth in part in the description which follows, and in part will be obvious from the description. The advantages of the disclosed compositions, devices, and methods will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosed devices and methods, as claimed. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE FIGURES The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects of the disclosure, and together with the description, serve to explain the principles of the disclosure. Figure 1A-Figure 1C. SERS spectra of the calcium ionophore-based optode (Figure 1A), protonation degree (1-^) and SERS intensity at 592 cm−1of chromoionophore I when exposed to various concentrations of Ca2+(Figure 1B). SERS pH response of the chromoionophore I (Figure 1C). Figure 2. Normalized spectra for Raman (blue) and SERS (red) of chromoionophore I and proportional spectrum for protonated chromoionophore I (black). The prominent SERS peaks at 511 cm-1(A), 592 cm-1(B), 672 cm-1(C), 1184 cm-1(D), and 1638 cm-1(E) can be attributed to the aromatic ring deformation. The modes in the range of 1255-1495 cm-1correspond to the vibration of the carbon chain. Figure 3. The dipole direction (red arrow) of modes A, B, and E in relation to the aromatic plane. The dashed arrow represents the direction perpendicular to the aromatic plane (out-of-plane), and the solid arrow denotes the direction within the aromatic plane. Figure 4A-Figure 4B. Correlations between the SERS intensity at 595 cm-1and pH (Figure 4A), as well as Ca2+(Figure 4B), using chromoionophore III as an indicator. Figure 5A-Figure 5B. SERS spectra of the sodium ionophore-based optode in the presence of various concentrations of Na+(Figure 5A). Response curve depicting its reaction to Na+and interfering cations (Figure 5B). Figure 6. Chromoionophore reorientation is reported to be associated with its protonation state modulated by the target cation within an ionophore-based ion-selective sensing framework. Figure 7: SEM image of AgNPs treated with HCl on filter paper (scale bar 500 nm). Figure 8: Sensing principle of the ion-selective optode containing chromoionophore, ionophore, and ion exchanger. Figure 9: SERS spectra of the calcium ionophore-based optode exposed to pH levels ranging from 3 to 12. Figure 10: Absorption spectra of protonated chromoionophore I (A), deprotonated chromoionophore I (B), protonated chromoionophore III (C), and deprotonated chromoionophore III (D). Figure 11: The approximate distances of 0.717 nm and 0.505 nm across the aromatic plane of the chromoionophore I were acquired from ChemSpider, a database maintained by the UK Royal Society of Chemistry. Figure 12: The dipole direction (red arrow) of modes C and D relative to the aromatic plane. The solid arrow denotes the direction within the aromatic plane. Figure 13: Zeta potential distribution of the Ag / AgCl nanocolloids utilized in the preparation of the SERS substrate. Figure 14: SERS spectra of the calcium ionophore-based optode using chromoionophore III as indicator when exposed to various concentrations of Ca2+. Figure 15A-Figure 15B: Response curves of the calcium ionophore-based optode's reaction to various concentrations of Ca2+and interfering cations using chromoionophore I (Figure 15A) and chromoionophore III (Figure 15B) as indicators, respectively. Figure 16. Schematic diagram of ion detection methods. Figure 17A. Schematic illustration of the preparation of the Ti3C2 MXene-AgNPs-based SERS substrate. Figure 17B. Schematic illustration of the sensing mechanism of the SERS response for target ion. Figure 18. Cyclic voltammogram (CV) curves of the prepared Ti3C2 MXene nanosheets (Mxene), [Ag(NH3)2]+solution (Ag), and their mixture (MXene-Ag). Figure 19A- Figure 19D. TEM image ( Figure 19A) and AFM ( Figure 19B) of Ti3C2MXene nanosheet. SEM images of the fabricated Ti3C2MXene-AgNPs SERS substrate ( Figure 19C) and Ag deposition ( Figure 19D) on FTO conductive glass. Figure 20. SERS spectrum of Rhodamine 6G on the prepared Ti3C2MXene-AgNPs substrate and the Raman spectrum of Rhodamine 6G powder. Figure 21. UV−vis spectra of protonated CHI (a), protonated CHI and Ti3C2 MXene- CHI mixture (b), deprotonated CHI (c), and the mixture of deprotonated CHI and Ti3C2 MXene solution (d). Figure 22A- Figure 22D. SERS spectra of the Ti3C2 MXene-AgNPs-based Ca²^ sensing platform at various Ca²^ concentrations in a pH 7.4 Tris-HCl buffer ( Figure 22A). SERS intensity at 592 cm-1across different ion concentrations ( Figure 22B). The theoretical response curves ( Figure 22C) and calibration of the experimental SERS intensity at 592 cm^¹ of the ionophore-based ion selective system ( Figure 22D) with the CHI, TFPB^, and calcium ionophore ratios set at 1:1:3 and 1:2:3. Figure 23A- Figure 23B. SERS response curves of Ti3C2MXene-AgNPs-based SERS sensing platform for Na+( Figure 23A) and K+( Figure 23B) to various concentrations of ions at pH 7.4. Figure 24A. SERS spectra of Rhodamine 6G on the Ti3C2 MXene-AgNPs substrate. Figure 24B. Histograms of the SERS peak intensity at 1509 cm-1acquired from 30 spots on the Rhodamine 6G on the Ti3C2 MXene-AgNPs substrate. Figure 25. SERS spectra of the Ti3C2 MXene-AgNPs-based Ca²^ sensing platform at various Ca²^ concentrations in a pH 7.4 Tris-HCl buffer when CHI, TFPB^, and calcium ionophore ratios set at 1:2:3. Figure 26A. SERS spectra of the Ti3C2MXene-AgNPs-based Na^ sensing platform in the presence of various Na^ concentrations in a pH 7.4 Figure 26B. SERS spectra of the Ti3C2MXene-AgNPs-based K^ sensing platform exposed to various K^ concentrations in a pH 7.4 Figure 27A- Figure 27F. The standard curve for measuring Ca2+and Na+in real samples: Ca2+in human blood serum ( Figure 27A), Na+in human blood serum ( Figure 27B), Ca2+in river water ( Figure 27C), Na+in river water ( Figure 27D), Ca2+in estuary water ( Figure 27E), Na+in estuary water ( Figure 27F). DETAILED DESCRIPTION The compositions, methods, and devices described herein may be understood more readily by reference to the following detailed description of specific aspects of the disclosed subject matter and the Examples included therein. Before the present compositions, methods, and devices are disclosed and described, it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Also, throughout this specification, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the disclosed matter pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. General Definitions In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings. Throughout the description and claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps. As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an agent” includes mixtures of two or more such agents, reference to “the component” includes mixtures of two or more such components, and the like. “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. By “about” is meant within 5% of the value, e.g., within 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Values can be expressed herein as an “average” value. “Average” generally refers to the statistical mean value. By “substantially” is meant within 5%, e.g., within 4%, 3%, 2%, or 1%. “Exemplary” means “an example of” and is not intended to convey an indication of a preferred or ideal embodiment. “Such as” is not used in a restrictive sense, but for explanatory purposes. It is understood that throughout this specification the identifiers “first” and “second” are used solely to aid in distinguishing the various components and steps of the disclosed subject matter. The identifiers “first” and “second” are not intended to imply any particular order, amount, preference, or importance to the components or steps modified by these terms. References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound. A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included. The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context. As used herein, by a “subject” is meant an individual. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. The term “inhibit” refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This can also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control. By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. For example, the terms “prevent” or “suppress” can refer to a treatment that forestalls or slows the onset of a disease or condition or reduced the severity of the disease or condition. Thus, if a treatment can treat a disease in a subject having symptoms of the disease, it can also prevent or suppress that disease in a subject who has yet to suffer some or all of the symptoms. The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. The term “artificial intelligence” is defined herein to include any technique that enables one or more computing devices or comping systems (i.e., a machine) to mimic human intelligence. Artificial intelligence (AI) includes, but is not limited to, knowledge bases, machine learning, representation learning, and deep learning. The term “machine learning” is defined herein to be a subset of AI that enables a machine to acquire knowledge by extracting patterns from raw data. Machine learning techniques include, but are not limited to, logistic regression, support vector machines (SVMs), decision trees, Naïve Bayes classifiers, and artificial neural networks. The term “representation learning” is defined herein to be a subset of machine learning that enables a machine to automatically discover representations needed for feature detection, prediction, or classification from raw data. Representation learning techniques include, but are not limited to, autoencoders. The term “deep learning” is defined herein to be a subset of machine learning that that enables a machine to automatically discover representations needed for feature detection, prediction, classification, etc. using layers of processing. Deep learning techniques include, but are not limited to, artificial neural network or multilayer perceptron (MLP). Machine learning models include supervised, semi-supervised, and unsupervised learning models. In a supervised learning model, the model learns a function that maps an input (also known as feature or features) to an output (also known as target or target) during training with a labeled data set (or dataset). In an unsupervised learning model, the model learns a function that maps an input (also known as feature or features) to an output (also known as target or target) during training with an unlabeled data set. In a semi-supervised model, the model learns a function that maps an input (also known as feature or features) to an output (also known as target or target) during training with both labeled and unlabeled data. Chemical Definitions Unless otherwise defined, 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. The organic moieties mentioned when defining variable positions within the general formulae described herein (e.g., the term “halogen”) are collective terms for the individual substituents encompassed by the organic moiety. The prefix Cn-Cm preceding a group or moiety indicates, in each case, the possible number of carbon atoms in the group or moiety that follows. The term “ion,” as used herein, refers to any molecule, portion of a molecule, cluster of molecules, molecular complex, moiety, or atom that contains a charge (positive, negative, or both at the same time within one molecule, cluster of molecules, molecular complex, or moiety (e.g., zwitterions)) or that can be made to contain a charge. Methods for producing a charge in a molecule, portion of a molecule, cluster of molecules, molecular complex, moiety, or atom are disclosed herein and can be accomplished by methods known in the art, e.g., protonation, deprotonation, oxidation, reduction, alkylation, acetylation, esterification, de-esterification, hydrolysis, etc. The term “anion” is a type of ion and is included within the meaning of the term “ion.” An “anion” is any molecule, portion of a molecule (e.g., zwitterion), cluster of molecules, molecular complex, moiety, or atom that contains a net negative charge or that can be made to contain a net negative charge. The term “anion precursor” is used herein to specifically refer to a molecule that can be converted to an anion via a chemical reaction (e.g., deprotonation). The term “cation” is a type of ion and is included within the meaning of the term “ion.” A “cation” is any molecule, portion of a molecule (e.g., zwitterion), cluster of molecules, molecular complex, moiety, or atom, that contains a net positive charge or that can be made to contain a net positive charge. The term “cation precursor” is used herein to specifically refer to a molecule that can be converted to a cation via a chemical reaction (e.g., protonation or alkylation). Assays Disclosed herein are assays, methods, and devices for detecting a target ion via ionophore-based ion-selective sensing using Surface Enhanced Raman Spectroscopy (SERS). For example, disclosed herein are assays for detection of a target ion via Surfaced Enhanced Raman Spectroscopy (SERS), the assay comprising: an ionophore, a nanostructured metal, an ion exchanger, and an indicator, such as a chromoionophore, wherein the ionophore comprises a binding portion configured to bind with the target ion, wherein the nanostructured metal is configured to enhance a Raman signal of at least a portion of the indicator, wherein the Raman signal changes when the target ion is bound to the ionophore. In some examples, at least a portion of the indicator changes orientation in the presence of the target ion (e.g., when the target ion is bound to the ionophore), and said change in orientation results in a detectable change in the Raman signal. In some examples, the indicator undergoes a change in protonation and orientation when the target ion is bound to the ionophore, which results in a detectable change in the Raman signal. In some examples, the assay comprises the ion exchanger for charge balancing. Any suitable ion exchanger can be used. For example, the ion exchanger can comprise tetrakis[3,5- bis(trifluoromethyl)phenyl]borate (TFPB). In some examples, the ion exchanger can comprise sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), potassium tetrakis[3,5- bis(trifluoromethyl)phenyl]borate (KTFPB), or a combination thereof. In some examples, the indicator comprises a chromoionophore. Any suitable chromoionophore can be used. Examples of chromoionophores include, but are not limited to, chromoionophore I, chromoionophore II, chromoionophore III, chromoionophore IV, chromoionophore V, chromoionophore VI, chromoionophore VII, chromoionophore VIII, chromoionophore XI, chromoionophore XVII, and combinations thereof. In some examples, the chromoionophore comprises chromoionophore I, chromoionophore III, or a combination thereof. The ionophore can comprise any suitable ionophore. In some examples, the ionophore selectively binds with the target ion. In some examples, the ionophore is selected in view of the target ion. In some examples, the ionophore comprises a calcium ionophore, a sodium ionophore, a potassium ionophore, a magnesium ionophore, a lithium ionophore, a copper(II) ionophore, a lead ionophore, an ammonium ionophore, a chloride ionophore, a carbonate ionophore, a nitrate ionophore, or a combination thereof. In some examples, the ionophore comprises a calcium ionophore, a sodium ionophore, a potassium ionophore, or a combination thereof. In some examples, the ionophore comprises calcium ionophore I, calcium ionophore II, calcium ionophore IV, calcium ionophore V, sodium ionophore III, sodium ionophore IV, sodium ionophore VI, sodium ionophore X, potassium ionophore I , potassium ionophore III, magnesium ionophore I, magnesium ionophore III, magnesium ionophore IV, magnesium ionophore VII, lithium ionophore VI, lithium ionophore VIII, copper(II) ionophore I, lead ionophore IV, ammonium ionophore I, chloride ionophore II, chloride ionophore III, chloride ionophore IV, carbonate ionophore VII, nitrate Ionophore VI, or a combination thereof. In some examples, the ionophore comprises calcium ionophore I, sodium ionophore X, potassium ionophore I , or a combination thereof. In some examples, the ionophore comprises calcium ionophore I, sodium ionophore X, or a combination thereof. The target ion can comprise any suitable ion. In some examples, the target ion comprises a cation, an anion, a polyion, a zwitterion, or a combination thereof. In some examples, the target ion comprises a cation, an ion, or a combination thereof. In some examples, the target ion comprises a sodium ion, a calcium ion, a potassium ion, a magnesium ion, a lithium ion, an ammonium ion, a chloride ion, a carbonate ion, a nitrate ion, a copper(II) ion, a lead ion, or a combination thereof. In some examples, the target ion comprises an anion. In some examples, the target ion comprises a chloride ion, a carbonate ion, a nitrate ion, or a combination thereof. In some examples, the target ion comprises a cation. In some examples, the target ion comprises a sodium ion, a calcium ion, a potassium ion, a magnesium ion, a lithium ion, an ammonium ion, a copper(II) ion, a lead ion, or a combination thereof. In some examples, the target ion comprises a metal ion, such as an alkali metal ion, an alkaline earth metal ion, a transition metal ion, a heavy metal ion, or a combination thereof. In some examples, the target ion comprises an alkali metal ion, an alkaline earth metal ion, or a combination thereof. In some examples, the target ion comprises a sodium ion, a calcium ion, a potassium ion, a magnesium ion, a lithium ion, or a combination thereof. In some examples, the target ion comprises a sodium ion, a calcium ion, a potassium ion, or a combination thereof. In some examples, the target ion comprises a sodium ion, a calcium ion, or a combination thereof. In some examples, the target ion comprises a transition metal ion, a heavy metal ion, or a combination thereof. In some examples, the target ion comprises a copper(II) ion, a lead ion, or a combination thereof. As used herein, “nanostructured” means any structure with one or more nanosized features. A nanosized feature can be any feature with at least one dimension less than 1 micrometer (µm) in size. For example, a nanosized feature can comprise a nanowire, nanotube, nanoparticle, nanopore, and the like, or combinations thereof. As such, the nanostructured metal can comprise, for example, a nanowire, nanotube, nanoparticle, nanopore, or a combination thereof. In some examples, the nanostructured metal can comprise a metal that is not nanosized but has been modified with a nanowire, nanotube, nanoparticle, nanopore, or a combination thereof. The nanostructured metal can comprise any metal suitable for Surface Enhanced Raman Spectroscopy (SERS). The nanostructured metal can comprise, for example, a metal selected from the group consisting of Ru, Rh, Pd, Os, Ir, Pt, Au, Ag, Cu, Al, and combinations thereof. In some examples, the nanostructured metal comprises a metal selected from the group consisting of Pt, Au, Ag, Cu, Al, and combination thereof. In some examples, the nanostructured metal comprises a metal selected from the group consisting of Au, Ag, and combinations thereof. In some examples, the nanostructured metal comprises Ag. In some examples, the nanostructured metal comprises a plurality of metal particles. In some examples, the nanostructured metal comprises a plurality of metal particles, wherein the metal selected from the group consisting of Ru, Rh, Pd, Os, Ir, Pt, Au, Ag, Cu, Al, and combinations thereof. In some examples, the nanostructured metal comprises a plurality of metal particles, wherein the metal selected from the group consisting of Pt, Au, Ag, Cu, Al, and combination thereof. In some examples, the nanostructured metal comprises a plurality of metal particles, wherein the metal selected from the group consisting of Au, Ag, and combinations thereof. In some examples, the nanostructured metal comprises a plurality of metal particles, wherein the metal is Ag (e.g., a plurality of Ag particles). The plurality of metal particles can comprise particles of any shape, such as a polyhedron (e.g., a platonic solid, a prism, a pyramid), a stellated polyhedron (e.g., a star), a cylinder, a hemicylinder, an elliptical cylinder, a hemi-elliptical cylinder, a sphere, a hemisphere, a cone, a semicone, etc. In some examples, the plurality of metal particles can have a regular shape, an irregular shape, an isotropic shape, an anisotropic shape, or a combination thereof. In some examples, the plurality of metal particles can have an isotropic shape or an anisotropic shape. In some examples, the plurality of metal particles can have a shape that is substantially spherical. The plurality of metal particles can have an average particle size. “Average particle size” and “mean particle size” are used interchangeably herein, and generally refer to the statistical mean particle size of the particles in a population of particles. For example, the average particle size for a plurality of particles with a substantially spherical shape can comprise the average diameter of the plurality of particles. For a particle with a substantially spherical shape, the diameter of a particle can refer, for example, to the hydrodynamic diameter. As used herein, the hydrodynamic diameter of a particle can refer to the largest linear distance between two points on the surface of the particle. Mean particle size can be measured using methods known in the art, such as evaluation by scanning electron microscopy, transmission electron microscopy, and / or dynamic light scattering. The plurality of metal particles can, for example, have an average particle size of 5 nanometers (nm) or more (e.g., 10 nm or more, 15 nm or more, 20 nm or more, 25 nm or more, 30 nm or more, 35 nm or more, 40 nm or more, 45 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, 125 nm or more, 150 nm or more, 175 nm or more, 200 nm or more, 225 nm or more, 250 nm or more, 300 nm or more, 350 nm or more, 400 nm or more, 450 nm or more, 500 nm or more, 600 nm or more, 700 nm or more, 800 nm or more, or 900 nm or more). In some examples, the plurality of metal particles can have an average particle size of 1 micrometer (micron, µm) or less (e.g., 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 225 nm or less, 200 nm or less, 175 nm or less, 150 nm or less, 125 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 45 nm or less, 40 nm or less, 35 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, or 10 nm or less). The average particle size of the plurality of metal particles can range from any of the minimum values described above to any of the maximum values described above. For example, the plurality of metal particles have an average particle size of from 5 nanometers (nm) to 1 micrometer (micron, µm) (e.g., from 5 nm to 500 nm, from 500 nm to 1 µm, from 5 nm to 200 nm, from 200 nm to 400 nm, from 400 nm to 600 nm, from 600 nm to 800 nm, from 800 nm to 1 µm, from 10 nm to 1 µm, from 5 nm to 900 nm, from 10 nm to 900 nm, from 5 nm to 400 nm, or from 20 nm to 200 nm). In some examples, the plurality of metal particles can be substantially monodisperse. “Monodisperse” and “homogeneous size distribution,” as used herein, and generally describe a population of particles where all of the particles are the same or nearly the same size. As used herein, a monodisperse distribution refers to particle distributions in which 80% of the distribution (e.g., 85% of the distribution, 90% of the distribution, or 95% of the distribution) lies within 25% of the median particle size (e.g., within 20% of the median particle size, within 15% of the median particle size, within 10% of the median particle size, or within 5% of the median particle size). In some examples, the nanostructured metal comprises a metal modified with a nanostructure, such as a metal film or layer with one or more nanosized features. In some examples, the nanostructured metal comprises a film or layer modified with a nanostructure, such as a film or layer modified with a plurality of metal particles. For example, the nanostructured metal can comprise a two-dimensional material modified with a plurality of metal particles. Examples of two-dimensional materials include, but are not limited to, two- dimensional transition metal carbides, nitrides, and carbonitrides. In some examples, the nanostructured metal comprises a two-dimensional transition metal carbide, nitride, and / or carbonitride modified with a plurality of metal particles. In some examples, the nanostructured metal comprises a two-dimensional transition metal carbide, nitride, and / or carbonitride modified with a plurality of metal particles, wherein the plurality of metal particles comprise Ru, Rh, Pd, Os, Ir, Pt, Au, Ag, Cu, Al, or a combination thereof. In some examples, the nanostructured metal comprises a two-dimensional transition metal carbide, nitride, and / or carbonitride modified with a plurality of metal particles, wherein the plurality of metal particles comprise Pt, Au, Ag, Cu, Al, or a combination thereof. In some examples, the nanostructured metal comprises a two-dimensional transition metal carbide, nitride, and / or carbonitride modified with a plurality of metal particles, wherein the plurality of metal particles comprise Au, Ag, or a combination thereof. In some examples, the nanostructured metal comprises a two-dimensional transition metal carbide, nitride, and / or carbonitride modified with a plurality of metal particles, wherein the plurality of metal particles comprise Ag (e.g., a plurality of Ag particles). In some examples, the ionophore, the nanostructured metal, the ion-exchanger, the indicator, or a combination thereof are at least partially dispersed in a solvent. The solvent can, for example, comprise water, ethylene glycol, polyethylene glycol, glycerol, alkane diol, ethanol, methanol, propanol, isopropanol, dimethyl sulfoxide (DMSO), acetonitrile, methylene chloride, tetrahydrofuran (THF), or combinations thereof. In some examples, the solvent comprises water. In some examples, the ionophore, the nanostructured metal, the ion-exchanger, the indicator, or a combination thereof are disposed on a substrate. Examples of substrates include, but are not limited to, glass, quartz, silicon, silicon dioxide, nitrides (e.g., silicon nitride), polycarbonate, polydimethylsiloxane (PDMS), a cellulosic / lignin based substrate (e.g., wood, paper (such as chromatography paper), etc.), and combinations thereof. In some examples, the substrate comprises glass, such as conductive glass. In some examples, the substrate comprises conductive glass, such as fluoride tin oxide (FTO) conductive glass. In some examples, the ionophore, the ion-exchanger, the indicator, or a combination thereof is attached to a surface of the nanostructured metal, such that the assay comprises a modified nanostructured metal comprising the nanostructured metal having the ionophore, the ion-exchanger, the indicator, or a combination thereof attached to the surface thereof. In some examples, the modified nanostructured metal is at least partially dispersed in a solvent. The solvent can, for example, comprise water, ethylene glycol, polyethylene glycol, glycerol, alkane diol, ethanol, methanol, propanol, isopropanol, dimethyl sulfoxide (DMSO), acetonitrile, methylene chloride, or combinations thereof. In some examples, the solvent comprises water. In some examples, the modified nanostructured metal is disposed on a substrate. Examples of substrates include, but are not limited to, glass, quartz, silicon, silicon dioxide, nitrides (e.g., silicon nitride), polycarbonate, polydimethylsiloxane (PDMS), a cellulosic / lignin based substrate (e.g., wood, paper (such as chromatography paper), etc.), and combinations thereof. In some examples, the substrate comprises glass, such as conductive glass. In some examples, the substrate comprises conductive glass, such as fluoride tin oxide (FTO) conductive glass. Methods Also disclosed herein are methods of making any of the assays disclosed herein. Also disclosed herein are methods of use of any of the assays disclosed herein. Also disclosed herein are methods for detecting a target ion via ionophore-based ion- selective sensing using Surface Enhanced Raman Spectroscopy (SERS). In some examples, the methods can comprise: contacting any of the assays disclosed herein with a liquid sample; subsequently collecting a surface enhanced Raman signal from the liquid sample and the assay; and processing the surface enhanced Raman signal to determine a property of the liquid sample. The liquid sample can comprise any liquid sample of interest. By way of example the liquid sample can comprise a bodily fluid. "Bodily fluid", as used herein, refers to a fluid composition obtained from or located within a human or animal subject. Bodily fluids include, but are not limited to, urine, whole blood, blood plasma, serum, tears, semen, saliva, sputum, exhaled breath, nasal secretions, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, interstitial fluid, lymph fluid, meningeal fluid, amniotic fluid, glandular fluid, feces, perspiration, mucous, vaginal or urethral secretion, cerebrospinal fluid, and transdermal exudate. Bodily fluid also includes experimentally separated fractions of all of the preceding solutions, as well as mixtures containing homogenized solid material, such as feces, tissues, and biopsy samples. In some examples, the liquid sample comprises a bodily fluid and the bodily fluid comprises saliva, sputum, nasal secretions, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, mucous, blood, serum, sweat, urine, cerebrospinal fluid, or a combination thereof. In some examples, the liquid sample comprises a bodily fluid and the bodily fluid comprises saliva, sputum, nasal secretions, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, mucous, or a combination thereof. In some examples, the liquid sample comprises a bodily fluid, and the bodily fluid comprises serum. In some examples, the liquid sample comprises an environmental sample. For example, the environmental sample can comprise water, such as river water, estuary water, sea water, lake water, contaminated water, tap water, etc. In some examples, the method is performed in vitro. In some examples, the method is performed in vivo. In some examples, the property of the liquid sample comprises the presence of the target ion in the liquid sample, the concentration of the target ion in the liquid sample, the identity of the target ion, or a combination thereof. Also disclosed herein are methods of use of any of the assays and / or any of the methods disclosed herein. In some examples, the assay or method is used for biological research, medical diagnostics, environmental analysis, pharmaceutical research, food and / or beverage industry, quality control, or a combination thereof. In some examples, the assay or method is used for bioimaging. In some examples, the assay or method is used for Intracellular ion sensing, extracellular ion sensing, or a combination thereof. Devices Also disclosed herein are devices, for example for performing any of the methods described herein. For example, also disclosed herein are devices comprising: a receptacle configured to at least partially contain any of the assays disclosed herein; an excitation source; a detector; and a computing device. The receptacle is further configured to position the assay such that the assay is in optical communication with the excitation source and the detector. The computing device is configured to receive and process an electromagnetic signal from the detector. When the device is assembled together with a liquid sample, then: the receptacle is configured to at least partially contain the assay in contact with the liquid sample and position the assay in contact with the liquid sample such that the assay and the liquid sample are in optical communication with the excitation source and the detector; the excitation source is configured to apply an excitation signal to the liquid sample and the assay; the detector is configured to collect a surface enhanced Raman signal from the liquid sample and the assay; and the computing device is configured to process the surface enhanced Raman signal to determine a property of the liquid sample. The excitation source and / or the detector can, for example, comprise a Raman spectrometer. In some examples, the device is further configured to output the property of the liquid sample and / or a feedback signal based on the property of the liquid sample. In some examples, the device can further comprise one or more output devices (e.g., a display, speakers, printer, LED, etc.) configured to output the property of the liquid sample and / or a feedback signal based on the property of the liquid sample. The feedback signal can, for example, comprise haptic feedback, auditory feedback, visual feedback, or a combination thereof. In some examples, the property of the liquid sample comprises the presence of the target ion in the liquid sample, the concentration of the target ion in the liquid sample, the identity of the target ion, or a combination thereof. In some examples, the device comprises a microfluidic device. A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. The examples below are intended to further illustrate certain aspects of the devices and methods described herein, and are not intended to limit the scope of the claims. EXAMPLES The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of measurement conditions, e.g., component concentrations, temperatures, pressures and other measurement ranges and conditions that can be used to optimize the described process. Example 1 - Cations Induced Chromoionophore Reorientation and Its Application for Cation Selective SERS Detection Abstract: Chromoionophore reorientation is reported to be associated with its protonation state modulated by the target cation within an ionophore-based ion-selective sensing framework (Figure 6). Surface-enhanced Raman spectroscopy (SERS) was utilized to monitor this change in molecular configuration. Upon exposure to cations, leading to deprotonation, the aromatic plane of the chromoionophore molecule shifts from an endwise orientation perpendicular to the SERS substrate's surface to a tilted edgewise absorption. This reorientation improves the coupling of the induced dipole within molecule and the induced electromagnetic field normal to the surface, leading to a heightened and more discernible SERS signal. The vibrational fingerprint spectroscopic technique SERS has been incorporated into ionophore- based ion-selective sensing for the first time. Introduction. As counterparts to ion-selective electrodes (ISEs) widely used commercially, ionophore-based ion-selective optodes (ISOs) have shown substantial potential in diverse applications such as ion bioimaging and ultra-sensitive determination [1-4], benefiting from inherent high selectivity and versatility like ion-selective electrodes [5,6]. Ion-selective optodes have been developed across various platforms, including polymeric membranes [7-10], micro / nanosensors [11-14], microfluidic chips [15-18], and paper-based optodes [19-26]. Chromoionophores, notably chromoionophore I (CHI) and chromoionophore III (CHIII), serve as prevalent optical indicators in ion-selective optodes. Presently, the field of ionophore-based ion-selective sensing predominantly relies on the electronic absorption (UV / Vis) and emission spectroscopy (fluorescence) sensitivity of these optical indicators to protonation state changes. However, the exploration of molecular fingerprint information has been largely overlooked, and there remains a significant gap in understanding its changes during the sensing process. Results and Discussion. Surface-enhanced Raman spectroscopy (SERS) represents one of the most sensitive analytical techniques currently available

[0027] . By harnessing the enhanced electromagnetic fields generated by noble metal nanostructures, SERS delivers precise and comprehensive molecular fingerprint information, enabling the detailed identification and characterization of molecular structures [28-34]. To closely examine the SERS signal of the chromoionophore in ion-selective optode system, a paper-based SERS detection platform utilizing Ag / AgCl nanocolloids was initially developed. This classical substrate is compatible with ionophore-based ion-selective sensing systems and enables feasible SERS detection [35- 41]. The sensing components in a conventional ion-selective optode, comprising an ionophore for target ion binding, an ion exchanger for charge balancing, and an indicator chromoionophore, were applied onto the substrate. The sensing principle has been previously described in relevant publications

[0042] . In brief, the ionophore selectively interacts with the target ion, modulating the protonation alteration of the chromoionophore, consequently eliciting discernible changes in the signal. A schematic is provided in Figure 8. A distinctive feature of the ionophore-based ion sensing system lies in its adaptable ionophore selection enabling the targeting of various ions

[0043] . This versatility allows the detection of other target ions under the ion-selective optode sensing framework. SERS signal from the calcium ionophore-based ion-selective optode using chromoionophore I as indicator, along with its variations corresponding to different concentrations of Ca2+, are depicted in Figure 1A. The SERS spectra demonstrate heightened intensity with rising Ca2+concentrations. Ionophore and ion exchanger (TFPB-) do not exhibit detectable SERS signal in current test setting (data not shown). According to the ion-selective optodes sensing principle, the ionic complexation process between the ionophore and target ion causes the deprotonation of the chromoionophore. An increase in Ca2+concentration leads to a reduced fraction of protonated chromoionophore molecules to maintain the electroneutrality. The protonation degree (1-^) can be calculated using the following equation [5, 42]: SH and SdH refer to the SERS intensity of fully protonated and deprotonated chromoionophore, while S represents the SERS intensity at different concentrations of Ca2+. Upon plotting the chromoionophore protonation degree (1-^) and the intensity of the SERS peak at 592 cm−1against Ca2+concentration, as shown in Figure 1B, a clear correlation emerges: the deprotonated chromoionophore exhibits a higher SERS intensity, which can be modulated by engaging the target cation within the ionophore-based ion sensing system. For additional verification, buffers with varying pH were employed to directly modulate chromoionophore I's protonation degree. Figure 1C shows the intensity at 592 cm-1as a pH function. A notable signal enhancement is evident within the basal range. The associated SERS spectra are shown in Figure 9. It is known that under resonant Raman conditions, where the excitation laser is close to the maximum absorption of the Raman reporter, a considerable amplification of Raman enhancement occurs compared to conventional SERS [44, 45]. The protonated chromoionophore, with a maximum absorption of 639 nm, is closer to the excitation laser (647 nm) than the deprotonated chromoionophore at 520 nm (Figure 10). Despite this proximity, it exhibits SERS intensity significantly lower than the deprotonated chromoionophore. Therefore, the resonant Raman process didn’t make a substantial contribution to the signal enhancement. Distinctive peaks were attributed by referencing prior publications and density functional theory (DFT) calculations for a more comprehensive elucidation of the spectral features [46, 47]. The molecule structure of chromoionophore comprises a heterocyclic plane and a lengthy carbon chain that heightens their hydrophobicity. The primary Raman shifts arise from the deformation of the aromatic ring plane and the -CH2 bending, as well as the C-C stretching within the carbon chain (Figure 2). Detailed analysis can be found in Table 1.

[0002] TABLE 1: Theoretical and experimental frequencies of the fundamental vibrational modes of chromoionophore I (CHI) within the range of 500-1800 cm-1. A scaling factor of 0.9813 was applied. Surface-enhanced Raman scattering occurs as the incident light directly induces an oscillating dipole within the molecules absorbed on the surface; meanwhile, it generates surface plasmon resonances in highly localized surface regions (hot spots), fostering intense induced electromagnetic fields (Eind). The Eind again polarize the molecule, leading to the emission of significantly enhanced Raman-shifted radiation [48-52]. These results reveal that the enhancement effect varies across different vibrational modes within the chromoionophore molecule. The aromatic plane serves as the primary origin of the SERS signal, while the vibrations of the extended carbon chain did not exhibit a comparable enhancement in intensity as observed in the fundamental ring vibrations. As shown in Figure 2, the aromatic ring-associated vibration modes, especially B at 592 cm-1, displayed low activity in Raman spectroscopy, they exhibited significant enhancement in SERS. Conversely, the extended carbon chain related vibration modes showed high activity in Raman spectroscopy but did not present clear SERS signals. The proximity of the metal surface is important for augmenting molecular polarizability via the electromagnetic enhancement mechanism, typically occurring within a range of 0.1-0.5 nm

[0053] . The dimensions of the aromatic ring plane measure approximately 0.7 nm in length and 0.5 nm in width (Figure 11). Prior studies have also consistently shown the reduced reactivity of alkyl groups toward metal surfaces compared to aromatic rings [54,55]. Thus, it can be deduced that chromoionophore molecules attach the aromatic plane to the Ag substrate while positioning the carbon chain group farthest from the surface. The SERS intensity associated with the aromatic plane undergoes a notable transition, starting from a lower level in the protonated state and escalating to a significantly stronger signal in its deprotonated form. Classical electrostatics suggests that a charge positioned close to a metal surface with a constant potential will cause the free electrons within the metal to polarize, so the dipole of the molecule induces image dipole within the metal surface

[0056] . Raman scattering originates from the induced dipole within the molecule and the corresponding image dipole formed on the metal surface. The parallel components of both dipoles nullify each other at large distance owing to their out-of-phase oscillation, while the in-phase perpendicular components actively contribute to amplifying signal intensity [57-59]. As a result, the radiating dipoles with perpendicular components to the metal surface couple the plasma oscillations in the induced electromagnetic fields normal to the surface, yielding significant enhancement in the resultant signal, where the extent of enhancement correlates proportionally with the dipole's projection onto the surface normal [60-64]. The dipole derivative unit vectors of distinct vibrational modes in both protonated and deprotonated chromoionophore I are determined by DFT, revealing consistent behavior across these two states. The red arrows in Figure 3 depict the calculated results for modes A, B, and E, while the corresponding vibrational component of each mode is highlighted in yellow. Further results for modes C and D are provided in Figure 12 for reference. The highest SERS intensity is mode B, likely due to its larger polarization volume. The dipole moment of this mode lies within the aromatic plane and spans the short cross-section of the plane (Figure 3). This configuration suggests a parallel orientation with the metal surface when the molecule stands endwise perpendicular to the surface. Conversely, upon reorienting to an edgewise vertical position, the dipole moment becomes normal to the surface. Like mode B, modes C and D exhibit an increased dipole projection onto the surface normal when transitioning from an endwise structure to an edgewise perpendicular configuration. According to the image-dipole theory, the intensity enhancements are more prominent for modes exhibiting a larger projection onto the surface normal. Hence, these observations suggest a reorientation of the aromatic plane in chromoionophore molecules during deprotonation. This transition involves shifting from an endwise perpendicular alignment to an edgewise orientation with the Ag substrate surface, positioning the carbon chain group farthest from the surface, as depicted in Figure 3. Mode E did not exhibit a substantial increase, as the dipole remains within the plane of the aromatic ring at an approximately 45° angle concerning the metal surface normal and remains constant at 45° after the reorientation (Figure 3). The dipole moment of Mode A, originating from rings III and IV in a perpendicular orientation to the aromatic plane (Figure 3), maintains parallelism with the metal surface during molecule reorientation within the plane. The increased intensity observed in the deprotonated state may be attributed to the proximity of rings nearing the metal surface in an edgewise vertical position, as incomplete dipole cancellation occurs when utilizing visible light as the excitation source. Furthermore, the aromatic plane exhibits a partial inclination rather than being entirely perpendicular to the metal surface, with an average calculated tilt of 12° from the surface normal. Detailed calculations are provided below. This slight plane tilt results in the dipole projection towards the surface normal, thereby presenting an additional factor that enhances the Raman signal. NMR shows that the tertiary amine at the end of the chromoionophore is where accepting and donating proton. The Zeta potential of Ag-based SERS substrate surface is -43 mV (Figure 13), leading to a strong attraction of positive charged protonated chromoionophore [65, 66]. The chromoionophore molecules adopt a perpendicular endwise orientation relative to the surface to achieve a highest possible surface number density, which is consistent with literature that perpendicular configuration is preferred in the compressed phase and stabilized by the intermolecular interactions [67-70]. Upon deprotonation, the electrostatic interaction affinity diminishes, and the electron density of the aromatic moiety is elevated, strengthening ^-stacking interactions with the metal surface [55, 71, 72]. These two elements induce the molecule to reorient itself to a tilted edgewise configuration. Figure 4A-Figure 4B illustrates that the deprotonation of chromoionophore III induced by the increase in pH and [Ca2+] results in heightened SERS signals (Figure 14), like those observed for chromoionophore I. The observed lower pKa of chromoionophore I (Figure 1C) compared to chromoionophore III (Figure 4A), consistent with previously reported findings [73- 75], is likely attributed to the destabilization of chromoionophore I's monocationic state caused by the electron-withdrawing effect of the carbonyl substituent on the imine nitrogen

[0076] . The lower pKa value of chromoionophore I also accounts for the chromoionophore I-based sensing system operating at lower Ca2+concentrations (Figure 1B) than the chromoionophore III-based system (Figure 4B). The high selectivity of Ca2+over several common interfering ions has been confirmed when using chromoionophore I and chromoionophore III as SRES indicators, respectively, and is illustrated in Figure 15A-Figure 15B. To further validate the compatibility of SERS with ionophore-based ion-selective sensing system, the SERS response of a sodium sensing system was investigated by employing sodium ionophore X and ion exchangers (TFPB-) to cooperate with chromoionophore I. As the response curves shown in Figure 5A-Figure 5B, the SERS signal increased as the [Na+] increasing with good selectivity over other common interreference ions. The demonstrated turn-on SERS response to ions lay the groundwork for integrating SERS technology with ionophore-based ion-selective sensing. This integration holds the potential to significantly advance ion detection by merging the high selectivity and versatility inherent in ionophore-based ion sensing principles with the heightened sensitivity, abundant vibrational fingerprint spectrum data, and non-destructive detection capabilities offered by SERS. Experimental section Chemicals and materials: calcium ionophore I, sodium ionophore X, chromoionophore I (CHI), chromoionophore III (CHIII), sodium or potassium tetrakis[3,5- bis(trifluoromethyl)phenyl]borate (TFPB), tri-sodium citrate, Trizma base, AgNO3, CaCl2, NaCl, MgCl2, KCl, LiCl, NaOH, methanol, hydrochloric acid (HCl) were purchased from Sigma- Aldrich. All solutions were prepared by dissolving appropriate salts into deionized water purified by Milli-Q Integral 5. Whatman qualitative filter paper (grade 5) was purchased from Fisher Scientific. Preparation of paper-based SERS substrate: AgNPs was fabricated according to previously reported method [77, 78]. A 20 ml 0.003 M AgNO3 solution is heated on a hot plate with vigorous stirring. After reaching boiling point, 1 ml of 1% tri-sodium citrate is added dropwise with continued vigorous stirring. Within a minute of adding the tri-sodium citrate, a noticeable change occurs in the solution's color, transitioning from colorless to a pale yellow. After four minutes from the start of boiling, the solution is removed from the heat source and allowed to cool down to room temperature. After synthesis, the AgNPs were mixed with HCl (10 mM) to generate Ag / AgCl nanocolloids. Following this, the Ag / AgCl nanocolloids were washed and then dropped onto filter paper, allowing it to dry for future use (Figure 7). Instrumentation and measurements: The Raman analysis was conducted using a Horiba LabRam HR-800, which is equipped with an Olympus IX71 inverted microscope and scanning stage, and a 647 nm laser was selected as the excitation source. For Ca2+selective SERS analysis, calcium ionophore I (1.5 mM), NaTFPB (0.5 mM), chromoionophore I or chromoionophore III (0.5 mM) were dissolved in methanol to form a homogeneous cocktail solution. To conduct Ca2+selective SERS analysis, a homogeneous cocktail solution was prepared by dissolving calcium ionophore I (1.5 mM), NaTFPB (0.5 mM), and either chromoionophore I or chromoionophore III (0.5 mM) in methanol. The formulation for detecting Na+includes a cocktail solution composed of sodium ionophore X (1.5 mM), KTFPB (0.5 mM), and chromoionophore I (0.5 mM). For SERS detection, ten microliters of the cocktail solution were applied onto the previously prepared substrate to create the relevant optodes. After drying, the optodes were submerged in a 10 mM Tris-HCl buffer at pH 7.4, with the salt concentrations varying. pH responses were assessed by using buffer solutions containing 2.5 mM citric acid, boric acid, and NaH2PO4, adjusted to the desired pH levels. A pH 3 universal buffer and 0.1 M NaOH were employed to ensure complete protonation and deprotonation. It's important to note that all optodes utilized in this study were dried before testing. The absorption spectra were obtained using an Agilent Cary 3500 Compact Peltier UV- Vis spectrophotometer. HCl and NaOH were introduced into chromoionophore I and chromoionophore III methanol solutions, respectively, to achieve full protonation and deprotonation. Zeta potential measurements were conducted utilizing a Zetasizer Nano ZS instrument from Malvern Instruments (Worcestershire, UK). A disposable polycarbonate folded capillary cell (model DTS1070) with a path length of 4 mm was employed for the measurements. Scanning electron microscope (SEM) micrographs was acquired by using a JEOL JSM 5900LV scanning electron microscope operated at an acceleration voltage of 5 kV. DFT methods: Structures of chromoionophores were optimized and vibrational frequencies were calculated by Density Functional Theory (DFT) using Gaussian 16 (Rev. C.01) for Linux

[0079] . All structures were in their singlet ground state, with the deprotonated molecules having no charge, and the protonated molecules with a +1 charge. Optimizations and harmonic frequency calculations were carried out at the B3LYP / 6-311G+(d, p) level of theory (gas-phase). Theoretical Raman spectra were generated with GaussView 6 (Rev.6.1.1) with (blank)cm-1band half-width-half-maximum

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Chem, 1992, 96 (9), 3776-3782. Example 2 - Ionophore-Based Ion-Selective Sensing Utilizing Surface-Enhanced Raman Spectroscopy Described herein is an ionophore-based ion-selective sensing method developed by harnessing the power of surface-enhanced Raman spectroscopy (SERS). Traditionally, ionophore-based ion-selective sensing has been extensively explored through techniques such as electronic absorption (UV / Vis), emission spectroscopy (e.g., fluorescence), and ion-selective electrode (ISE) methods (Figure 16). However, the inherent limitations of these conventional approaches, including issues like broad emission profiles, photobleaching, significant background interference, and low sensitivity, have presented substantial challenges when deploying ionophore-based ion-selective sensors for practical applications. These challenges are particularly pronounced in biological research, medical diagnostics, and environmental analysis. The method described herein represents a milestone, enabling the detection of various ions, including cations, anions, and polyions, through the principles of ionophore-based ion- selective sensing using surface-enhanced Raman spectroscopy (SERS). SERS offers unparalleled capabilities by meticulously characterizing the vibrational fingerprint spectrum of molecules with hyperspectral precision. This attribute equips the developed sensing method with heightened sensitivity and specificity, rendering it exceptionally well-suited for the comprehensive analysis of complex samples. Moreover, SERS provides non- destructive detection and multifaceted analytical capabilities rooted in well-defined vibrational Raman peaks. These unique advantages mean that the developed sensing method can precisely detect ions in biological and environmental samples. Described herein are ionophore-based ion-selective sensing utilizing Surface-Enhanced Raman Spectroscopy (SERS). The selective detection capability of this method for calcium and sodium ions (Ca2+, Na+) has been confirmed using filter paper as a sensing substrate. For other ions, as the adhere to a similar sensing principle, the same general methods can be adapted and optimized for said other ions. This technology can further be integrated into nanoparticles, as a nanoparticle-based sensing platform is more suitable and practical for biological samples, especially for bioimaging. For example, nanomaterials can be developed and used as sensors (e.g., nanosensors) based on the methods and results described herein. The sensing chemicals for the selective detection of various target ions can be further investigated. These results and sensors can be used, for example, for bioimaging, such as for in vitro intracellular and extracellular ion sensing. These results and sensors can also be combined with a local delivery technique for in vivo ion sensing. The methods described herein can be applied in 1. Biological Research: detecting and analyzing ions within biological samples to study cellular processes, ion transport, and various biochemical reactions. 2. Medical Diagnosis: detecting ions relevant to medical conditions. It could be used to diagnose diseases early or to monitor specific ion concentrations in clinical samples, for instance, measuring Ca2+concentration in blood samples for monitoring hyperparathyroidism, Na+concentration in Cardiomyocytes for screening cardiac arrhythmias drugs, and tracking K+levels in tumors, which are typically higher than in other areas. 3. Environmental Analysis: Monitoring ion concentrations in environmental samples, such as soil, water, and air. It can aid in assessing pollution levels and environmental health. 4. Pharmaceutical Research: Investigating the interactions of ions with drugs and their impact on drug formulations and pharmacokinetics. 5. Food and Beverage Industry: Quality control and safety monitoring in the food and beverage industry can benefit from the precise detection of ions that may impact product quality and safety. Uses also include: 1. Research both in academic and industrial settings, working in ion-associated biological, medical, and environmental fields. 2. Healthcare facilities, hospitals, clinics, and medical laboratories when applied to medical diagnostics and monitoring of ion levels in patients. 3. Biotechnology companies can utilize the technology for various applications, including the development of biosensors and the monitoring of bioprocesses. 4. Government agencies and organizations involved in environmental monitoring and regulation can deploy this technology for assessing water quality, air quality, and soil conditions. Example 3 - Mxene-AgNPs based SERS platform for ion sensing Abstract: Surface-enhanced Raman spectroscopy (SERS) has advanced as an important analytical technology, benefiting numerous fields with its high sensitivity and versatility. However, its application in detecting ions has been underexplored. In this study, a SERS sensing platform was developed for ion detection by creating a Ti3C2Mxene-AgNPs substrate and integrating it with the ionophore-based ion-selective sensing framework. The Ti3C2Mxene- AgNPs substrate was successfully prepared via electrodeposition on conductive glass, achieving an enhancement factor of 0.64 × 108and demonstrating good signal repeatability (RSD = 4.92%). The AgNPs ensure amplified and stable SERS activity, while large surface area of the MXene nanosheets offers a significant advantage for AgNPs and sensing chemical loading. The Ti3C2 Mxene-AgNPs SERS substrate enables ionophore-based ion-selective sensing with exceptional selectivity and high sensitivity for target ion detection, eliminating the need for plasticizers. A selective turn-on SERS response of this platform to various concentrations of K+, Na+, and Ca2+was exemplified in this study, further revealing its excellent generalizability. Moreover, the applicability of this method was preliminarily demonstrated by its successful application in determining Na+and Ca2+in human blood serum and environmental water samples (river and estuary). The successful integration of the versatile and rapidly progressing SERS technique, enabled by the Ti3C2Mxene-AgNPs substrate, for electrolyte cation detection offers a promising avenue for enhancing current ion detection capabilities in biological and environmental samples. Introduction. Surface-enhanced Raman spectroscopy (SERS) has made significant advancements in both fundamental research and practical applications over the past half-century. Its ability to deliver precise and comprehensive molecular fingerprint information makes it one of the most powerful and sensitive analytical techniques [A1]. SERS offers unique features such as flexible excitation wavelengths from the visible to infrared region, resistance to photobleaching, negligible interference from the autofluorescence of biological systems while also ensuring high biocompatibility and prompt results. Moreover, SERS demonstrates strong resistance to water and air and does not require tedious sample pretreatment [A2]. Many companies now offer portable and handheld Raman spectrometers comparable in size to cell phones. Continued efforts are being made to make these devices even more convenient while enhancing their test performance. All these unique properties of SERS have led to its extensive application in various fields, including clinical diagnosis, environmental analysis, and food safety detection. Using SERS to detect small inorganic ions has attracted considerable efforts, and significant progress has been made in detecting heavy metal ions [A3]. Since metal ions do not exhibit vibrational energy modes, SERS detection of ions has relied on indirect approaches. Thymine has served as a chemoreceptor for Hg2+ions [A4]. The reversible interaction between Ag+and glucose oxidase (GOD) has been utilized for Ag+detection via SERS [A5]. Additionally, the binding of a dipicolylamine-based ligand to Cu²^ and the interaction of glutathione (GSH) with As³^ ions have been applied in the SERS detection of Cu²^ and As³^ [A6, A7]. It has been widely demonstrated that SERS provides the high selectivity, excellent portability, and ultrasensitivity essential for the detection of heavy metal ions. Electrolyte cation detection is important in physiology, biochemical science, environmental monitoring, and others [A8, A9]. The traditional instrument methods, such as ion chromatography, atomic spectroscopy, and inductively coupled plasma mass spectrometry, are destructive and require trained personnel and complex sample preparation, resulting in increased costs and prohibiting rapid in-situ monitoring. Given the advantages of SERS discussed above, it has significant potential to make important contributions to ion detection. However, applying SERS to detect electrolyte cations presents substantial challenges and remains largely unexplored. Ionophore-based ion-selective sensors offer high selectivity and response flexibility similar to the extensively commercialized ion-selective electrodes (ISEs). Ionophore-based ion- selective sensors include, in addition to an ionophore for target ion binding, an ion exchanger for charge balancing, and an indicator [A10, A11]. These components typically cooperate in a plasticized hydrophobic sensing phase, a polymeric film, or small particles. This interaction modulates changes in the signal of the indicator, which are commonly assessed by measuring the fluorescence or absorbance (colorimetric) spectra. Using various ionophores enables the detection of a wide variety of organic and inorganic ions with high selectivity [A12]. Integrating SERS with ionophore-based ion-selective sensing not only leverages the high selectivity and flexibility of ionophore principles but also enhances sensitivity, portability, detailed vibrational fingerprint data, and non-destructive detection capabilities unique to SERS. Two-dimensional transition-metal carbides, nitrides, or carbonitrides (MXenes) have emerged as a new class of SERS substrates, exhibiting a range of intriguing properties, including tunable electronic structures, cost-effective fabrication, outstanding flexibility, good biocompatibility, and relatively strong plasmon resonance effect in the visible and near-infrared ranges [A13-A15]. In particular, Ti3C2 MXene, which is easy to synthesize and highly stable, is currently the most studied. Its atomic-scale thickness effectively preserves the enhancement capabilities of noble-metal nanoparticles while minimally disrupting the plasmonic field [A16]. Ti3C2 MXene-noble-metal nanoparticle (Au / Ag)-based SERS-active substrates have been prepared through a self-assembly process, offering enhanced sensitivity and reliability for detecting toxic mycotoxin Ochratoxin A (OTA) [A17], dopamine in serum [A18], organic pollutants [A19], trinitrotoluene (TNT) molecules [A20], and bacteria [A21]. In addition to the advantages of Ti3C2MXene as a SERS substrate, its unique planar structure, large specific surface area, and high conductivity enable the firm and stable binding of probe molecules and nanoparticles to its surface [A22]. These properties make it particularly well-suited for loading ionophore-based ion-selective sensing chemicals, enabling SERS-based ion detection. In this study, the Ti3C2 MXene-AgNPs substrate was developed for the first time to integrate with the ionophore-based ion-selective sensing system for ion SERS detection. Electrodeposition process was employed to synthesize the Ti3C2 MXene-AgNPs substrate on fluoride tin oxide (FTO) conductive glass. Conductive glass is ideal for electrodeposition due to its affordability, excellent electrical conductivity, and high stability. Additionally, its surface roughness reduces surface energy, ensuring efficient electrodeposition. Electrodeposition is an effective method for the direct growth of nanostructures on flat substrates, offering superior adhesion and ensuring good substrate-to-substrate reproducibility of SERS spectra. The feasibility of the Ti3C2MXene-AgNPs substrate-based ion SERS sensing method were demonstrated using chromoionophore I as the SERS indicator for K^, Na^, and Ca²^ model ions. High selectivity was achieved through the use of their corresponding ionophores. As a preliminary application, this new SERS sensing method was successfully used to determine the Na^ and Ca²^ levels in human serum and environmental water samples (river and estuary). Experimental Section Chemicals and reagents. Chromoionophore I (CHI), sodium tetrakis[3,5- bis(trifluoromethyl)phenyl]borate (NaTFPB), potassium tetrakis[3,5- bis(trifluoromethyl)phenyl]borate (KTFPB), potassium ionophore I (KI), calcium ionophore I (CaI), sodium ionophore X (NaX), lithium fluoride, hydrochloride, potassium nitrate, silver nitrate, tetrachloroauric acid (Tris), ammonia solution, tetrahydrofuran (THF), titanium aluminum carbide (Ti3AlC2, 90%), human blood serum and all other salts were purchased from Sigma-Aldrich. FTO glasses with a sheet resistance of 7 ^ / square were bought from Shenzhen Hua-nan technology Co. Ltd. All chemicals were used without further purification, and all solutions were prepared in 100 mM Tris-HCl buffer at pH 7.4 with distilled water (18.2 M^·cm) obtained from a Milli-Q purification system. Ti3C2 MXene-AgNPs substrate preparation. The synthesis procedure for Ti3C2 MXene was adapted from previous study [A22]. Briefly, 1.6 g of LiF and 20 mL of 9 M concentrated HCl were mixed by stirring for several minutes, then placed in an oil bath to maintain a temperature of 35°C. Next, 1 g of Ti3AlC2 was slowly added to the mixture, which was allowed to react for 24 hours. The mixture was then centrifuged at 3500 rpm for 30 minutes, and the suspension was washed with ultrapure water until the pH of the supernatant reached 6. The mixture was then re-dispersed in 100 mL of DI water, and the obtained black suspension was subjected to sonication for 30 minutes (5800-MTH, Branson Ultra Sonic Bath, US), followed by another round of centrifugation at 3500 rpm for 60 minutes (5425R, Eppendorf, Germany). The supernatant was collected, yielding monolayer Ti3C2MXene. For the electrodeposition of MXene / Ag NPs on FTO, 50 mL of an electrolyte solution containing 0.01 M KNO^, 10 mg / L Ti3C2Tx, and 0.5 mM [Ag(NH3)2]+was introduced into an electrochemical cell. A three-electrode system was employed, with FTO glass as the working electrode, a Pt sheet as the counter electrode, and Ag / AgCl as the reference electrode. The deposition was conducted using an electrochemical workstation (SP-150e, Bio-logic SAS, France). The applied bias voltage was controlled in two stages: the first stage at -1.0 V for 10 seconds, followed by a second stage at -0.2 V for 10 minutes. After deposition, the FTO- MXene / Ag NPs electrode was rinsed with distilled water and allowed to dry naturally. Ti3C2MXene-AgNPs substrate characterization. Transmission electron microscopy (TEM) was conducted using a JEOL JEM-1400 Flash (Japan) instrument operated at 120 kV to capture the nanostructure of the samples. Surface morphology was analyzed with an atomic force microscope (AFM, TT-2 AFM Workshop, UK), and scanning electron microscopy (SEM) images were obtained using a JEOL Neoscope scanning electron microscope (US). Raman spectra of samples exposed to various ion solutions were collected with a Horiba XploRa Raman microscope, equipped with an Olympus BX51 upright microscope, a scanning stage, and a 785 nm laser as the excitation source. The SERS enhancement factor (EF) and reproducibility of the as-prepared Ti3C2 MXene- AgNPs substrate were evaluated using Rhodamine 6G (R6G).20 µL of 0.1 mM R6G solution was applied onto the Ti3C2MXene-AgNPs substrate, which is placed on a magnetic stirrer disk and heated to 60°C to prevent the formation of coffee rings. The enhancement factor is calculated based on the intensity of the characteristic peak around 1356 cm-1. For reproducibility testing, 20 µL of the R6G solution is applied to the Ti3C2MXene-AgNPs substrate. A total of three samples are tested, with ten distinct points measured per sample, resulting in 30 data points. By comparing the Raman spectra from each point, the reproducibility of the experimental results across the samples is assessed. Enhancement Factor (EF) calculation. The calculation of the enhancement factor (EF) for a SERS substrate was thoroughly demonstrated in a previous study. Briefly, the enhancement factor can be evaluated using the formula provided below: where ISERS is the intensity of SERS, and IRaman is the intensity of Raman. NSERS and NRaman represent the number of Rhodamine 6G molecules within the laser beam area on prepared Ti3C2 MXene-AgNPs substrate and the powder sample, respectively. The intensity of the peak at 1365 cm-1position was chosen to calculate the enhancement factor here. Ion SERS sensing platform preparation and application. A homogeneous cocktail solution for Ca²^ sensing was prepared by mixing the sensing components CHI (0.5 mM), TFPB- (0.5 mM), and CaI (1.5 mM) in a concentration ratio of 1:1:3 in THF. Similarly, cocktail solutions for Na^ and K^ were prepared using NaX and KI, respectively, under identical conditions. The Ti3C2 MXene-AgNPs substrate was placed on a magnetic stirrer and heated to 60°C.20 µL of the prepared sensing cocktail solution was applied to the FTO-MXene / Ag NP substrate. At this temperature, rapid evaporation of the organic solvent minimized coffee-ring formation of the sensing layer. After the substrate dried, it was immersed in a solution containing varying ion concentrations for 10 minutes. The performance of the prepared ion SERS sensing platform for real sample applications was evaluated by testing Ca²^ and Na^ concentrations in samples from human blood serum, natural river water (near Pioneer Park, Deerfield Beach, FL, 2.5 km away from the coastline), and estuary water (near Sullivan Park, Deerfield Beach, FL; 1 km away from the coastline) using a standard addition method. Human blood serum, river water, and estuary water samples were diluted by factors of 100, 2, and 100, respectively, using Tris-HCl buffer for Ca2+concentration testing. Additionally, each sample was diluted 100-fold with Tris-HCl buffer to analyze Na+concentration. Prior to dilution, river and estuary water samples were filtered through a 0.45 µm filter. The results obtained from the developed SERS-based method were compared with measurements using microwave plasma atomic emission spectroscopy (MP-AES, model 4210, Agilent). Results and discussion Ti3C2MXene-AgNPs-based ion SERS sensing substrate preparation and sensing principal elucidation. As illustrated in Figure 17A, the Ti3C2 MXene-AgNPs-based SERS substrate was synthesized via electrodeposition by reducing Ti3C2 MXene nanosheets and silver ammonia complex ([Ag(NH3)2]+) on the surface of FTO conductive glass. Ti3C2 MXene nanosheets were prepared by selectively etching aluminum atoms from the precursor MAX phase Ti3AlC2 using a mixture of HCl and LiF, followed by repeated washing to remove excess etchant and residual aluminum. The electrochemical reduction potentials of the prepared Ti3C2 MXene nanosheets, [Ag(NH3)2]+solution, and their mixture were determined to be -0.35 V, +0.23 V, and +0.17 V, respectively, as shown in Figure 18. Compared to the [Ag(NH3)2]+solution, the mixture of Ti3C2MXene nanosheets and [Ag(NH3)2]+exhibited a lower oxidation potential and a reduced reduction potential, indicating it became easier to oxidize and more difficult to reduce. In the two-step chronoamperometry electrodeposition method used here, -1 V was initially applied to deposit Ti3C2MXene-AgNPs nuclei, followed by a second step at -0.2 V to promote the growth of fine and uniform Ti3C2 MXene-AgNPs. This hybrid Ti3C2 MXene- AgNPs substrate is particularly well-suited for SERS-based ion sensing, combining high SERS activity with a large specific surface area that effectively hosts components for ionophore-based ion-selective sensing frameworks. The ionophore-based ion-selective sensing system comprises three key components: an ionophore, an ion exchanger, and an indicator. The ion exchanger, typically a hydrophobic compound, provides lipophilic anionic sites within the sensing phase [A23]. A commonly used ion exchanger is the salt of tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (TFPB^). Ionophores selectively bind to target ions, which triggers the indicator molecule's deprotonation to maintain charge balance in the sensing phase. In previous research, the feasibility of using SERS for ion sensing was demonstrated with Chromoionophore I (CHI) as the indicator [A24]. The mechanism behind the variation in SERS intensity of CHI during the sensing process was also elucidated based on electromagnetic enhancement and the image-dipole theory. Specifically, when the CHI molecule undergoes deprotonation induced by the target ion, its aromatic plane shifts from an initial endwise orientation on the SERS substrate's surface to an edgewise alignment. This reorientation enhances the coupling between the induced dipole within the CHI molecule and the electromagnetic field normal to the SERS substrate surface. This leads to a stronger and more distinct SERS signal, as shown in Figure 17B. Characterizations of Ti3C2MXene-AgNPs substrate. The structure and morphology of the synthesized Ti3C2MXene nanosheets and Ti3C2MXene-AgNPs SERS substrate were further characterized. As shown in the transmission electron microscopy (TEM) image in Figure 19A, the monodisperse Ti3C2MXene nanosheet exhibits a thin planar 2D structure. Figure 19B displays the atomic force microscopy (AFM) image of the flat surface of the Ti3C2 MXene nanosheets, with the corresponding height profile along the white dotted line revealing a thickness of no more than 2 nm. This indicates the successful fabrication of a single, large layer of Ti3C2 MXene nanosheets. The surface structure of the Ti3C2 MXene-AgNPs SERS substrate was studied using scanning electron microscopy (SEM) and compared to the substrate fabricated using only the silver ammonia solution. As shown in Figure 19C and Figure 19D, the inclusion of Ti3C2MXene nanosheets resulted in a denser and more uniform structure. The Ti3C2MXene nanosheets prepared using above method are negatively charged (Zeta potential: -21.17 mV) due to the presence of C-Ti-OH groups, which have been shown to adsorb positively charged ions or nanoparticles through electrostatic attraction. Moreover, MXene can also serve as a reductant, reducing metal cations to metallic nanoparticles through the spontaneous donation of electrons [A25]. This process results in a more strongly binding MXene-nanoparticle SERS substrate, attributed to the unsaturated oxidation state of its terminal metal atoms. Therefore, the electrostatic adsorption occurred between the Ti3C2 MXene nanosheets and [Ag(NH3)2]+before electrodeposition began, leading to the partial reduction of [Ag(NH3)2]+on the surface of the Ti3C2 MXene nanosheets. This also explains why the mixture of Ti3C2 MXene nanosheets and [Ag(NH3)2]+exhibited lower reducibility, as demonstrated by the cyclic voltammetry (CV) in Figure 18. The reduced reducibility slowed the electrodeposition process, resulting in a denser and more uniform structure. The SERS properties of the prepared Ti3C2MXene-AgNPs substrate were evaluated using Rhodamine 6G, a commonly used Raman reporter. The SERS spectrum of Rhodamine 6G on the Ti3C2MXene-AgNPs substrate, shown in Figure 20, displays clear characteristic peaks consistent with previous results. Additionally, Figure 20 depicts the Raman spectrum of Rhodamine 6G powder, and the SERS enhancement factor (EF) was calculated by the universalformula: where I and N denote the intensity of the Raman signal and thenumber of molecules, respectively. ISERSand IRamanwere obtained experimentally. Accurate values of NSERS and NRaman were determined by carefully considering the test conditions and the concentrations of the Rhodamine 6G molecules. The enhancement factor of the Ti3C2 MXene- AgNPs substrate was estimated to be 0.64 × 108. The uniformity and reproducibility of the as- prepared Ti3C2 MXene-AgNPs substrate were quantitatively evaluated by measuring ten random points on a single substrate surface across three different production batches. A total of 30 measurements yielded nearly identical SERS spectra ( Figure 24A- Figure 24B), with the relative standard deviation (RSD) values of the signals calculated to be 4.92%, indicating excellent uniformity and reproducibility of the Ti3C2MXene-AgNPs substrate for SERS measurements. SERS responses of the Ti3C2MXene-AgNPs-based ion sensing platform for target ion detection. The Ti3C2MXene-AgNPs substrate was designed and developed in this work to host the sensing components for SERS-based ion sensing, leveraging the high SERS activity of the AgNPs, the large specific surface area of the Ti3C2 MXene, and its stable interaction with the sensing components. To confirm the interaction and photoinduced charge transfer between the indicator CHI and Ti3C2 MXene, the UV-vis absorption spectra of pure CHI and Ti3C2 MXene- CHI mixture solutions are presented in Figure 21. The main absorption peak of deprotonated CHI shifts from 526 nm to 543 nm, while the protonated CHI peak shifts from 610 nm to 620 nm under similar conditions when adsorbed on Ti3C2 MXene. These redshifts suggest a strong interaction and photoinduced charge transfer between the CHI molecules and the Ti3C2 MXene substrate [A20, A26]. For Ca2+sensing, the Ti3C2MXene-AgNPs-based ion sensing platform were prepared with calcium ionophore I, TFPB-, and CHI. In traditional ionophore-based ion- selective sensing systems, the sensing components ionophore, ion exchanger, and indicator work together within a plasticized PVC film or nanoparticles to isolate the sensing components from the aqueous sample while allowing ion transfer. The Xie group developed a series of plasticizer- free ionophore-based ion-selective nanosensors [A27], demonstrating that this sensing system functions effectively in a hydrophobic microenvironment without the need for plasticizer. The Meyerhoff and Wang groups further demonstrated the feasibility of this sensing system in matrices that are generally considered hydrophilic, such as cellulose paper and fabric, by depositing the sensing components onto these materials without the use of a plasticizer [A28- A30]. These research outcomes have expanded the applicability of the ionophore-based ion- selective sensing system by allowing flexible adoption of various matrices. Figure 22A shows the SERS responses of the Ti3C2 MXene-AgNPs-based Ca²^ sensing platform to various concentrations of Ca²^ solutions, with the CHI, TFPB, and calcium ionophore ratio set at 1:1:3. The intensity of the SERS peak at 592 cm−1demonstrated a response range from 1 µM to 0.1 M of Ca²^, with increased intensity corresponding to rising Ca²^ concentrations, and exhibited good selectivity over potential interfering cations (K^, Na^, Li^, and Mg²^) across concentrations from 10-4to 0.1 M ( Figure 22B). Based on the principle of ionophore-based ion-selective sensing, the complexation between the ionophore and the target ion leads to the deprotonation of the CHI indicator. (1-^) represent the protonated degree of indicators, which could be describe by Equation 1, and the equilibrium of ion transfer in the sensing process is described by Equation 2, where HInd^ and Ind represent the protonated and deprotonated forms of CHI, respectively. R^ denotes the lipophilic anionic site TFPB^, L represents the calcium ionophore I, and L^Ca²^ represents the formed adduct. The aq and org subscripts designate the surrounding aqueous phase and organic sensing phase, respectively. Therefore, the equilibrium constant can be expressed with Equation 3, where the terms in brackets are the concentrations of each species. According to the law of conservation of electric charge in the organic sensing phase: 4) where the [R0-] is the total concentration of ion-exchanger in the system, and Vorg is the volume of sensing phase. According to the law of mass conservation for ionophores and indicators, the following relationship should generally apply: where the [L0] and [Ind0] are the total concentration of ionophore, indicator CHI, respectively. According to Equations 3 through 6, the Ca²^ concentration can be determined using the following equation: The theoretical response curves of this sensing system, with CHI, TFPB^, and calcium ionophore ratios set at 1:1:3 and 1:2:3, were calculated based on Equation 7 and are depicted in Figure 22C. The results reveal a sigmoidal relationship between the protonation degree of the indicators (1-^) and the concentration of the target ion, with a higher TFPB^ ratio theoretically corresponding to a higher ion concentration range. The experimental findings aligned well with the theoretical response curves. As shown in Figure 22D, increasing the TFPB- ratio to 1:2:3 shifts the sigmoidal response curve to a higher Ca²^ concentration range, from 0.1 mM to 0.1 M. The corresponding SERS spectra are shown in Figure 25. Notably, the target ions caused a decrease in the protonation degree of the indicators (1-^), which led to an increase in the SERS signal. This suggests that the newly developed Ti3C2MXene-AgNPs-based ion SERS sensing platform adheres to the ionophore-based ion-selective sensing principle and offers flexibility in adjusting the response range by varying the ratios of CHI, TFPB-, and calcium ionophore. Moreover, while a decrease in protonation typically results in a turn-off fluorescence response, the turn-on SERS response observed here is a new addition to the ionophore-based ion-selective sensing system. To assess the applicability of the Ti3C2 MXene-AgNPs-based ion SERS sensing platform for other ions, the SERS responses to Na+and K+were selectively investigated by replacing calcium ionophore I with sodium ionophore X and K+selective ionophore valinomycin, in conjunction with TFPB- and CHI. In both Na+and K+sensing systems, the SERS signal intensity exhibited a positive sigmoidal relationship with the logarithmic concentration of the target ions, starting from 10 µM and 1 µM, respectively. Moreover, both systems demonstrated strong selectivity against other interfering ions. The response curves are illustrated in Figure 23A- Figure 23B, and the corresponding SERS spectra are depicted in Figure 26A- Figure 26B. Therefore, the compatibility of SERS with ionophore-based ion-selective sensing systems is further validated, and the versatility of the Ti3C2 MXene-AgNPs SERS substrate for ion detection is confirmed. Human blood serum and environmental water analysis. To evaluate the practical potential of the Ti3C2 MXene-AgNPs-based ion SERS sensing platform, this new detection method was preliminarily applied to measure Ca²^ and Na^ levels in representative biological and environmental samples, including human blood serum, river water, and estuary water. Detecting serum ion levels is vital for routine health monitoring, accurate disease diagnosis, and effective management. Similarly, monitoring ion concentrations in environmental water samples is crucial for safeguarding environmental health and ensuring ecological balance. To quantify the Ca²^ and Na^ levels in the designated real samples, each sample was tested at six different spots using mapping scanning mode under consistent conditions. After averaging the acquired spectra, standard curves were established by plotting the intensities of the SERS peak at 592 cm^¹ against a series of Ca²^ and Na^ concentrations. The standard curves for samples with varying ion concentrations are shown in Figure 27A- Figure 27F. Ca²^ concentrations in human blood serum, river water, and estuary water were measured at 2.57 mM, 4.78 mM, and 6.82 mM, respectively, while Na^ concentrations were 142 mM, 24 mM, and 320 mM, respectively. These results were further validated using MP-AES, with the findings summarized in Table 2, showing an accuracy range of 90% to 114.2%. Thus, the developed Ti3C2 MXene-AgNPs-based ion SERS sensing method enables the detection of target ions in various samples with high selectivity and accuracy. The ion SERS sensing substrate can be integrated into microfluidic chips. It has been confirmed that the commonly used PDMS microfluidic chip cover can effectively encapsulate the surface of FTO conductive glass following a simple plasma cleaning process (data not shown). Moving forward, efforts will focus on incorporating the developed ion SERS sensing substrate into microfluidic systems to enable more user-friendly, multi-detection capabilities at a lower cost. Table 2. Results for measurements of Ca2+and Na+in real samples with MP-AES and the proposed methods. Conclusions: In summary, a versatile SERS-based ion sensing platform was developed via the electrodeposition of monolayer MXene nanosheets and silver nanoparticles (AgNPs) onto conductive glass. The resulting Ti3C2MXene-AgNPs substrate exhibited an enhancement factor (EF) of 0.64 × 10^, with excellent uniformity and reproducibility. This substrate not only offers high SERS sensitivity but is also compatible with ionophore-based ion-selective sensing systems, enabling efficient SERS-based ion detection. This approach demonstrated excellent selectivity, benefiting from the unique ionophore-target ion recognition. Additionally, the flexibility to change the ionophore and adjust the ratio of sensing components enables the detection of various target ions and fine-tuning of the sensing ranges. As a preliminary application, the concentrations of Ca²^ and Na^ were successfully detected in typical biological and environmental samples, including human blood serum, river water, and estuary water. The Ti3C2MXene-AgNPs-based ion SERS sensing platform opens new frontiers in ion sensing, significantly enhancing detection capabilities across diverse sample types. Reference: [A1] Wang J et al. Advanced Science 2019, 6, 1900730. [A2] Pérez-Jiménez AI et al. Chem Sci 2020, 11, 4563–77. [A3] Alvarez-Puebla RA et al. Angewandte Chemie Int. Ed.2012, 51, 11214–23. [A4] Song C et al. Biosens Bioelectron 2017, 87, 59–65. [A5] Sun D et al. Talanta 2017, 171, 159–65. [A6] Dugandži^ V et al. Sens Actuators B Chem 2019, 279, 230–7. [A7] Li J et al. ACS Appl Mater Interfaces 2011, 3, 3936–41. [A8] Bruce Alberts et al. 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[A25] Satheeshkumar E et al. Sci Rep 2016, 6, 32049. [A26] Li J et al. ACS Nano 2022, 16, 1160–9. [A27] Du X et al. Anal Chem 2018, 90, 5818–24. [A28] Zhang Q et al. ACS Appl Mater Interfaces 2020, 12, 25616–24. [A29] Wang X et al. Anal Chem 2017, 89, 12334–41. [A30] Cui Y et al. Anal Bioanal Chem 2022, 414, 7585–95. EXEMPLARY ASPECTS In view of the described compositions and methods, herein below are described certain more particularly described aspects of the inventions. The particularly recited aspects should not, however, be interpreted to have any limiting effect on any different claims containing different or more general teachings described herein or that the “particular” aspects are somehow limited in some way other than the inherent meanings of the language and formulas literally used therein. Example 1: An assay for detection of a target ion via Surfaced Enhanced Raman Spectroscopy (SERS), the assay comprising: an ionophore, a nanostructured metal, an ion exchanger, and an indicator, such as a chromoionophore, wherein the ionophore comprises a binding portion configured to bind with the target ion, wherein the nanostructured metal is configured to enhance a Raman signal of at least a portion of the indicator, wherein the Raman signal changes when the target ion is bound to the ionophore. Example 2: The assay of any examples herein, particularly example 1, wherein at least a portion of the indicator changes orientation in the presence of the target ion (e.g., when the target ion is bound to the ionophore), and said change in orientation results in a detectable change in the Raman signal. Example 3: The assay of any examples herein, particularly example 1 or example 2, wherein the indicator undergoes a change in protonation and orientation when the target ion is bound to the ionophore, which results in a detectable change in the Raman signal. Example 4: The assay of any examples herein, particularly examples 1-3, wherein the ion exchanger comprises tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (TFPB). Example 5: The assay of any examples herein, particularly examples 1-4, wherein the ion exchanger comprises sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), potassium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (KTFPB), or a combination thereof. Example 6: The assay of any examples herein, particularly examples 1-5, wherein the indicator is a chromoionophore. Example 7: The assay of any examples herein, particularly example 6, wherein the chromoionophore comprises chromoionophore I, chromoionophore III, or a combination thereof. Example 8: The assay of any examples herein, particularly examples 1-7, wherein the ionophore selectively binds with the target ion. Example 9: The assay of any examples herein, particularly examples 1-8, wherein the ionophore is selected in view of the target ion. Example 10: The assay of any examples herein, particularly examples 1-9, wherein the ionophore comprises a calcium ionophore, a sodium ionophore, a potassium ionophore, a magnesium ionophore, a lithium ionophore, a copper(II) ionophore, a lead ionophore, an ammonium ionophore, a chloride ionophore, a carbonate ionophore, a nitrate ionophore, or a combination thereof. Example 11: The assay of any examples herein, particularly examples 1-10, wherein the ionophore comprises calcium ionophore I, calcium ionophore II, calcium ionophore IV, calcium ionophore V, sodium ionophore III, sodium ionophore IV, sodium ionophore VI, sodium ionophore X, potassium ionophore I , potassium ionophore III, magnesium ionophore I, magnesium ionophore III, magnesium ionophore IV, magnesium ionophore VII, lithium ionophore VI, lithium ionophore VIII, copper(II) ionophore I, lead ionophore IV, ammonium ionophore I, chloride ionophore II, chloride ionophore III, chloride ionophore IV, carbonate ionophore VII, nitrate Ionophore VI, or a combination thereof. Example 12: The assay of any examples herein, particularly examples 1-11, wherein the ionophore comprises a calcium ionophore, a sodium ionophore, a potassium ionophore, or a combination thereof. Example 13: The assay of any examples herein, particularly examples 1-12, wherein the ionophore comprises calcium ionophore I, sodium ionophore X, potassium ionophore I , or a combination thereof. Example 14: The assay of any examples herein, particularly examples 1-13, wherein the ionophore comprises calcium ionophore I, sodium ionophore X, or a combination thereof. Example 15: The assay of any examples herein, particularly examples 1-14, wherein the target ion comprises a cation, an anion, a polyion, a zwitterion, or a combination thereof. Example 16: The assay of any examples herein, particularly examples 1-15, wherein the target ion comprises a cation, an anion, or a combination thereof. Example 17: The assay of any examples herein, particularly examples 1-16, wherein the target ion comprises a sodium ion, a calcium ion, a potassium ion, a magnesium ion, a lithium ion, an ammonium ion, a chloride ion, a carbonate ion, a nitrate ion, a copper(II) ion, a lead ion, or a combination thereof. Example 18: The assay of any examples herein, particularly examples 1-17, wherein the target ion comprises an anion. Example 19: The assay of any examples herein, particularly examples 1-18, wherein the target ion comprises a chloride ion, a carbonate ion, a nitrate ion, or a combination thereof. Example 20: The assay of any examples herein, particularly examples 1-19, wherein the target ion comprises a cation. Example 21: The assay of any examples herein, particularly examples 1-20, wherein the target ion comprises a sodium ion, a calcium ion, a potassium ion, a magnesium ion, a lithium ion, an ammonium ion, a copper(II) ion, a lead ion, or a combination thereof. Example 22: The assay of any examples herein, particularly examples 1-21, wherein the target ion comprises a metal ion, such as an alkali metal ion, an alkaline earth metal ion, a transition metal ion, a heavy metal ion, or a combination thereof. Example 23: The assay of any examples herein, particularly examples 1-22, wherein the target ion comprises an alkali metal ion, an alkaline earth metal ion, or a combination thereof. Example 24: The assay of any examples herein, particularly examples 1-23, wherein the target ion comprises a sodium ion, a calcium ion, a potassium ion, a magnesium ion, a lithium ion, or a combination thereof. Example 25: The assay of any examples herein, particularly examples 1-24, wherein the target ion comprises a sodium ion, a calcium ion, a potassium ion, or a combination thereof. Example 26: The assay of any examples herein, particularly examples 1-25, wherein the target ion comprises a sodium ion, a calcium ion, or a combination thereof. Example 27: The assay of any examples herein, particularly examples 1-26, wherein the target ion comprises a transition metal ion, a heavy metal ion, or a combination thereof. Example 28: The assay of any examples herein, particularly examples 1-27, wherein the target ion comprises a copper(II) ion, a lead ion, or a combination thereof. Example 29: The assay of any examples herein, particularly examples 1-28, wherein the nanostructured metal comprises a metal selected from the group consisting of Ru, Rh, Pd, Os, Ir, Pt, Au, Ag, Cu, Al, and combinations thereof. Example 30: The assay of any examples herein, particularly examples 1-29, wherein the nanostructured metal comprises a metal selected from the group consisting of Pt, Au, Ag, Cu, Al, and combination thereof. Example 31: The assay of any examples herein, particularly examples 1-30, wherein the nanostructured metal comprises a metal selected from the group consisting of Au, Ag, and combinations thereof. Example 32: The assay of any examples herein, particularly examples 1-31, wherein the nanostructured metal comprises Ag. Example 33: The assay of any examples herein, particularly examples 1-32, wherein the nanostructured metal comprises a plurality of metal particles. Example 34: The assay of any examples herein, particularly example 33, wherein the metal particles have an isotropic shape or an anisotropic shape. Example 35: The assay of any examples herein, particularly example 33 or example 34, wherein the metal particles have an average particle size of from 5 nanometers (nm) to 1 micrometer (micron, µm). Example 36: The assay of any examples herein, particularly examples 1-35, wherein the nanostructured metal comprises a metal modified with a nanostructure. Example 37: The assay of any examples herein, particularly examples 1-36, wherein the nanostructured metal comprises a film or layer modified with a nanostructure, such as a film or layer modified with a plurality of metal particles. Example 38: The assay of any examples herein, particularly examples 1-37, wherein the nanostructured metal comprises a two-dimensional material modified with a plurality of metal particles. Example 39: The assay of any examples herein, particularly example 38, wherein the two-dimensional material comprises a two-dimensional transition metal carbide, a two- dimensional transition metal nitride, a two-dimensional transition metal carbonitride, or a combination thereof. Example 40: The assay of any examples herein, particularly examples 1-39, wherein the nanostructured metal comprises a two-dimensional transition metal carbide, a two-dimensional transition metal nitride, and / or a two-dimensional transition metal carbonitride modified with a plurality of metal particles. Example 41: The assay of any examples herein, particularly examples 1-40, wherein the nanostructured metal comprises a two-dimensional material modified with a plurality of metal particles; the two-dimensional material comprising a two-dimensional transition metal carbide, a two-dimensional transition metal nitride, a two-dimensional transition metal carbonitride, or a combination thereof; and the plurality of metal particles comprising Au, Ag, or a combination thereof. Example 42: The assay of any examples herein, particularly examples 1-41, wherein the ionophore, the nanostructured metal, the ion-exchanger, the indicator, or a combination thereof are at least partially dispersed in a solvent. Example 43: The assay of any examples herein, particularly examples 1-42, wherein the ionophore, the nanostructured metal, the ion-exchanger, the indicator, or a combination thereof are disposed on a substrate. Example 44: The assay of any examples herein, particularly examples 1-43, wherein the ionophore, the ion-exchanger, the indicator, or a combination thereof is attached to a surface of the nanostructured metal, such that the assay comprises a modified nanostructured metal comprising the nanostructured metal having the ionophore, the ion-exchanger, the indicator, or a combination thereof attached to the surface thereof. Example 45: The assay of any examples herein, particularly example 44, wherein the modified nanostructured metal is at least partially dispersed in a solvent. Example 46: The assay of any examples herein, particularly example 44, wherein the modified nanostructured metal is disposed on a substrate. Example 47: A method of making the assay of any examples herein, particularly examples 1-46. Example 48: A method for detecting a target ion via ionophore-based ion-selective sensing using Surface Enhanced Raman Spectroscopy (SERS). Example 49: The method of any examples herein, particularly example 48, wherein the method comprises: contacting the assay of any examples herein, particularly examples 1-46 with a liquid sample; subsequently collecting a surface enhanced Raman signal from the liquid sample and the assay; and processing the surface enhanced Raman signal to determine a property of the liquid sample. Example 50: The method of any examples herein, particularly example 49, wherein the liquid sample comprises a bodily fluid. Example 51: The method of any examples herein, particularly example 49, wherein the liquid sample comprises an environmental sample. Example 52: The method of any examples herein, particularly examples 48-51, wherein the method is performed in vitro. Example 53: The method of any examples herein, particularly examples 48-52, wherein the method is performed in vivo. Example 54: The method of any examples herein, particularly examples 48-53, wherein the property of the liquid sample comprises the presence of the target ion in the liquid sample, the concentration of the target ion in the liquid sample, the identity of the target ion, or a combination thereof. Example 55: A method of use of the assay of any examples herein, particularly examples 1-46 and / or the method of any examples herein, particularly examples 48-54. Example 56: The method of any examples herein, particularly example 55, wherein the assay or method is used for biological research, medical diagnostics, environmental analysis, pharmaceutical research, food and / or beverage industry, quality control, or a combination thereof. Example 57: The method of any examples herein, particularly example 55 or example 56, wherein the assay or method is used for bioimaging. Example 58: The method of any examples herein, particularly examples 55-57, wherein the assay or method is used for Intracellular ion sensing, extracellular ion sensing, or a combination thereof. Example 59: A device comprising: a receptacle configured to at least partially contain the assay of any examples herein, particularly examples 1-46; an excitation source; a detector; and a computing device; wherein the receptacle is further configured to position the assay such that the assay is in optical communication with the excitation source and the detector; and wherein the computing device is configured to receive and process an electromagnetic signal from the detector; wherein, when the device is assembled together with a liquid sample, then: the receptacle is configured to at least partially contain the assay in contact with the liquid sample and position the assay in contact with the liquid sample such that the assay and the liquid sample are in optical communication with the excitation source and the detector; the excitation source is configured to apply an excitation signal to the liquid sample and the assay; the detector is configured to collect a surface enhanced Raman signal from the liquid sample and the assay; and the computing device is configured to process the surface enhanced Raman signal to determine a property of the liquid sample. Example 60: The device of any examples herein, particularly example 59, wherein the excitation source and / or the detector comprise a Raman spectrometer. Example 61: The device of any examples herein, particularly example 59 or example 60, wherein the device is further configured to output the property of the liquid sample and / or a feedback signal based on the property of the liquid sample. Example 62: The device of any examples herein, particularly example 61, wherein the feedback signal comprises haptic feedback, auditory feedback, visual feedback, or a combination thereof. Example 63: The device of any examples herein, particularly examples 59-62, wherein the property of the liquid sample comprises the presence of the target ion in the liquid sample, the concentration of the target ion in the liquid sample, the identity of the target ion, or a combination thereof. Example 64: The device of any examples herein, particularly examples 59-63, wherein the device comprises a microfluidic device. Other advantages which are obvious and which are inherent to the invention will be evident to one skilled in the art. It will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense. The methods of the appended claims are not limited in scope by the specific methods described herein, which are intended as illustrations of a few aspects of the claims and any methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative method steps disclosed herein are specifically described, other combinations of the method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

Claims

CLAIMS What is claimed is:

1. An assay for detection of a target ion via Surfaced Enhanced Raman Spectroscopy (SERS), the assay comprising: an ionophore, a nanostructured metal, an ion exchanger, and an indicator, such as a chromoionophore, wherein the ionophore comprises a binding portion configured to bind with the target ion, wherein the nanostructured metal is configured to enhance a Raman signal of at least a portion of the indicator, wherein the Raman signal changes when the target ion is bound to the ionophore.

2. The assay of claim 1, wherein at least a portion of the indicator changes orientation in the presence of the target ion (e.g., when the target ion is bound to the ionophore), and said change in orientation results in a detectable change in the Raman signal.

3. The assay of claim 1 or claim 2, wherein the indicator undergoes a change in protonation and orientation when the target ion is bound to the ionophore, which results in a detectable change in the Raman signal.

4. The assay of any one of claims 1-3, wherein the ion exchanger comprises tetrakis[3,5- bis(trifluoromethyl)phenyl]borate (TFPB).

5. The assay of any one of claims 1-4, wherein the ion exchanger comprises sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), potassium tetrakis[3,5- bis(trifluoromethyl)phenyl]borate (KTFPB), or a combination thereof.

6. The assay of any one of claims 1-5, wherein the indicator is a chromoionophore.

7. The assay of claim 6, wherein the chromoionophore comprises chromoionophore I, chromoionophore III, or a combination thereof.

8. The assay of any one of claims 1-7, wherein the ionophore selectively binds with the target ion.

9. The assay of any one of claims 1-8, wherein the ionophore is selected in view of the target ion.

10. The assay of any one of claims 1-9, wherein the ionophore comprises a calcium ionophore, a sodium ionophore, a potassium ionophore, a magnesium ionophore, a lithium ionophore, a copper(II) ionophore, a lead ionophore, an ammonium ionophore, a chloride ionophore, a carbonate ionophore, a nitrate ionophore, or a combination thereof.

11. The assay of any one of claims 1-10, wherein the ionophore comprises calcium ionophore I, calcium ionophore II, calcium ionophore IV, calcium ionophore V, sodium ionophore III, sodium ionophore IV, sodium ionophore VI, sodium ionophore X, potassium ionophore I , potassium ionophore III, magnesium ionophore I, magnesium ionophore III, magnesium ionophore IV, magnesium ionophore VII, lithium ionophore VI, lithium ionophore VIII, copper(II) ionophore I, lead ionophore IV, ammonium ionophore I, chloride ionophore II, chloride ionophore III, chloride ionophore IV, carbonate ionophore VII, nitrate Ionophore VI, or a combination thereof.

12. The assay of any one of claims 1-11, wherein the ionophore comprises a calcium ionophore, a sodium ionophore, a potassium ionophore, or a combination thereof.

13. The assay of any one of claims 1-12, wherein the ionophore comprises calcium ionophore I, sodium ionophore X, potassium ionophore I , or a combination thereof.

14. The assay of any one of claims 1-13, wherein the ionophore comprises calcium ionophore I, sodium ionophore X, or a combination thereof.

15. The assay of any one of claims 1-14, wherein the target ion comprises a cation, an anion, a polyion, a zwitterion, or a combination thereof.

16. The assay of any one of claims 1-15, wherein the target ion comprises a cation, an anion, or a combination thereof.

17. The assay of any one of claims 1-16, wherein the target ion comprises a sodium ion, a calcium ion, a potassium ion, a magnesium ion, a lithium ion, an ammonium ion, a chloride ion, a carbonate ion, a nitrate ion, a copper(II) ion, a lead ion, or a combination thereof.

18. The assay of any one of claims 1-17, wherein the target ion comprises an anion.

19. The assay of any one of claims 1-18, wherein the target ion comprises a chloride ion, a carbonate ion, a nitrate ion, or a combination thereof.

20. The assay of any one of claims 1-19, wherein the target ion comprises a cation.

21. The assay of any one of claims 1-20, wherein the target ion comprises a sodium ion, a calcium ion, a potassium ion, a magnesium ion, a lithium ion, an ammonium ion, a copper(II) ion, a lead ion, or a combination thereof.

22. The assay of any one of claims 1-21, wherein the target ion comprises a metal ion, such as an alkali metal ion, an alkaline earth metal ion, a transition metal ion, a heavy metal ion, or a combination thereof.

23. The assay of any one of claims 1-22, wherein the target ion comprises an alkali metal ion, an alkaline earth metal ion, or a combination thereof.

24. The assay of any one of claims 1-23, wherein the target ion comprises a sodium ion, a calcium ion, a potassium ion, a magnesium ion, a lithium ion, or a combination thereof.

25. The assay of any one of claims 1-24, wherein the target ion comprises a sodium ion, a calcium ion, a potassium ion, or a combination thereof.

26. The assay of any one of claims 1-25, wherein the target ion comprises a sodium ion, a calcium ion, or a combination thereof.

27. The assay of any one of claims 1-26, wherein the target ion comprises a transition metal ion, a heavy metal ion, or a combination thereof.

28. The assay of any one of claims 1-27, wherein the target ion comprises a copper(II) ion, a lead ion, or a combination thereof.

29. The assay of any one of claims 1-28, wherein the nanostructured metal comprises a metal selected from the group consisting of Ru, Rh, Pd, Os, Ir, Pt, Au, Ag, Cu, Al, and combinations thereof.

30. The assay of any one of claims 1-29, wherein the nanostructured metal comprises a metal selected from the group consisting of Pt, Au, Ag, Cu, Al, and combination thereof.

31. The assay of any one of claims 1-30, wherein the nanostructured metal comprises a metal selected from the group consisting of Au, Ag, and combinations thereof.

32. The assay of any one of claims 1-31, wherein the nanostructured metal comprises Ag.

33. The assay of any one of claims 1-32, wherein the nanostructured metal comprises a plurality of metal particles.

34. The assay of claim 33, wherein the metal particles have an isotropic shape or an anisotropic shape.

35. The assay of claim 33 or claim 34, wherein the metal particles have an average particle size of from 5 nanometers (nm) to 1 micrometer (micron, µm).

36. The assay of any one of claims 1-35, wherein the nanostructured metal comprises a metal modified with a nanostructure.

37. The assay of any one of claims 1-36, wherein the nanostructured metal comprises a film or layer modified with a nanostructure, such as a film or layer modified with a plurality of metal particles.

38. The assay of any one of claims 1-37, wherein the nanostructured metal comprises a two- dimensional material modified with a plurality of metal particles.

39. The assay of claim 38, wherein the two-dimensional material comprises a two- dimensional transition metal carbide, a two-dimensional transition metal nitride, a two- dimensional transition metal carbonitride, or a combination thereof.

40. The assay of any one of claims 1-39, wherein the nanostructured metal comprises a two- dimensional transition metal carbide, a two-dimensional transition metal nitride, and / or a two- dimensional transition metal carbonitride modified with a plurality of metal particles.

41. The assay of any one of claims 1-40, wherein the nanostructured metal comprises a two- dimensional material modified with a plurality of metal particles; the two-dimensional material comprising a two-dimensional transition metal carbide, a two-dimensional transition metal nitride, a two-dimensional transition metal carbonitride, or a combination thereof; and the plurality of metal particles comprising Au, Ag, or a combination thereof.

42. The assay of any one of claims 1-41, wherein the ionophore, the nanostructured metal, the ion-exchanger, the indicator, or a combination thereof are at least partially dispersed in a solvent.

43. The assay of any one of claims 1-42, wherein the ionophore, the nanostructured metal, the ion-exchanger, the indicator, or a combination thereof are disposed on a substrate.

44. The assay of any one of claims 1-43, wherein the ionophore, the ion-exchanger, the indicator, or a combination thereof is attached to a surface of the nanostructured metal, such that the assay comprises a modified nanostructured metal comprising the nanostructured metal having the ionophore, the ion-exchanger, the indicator, or a combination thereof attached to the surface thereof.

45. The assay of claim 44, wherein the modified nanostructured metal is at least partially dispersed in a solvent.

46. The assay of claim 44, wherein the modified nanostructured metal is disposed on a substrate.

47. A method of making the assay of any one of claims 1-46.

48. A method for detecting a target ion via ionophore-based ion-selective sensing using Surface Enhanced Raman Spectroscopy (SERS).

49. The method of claim 48, wherein the method comprises: contacting the assay of any one of claims 1-46 with a liquid sample; subsequently collecting a surface enhanced Raman signal from the liquid sample and the assay; and processing the surface enhanced Raman signal to determine a property of the liquid sample.

50. The method of claim 49, wherein the liquid sample comprises a bodily fluid.

51. The method of claim 49, wherein the liquid sample comprises an environmental sample.

52. The method of any one of claims 48-51, wherein the method is performed in vitro.

53. The method of any one of claims 48-52, wherein the method is performed in vivo.

54. The method of any one of claims 48-53, wherein the property of the liquid sample comprises the presence of the target ion in the liquid sample, the concentration of the target ion in the liquid sample, the identity of the target ion, or a combination thereof.

55. A method of use of the assay of any one of claims 1-46 and / or the method of any one of claims 48-54.

56. The method of claim 55, wherein the assay or method is used for biological research, medical diagnostics, environmental analysis, pharmaceutical research, food and / or beverage industry, quality control, or a combination thereof.

57. The method of claim 55 or claim 56, wherein the assay or method is used for bioimaging.

58. The method of any one of claims 55-57, wherein the assay or method is used for Intracellular ion sensing, extracellular ion sensing, or a combination thereof.

59. A device comprising: a receptacle configured to at least partially contain the assay of any one of claims 1-46; an excitation source; a detector; and a computing device; wherein the receptacle is further configured to position the assay such that the assay is in optical communication with the excitation source and the detector; and wherein the computing device is configured to receive and process an electromagnetic signal from the detector; wherein, when the device is assembled together with a liquid sample, then: the receptacle is configured to at least partially contain the assay in contact with the liquid sample and position the assay in contact with the liquid sample such that the assay and the liquid sample are in optical communication with the excitation source and the detector; the excitation source is configured to apply an excitation signal to the liquid sample and the assay; the detector is configured to collect a surface enhanced Raman signal from the liquid sample and the assay; and the computing device is configured to process the surface enhanced Raman signal to determine a property of the liquid sample.

60. The device of claim 59, wherein the excitation source and / or the detector comprise a Raman spectrometer.

61. The device of claim 59 or claim 60, wherein the device is further configured to output the property of the liquid sample and / or a feedback signal based on the property of the liquid sample.

62. The device of claim 61, wherein the feedback signal comprises haptic feedback, auditory feedback, visual feedback, or a combination thereof.

63. The device of any one of claims 59-62, wherein the property of the liquid sample comprises the presence of the target ion in the liquid sample, the concentration of the target ion in the liquid sample, the identity of the target ion, or a combination thereof.

64. The device of any one of claims 59-63, wherein the device comprises a microfluidic device.

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