Group 14 Metallafluorenes as Sensitive Luminescent Probes of Surfactants in Aqueous Solution

Sila- and germafluorenes containing alkynyl(aryl) substituents at the 2,7- position are strongly emissive with high quantum yields in organic solvents. Provided they are sufficiently soluble in water, their hydrophobic structures have the potential for many biological and industrial applications in the detection and characterization of lipophilic structures. To that end, the emission behaviors of previously synthesized 2,7- bis[alkynyl(biphenyl)]-9,9-diphenylsilafluorene (1), 2,7- bis[alkynyl(methoxynaphthyl)]-9,9-diphenylgermafluorene (2), 2,7- bis[alkynyl(p-tolyl)]-9,9-diphenylsilafluorene (3), and 2,7- bis[alkynyl(m-fluorophenyl)]-9,9-diphenylsilafluorene (4) were characterized in aqueous solution and in the presence of various surfactants. Despite a high degree of hydrophobicity, all of these metallafluorenes (MFs) are soluble in aqueous solution at low micromolar concentrations and luminesce in a common aqueous buffer. Further, the 2,7 substituent makes the emission behavior tunable (up to 30 nm). Fold emission enhancements in the presence of various surfactants are highest toward Triton X-100 and CTAB (ranging from 5 to 25 fold) and are lowest for the anionic surfactants SDS and SDBS. These enhancements are competitive with existing probes of surfactants. Quantum yields in buffer range from 0.11 to 0.34, competitive with many common fluorophores in biological use. Strikingly, MF quantum yields in the presence of TX-100 and CTAB approach 100 % quantum efficiency. MF anisotropies are dramatically increased only in the presence of TX-100, CTAB, and CHAPS. Coupled with the above data, this suggests that MFs associate with neutral and charged surfactant aggregates. Interactions with the anionic surfactants are weaker and/or leave MFs solvent exposed. These properties make metallafluorenes competitive probes for surfactants and their properties and behaviors, and thus could also have important biological applications.


Introduction
Sila-and germa-fluorenes, also known as dibenzosiloles or dibenzogermoles, are a class of photoluminescent compounds with high quantum yields in organic solvents [1,2]. Interest in their potential applications as OLEDs recently drove us to synthetically explore 2,7-alkynyl(aryl) substitutions (Fig. 1a), which could be exploited to tune spectral behavior by extending the high degree of conjugation [1,2]. Such substitutions can also attenuate solubility in various solvents.
These optical properties and the hydrophobic nature of the structures suggest that if they are sufficiently soluble in aqueous solution, the utility of these compounds could be expanded to detect surfactants, common contaminants in wastewater [3][4][5], and could also have applications in the probing of lipid structures and behaviors [3][4][5][6].
Metallafluorenes (MFs) of sufficient solubility in aqueous solution and sensitivity to surfactants would represent a new class of fluorescent detectors for the detection and study of surfactants and relevant biological structures. Here we characterize the aqueous solution emission behaviors of four previously synthesized metallafluorenes [1,2] and their interactions with a variety of surfactant solutions. Indeed, we demonstrate here that these MFs are soluble in aqueous solution at low µM concentrations and luminesce under these conditions with competitive quantum yields. Further, dramatic spectral changes, increases in quantum yield and anisotropies occur in the presence of some surfactants, which demonstrate their potential as fluorescent probes of lipophilic structures in aqueous environments.

Materials
Coumarin-102 dye (99 %), Triton X-100, sodium d o d e c y l b e n z e n e s u l f o n a t e ( S D B S ) , a n d hexadecyltrimethylammonium bromide (C-TAB) were purchased from Sigma-Aldrich (St. Louis, MO). Dodecyl sulfate sodium salt (SDS) was purchased from Fisher Scientific (Waltham, MA). CHAPS was purchased from Anatrace (Maumee, OH) and 200 proof ethanol was purchased from Decon Labs (King of Prussia, PA). All chemicals used were of reagent grade and were used as received without further purification.

Spectroscopy
Absorbance spectra were recorded on a Shimadzu 1800 with slits set to 1 nm.
Luminescence experiments were performed in acidwashed quartz cuvettes at 10 mM Tris, pH 8.0 on a Tformatted Fluorolog-3 (SPEX) spectrofluorimeter equipped with a polarization assembly. The temperature was maintained 25°C with a thermostatted cell holder equipped with a magnetic stirrer. All intensities were repeated at least three times on different days and the results averaged.
For fluorescence anisotropy, at least three readings were collected over a 0.1 s integration time each and averaged. Anisotropy values were obtained in triplicate and automatically calculated from Eq. 1: b Examples of fluorescent surfactant indicators [3,[7][8][9] where I is the recorded intensity at the indicated polarizer orientations and A is the anisotropy. This experiment was repeated at least three times on different days and the results averaged. Emission spectra collected in the presence of salts (Na 2 HPO 4 , K 2 HPO 4 , CaCl 2 , ZnCl 2 , NH 4 Cl, (NH 4 ) 2 SO 4 , MgSO 4 ) were conducted at 1 mM salt (a common concentration used in the literature [6] and 1 µM MF. ZnCl 2 results in MF precipitation and therefore these data were not reported.
Quantum yield was measured using the previously reported relative method [11] at an excitation wavelength of 350 nm using a range of absorbances. Absorbances were kept below 0.1 to minimize non-linear effects [12]. The fluorescence spectra were recorded from 375 nm to 625 nm at the excitation wavelength of 350 nm. An excitation and emission slit width of 1.0 nm was used. The spectra of the reference standard and the unknown were measured under identical conditions. The slope of the integrated fluorescence intensity versus absorbance was used to calculate the quantum yield using Eq. 2. Coumarin 102 dye in ethanol, with a f of 0.764 [13], was used as the reference standard for all determinations.
where is the quantum yield, m is the slope of integrated fluorescence intensity against absorbance, and n is the refractive index of the solvent.

Structural Features of Metallafluorenes in This Study
The available, previously reported metallafluorenes provide for an initial exploration of aqueous solution behavior (Fig. 1a). MFs with both Si (1, 3 and 4) and Ge centers (2) are included; both mono (3, 4) and bicyclic (1, 2), fused (2) and not fused (1) 2,7 substituents are represented. Importantly for aqueous applications, there is variety in the polarity of this group among these compounds. Of particular interest is 2, which features a 6-methoxy naphthyl substitution, as naphthyl groups are common in fluorophores that interact with surfactants [3,4,7,8].

Group 14 Metallafluorenes are Soluble and Luminescent in Aqueous Solution
Stock solutions of compounds 1-4 were prepared in DMSO as determined by mass. Extinction coefficients were calculated from absorbance spectra and confirmed from the slope of a line fit to absorbance data as a function of concentration. See Table 1. These extinction coefficients were then used to prepare solutions for the remainder of the study. Despite significant hydrophobic character, if dispensed from a DMSO stock near 1 mM, these MFs are water soluble in the low micromolar range, sufficient for spectroscopic work. From DMSO stocks, aqueous solutions were prepared in 10 mM Tris buffer, pH 8.0 (a common biological buffer) with 5 % or less of DMSO and both absorbance and emission spectra obtained. It is clear from Fig. 2 that the 2,7 substituent provides an optical tunability of spectra in aqueous solution of up to 14 nm (376-390 nm) at absorption. The same is true of emission, but here the range is 30 nm. 1 is the most blue shifted of the group, while 2 is the most red shifted. Indeed, using https://www.molinspiration.com/cgi-bin/properties to compute logP for the 2,7 substituents confirms that the biphenyl substituent is the most lipophilic (hydrophobic) with a logP of 4.31 and the tolyl and phenylfluorine substituents are the least lipophilic (2.96 and 2.65, respectively). While the methoxynaphthyl substituent has a high logP value (3.73), it is the only one of this series with a Ge center. Stokes shifts range from 66 to 89 nm. Spectroscopic parameters are summarized in Table 1.

Absorption Behavior
The hydrophobicity of the MFs suggests that they could interact with detergents/surfactants. To explore this, absorption spectra were collected for 1-4 in the presence of 5 surfactants that vary with respect to charge at a concentration above their respective CMC values (TX, CHAPS, CTAB, SDS, and SDBS; see Table S1 for a summary of properties). In all cases, the spectrum of the surfactant itself is subtracted from that of the mixture (Fig. 3). TX elicited the largest enhancements in absorbance, and in the case of 2 and 3, the absorbance approached that obtained in DMSO. The spectral absorbance changes are the most interesting with 2 and 4. For these MFs, changes in peak shape are also observed. CTAB introduces scatter for 4 (see intensity between 425 and 450 nm) relative to CTAB itself, the contribution of which has been subtracted. This suggests a change in particle size and thus a direct interaction that affects both the MF and the surfactant (see "Probing Metallafluorene-surfactant Interactions" section for more detail). In contrast, the spectral responses to the anionic surfactants SDS and SDBS were very small to insignificant for all four MFs. This is typical among fluorescent surfactant indicators [3][4][5]9].  Table 2 for all four compounds. In general, the spectral responses to TX are the most dramatic: a large blue shift is observed in all cases (37-63 nm of lambda max), which is consistent with an interaction between the MF and the surfactant that reduces exposure to aqueous solvent and its relaxation effects. In addition, probably due to the same effect, the electronic transitions are quite distinct in the spectra. Further, the fold emission enhancements are large (6-19 fold). Given that TX is a neutral and aromatic surfactant, this seems reasonable and is comparable to that observed with other indicators [8,14].
Similar effects are also observed for 2 and 3 towards CHAPS and CTAB, and fold enhancements are similar to that for TX. A standout is 1, which shows a 25-fold enhancement in the presence of CTAB. In contrast, λ max shifts and emission enhancements are small or negligible upon the addition of anionic surfactants SDS and SDBS to all compounds. This is consistent with the literature [4,6,9,10].

Effect of Surfactants on Metallafluorene Quantum Yields
Quantum yields ( ) were measured using coumarin 102 in ethanol as a standard as described in Materials and Methods section. As summarized in Table 3, values in Tris buffer range from 0.09 to 0.34. On the low end are 2 and 4. values for these in water fall in a similar range with common probes 8-Anilinonaphthalene-1-sulfonic acid (ANS; 0.004; [15]), tryptophan (0.13; [16]), and coumarin (0.09; [17]), as well as with DMNDC (0.01; [8]). However, for 3 and 4, in water is relatively high (near 0.3). Common xanthene dyes like Rhodamine B and Texas Red have quantum yields of 0.31 and 0.35 in water [18], which is considered respectable for biological probes. 4 has a comparable quantum yield to this family of dyes and a greater quantum yield than dyes like Cy3 and Cy5 (quantum yields 0.04 and 0.27, respectively) [19] and the surfactant indicator N-n-octyl-4-(1-methylpiperazine)-1,8-naphthalimide iodide (0.24; [3]).
values are at almost 100 % quantum efficiency in the presence of TX and CTAB, indeed higher than in the organic solvent DCM, and values for all but 2 are above 0.8 in CHAPS. The values in the presence of anionic surfactants are much lower (0.38-0.55), but all of these are higher than any observed in buffer alone.

Effect of Ions on Metallafluorene Emission Behavior
To address specificity of emission enhancements induced by surfactants, the effects of various ions on MF emission were examined. As summarized in Fig. 7, generally very small reductions in emission were observed. And while they are the same scale as the enhancements observed in the presence of anionic surfactants, they are dwarfed by the changes upon the addition of TX, CTAB, and CHAPS. Thus the response to ions is very small and in an opposite direction to those observed for surfactants, indicating a high degree of specificity toward surfactants.

Probing Metallafluorene-surfactant Interactions
The above data suggest an interaction between MFs and surfactant micelles. To probe this possibility, MF anisotropies     were collected in the presence and absence of surfactant (Fig. 8). When MFs are added to TX, CHAPS, and CTAB, there is an obvious large increase in anisotropy. This is consistent with association of MF with the surfactant micelle. Coupled with spectral changes (blue shifts), fold enhancements and quantum yields, these data are consistent with substantial protection of MFs from polar solvent and its relaxation effects. This is in sharp contrast to the effect of anionic surfactants, which generally result in lower fold-enhancements, quantum yields, and anisotropies. This latter pattern is consistent with either weak interactions with aggregates or an association with smaller surfactant structures that provide little to no protection from solvent relaxation and no evidence that MFs are highly sequestered in hydrophobic environments.
To better understand the interaction of MF with a surfactant micellar surface, TX was titrated into 4. As shown in Fig. 9, emission increases dramatically at 200 µM, commonly reported as the CMC of TX [8,20]. This is also consistent with a sensitivity toward aggregates surfaces and thus a direct interaction with them.

Conclusions
The results here demonstrate the potential of MFs as probes of surfactants. First, substitution at the 2,7 position offers an opportunity to tune desirable properties that could include water solubility as well as electronic properties that could include l max and quantum yield. Second, they have sufficient water solubility and favorable luminescence behaviors for use in aqueous solution. Third, the differences in MF responses to various surfactants suggest sensitivities to surfactant charge, aggregate size, and CMC [3,5,7], any of which could make them useful probes of these properties. 1 exhibits the highest fold enhancements toward neutral and cationic surfactants; 2 exhibits the most distinct spectral responses to the various surfactants. Both have bicyclic 2,7 substituents and suggest a pathway for the development of second-generation MF probes.
We hypothesize that these compounds are intramolecular charge transfer (ICT) dyes that contain an electron rich moiety and electron deficient moiety connected by conjugated bonds. As an ICT dye, they may have future applications as aggregation-induced emission enhancing probes for protein, nucleic acid, and polysaccharide detection [21]. Given their  Table 2   This could include detecting changes in local membrane lipid composition in response to a molecular or cellular stimulus [22] as well as imaging cellular structures that have differing lipid compositions [22]. There are also potential applications in probing protein behavior through the detection of conformational changes (that often involve changes in accessible nonpolar surface area) [23]. Finally, through conjugation with a biomolecule of interest, MFs could be used to measure proximity or distance via FRET with chromophores located on biomolecular binding partners (e.g., lipids, proteins, and nucleic acids) [24]. Investigations into the electronic mechanisms of these MFs and their potential applications are ongoing. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Helena Spikes, Shelby Jarrett-Noland and Stephan Germann. The first draft of the manuscript was written by Cynthia Dupureur and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding This work was supported by the National Science Foundation (to Janet Braddock-Wilking) CHE-1362431.
Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations
Conflict of Interest The authors have no conflicts of interest to declare that are relevant to the content of this article.