Engaging Dynamic Surfactant Assemblies in Improving Metal Ion

Article Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Engaging Dynamic Surfactant Assemblies in Improving Metal Ion Sensitivity of a 1,4,7-Triazacyclononane-Based Receptor: Differential Optical Response for Cysteine and Histidine Bappa Maiti,†,‡,∥ Nilanjan Dey,†,∥ and Santanu Bhattacharya*,†,‡ †

Department of Organic Chemistry, Indian Institute of Science, Bangalore 560012, India School of Applied & Interdisciplinary Sciences, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India

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Downloaded by BUFFALO STATE at 20:24:45:780 on May 23, 2019 from https://pubs.acs.org/doi/10.1021/acsabm.9b00083.

S Supporting Information *

ABSTRACT: Self-assembly as well as metal ion binding property of an amphiphilic, dansylated 1,4,7-triazacyclononane (TACN) probe have been investigated in the presence of various surfactant assemblies in aqueous media. As expected, the receptor molecule shows highly sensitive, but rather nonspecific, interaction with metal ions in the bulk water medium. Thus, to achieve the good specificity without dampening the sensitivity of the probe, we embedded the sensor in different surfactant assemblies, such as micelles and vesicles, and explored their metal ion sensing ability. Change in microenvironment by restricting conformational mobility and increasing local hydrophobicity renders a drastic improvement in selectivity and also sensitivity toward Cu2+. Further, the preformed Cu2+ complex of the probe was utilized for exclusive “turn-on” detection of both cysteine (green fluorescence) and histidine (blue fluorescence). The diverse complexation mode of interactions with these amino acids caused a distinct change in the monomer to aggregate ratio, which was reflected in different spectral response. Furthermore, the dansylated probe was involved in developing reusable paper strips for rapid on-site detection of both Cu2+ and cysteine. KEYWORDS: surfactant assemblies, selectivity and sensitivity, Cu2+ recognition, tandem sensing of amino acids, paper strips

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INTRODUCTION Polyaza macrocyclic ligands are well-known in the literature for their excellent metal ion binding ability owing to macrocyclic effects.1 In addition, these receptor compounds also show superior water solubility as well as pH-sensitive, reversible metal ion chelating property, which further contribute to their popularity. Various purposes such as metal carriers for diagnostic or therapeutic, orientating proteins at interfaces, metalloenzyme biosite, and visual metal ion sensors are served by these ligands.2−6 In spite of their huge applicability in the field of supramolecular chemistry and biomedical sciences, the polyaza macrocyclic-based probes are not still well explored in bioanalytical estimations, mostly due to their nonspecific behavior. Polyaza macrocyclic probes are known to interact with various transition metal ions, like Cu2+, Hg2+, Pb2+, or Zn2+ etc., to a comparable extent.7 Therefore, the major challenge that lies here is to improve their metal ion recognition ability without disturbing sensitivity. On the other hand, it is known in the literature that the microenvironment of the embedded dyes changes drastically inside the supramolecular hosts as compared to the bulk medium.8,9 Microenvironment, as defined by micropolarity, microviscosity, local pH, and extent of hydration, alone or collectively, could influence the photophysical properties of the © XXXX American Chemical Society

receptor compounds, such as pH sensitivity, metal ion binding affinity, etc. Interestingly, the microenvironment of the probe molecules inside the surfactant assembly depends on the order, interfacial hydration, as well as polarity of the aggregates.10−15 In this context, micelles and vesicles are the popular supramolecular host for the encapsulation of various dyes as well as drug molecules.16−21 These microscopic assemblies could also accommodate a very small amount of functionalized amphiphiles with matching hydrophobic tail and a sufficiently polar headgroup, which mainly resides at the surfactant−water interface. In the functionalized micelle or vesicle mostly, the headgroups represent the recognition site (or binding site), responsible for interacting with target analytes.22−27 Specific molecular-level interactions of the functionalized polar headgroup with different analytes like protein and metal ions at a hydrophilic−hydrophobic interface are driven by the local environment of the head groups which is different for different surfactant systems. Several research groups reported an alkylated metal ion binding moiety which could be easily dispersed in micelle or Received: January 30, 2019 Accepted: March 20, 2019

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DOI: 10.1021/acsabm.9b00083 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

Figure 1. Molecular structures of dansylated TACN chelator amphiphile and their aggregation in water.

Figure 2. (a) Fluorescence color changes of 1 (10 μM) upon addition of different metal ions (50 μM) in water, observed under the long UV lamp. (b) Interaction of probe 1 (2 μM, λex = 340 nm) with different metal ions (10 μM) in water medium (considering 475 nm wavelength). (c) Mode of interaction of 1 with metal ions in water.

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RESULTS AND DISCUSSION Interaction with Metal Ions in the Aqueous Medium. Due to an excellent balance between the hydrophilic (binding site) and hydrophobic units (long alkyl chains), probe 1 (Scheme S1, Figures 1 and S1−2; for synthetic procedure and characterizations, see the Supporting Information) indeed forms self-assembled nanoaggregates in water, as evident from its low critical aggregation concentration (∼0.2 μM) in water (Figure S3a,b). The dynamic light-scattering (DLS) studies indicated the formation of aggregates of diverse sizes in water (Figure S3c). Further, the metal-ion-chelating property of this receptor lipid 1 was explored in water. Addition of different transition metal ions (Ag+, Cd2+, Cu2+, Co2+, Hg2+, Mg2+, Mn2+, Pb2+, Zn2+) induced quenching of cyan-colored fluorescence to different extents (Figure 2a and 2b). Though the degree of quenching was maximum for Cu2+ (∼3.55-fold), a substantial reduction of intensity was also observed with Hg2+ and Pb2+ (Figure S4c).35 On the contrary, the presence of Ni2+ and Zn2+ resulted in the enhancement in fluorescence with prominent blue shifts in the emission maxima (∼25 nm for Ni2+ and ∼34 nm for Zn2+) (Figure S4a,b). The Ni2+- or Zn2+-induced turn-on emission response could be explained by a chelation-enhanced fluorescence (CHEF) mechanism involving both the pendant sulfonamide N-anion and the macrocyclic nitrogen centers (Figure 2c).36 On the other hand,

lipid. Bispicolyl, dipyridyl, and crown ether are commonly used as binding sites in these types of metal ion binding lipids.28−34 Upon embedment in the micellar or vesicular environment, these chelating moieties showed a differential affinity toward metal ions, and also their metal complexes were used in functionalized micelle or vesicle. Considering this, here we have designed an amphiphilic probe with 1,4,7-triazacyclononane (TACN) as the receptor moiety and dansyl as the signaling unit (Figure 1). Long aliphatic chains were attached intentionally for proper embedment of the probe in the micelle or lipid bilayer where the binding moiety should be exposed at the Stern layer region or at the outer leaflet of the surface for interaction with extraneously added bulk metal ions. Therefore, here we have demonstrated a new strategy of improving metal ion selectivity of a TACN-based probe by embedding into different supramolecular host materials like micelles or vesicles which would sufficiently alter the microenvironment around the binding site. Further, the preformed copper complex of the sensor was found to exhibit differential signals for cysteine (appearance of green fluorescence) and histidine (appearance of blue fluorescence). This ensured simultaneous nanomolar detection of both the amino acids at biological pH. In parallel, we have also developed inexpensive, reusable paper strips for on-site detection of Cu2+ in remote areas without engaging any costly instruments. B


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Figure 3. (a) Plausible aggregation of probe 1 in different medium, water, micelle, and vesicle. (b) Fluorescence spectra of 1 (2 μM, λex = 340 nm) in water, doped in micelle and DOPC vesicle (5 mol %) at pH 7.4. (c) Fluorescence color changes of 1 (10 μM) upon addition of different metal ions (50 μM) in diverse media (Brij-58 and DOPC vesicle), observed under the long UV lamp.

Figure 4. (a) Change in emission intensity of 1 (2 μM, λex = 340 nm) at 513 nm upon addition of different metal ions in (a) Brij-58 micelle and (b) DOPC vesicle.

the Cu2+ ion, owing to the open-shell electronic configuration (d9 system), induced emission quenching as a result of photoinduced electron transfer (PET). Similarly, emission quenching by Hg2+/Pb2+ ions can be explained in terms of a heavy metal ion effect. Design Probe-Surfactant Assemblies for Metal Ion Interaction. In order to improve the selectivity of the probe molecule, we have incorporated various types of surfactant assemblies, such as micelles (Brij-58; SDS, sodium dodecyl sulfate; and CTAB, cetyltrimethylammonium bromide) and vesicles (DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine and DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine) in the studies, which could affect the photophysical response of the dye by modifying the microenvironment of the chelator moiety (Figure S5). The location of the chelator moiety in micelle and vesicle is expected to be at the hydrophilic−hydrophobic interface (micellar or vesicular surface) and therefore will remain accessible to interact with bulk metal ions (Figure 3a). Metal Ion Interaction at the Mesoscopic Interface. Case Study at the Micellar Interface. Emission spectra of 1 in a neutral micelle (Brij-58) showed a prominent enhancement in the aggregate fluorescence (∼2-fold) with an ∼23 nm red

shift (from 490 to 513 nm) in emission maxima, which was also evident from the substantial change in emission color from cyan to bright green (Figure 3b,c). The larger aggregate emission (Φ = 0.040) in the presence of surfactant aggregates suggested the local accumulation of a dansyl fluorophore as well as a TACN unit (i.e., probe molecules) at the micellar interface. An incubation time of ∼10 min was given to ensure the complete embodiment of the guest molecules at the micellar interface. Addition of Cu2+ in this situation showed a change in fluorescence color from green to faint blue with quenching of aggregate emission by ∼4.92-fold (Figures 3c and 4a and S6a-c). This phenomenon indicates that the positive charge at the TACN moiety enhances upon coordination with Cu2+, which induces dispersion of the probe molecules on the micellar surface. As a result, the effective local concentration of dye at the interface reduces, and the intensity of the aggregate emission decreases (Figure 5). Further, selectivity studies in this condition indicate the probe is fairly selective for Cu2+. However, a significantly large interference was still observed from the Hg2+ (∼1.85-fold) ion (Figures 4a and S6a). Incorporation of charged surfactants (like SDS and CTAB) significantly reduced the extent of response toward Cu2+. The C


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Figure 5. Schematic representation of the plausible molecular organizations of 1 in water and different supramolecular hosts (micelles and vesicles) both before and after interaction with metal ion (Cu2+).

presence of charged head groups (−SO3− for SDS and −NMe3+ for CTAB) might resist the approach of the metal ions toward the binding site (TACN) either via electrostatic repulsion (in case of CTAB) or via competitive binding (in case of SDS) (Figure S7). Case Study at the Membranous Interfaces. Similarly, the emission spectrum of 1 in the DOPC membrane (5 mol % doped) predominantly showed an aggregate (λmax = 513 nm) emission band from the dansyl unit (Figure 3b). Here also, a relatively higher aggregate emission (Φ = 0.054) suggested a comparatively larger self-assembly of probe molecules at the bilayer interface than that observed in the micelle or water medium. As expected, the intense green emission of the compound was found to be quenched (faint blue emission was visible) in the presence of Cu2+ (Figures 3c and 4b). The fluorescence spectrum of probe 1 in the presence of Cu2+ showed an ∼20-fold diminution of aggregate emission, which is massive compared to that observed in the water/micelle medium (Figure S8b,c). The increase in electrostatic repulsion between the metal-bound TACN chelator head groups might have caused dispersion of initially aggregated receptor lipids in the phospholipid membrane (Figure 5). This kind of dispersion of functionalized amphiphiles in the lipid bilayer upon metal ion chelation is known in the literature in the case of the phospholipid membrane.34 The unaffected emission profile upon addition of other transition metal ions (including Hg2+, Pb2+, Ni2+, and Zn2+) ensured the excellent selectivity of the present system toward Cu2+ (Figures 3c, 4b, and S8a). The saturation in optical response was observed upon addition of only ∼9 μM of Cu2+, indicating a considerably low detection limit for Cu2+ in water (∼24 nM) (Figure S9a,b). Further, to confirm the metal ion induced dispersion of the chelator probes, another lipid DPPC with a relatively higher order of organization at room temperature was considered (Figure S10a). Unlike the fluid-like nature of the DOPC (Tm = −16.5

°C) bilayer, DPPC endorses a mostly solid-like gel phase at room temperature (Tm = 41.2 °C).37 As expected, a comparatively poor response was observed for Cu2+ in the DPPC membrane (Figure S10b). The mobility of the chelator probe 1 in the DPPC vesicle is limited, which effects the metal ion induced dispersion of the chelator probes and also subsequent metal coordination to the neighboring molecules. Tuning of Selectivity and Sensitivity Deciding the Role of the Microenvironment. Upon scanning a wide range of supramolecular hosts (on moving from bulk water to micelle to vesicle), we finally achieved selective response from a notoriously “nonspecific” TACN derivative (1) toward Cu2+. At the same time, the extent of interaction with Cu2+ was also found to be improved when incorporated at the bilayer surface of the phospholipid membrane. In order to explain these observations, we focused on the changes in the microenvironment of the TACN moiety of probe 1 in various media and correlate them with its sensitivity as well as selectivity toward metal ions as reported by fluorescent signal of the dansyl moiety. Though probe 1 forms relatively unorganized random aggregates in water, in surfactant assemblies, the probe is expected to be present in the well-organized aggregated form. Particularly in the bilayer membrane, the compound can align itself in such a way that the long aliphatic chains remain embedded in the hydrophobic region, while the TACN unit is localized at the surfactant/water interface. As expected, the random aggregate formation in bulk water medium resists the approach of the ionic analytes (metal ions) toward the TACN binding sites of 1 and therefore exhibits poor sensitivity (Figure 5). Also, the fluorescent signal intensity of the dansyl moiety of 1 was found to be dependent on the polarity of the medium, as evident by spectral analysis in a different dioxane−water mixture medium (Figure 6a). So, it is also expected that one of the important microenvironment properties, i.e., micropolarity D


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Figure 6. (a) Fluorescence intensities of 1 (2 μM, λex = 340 nm) in medium with different polarity (ratios of dioxane−water). (b) Change in Cu2+ ion sensitivity of 1 in different organic medium showing the effect of micropolarity. (c) Effect of Triton-X micelles on metal ion selectivity of 1 (2 μM, λex = 340 nm) in the DOPC vesicles, monitored at 513 nm.

Figure 7. (a) Fluorescence spectra of a copper complex of 1 (2 μM, λex = 340 nm) upon addition of cysteine and histidine (8 μM) in the DOPC vesicles. (b) Reversibility check for cysteine and histidine interaction with 1 in DOPC vesicles and Cu2+, depicting different modes of interactions.

mediums (acetonitrile and THF) having different polarity indices. As intended, probe 1 showed superior interaction with Cu2+ in the organic solvent having lower polarity value, THF (Figure 6b). In addition, the presence of polar head groups (−NMe3+ and phosphate) in DOPC not only influences the microenvironment around the TACN binding site of the compound but also ensures that only the metal ions with very high binding affinity toward the TACN moiety (with respect to the supramolecular host) could be able to render the spectral changes, i.e., Cu2+. In the case of a neutral micelle (Brij-58), a dynamic aggregate is formed that falls between bulk water and a well-defined bilayer

around the TACN binding site of probe 1, may be altered in different surfactant systems (Figure 3b). Indeed, lowering of micropolarity value around the TACN moiety of the probe on moving from water (εT = 62.8) to Brij-58 (εT = 55.2) to the DOPC membrane (εT = 47.8) was observed and ensured the minimal hydration of the TACN binding site in DOPC, which may in turn result in higher sensitivity toward Cu2+. It is known in the literature that the low apparent micropolarity inside the surfactant assembly can enhance the possibility of noncovalent interactions, like hydrogen bonding, charge pairing, etc.38,39 To experimentally prove the above speculations, we have studied Cu2+ sensitivity of the probe 1 in two different organic E


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Figure 8. Schematic representation of the plausible change in aggregation pattern of the 1−Cu2+ complex in a phospholipid vesicle (DOPC) during interaction with cysteine and histidine.

“turn-on” response with both cysteine (λmax = 513 nm) and histidine (λmax = 435 nm) in water. However, both of these amino acids could be detected simultaneously through an easily distinguishable optical signature. Cysteine enhanced the aggregate emission by ∼16-fold, whereas there was a histidineinduced increase in monomeric emission by ∼6-fold (Figure S12a,b). Titration studies showed that in both the cases spectral saturations could be obtained upon addition of only ∼10 μM of analyte, which was also supported by their significantly lower detection limits (∼32 nM for cysteine and ∼54 nM for histidine) (Figure S13a,b). The diverse spectral behavior of the metal conjugate (1 + Cu2+) in the DOPC bilayer with histidine and cysteine might be attributed by their different mode of interactions (Figure 8). Reversibility of amino acid coordination was established through sequential addition of a metal ion (Cu2+) and the respective amino acid to the aqueous solution of 1 doped in DOPC. In the case of cysteine, we could witness the recovery of molecular emission multiple times, whereas no such reversibility was traced with histidine (Figure 7b). This indicates that the histidine probably forms a ternary conjugate with the copper complex of 1. Again, a similar conclusion was drawn from ESI-MS mass spectrum analysis upon addition of amino acids into the solution of the preformed copper complex of 1. Incorporation of histidine exhibited the formation of a sharp peak at 535.84 corresponding to a 1:1:1 ternary complex (Figure S14). The

interface. As a result, the TACN moiety in 1 doped in Brij-58 interacts not only with Cu2+ but also slightly with Hg2+. Further, to ensure the importance of microenvironment provided by supramolecular hosts on the selectivity process, we had treated 1 doped in the DOPC vesicle with triton-X (0.15% wt/v) prior to the metal ion addition. Triton-X is capable of rupturing the phospholipid vesicles. As expected, the addition of metal ions in this condition clearly depicts poor selectivity of 1 toward Cu2+ (Figure 6c). Tandem Sensing of Amino Acids at the Bilayer Surface. Recently, metal complexes have gotten immense attention in biomedical sciences because of their “targetspecific” interactions with biologically relevant analytes. In this context, copper complexes appeared to be one of the most promising templates due to their affinity toward versatile analytes including amino acids, cyanide, and phosphates, etc.40−46 Considering this fact, here we have employed the preformed Cu2+ complex of 1 in a DOPC vesicle (5 mol % doped) as the template for amino acid sensing. Formation of the metal complex was verified by ESI-MS mass spectral and elemental analysis (Figure S11). Among all the tested amino acids, there was a cysteine-induced appearance of green emission like 1, while incorporation of histidine resulted in the blue-colored fluorescence (Figure 7a). Thus, the emission spectra of the liposome (5 mol % copper complex of 1 in DOPC) showed a F


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Figure 9. (a) Test strips (under UV lamp) depicted fast-track detection of Cu2+ and cysteine. (b) DFT-optimized structures of 1, 1 + Cu2+, and ternary complex with histidine (6-31G* method for C, H, N, S, and O and LANL2DZ for Cu).

for Cu2+ sensing. Thus, a single TLC strip can be used multiple times, which will make the protocol more convenient and significantly lower its cost. Since in this case no sophisticated instrument is required (only a hand-held UV torch), people with limited knowledge in science will be able to use this without much difficulty. Theoretical Insight into Mechanism of Analyte Binding. To rationalize the spectral behavior of 1 in the presence of Cu2+ and histidine (cysteine regenerates the molecular signal), computational studies were performed at the B3LYP level of theory considering the 6-31G* basis set for C, H, N, and O atoms and LANL2DZ for Cu. In the optimized structure, Cu2+ was found to be closely associated with the TACN unit (Figure 9b). The close proximity of Cu2+ with the TACN nitrogen atoms was prominent from the short Cu−N distances obtained as 1.78 Å (with N1), 1.92 Å (with N2), and 2.01 Å (with N3). Time-dependent DFT studies showed that the spectral signal of 1 was mainly dominated by the transition from HOMO to LUMO with oscillator strength 0.101 (Figure S15). Incorporation of Cu2+ increases the effective electron transfer (PET) from the dansyl moiety to fragment TACN− Cu2+. This was also evident from the comparatively nonlocalized distribution of frontier molecular orbitals (FMOs) over the entire molecular framework. Here, TD-DFT calculations identified HOMO−2 (β) to LUMO (β) excitation as the predominant electronic transition (f = 0.064) (Figure S16a). Again, in the histidine complex, the distance of Cu2+ to imidazole nitrogen was found to be 2.49 Å.

Engaging Dynamic Surfactant Assemblies in Improving Metal Ion

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