Group of Uniform Materials Based on Organic Salts (GUMBOS): A Review of Their Solid State Properties and Applications

Ionic liquids (ILs) are defined as organic salts with melting points below 100 °C. Such ionic compounds are typically formed using bulky cations and/or bulky anions in order to produce liquids or lower melting solids. ILs have been widely explored in several research areas including catalysis, remediation, solvents, separations, and many others. The utility of such compounds has also been recently broadened to include solid phase ionic materials. Thus, researchers have pushed the boundaries of ILs chemistry toward the solid state and have hypothesized that valuable properties of ILs can be preserved and fine-tuned to achieve comparable properties in the solid state. In addition, as with ILs, tunability of these solid-phase materials can be achieved through simple counterion metathesis reactions. These solid-state forms of ILs have been designated as a g roup of u niform m aterials b ased on o rganic s alts (GUMBOS). In contrast to ILs, these materials have an expanded melting point range of 25 to 250 °C. In this chapter, we focus on recent developments and studies from the literature that provide for fine tuning and enhancing properties through transformation and recycling of diverse ionic compounds such as dyes, antibiotics, and others into solid state ionic materials of greater utility. low incubated further with FRET nanoparticles normal HeLa cells incubated FRET nanoparticles a microfluidic an Resultant emission


Introduction
In recent years, many different kinds of materials and techniques have been developed for improved analytical measurements [1][2][3][4][5][6][7]. However, in order to be generally applicable, most materials should have several key properties. These desired properties include, but are not limited to (1) simplicity of preparation (e.g. development involves simply mixing two chemical solutions), (2) tunability (easy introduction of uniform multifunctionality through simple variations), and (3) limited or no toxicity (can be easily designed using materials that are already Food and Drug Administration (FDA) approved). As an example of the latter material, the near infrared (NIR) dye, indocyanine green, for near infrared fluorescence measurements has received early approval by the FDA [7].
On the basis of the above considerations, a wide variety of materials and nanomaterials have been developed and employed for bioanalytical and environmental measurements. In regard to nanomaterials, studies reveal that in general primary properties such as spectra, colorimetric response, and magnetism are size dependent and somewhat tunable. Some of these materials, including carbon dots and silicon dots, exhibit very low cytotoxicities. However, other nanomaterials such as carbon nanotubes and quantum dots have considerably higher toxicities. In some cases, e.g. P-dots and nanogels [8][9][10], toxicity depends on the type of polymer used. Aqueous co-ordination complexes are another category of materials and nanomaterials with variable toxicities that have recently been used for analytical and environmental applications [11,12].
We believe that when one does an exhaustive examination of the literature and considers the inherent properties identified above for improved analytical measurements, a logical conclusion is that ILs, GUMBOS, and nanomaterials derived from GUMBOS (nanoGUMBOS) represent novel classes of materials that best satisfies all of the above properties. Both ILs and GUMBOS are based on use of organic salts. Examples of typical ions used in these salts (ILs and GUMBOS) are shown in Figure 1.
We note that GUMBOS and nanoGUMBOS are solid phase organic salts (m.p. > 25 °C and < 250 °C) and ILs are typically liquids or low melting solids (m.p. < 100 °C). See Figure 3 below for differentiation between ILs and GUMBOS in terms of melting points. Therefore, some GUMBOS (and nanoGUMBOS) materials fit into the general category of frozen ILs since ILs from 100 °C down to 25 °C are solids. However, many GUMBOS materials are outside the generally accepted temperature range for ILs. Accordingly, a new, more general term of GUMBOS, as defined above, was adopted to apply to this entire class of solid phase organic salts. To date, numerous strategies for this kind of chemistry have been developed. In this chapter, we desire to discuss some of these applications in detail, particularly as applied to the general area of analytical and environmental chemistry.

Biological applications
ILs have been recognized for their properties such as non-volatility, viscosity, negligible vapor pressure, high ionic strength, thermal stability, and low toxicity, among others [79]. As a result of these important properties, ILs were initially designated as green and designer solvents (i.e. first generation ILs) [80]. Eventually, due to their high tunability, new ILs were strategically designed for a variety of functional materials, including lubricants, catalysts, energy materials, etc. [81][82][83][84][85][86]. These types of ILs are known as second generation ILs. Finally, major interest has focused on development of new ILs (third generation ILs) for biological applications to achieve biocompatible and low toxic compounds through use of bio-counterions [87,88]. Moving forward, more attention from the scientific community has focused on development, or recycling of, various molecules into solid phase materials (frozen ILs or GUMBOS) for several biological applications [17, [89][90][91]. In this section, the use of frozen ILs and GUMBOS for biological applications such as cancer and antibiotic therapies are discussed.

GUMBOS and nanoGUMBOS as chemotherapeutic agents
Cancer is the second leading cause of death in the United States and is a major health concern worldwide [92]. Treatment of cancer typically includes surgery, radiotherapy, hormone therapy, immunotherapy and/or chemotherapy [93]. Effectiveness of these treatments depends upon several factors, such as stage of cancer at the moment of diagnosis, general health of the individual, size and type of tumor, among others. In general, treatment of a person with cancer will involve a combination of therapies as a result of these several factors, and chemotherapy is the most commonly employed treatment. Unfortunately, chemotherapy will often be accompanied with several adverse effects, such as nausea, vomiting, diarrhea, fatigue, malnutrition, anemia, hepatotoxicity, nephrotoxicity, among others [94][95][96]. These side effects are the result of high toxicity of typical chemotherapeutic agents that generally lack selectivity toward carcinogenic cells. For all of these reasons, over the past decades major attention has focused on developing new chemotherapeutic agents that are selectively toxic to cancer cells [97][98][99]. Moreover, investigations have also focused on early detection methods that involve use of tumor-targeting dyes, as well as near infrared (NIR) dyes for detection, photothermal therapy (PTT) and photodynamic therapy (PDT) [100][101][102][103].
Due to their relatively high division rate and subsequent growth relative to normal cells, cancer cells use more energy [104][105][106]. It is well established that the mitochondria are organelles that synthesize adenosine triphosphate (ATP), which is the energy source of cells. As a result, mitochondria in tumor cells have a higher negative mitochondrial membrane potential as compared to normal cells. For this reason, major interest has been directed toward study of cationic compounds as well as positively charged vesicles as chemotherapeutic agents. Several publications from the literature document that these type of compounds are attracted to, and accumulate more selectively, in this organelle of cancer cells, resulting in disruption of ATP synthesis and subsequent induction of cell death [16,17,22,89,107,108].
Cationic rhodamine dyes have been studied as mitochondrial targeting agents as early as the 1970s [109][110][111][112][113][114]. Furthermore, studies with rhodamine dyes demonstrate that these dyes are toxic to cells above certain concentrations [115,116]. In contrast, it has been previously reported that hydrophobicity of drugs may improve cellular uptake and distribution inside cancer cells [117] [BETI] was observed in this study. Clearly, anion variation affected and tuned hydrophobicity, along with other physico-chemical properties, of the parent dye. The low water solubility of these compounds allowed synthesis of nanoGUMBOS through a simple reprecipitation method. In vitro cellular cytotoxicity of these GUMBOS and nanoGUMBOS towards normal breast cells (Hs578T), hormone-independent human breast adenocarcinoma (MDA-MB-231) and hormone-dependent human breast adenocarcinoma (MCF7) cell lines using an MTT assay was evaluated. that these compounds were more selectively toxic toward cancer cell lines than normal cells. Moreover, these GUMBOS were more toxic against MDA-MB-231 cancer cells, which were the most aggressive cancer cell lines evaluated using 50% inhibition concentration (IC 50  These experiments allowed the investigators to conclude that these nanoGUMBOS were internalized into cancer cells through a clathrin mediated endocytosis pathway. In contrast, these nanoGUMBOS were found to be internalized in normal cells through an independent endocytic route. Interestingly, it was further demonstrated that [R6G][BETI] nanoGUMBOS passed through lysosome vesicles before reaching the mitochondria. For this reason, Bhattarai and coworkers investigated the integrity of these nanoparticles at lysosomic pH (pH = 4) and normal pH (7.4) values using transmission electron microscopy (TEM) and dynamic light scattering (DLS) experiments. Evaluation of these results demonstrated that nanoparticles lost integrity under acidic pH similar to those in the lysosome, thus releasing [R6G] + that subsequently entered the mitochondria, and inhibited ATP synthesis and eventually causing apoptosis of cancer cells. Internalization in normal cells did not involve entry through the lysosome. Thus, these investigators concluded that this differential internalization route was the primary reason for selectivity of [R6G][BETI] nanoGUMBOS towards cancer cells. Finally, Bhattarai and co-workers evaluated in vivo efficacy of nanoGUMBOS in tumor size reduction in an athymic nude mouse model. Evaluation of these results demonstrated that nanoGUMBOS inhibited tumor growth and decreased tumors size by 50% making this material a good candidate for in vivo chemotherapeutic applications [18].
It has been previously reported in the literature that there is a strong correlation between size, hydrophobicity, and surface charge of nanomaterials as related to resultant toxicity against cancer cells [118,119]. Moreover, in vivo studies have demonstrated that nanoparticle size has an important effect in increasing cellular uptake into tumor tissue via leaky tumor vasculature through a phenomenon known as enhanced permeability and retention (EPR) effect [120]. In this regard, Hamdan et al. have shown that use of cyclodextrins (CDs) in nanoGUMBOS synthesis results in more uniform and smaller nanoparticles [45]. CDs are cyclic oligosaccharides having conical shapes, with an inner hydrophobic cavity and an external hydrophilic surface. This characteristic structure of CDs allows interaction with some hydrophobic compounds to provide encapsulation and increased water solubility [121,122] [TPB] nanoGUMBOS in the presence of three different CDs: 2-hydroxypropyl-αCD (HP-αCD), 2-hydropropyl-βCD (HP-βCD), and γ-CD in order to optimize nanoparticle size, uniformity and stability [17]. In this report, nanoparticle synthesis was performed by directly mixing stoichiometric quantities of each parent compound in the presence of predetermined concentrations of each CD until synthetic conditions were optimized. These researchers noticed, from TEM and zeta potential data, that CD-templated nanoparticles presented lower size and higher zeta potentials as compared to control nanoGUMBOS (Figure 4). In Table 1, size, zeta potential and cytotoxicity of CD-templated and control nanoGUMBOS are summarized. Based on evaluation of results obtained using cytotoxicity studies performed with MDA-MB-231 and pancreatic cancer (MiaPaca) cell lines, these researchers noticed a significant decrease in IC 50 values, suggesting that CD-templating enhances toxicity of these nanoparticles.
Another approach from this laboratory involved use of an IR-780 dye to synthesize GUMBOS and nanoGUMBOS [16]. IR-780 is a NIR fluorescent dye that has been studied as a possible theranostic agent since it can be employed as an imaging agent as well as a photothermal and photodynamic agent [123][124][125]. In this regard, Chen et al. synthesized three IR-780-based GUMBOS through a simple metathesis   50 values against MDA-MB-231, which was the most invasive and aggressive cancer cell line evaluated. These findings indicate that a simple anion variation in a parent compound can selectively change its cytotoxicity towards cancer cell lines. Relative cell viability was evaluated for each nanoGUMBOS in normal breast cells. These researchers found that all nanoGUMBOS studied were more selectively cytotoxic against cancer cell lines. Results observed after cellular uptake and fluorescence microscopy studies of each nanomaterial allowed these researchers to conclude that nanoGUMBOS, especially [BETI], were internalized and accumulated within the mitochondria in higher amounts than with the parent compound. It has been previously reported in the literature that mitochondrial accumulation of [IR-780][I] is followed by cellular apoptosis [126]. In addition, these researchers investigated nanoGUMBOS as inducers of necrosis or mitochondrial disruptors, by employing a mitochondrial toxicity assay. Evaluation of these results showed that nanoGUMBOS presented behavior similar to [I] nanomaterials and acted as mitochondrial toxins by inhibiting oxidative phosphorylation. In summary, nanoGUMBOS synthesized in this work represented great potential as possible chemotherapeutic agents along with a strategic advantage as compared to other reported nanomaterials that require complicated synthetic procedures and labels to increase selectivity against cancer cells [127][128][129][130].
In another work, Chen and coworkers evaluated in vitro and in vivo cytotoxicity and photothermal properties of CD-[IR-780][TPB] complexed nanoGUMBOS [89]. In this work, [TPB] GUMBOS were synthesized and nanoGUMBOS were obtained in using HP-β-CD. In this case, CD-[IR-780][TPB] nanoGUMBOS represented larger diameters than nanoGUMBOS without CD (Figure 5a and b). These results were different from those obtained by Bhattarai et al. [17], in which CDs acted as a template. Based on analyses of TEM and differential scanning calorimetry results, along with computational modelling, the authors demonstrated that a stable complex was formed between HP-β-CD and  [131]. Redox values, zeta potentials, HOMO energy level, as well as optical properties of all [Cy]-based organic salts were determined in that study. These results demonstrated that counterion exchange allowed tuning of HOMO energy levels of [Cy] + . However, absorbance of all synthesized Cy-based organic salts spectra remained essentially the same.
The above cited authors then synthesized nanoparticles through a simple reprecipitation method. All Cy-based organic salts nanoparticles were determined to have an average size between 5-9 nm and were stable for 22 days. Following these experiments, Broadwater and collaborators evaluated cytotoxicity and phototoxicity of Cy-based organic salt nanoparticles against two different cell lines: human lung carcinoma (A549) and metastatic human melanoma (WM1158) in the absence and presence of 850 nm light. These results indicated that [I] − , [SbF6] − , and  Finally, these authors evaluated in vitro imaging properties of all Cy-based organic salts nanoparticles. Analyses of these results demonstrated that non-toxic Cy-based organic salts could be employed at higher concentrations that resulted in a higher fluorescence intensity. Additionally, an in vivo evaluation of these compounds demonstrated that these compounds were accumulated in tumor sites.

Recycling of antimicrobial agents and other medicines
Antibiotics were introduced into modern medical practices in the 19th century with the discovery of sulfonamides in the 1930s and penicillin in the 1940s [132][133][134]. Without such medication, people often died from infections such as syphilis, gonorrhea, and pneumonia. Thus, use of these antibiotics represented the saving of many thousands of lives and a new era in medicine [135,136]. Nevertheless, over the years since such discoveries, increased production, indiscriminate use, and over consumption of antibiotics has created an unfortunate outcome of antibiotic and multi-antibiotic resistant bacteria [137][138][139]. For this reason, the scientific community has begun to focus on syntheses of new antibiotics that could provide alternative therapies in order to avoid bacterial resistance mechanisms. However, syntheses of completely new antibiotics require great ingenuity, intense synthetic prowess, excellent purification, and considerable resources [140][141][142]. As an alternative to the foregoing strategy, several research groups have applied an ion metathesis strategy for antibiotic renewal and enhancement. Thus, recycling of current antibiotics have become a reality.
Florindo et al. synthesized ampicillin based ILs employing triethylammonium ([TEA] + ), choline ([N 1112 OH] + ), trihexyltetradecylphosphium ([P 66614 ] + ), 1-ethyl-3-methylimidazolium [C 2 MIm] + , 1-hydroxy-ethyl-3-methylimidazolium [C 2 OHMIm] + and cetylpyridinium [C 16 Pyr] + as cations to tune crystalline forms and pharmaceutical properties [143]. In that study, water solubility, octanol/water partition coefficient (K o/w ), and phospholipid/water partition (K p ) of synthesized compounds were evaluated. Water solubility of active pharmaceutical ingredients (API) is of great importance because it determines the accessibility and distribution of API within the body. Using water solubility results at room temperature and at 37 °C, the following trend was observed: . All Amp-based ILs studied showed lower solubility as compared to [Na][Amp] at room temperature. However, cations with hydroxyl groups presented higher solubility at 37 °C than the parent compound. In contrast, Amp-ILs with longer carbon chains, such as [P 66614 ] + and [C 16 Pyr] + , showed K p values higher than the parent compound, indicating that these compounds could interact better with cellular membranes. Based on results obtained in this study, [N 1112 OH][Amp] provided the most promising pharmaceutical properties with higher solubility, lower cytotoxicity, lower inflammation response and similar K o/w relative to the parent compound. Thus, these researchers confirmed that pharmaceutical properties from [Na][Amp] could be finely tuned through simple counterion exchanges.
In another study, the same researchers synthesized ciprofloxacin and norfloxacin fluoroquinolones (FQ ) based protic ionic liquids (PILs) through reaction with mesylic acid (

[Mes][H]), gluconic acid ([Glu][H]), and glycolic acid ([Gly][H])
to tune their crystalline forms and pharmaceutical properties to enhance their bioavailability [144]. In this case, similar properties as in previous studies were evaluated [143]. The authors observed a clearly increasing trend in aqueous solubility depending on the anion present in FQ-PILs: These observations were in agreement with K o/w studies obtained by these researchers. Similar K p results for parent FQs were obtained for their respective organic salts, which indicated that interactions with cellular membranes were not affected. Based on these results, Florindo et al. concluded that FQ-based organic salts studied in this work presented high potential as alternatives to the original antibiotics.
Santos, et al. employed two FQs (ciprofloxacin and norfloxacin) to synthesize active pharmaceutical ingredient (API)-based ILs by combining their salts with the following cations: [145]. Water solubility of the synthesized compounds were evaluated, and the following trend was observed: This trend was similar to results reported in a previous study from the same group [145]. The authors determined IC 50 concentrations of these FQ-ILs in this study against three bacteria: Bacillus subtilis (B. subtilis), Staphylococcus aureus (S. aureus) and Klebsiella pneumoniae (K. pneumonia) and compared results with IC 50 values of the parent compounds. In order to evaluate if the synthesized compounds were more effective than the parent compounds, these researchers calculated the relative decrease of inhibitory concentration (RDIC) obtained by divided the minimum inhibitory concentration (MIC) values of each API-ILs by MIC of the corresponding API. Interestingly, most compounds evaluated in this work presented better antimicrobial activity than the original FQ with RDIC values higher than one [145].
Frizzo et al. employed sodium ibuprofen ([Na][Ibu]) and sodium docusate to synthesize API-based ILs [146]. Sodium cations in the parent compounds were replaced with ranitidine ([Ran] + ), diphenhydramine, glycine, or glycine ethyl cations. In this work, these researchers tested all synthesized compounds along with parent compounds against several types of bacteria and species of Candidas fungus. Evaluation of their results demonstrated that, in general, all API-ILs presented better antifungal activity than the precursors. For example, all ibuprofen-based ILs demonstrated antifungal activity. Interestingly, [Ran][Ibu] ILs presented antifungal activity when the parent compounds did not. In contrast, most API-ILs presented higher antibacterial activity than the parent compounds. These researchers synthesized ibuprofen-and docusate-based ILs that demonstrated high potential as antimicrobial and antifungal agents.
Cole and coworkers proposed recycling of antibiotics into GUMBOS [15]. In this work, ampicillin based GUMBOS (Amp-GUMBOS) were synthesized through a simple metathesis reaction where a sodium cation was replaced by hexadecylmethyl-imidazolium ([C 16 MIm] + ), hexadecyl-dimethyl-imidazolium ([C 16 M 2 Im] + ) and [C 16 Pyr] + . These Amp-ILs were tested against Gram negative and positive bacteria and compared to parent compounds. Interestingly, MIC values obtained for Amp-GUMBOS in these experiments demonstrated that these concentrations were between 2 to 43 times lower than MIC values determined for ampicillin.
Following their previous studies, Cole and co-workers employed chlorhexidine and ampicillin to synthesize antibacterial GUMBOS [14]. These two antibacterial agents are commonly used in veterinary practices to treat and/or prevent the presence of E. coli strains that are commonly found in cattle. The presence of E. coli strains could produce severe illness in humans, especially E. coli O157:H7, which is well-known because it produces bloody diarrhea, haemorrhagic colitis and haemolytic uraemic syndrome in humans [148][149][150]. For this reason, development of a prophylactic treatment for this type of bacteria are highly desirable for eradication or minimization in cattle to prevent human illnesses. Cole et al. evaluated the efficacy of this Chlorhexidine di-ampicillin GUMBOS to kill several E. coli O157:H7 strains isolated from different sources such as chicken, pork, beef, apple cider, burger and humans [14]. Chlorhexidine di-ampicillin GUMBOS was found to kill E. coli strains more effectively since these GUMBOS presented MIC values much lower than the parent compounds and their unreacted stoichiometric mixture. Moreover, interaction indices indicated that this antimicrobial GUMBOS presented a synergetic mechanism effect. Chlorhexidine is a commonly used antiseptic; however, it presents high cytotoxicity against normal cells. For this reason, these researchers studied cytotoxicity of GUMBOS, parent compounds and unreacted stoichiometric mixtures in Hela cells. Interestingly, GUMBOS were less toxic than parent chlorhexidine, reaching 93% cell viability.
In another work, Cole and coworkers recycled four β-lactam antibiotics (ampicillin, cephalothin, carbenicillin and oxicilin) into GUMBOS, by combining them with chlorhexidine diacetate [151]. Twenty-five bacteria isolates were obtained from several sources, where most of these were resistant or multi-resistant to antibiotics. These four β-lactam -based chlorhexidine GUMBOS were tested against these isolates. Results obtained by Cole and coworkers demonstrated that these β-lactam -based chlorhexidine GUMBOS were more effective against these isolates with MIC values in a range between 0.1 to 32 μM as compared to parent compounds with higher MIC (5 to >1250 μM). Moreover, in this report Cole et al. evaluated if these GUMBOS presented a synergetic, additive or antagonist effect relative to their unreacted mixtures of stoichiometric equivalents. Interestingly, these researchers found that for most GUMBOS studied, the observed effect was synergetic [152].
Neisseria gonorrhoeae is another bacterial target that is primarily sexually transmitted and responsible for the disease gonorrhea [153]. In recent years, N. gonorrhoeae resistance to current treatments have been isolated and reported around the world [154,155]. Lopez et al. have synthesized GUMBOS from an antiseptic octenidine and a discontinued antibiotic carbenicillin as a possible alternative to reduce and minimize N. gonorrhoeae transmission [156]. The zone of inhibition (ZOI) for N. gonorrhoeae strains and clinical isolates were studied in the presence of GUMBOS, parent compounds, and antibiotics currently employed for gonorrhea treatment. Evaluation of results obtained demonstrated that synthesized GUMBOS presented an additive effect as compared to the parent compounds as well as an equivalent antimicrobial activity like azithromycin.

Sensing materials
Sensing strategies for a variety of systems, from biological targets [157], environmental and regulatory applications [158,159], mechanical integrity of structures [160][161][162], and more [60,163], are continuously under investigation in the scientific community. In general, recognition can be categorized into two different methodologies: targeted and non-targeted [164]. Targeted strategies require materials that are designed to respond to specific analyte(s) and thus, require a high degree of specificity for singular analytes [63,159,165]. Differential strategies, however, can potentially provide information within convoluted and complex mixtures based on several non-specific sensors or one sensor with multi-layered responses to different analytes [166]. In the following sections, solid-state ionic materials for various sensing applications are discussed.

Ratiometric sensing: fluorescence imaging
Previous investigations using fluorescent imaging with solid-state ionic materials have undergone scrutiny to prevent or reduce self-quenching between dye molecules in order to enhance properties such as excitation energy transfer and achieve on/off switching in nanoparticle structures [167][168][169][170][171][172]. Traditionally, dye selfquenching has been rectified by introducing bulky side-chains into the molecular structure via synthetic organic chemistry [172][173][174]. However, this type of strategy requires several synthetic and purification steps that result in increased expense. In contrast, large counterions were observed to also inhibit this self-quenching phenomenon in a much more facile manner through a simple ion metathesis reaction [28,50]. Several research groups have capitalized on this strategy to study polymeric nanoparticle encapsulated rhodamine-derived GUMBOS, respective photophysical properties, and cellular uptake ability for imaging applications along with targeting agents to provide organelle contrast [28,172,175,176].
More recently, researchers have diversified beyond cellular imaging techniques. For example, Severi et al. have explored polymer encapsulation of nanoprobes that undergo efficient Förster resonance energy transfer (FRET) for potential pointof-care applications with smartphones [177]. In this study, ester-modified cations rhodamine 110 and 6G cations ([R110] + and [R6G] + , respectively) were employed as FRET donor dyes with bulky tetrakis[3,5-bis (1,1,1,3,3,3-hexafluoro-2-methoxy-2-propyl)phenyl]borate trihydrate ([F12] − ) and tetrakis(perfluoro-tertbutoxy) aluminate ([F9-Al − ]) counterions [176]. These Ion pairs were encapsulated with DNA cancer marker (survivin) targeted polymer nanoparticles, which were also functionalized using a red-emitting oligonucleotide-functionalized dye as a FRET acceptor. After nanoparticle size, quantum yield (QY), FRET acceptor concentration optimization, and evaluation of FRET capabilities, encapsulated [R6G][F9-Al] nanoprobes were evaluated for red, green, blue (RGB) survivin DNA marker detection in solution using fluorescence spectroscopy. These researchers found that their designed system had a limit of detection of 3pM. Upon optimization of microscopic and digital imaging, these researchers also found that using an iPhone SE, [FL] triple GUMBOS. The resultant compound was then employed for nanoGUMBOS synthesis, and when precipitated from water with neutral pH, nanoparticles of approximately 4.4 nm ± 0.7 nm were obtained. However, when using other pH values for nanoGUMBOS synthesis, these researchers determined that nanoGUMBOS sizes and size distributions varied. Absorbance and fluorescence emission properties from low to high pH values were reported, and noticeable ratiometric changes in spectra were observed. A linear ratiometric trend corresponding to pH-dependent FRET responses was observed between moderate pH values (approximately pH 5.0 to 7.0), from which quantitative information may be derived. To further demonstrate the applicability of this three-component nanoGUMBOS system, these investigators also conducted fluorescence microscopy imaging studies with normal and cancerous breast cells. These studies demonstrated that nanoGUMBOS maintained clear selectivity for breast cancer cells since, as cells were illuminated. In contrast, normal cells remained dim. Therefore, three-component nanoGUMBOS were determined useful for both pH sensing and fluorescence imaging of breast cancer cells without the use of polymer encapsulation [27].
Another application for FRET-based sensing of solid-state ionic materials is described by Ashokkumar et al. [179]. In this work, oxygen sensing nanoparticle probes for cellular systems were developed using a polymer encapsulated novel cyanine dye called [BlueCy] + tetrakis(pentafluorophenyl) borate ([F5-TPB] − ) or [BlueCy][F5-TPB] that was also loaded with oxygen sensing platinum octaethylporphyrin (PtOEP) as a FRET acceptor. In this case, [BlueCy] + was designed as FRET donor and synthesized from two cyanine dyes: 2-methyl-3-octadecylbenzo[d] thiazol-3-ium iodide and 3-methyl-2-(methylthio)benzo[d]thiazol-3-ium iodide. After dye encapsulation into poly(methyl methacrylate-co-methacrylic acid) and poly(lactic-co-glycolic acid), PMMA-MA and PLGA, respectively, it was determined that both dyes were successfully incorporated into PMMA-MA. Nanoparticle sizes, PtOEP loading, and photophysical properties were evaluated. These investigators determined that dye encapsulated PMMA-MA nanoparticles were 40 nm in diameter with 17% QY after reprecipitation from dioxane. Moreover, after testing several ratios of donor dye loadings, ratios of 1:100 (PtOEP:[BlueCy][F6-TPB]) demonstrated good FRET efficiency. Solution based experiments for oxygen sensing were performed, and ratiometric trends were demonstrated for oxygen rich and poor environments. After confirming low phototoxicity when incubated with HeLa cells, the investigators conducted further studies with FRET nanoparticles in low and normal oxygen environments. HeLa cells were incubated with FRET nanoparticles in a microfluidic device, and an oxygen gradient was introduced by application of an oxygen scavenger. Resultant emission gradients were observed after fluorescent microscopic images were obtained. Ultimately, these researchers demonstrated the utility of their nanoprobe for detection of cancer cells via microfluidic application. As a result, the authors concluded that this probe could also be used to visualize oxygen gradients in cancerous cells.
In another study from the Warner research group, nanoGUMBOS were synthesized and evaluated as ratiometric sensors for reactive oxygen species (ROS) [42]. Cong et al. designed binary nanoGUMBOS using reprecipitation of 1,1′-diethyl-2,2′-cyanine and 1,1′-diethyl-2,2′-carbocyanine bis(perfluoroethylsulfonyl) imide ([PIC][NTf 2 ] and [PC][NTf 2 ], respectively). Optimal FRET efficiency was determined to be 10:1 [PIC]:[PC] molar ratio, and binary nanoparticles shapes were classified as nanodiamonds with spectrally consistent J-aggregation. Analysis of variance (ANOVA) was employed to investigate reactivity of ROS with nanoGUM-BOS. Significant differences were observed for hydroxyl radical ( • OH) over four other evaluated ROS, indicating selectivity of this binary nanoGUMBOS system toward • OH species. Moreover, these investigators observed a linear trend for ratiometric sensing of this probe at various concentrations of • OH in the presence of singlet oxygen ( 1 O 2 ). Further, potential applications in imaging were investigated, nanoGUMBOS were incubated with breast cancer cells and exposed to oxidative stress. Fluorescence emission changes before and after oxidative stress indicated results in agreement with solution-based studies. Therefore, a binary ratiometric nanoGUMBOS probe was developed for potential quantitative ROS imaging studies using a facile method.

Differential sensing: biological applications
Biosensing of mixtures of biomarkers and/or proteins is of particular interest for disease diagnosis and treatment [180][181][182]. Many current methods, such as enzyme-linked immunosorbent assay (ELISA) or polyacrylamide gel electrophoresis (PAGE) coupled to mass spectrometry, require expensive resources and labor intensive steps [182][183][184]. Organic salts are of increasing interest for development of fluorescent sensor arrays for protein detection and discrimination as they are easily tunable for increasing hydrophobicity, traditionally more stable upon ion exchange, and require little resources for purification [90].
As a result of notably larger sensor responses, [TNS]-based GUMBOS were determined to have highest sensitivity to HSA, α-Ant, and β-Lac proteins. For this reason, the investigators employed responses for sensor responses to different concentrations of HSA, α-Ant, and β-Lac for multivariate analysis. Both sensor response values and corresponding protein concentrations were employed to build a principal component analysis (PCA) model. By employing the first two principal components (PCs), which accounted for 99.72% of the variance, a linear discriminant analysis (LDA) model with cross-validation was constructed reaching 100% discrimination accuracy. These researchers noted that the highest sensor responses were obtained for HSA and α-Ant. Thus, these sensor responses were employed to generate anther PCA model in order to evaluate discrimination between these two proteins regardless of protein concentration. In this model, the first two PCs accounted for 99.91% variance, and LDA with cross-validation resulted in 91.7% accuracy. To improve this accuracy, these investigators normalized sensor responses for each protein, constructed a PCA model with the first three PCs corresponding to 98.29% variance. These three PCs were employed for LDA construction and, with cross-validation, accuracy resulted in 100% discrimination. Furthermore, five mixtures of different HSA: α-Ant ratios were evaluated for mixture discrimination analysis. In this case, PCA followed by LDA resulted in 100% discrimination accuracy. Thus, TNS-GUMBOS were evaluated and confirmed as useful materials for protein sensor arrays for analyses of serum proteins HSA, α-Ant, and β-Lac. In the above study, seven proteins were investigated, including the four most abundant serum proteins: HSA, IgG, transferrin (Trans), and fibrinogen (Fib), along with three non-serum proteins hemoglobin (Hb), cytochrome C (CytC), and lysozyme (Lys), with each protein exhibiting different physical characteristics. The investigators observed different response patterns for each protein. In this work, these researchers determined that employing raw data was optimal for constructing an LDA model, in which 100% discrimination accuracy of proteins was achieved. Among different protein concentrations, sensor responses were determined to be stable between 0.1 to 20 μg/mL. Mixtures of two proteins, HSA and Hb, were also investigated in this work. Various weight ratios of HAS:Hb mixtures from 100% HSA to 100% Hb, were evaluated and 100% accuracy was achieved when LDA was constructed using these response patterns. However, 80:20 HSA:Hb was observed to be an outlier with the lowest canonical score values, and further analysis using hierarchical cluster analysis determined this dataset to be less related to other ratios. Protein spiked artificial urine with 5 μg/mL protein concentration was employed to evaluate sensor array performance in real samples, and LDA model performance achieved 100% discrimination accuracy. Thus, a series of TC-based GUMBOS were successfully synthesized into nanoGUMBOS and microGUMBOS and developed as protein sensor arrays capable of 100% discrimination in complex mixtures.

Differential sensing: volatile organic compounds
ILs have been explored for quartz crystal microbalance (QCM) applications as chemosensors for detection and discrimination of volatile organic compounds (VOC) [185][186][187][188]. However, for these investigations, differentiation of VOCs sensor response relied on concentration and molecular composition of an analyte. In 2012, Regmi and coworkers developed a system to correlate sensor responses using GUMBOS-polymer composite [75]. In this work, investigators characterized and explored the responses of cellulose acetate and 1-butyl-2,3-dimethylimidazolium hexafluorophosphate (CA-[BM 2 IM][PF 6 ]). By carefully evaluating characteristic responses upon exposure to control sensors, composite material, and confirming results using molecular dynamic simulations, investigators determined that sensor response recorded as changes in frequency were directly proportional to changes in motional resistance. Thus, these researchers successfully derived molecular weight trends from their composite sensor.

Optoelectronic developments
With increasing global commercialization of state-of-the-art optoelectronic displays, the demand for higher performance and flexible materials has also increased [107][108][109][110]. In general, these devices are comprised of emissive layers between electrodes along with several other electronically active layers. Organic light emitting diodes (OLEDs) and organic photovoltaics (OPVs) have been the central target for a multitude of research groups, from organic emissive layer development to full device performance [107][108][109][110][111]. Counterion strategies using cations and anions within active layers for enhancement on optoelectronic device fabrication to effects on emission and device function will be discussed in the following sections.

Counterion strategies for light emissive layers in OLEDs
Scientists have optimized several characteristics for targeted OLED development where they require consistent uniformity of emissive layers for potential manufacturing production [190], low crystallinity to prevent non-linear optical activity [191,192], resistance to oxidation and water [193], and high thermal stability and optical purity [194]. Ionic transition metal complexes (ITMCs) are of huge interest as a wide range of emissive hues is easily achievable, synthesis is relatively simple, and they have desirable luminescent properties [195]. However, traditional methods of OLEDs fabrication involves vacuum evaporation deposition, or vacuum thermal evaporation (VTE) [196], which involves uniform coating of emissive layers [197].
In this regard, Dongxin Ma and coworkers have investigated four cationic iridium complexes as candidates for VTE through counterion control [195]. By incorporating large non-coordinating anions, these investigators achieved VTE iridium-based ionic emissive layers. The anions [PF 6 ] − , [TPFB] − , and tetrakis[3,5bis(trifluoromethyl)phenyl]borate ([BArF] − ) were employed for quantum chemical calculations, and the investigators determined that distances between iridium and boron atoms were larger than 8 Å with both [TPFB] − and [BArF 24 ] − counterions. In comparison, distances between iridium and phosphorous was determined to be 6 Å, as a result of a larger partial positive charge on the phosphorous atom. Compounds synthesized from metathesis with bulkier anions [TPFB] − and [BArF 24 ] − were employed for device fabrication using VTE as larger interatomic distances were presumed more suitable for phase transition, and device performance was evaluated. These investigators found devices ranged from blue to red-orange with external quantum efficiencies (EQEs) ranging from 1.2% (blue emission) to 8.1% (yellow emission) [195]. In 2018, these anions were also employed to produce two red-orange devices based on cationic iridium compounds, and these compounds were useful as dopants in 4,4′,4″-tris(carbazol-9-yl)triphenylamine emissive layers (TCTA) to produce white OLEDs with Commission Internationale de L'Eclairage (CIE) coordinate values equal to (0.33, 0.34), that were near to the required values (0.33, 0.33) [198].
Carbazole-containing compounds with expanded conjugation are known to provide efficient blue emission, although they often require high labor and resource costs. In this regard, Siraj [54]. These GUMBOS were then compared to parent [CI][I] to evaluate counterion effects on thermal and photochemical properties that relate to performance for blue-emitters for OLEDs. In this study, non-uniform packing was observed in all GUMBOS as a result of cation structure. All ion-exchanged GUMBOS also demonstrated significantly higher thermal stabilities with onset degradation temperatures ranging from 310 to 417 °C as determined by thermal gravimetric analysis (TGA), where increasing size of anion yielded increased degradation temperature ( While VTE has dominated OLED manufacturing, it often requires expensive equipment and is both energy and time consuming [200]. For this reason, several researchers have explored solution processing methods, such as spin coating [200], electrospray deposition [54], along with other methods [201,202] to provide faster, more inexpensive fabrication procedures [203]. In this report, [ [204], and uniform coating was achieved and confirmed by scanning electron microscopy and fluorescence microscopic analysis. Solid-state emission spectra displayed very slight red-shifting from methanolic spectra of ion-metathesis GUMBOS. In addition, photostabilities were investigated, and [CI][BETI] displayed an irradiation-induced increase in photostability. In contrast, [CI][OTf ] and [CI][NTf 2 ] were relatively stable while irradiated for 3000 s. Moreover, cyclic voltammetry and quantum chemical calculations further supported spectral properties of evaluated CI-based GUMBOS. In 2016, Zhang and coworkers designed a novel cyanopyridinium stilbene cation ([Py] + ) in order to examine the influence of counterion effects on solidstate photophysical properties [205]. Chloride ([Cl] − ), nitrate ([NO 3 ] − ), tosylate ([OTs] − ), and [TPB] − anions were employed in this study to form Ion pairs, and the resultant compounds showed little fluorescence in solution. When explored as films, blue-shifting of emission peaks occurred and increased with increasing hydrophobicity of counterions; QYs also increased following this trend. However, [Py][TPB] GUMBOS were non-emissive in solid-state. In order to understand this variance in trend, investigators used X-ray crystallography and quantum chemical calculations. From these studies, the authors determined that dimeric fluorophore aggregates were responsible for emission in GUMBOS. In [Py][TPB], fluorophores became dilute as a result of bulky anions, resulting in very weak fluorescence emission. This was also confirmed in quantum chemical studies, where intramolecular charge transfer characteristics were confirmed through prediction of frontier molecular orbital placement to reveal donor-σ-acceptor properties for dimeric stacking. In another study, expansion of applications of the propidium dication ([P] 2+ ) was investigated by exchanging iodide counterions for [OTf ] − , [NTf 2 ] − , and [BETI] − anions to generate P-based GUMBOS for potential solid-state applications [53]. Thermal, spectral, photo-physical, computational, and electrochemical properties were investigated for all P-based GUMBOS. While [P][OTF] retained physical properties similar to parent dye, such as solubility in more polar solvents, thermal degradation, and higher relative crystallinity, similar to the parent compound. In contrast, [ [206]. These investigators also performed cyclic voltammetry to determine oxidation and reduction potentials for each P-based GUMBOS, as well as solution-phase QY calculations. It was determined through computational experiments that electronic transitions would lead to an increased propensity for torsional twisting in the solid state [207]. From these studies, the authors concluded that by simple counterion exchange, applications for propidium dication may be expanded beyond biological probes to potential candidates in optoelectronic devices [53].

Recent advances in dye-sensitized solar cells
Dye-sensitized solar cells (DSSCs) are an emerging next-generation technology in OPVs [208]. Through intrinsic characteristics such as natural transparency, good efficiency in low light conditions, flexible substrate production and more, applications may be expanded to windows, indoor fixtures, and wearable electronics [209,210]. Incorporation of two or more complementary sensitizing dyes allows for potential absorption of all wavelengths of sunlight to achieve much higher power conversion efficiencies (PCEs) [208,[211][212][213][214]. When implemented in this field, tunable investigations of co-sensitizing dyes that are also ionic and primarily limited to structural variations of zwitterionic squaraine-heptamethine structures rather than ion pairs [215,216]. Polymethine, or cyanine dyes, however, have been extensively studied in OPV technologies, and several groups have begun explorations into counterion application in DSSCs [217][218][219][220][221][222].

Organic salts to reduce work function in optoelectronics
Work function (WF) is a metric by which charge transfer at electrodes is measured to determine electron injection efficiencies of optoelectronics [228]. For this reason, many groups have targeted improving device efficiency by optimizing charge transport at interfacial layers by including electroactive coatings [228][229][230]

Conclusions and future directions
Much like room temperature ILs, the ionic properties of frozen ILs and GUMBOS lend to high tunability as a result of the exponential combinations possible of known anions and cations. This important characteristic allows for strategic design of specific GUMBOS for a targeted analytical task. This chapter summarized a few examples of possible GUMBOS applications. Moreover, it has been demonstrated that several physico-chemical properties of these compounds are improved in solid state as compared to liquid phase organic salts. For these reasons, we hypothesize that implementation of solid-phase ILs and GUMBOS in the analytical and materials fields will increase in the future.   [76] Siraj, N.W., Isiah M. , Compositions including a ruthenium molecular dye-based GUMBOS, methods of making compositions, methods of use of compositions, and devices using the compositions. 2015.