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Multicomponent System of Single‐Walled Carbon Nanotubes Functionalized with a Melanin‐Inspired Material for Optical Detection and Scavenging of Metals

The accumulation of metal ions in organisms and the presence of heavy metals in water cause adverse effects on ecosystems and results in numerous human health issues such as cancer and neurogenerative diseases. Therefore, the development of novel platforms for metal‐scavenging and rapid metal detection for in situ applications are of high importance. Here, this challenge is tackled by taking advantage of the metal chelation ability of a melanin‐inspired material in combination with the near‐infrared (NIR) fluorescence response of single‐walled carbon nanotubes (SWCNTs) to surface binding. SWCNTs are functionalized by a melanin‐like substance, obtained by enzymatic oxidative polymerization of a fluorenylmethyloxycarbonyl‐tyrosine (FmocY) precursor. The resulting multicomponent system (SWCNT‐FmocYOx) serves as a metal‐ion scavenging platform that concurrently reports on metal binding with optical signal transduction. Upon binding of a library of mostly divalent transition metal‐ions, the fluorescence emission of the functionalized SWCNTs is modulated, showing a concentration‐dependent response with a limit of detection in the nanomolar range. Metal‐binding and removal from water of up to 98% is further shown via inductively coupled plasma mass spectrometry. The SWCNT‐FmocYOx hybrid system presents a novel platform with NIR optical signal for real‐time feedback on metal‐ion scavenging.


Introduction
Metal-ion sensors and scavengers are mainly applied to detect and remove heavy metal contaminants from water, that is not yet fully understood, [12,13] and the chemical synthesis of melanins by oxidation of tyrosine or 1-3,4-dihydroxyphenylalanine (l-DOPA) typically results in the formation of insoluble polymers. These challenges have led to increasing efforts to control the biomimetic synthesis and properties of melanin and melanin-like materials, [14][15][16][17][18] including those formed by enzymatic oxidation of short, tyrosine-containing self-assembling peptides. [15,17,18] The enzymatic oxidation of tyrosine or tyrosinederivatives leads to functional groups like catechols, and orthoquinones that are able to perform the metal-ion chelation. [10] Among different types of metal-ion sensor platforms, optical nanosensors offer numerous advantages, in particular, noninvasive, real-time read-out, small sampling volumes, and in situ application. Different types of optical metal-ion nanosensors are reported, which are either based on plasmonic effects of noble metal nanoparticles or fluorescence emission of organic fluorescence dyes or quantum dots. [19] Plasmonic nanoparticle metal sensors are mainly based on the effect of particle aggregation after metal addition inducing plasmon resonance wavelength changes. [20,21] This technique requires the application of many nanoparticles, to observe a signal. Organic fluorescent dyes have the disadvantage of suffering from photo bleaching, limiting their read-out and application time and quantum dots have limited biocompatibility, consisting of highly toxic transition metals (Cd, Zn, Pb) themselves. [19,20] Functionalized single-walled carbon nanotubes (SWCNTs) have emerged as excellent optical nanoprobes for numerous targets. [22,23] SWCNTs are graphene-based nanostructures, where different rolling orientations of graphene sheets give rise to a wide range of nanotube chiralities, [24,25] each of which has unique physical, chemical, and optical properties. [26][27][28][29] Semiconducting SWCNTs fluoresce in the near-infrared (NIR) range, which overlaps with the transparency window of biological samples, thus can be utilized for biotechnological and biomedical imaging and sensing applications, [23,[30][31][32][33][34][35][36][37] including sensing of small molecules, [38][39][40][41][42][43][44] reactive oxygen species, [45][46][47] microRNA, [48] lipids, [49] proteins, [50][51][52][53][54] enzymes, [55][56][57][58] volatiles, [59,60] and pathogens. [61,62] While previously, DNA functionalized fluorescent SWCNTs were utilized to detect arsenite within plants, [63] most SWCNT applications as metal sensors are based on oxidized (non-fluorescent) SWCNTs and do not exploit their optical properties. [64][65][66][67][68] Here, we present a multicomponent system composed of fluorescent SWCNTs and a melanin-like material that can perform metal-ion scavenging and functions as a fluorescence probe for metal-ions. To this end, we initially suspend SWCNTs with fluorenylmethyloxycarbonyl-tyrosine (SWCNT-FmocY). Subsequently, the enzymatic oxidative polymerization of Fmoc-tyrosine using tyrosinase leads to a stable suspension of SWCNTs (SWCNT-FmocY Ox ) with a melanin-like polymer bearing ortho-quinone and catechol functional groups. We show that metal-ions added to this SWCNT-suspension can bind to the polymer -a process we are able to monitor by a change in the fluorescence properties of the SWCNTs (Scheme 1). Further, we show the potential of our material to remove metal-ions from water.

Enzymatic Oxidative Polymerization of FmocY and SWCNT-FmocY
To obtain composites of SWCNTs and oxidized polymerized FmocY, we first suspended the SWCNTs with FmocY, and in a second step, we added the enzyme tyrosinase to induce enzymatic oxidative polymerization of the FmocY dispersant. We compared the enzymatic oxidative polymerization of a pure FmocY-solution and a FmocY-solution that contains the dispersed SWCNTs via absorption spectroscopy and transmission electron microscopy (TEM) (Figure 1). Figure 1a shows the time-dependent increase in absorption between 400 and 700 nm of FmocY after the addition of tyrosinase and the brown color of the resulting solution of FmocY Ox . This color change is typically observed following enzymatic oxidative polymerization of tyrosine or tyrosine-derivatives and indicates the formation of melanin-like materials. Similar to FmocY, the oxidation of SWCNT-FmocY to SWCNT-FmocY Ox , shows the same increase in absorption between 400 and 700 nm, while the absorption of the SWCNTs in the NIR-spectral wavelength range (800-1400 nm) stays stable, indicating that the SWCNTs do not aggregate during the oxidative polymerization of FmocY ( Figure 1b). Although FmocY can suspend SWCNTs, as shown by the absorption spectroscopy ( Figure 1b, initial time point), the suspension lacks long-term stability. Nevertheless, after enzymatic oxidative polymerization of FmocY, the suspension remains stable ( Figure S1, Supporting Information). In addition to the absorption spectroscopy, we performed NMR spectroscopy on FmocY and FmocY Ox , which shows shifts in the aromatic hydrogen atoms of the tyrosine moiety, confirming the oxidation process ( Figure S2, Supporting Information).
To characterize the morphological properties of our material, we analyzed FmocY in the presence or absence of SWCNTs, before and after enzymatic oxidation using TEM imaging. Similar to other Fmoc-conjugated amino acids, [18] FmocY self-assembles into fibrillary structures with a defined diameter of (10.3 ± 1.2) nm ( Figure 1c). However, after the suspension of SWCNTs with FmocY, the images reveal two distributions: thicker and less defined fibers with a diameter of (15.6 ± 4.1) nm, and long, much thinner tubular structures of (6.4 ± 2.0) nm in diameter (Figure 1e). The increase in dia meter and the deformation of the fibers compared to the fibers of FmocY alone is attributed to self-assembled hybrids of FmocY incorporating SWCNT, owing to the interaction of FmocY with the SWCNT-surface. The smaller diameter population, on the other hand, may be attributed to SWCNTs, as surfactant (sodium cholate) suspended SWCNTs show a similar diameter of (4.3 ± 1.0) nm ( Figure S3, Supporting Information). After the reaction with tyrosinase, we can observe the formation of spherical amorphous structures in both samples. In the case of FmocY, we also observe remaining fibers with the same diameters as before oxidation (9.3 ± 1.2) nm ( Figure 1d). In the case of SWCNT-FmocY Ox , the sample is imaged after filtration, such that the excess FmocY Ox that was not bound to the SWCNTs was filtered out (Figure 1f), and the FmocY Ox can be seen as spherical pigments attached to the SWCNTs. The diameter of the SWCNT in the SWCNT-FmocY Ox image was determined to be (5.6 ± 1.4) nm, similar to the diameter of the thin tubular structures in Figure 1e, before oxidation. These results show that FmocY can suspend SWCNTs and undergo enzymatic oxidative polymerization by tyrosinase in the presence, and with negligible influence, of the SWCNTs. While the SWCNT-FmocY suspension aggregates several hours after suspension, SWCNT-FmocY Ox is stable in suspension over several months.

Fluorescence Properties of SWCNT-FmocY During Oxidative Polymerization
The emission intensity and peak wavelength of the SWCNT NIR fluorescence are sensitive to changes in the dielectric environment in close proximity of the SWCNT surface. [69] Adv. Funct. Mater. 2022, 32, 2209688 These changes can be induced, e.g., via analyte adsorption or, as shown previously, by enzymatic reactions on the SWCNT corona phase. [58] Accordingly, we observe the modulation of the fluorescence emission intensity during oxidative polymerization of SWCNT-FmocY to SWCNT-FmocY Ox throughout the reaction under an excitation wavelength of λ ex = 730 nm (Figure 2a). The changes in the fluorescence intensity depend on the SWCNT chirality. While the (6,4) and (9,1) chiralities show an increase in fluorescence emission, the (8,3) and (6,5) chiralities show minor changes, and all other chiralities show an intensity decrease of up to 25% during the enzymatic oxidative polymerization of FmocY. Chirality-dependent response of SWCNTs undergoing the same reaction has been reported, and it was shown to result from the structural and chemical differences of the SWCNTchiralities. [29,51,52,70,71] These differences lead to variations in the SWCNT corona phase, which can lead to the fluorescence intensity decrease or increase. Figure 2b compares the oxidative polymerization measured via fluorescence spectroscopy of the (9,4) chirality of the SWCNT-FmocY (Figure 2a) and via absorption spectroscopy of FmocY at 400 nm (Figure 1a). The rate constants of the fluorescence decrease and the absorption increase are comparable: k SWCNT-fluorescence = 0.167 h −1 and k FmocY-absorption = 0.165 h -1 , indicating that the fluorescence modulations can be indeed attributed to the enzymatic oxidative polymerization of FmocY. Figure 2c shows the excitationemission map of the SWCNT-FmocY Ox sample with the (9,4)-chirality fluorescence emission highlighted, which was used for the measurements and quantification throughout this article. The characterization of the fluorescence properties of the SWCNT shows that we maintain fluorescence emission during enzymatic oxidative polymerization of the FmocY. Additionally, we can follow the process via fluorescence spectroscopy.

Fluorescence Intensity Changes Upon the Addition of Metal-Ions
Catechols and ortho-quinones, which are formed following the enzymatic oxidation of FmocY [72] and thus found on the SWCNT-surface, have been reported to be complexation sites for metal-ions, in particular for divalent heavy-metal-ions. [73] The accumulation of metal-ions in close proximity to the SWCNT-surface due to their binding to FmocY Ox around the SWCNTs has an impact on the dielectric environment of the SWCNT-surface, and thus can potentially induce a change in the SWCNT fluorescence. We tested the effect of metal-ions on SWCNT-FmocY Ox for a library of metal-ions, namely: Mg 2+ , Ca 2+ , Cu 2+ , Fe 2+ , Mn 2+ , Hg 2+ , Cr 3+ , Zn 2+ , and Pb 2+ . Figure 3a,b shows, as examples, the concentration-dependent intensity changes of the fluorescence spectra of SWCNT-FmocY Ox in water following the addition of Fe 2+ and Cu 2+ , respectively. Depending on the type of metal-ion, we observe an intensity increase (Fe 2+ ) or an intensity decrease (Cu 2+ ). SWCNTs fluorescent sensors have been shown to respond via intensity increase or decrease upon analyte binding, while the nature of the intensity change is still a matter of active research. [71] Several types of metal-ions in the presence of surfactant-dispersed SWCNTs have been reported to induce fluorescence intensity decrease, but with higher metal concentrations than used in this work. [74] To test the effect of metal-ions on the SWCNT-surface we used sodium cholate suspended SWCNTs (SWCNT-). Small surfactants, like sodium cholate, are able to suspend SWCNTs by weak hydrophobic interactions, [75] and can be easily replaced and leave the SWCNT-surface accessible. [76] We compare the intensity changes of SWCNT-SC to the intensity changes of SWCNT-FmocY Ox , both at a SWCNT-concentration of 1 µg mL −1 , upon the addition of the metal-ions at a concentration of 10 × 10 −6 m in water (Figure 3c). We observe an intensity decrease of the SWCNT-SC fluorescence in response to  the addition of Cu 2+ and Hg 2+ that can result from their direct binding of the SWCNT surface, whereas for all other metals, we see negligible effects. However, the addition of metal-ions to SWCNT-FmocY Ox induces a stronger intensity decrease for Cu 2+ and Hg 2+ compared to SWCNT-SC and a strong intensity increase for all other metals. These results show that the accumulation of metal-ions close to the SWCNT-surface via the binding to FmocY Ox plays a key role in the fluorescence intensity change, which cannot be solely attributed to direct binding of the metal-ions to the SWCNT surface. Due to the instability of the suspension of SWCNT with FmocY before oxidative polymerization, we cannot compare our results with SWCNT-FmocY ( Figure S1, Supporting Information). In the case of Cu 2+ and Hg 2+ , the intensity decrease is not a result of SWCNTs aggregation, as no change in the absorption is observed ( Figure S4, Supporting Information). Further, the fluorescence intensity change cannot be attributed to the negatively charged counter-ion of the metal, as proven by comparing CuCl 2 and CuSO 4 ( Figure S5, Supporting Information). Figure 3d shows the concentration-dependent fluorescence response for all metals tested and the corresponding fitting function to determine the dissociation constants (K d ) and the limit of detection (LOD). The results are summed up in Table 1.
The K d values are in the lower micromolar range with Zn 2+ <Cu 2+ <Cr 3+ <Ca 2+ <Mg 2+ <Pb 2+ <Hg 2+ <Mn 2+ <Fe 2+ , proving the ability of the SWCNT-FmocY Ox composite to bind metalions already in low concentrations and transduce this binding to a fluorescence signal. As the addition of metal-ions to water has an acidic effect, we determined the pH of the metal-ion solutions at a concentration of 300 × 10 −6 m (Table S1, Supporting Information). As expected, the pH values of the solutions differ for the different metal-ions; however, there is no correlation between the pH and the sensor response, i.e., the K D or the extent of intensity change. For a potential application   of the SWCNT-FmocY Ox as optical probes, the limit of detection is in the nanomolar concentration range reaching from 19 × 10 −9 m (Zn 2+ ) to 536 × 10 −9 m (Mn 2+ ).

Metal-Ion Binding and Removal with SWCNT-FmocY Ox
SWCNT-FmocY Ox are well suspended in aqueous media, but due to their long tubular structure, they are retained during centrifugal filtration even by a membrane with a high molecular weight cutoff (MWCO). To further confirm that metal-ions bind to SWCNT-FmocY Ox we incubate SWCNT-FmocY Ox (5 µg mL −1 ) with a metal-ion solution containing 1 ppm of metal. The suspension was incubated for 30 min and filtered through a centrifugal filter (Figure 4a). The concentration of metal-ions in the filtrates was measured via inductively coupled plasma mass spectrometry (ICP-MS) and compared with the metal concentration of a metal solution after filtration without the addition of SWCNT-FmocY Ox (Figure 4b). The percentage of metal-ions after filtration with and without SWCNT are summed up in These results show, first, that the metal-ions are indeed binding to the SWCNT-FmocY Ox and that the SWCNT-FmocY Ox can be used as scavengers of heavy metal-ions in aqueous solutions. Differences in metal-complexation and binding may be attributed to factors like electronegativity, hydration, or effective ionic radius. Still, a detailed rationalization of the reasons for different metal complexation is difficult due to the complexity of the binding material, consisting of catechol and quinone functional groups and the three-dimensional conformation of the polymer. In a recent study on metal absorption to melanin nanoparticles, the absorption capacities showed a similar trend of Pb<Cu<Zn, which was attributed to the solution phase electronegativity. [77]

Conclusion
We presented a hybrid multicomponent system of fluorescent SWCNTs and a melanin-inspired material, FmocY Ox that can act both as a metal-ion scavenger, and an optical reporter for metal-ions binding. We show that FmocY can suspend SWCNTs in water and undergo enzymatic oxidative polymerization mediated by tyrosinase. Characterizing the reaction by absorption and fluorescence spectroscopy, and the morphology of the resulting material using electron microscopy, showed that FmocY, in the presence of the SWCNTs, is oxidized with negligible influence of the SWCNTs. Moreover, the SWCNT NIR fluorescence can be used as a real-time optical reporter for the oxidative polymerization process, demonstrating a similar reaction rate as the traditional absorption spectroscopy. The resulting SWCNT-FmocY Ox suspension is highly stable in water, in contrast to SWCNT-FmocY, which aggregates if not oxidized. The SWCNT-FmocY Ox allows monitoring metal-ion binding through changes in the SWCNT fluorescence properties, which is demonstrated for a library of mainly transition metal-ions, but also divalent alkaline earth metal-ions like Mg 2+ and Ca 2+ . Confirming the binding of the metal-ions to the SWCNT-FmocY Ox , we show that the metal-ions could be removed from an aqueous solution with SWCNT-FmocY Ox by centrifugal filtration through a high-MWCO membrane. The SWCNT-FmocY Ox material presented in this study is able to bind metal-ions, and at the same time, function as a fluorescent reporter for metal-ion binding with a LOD in the nanomolar concentration range. This platform benefits from easy synthesis, high stability, the biocompatibility of the SWCNTs, [78][79][80] and the optical signal in the biologically transparent window. Metal removal from water, shown by the reduction of >90% of toxic metal-ions like Pb 2+ , Zn 2+ , and Cu 2+ , opens new possibilities for using SWCNT-FmocY Ox for metal scavenging applications from polluted water. Further, owing to   Table 2.

Experimental Section
Suspension of SWCNT with Fmoc-Tyrosine (SWCNT-FmocY): Fifteen milligrams of Fmoc-Tyrosine (FmocY, Sigma-Aldrich) were dissolved in 1 mL of water with the addition of sodium hydroxide (0.5 m, ≈120 µL). Two milligrams of HiPCO (high-pressure carbon monoxide) SWCNTs (NanoIntegris) were suspended in 1 mL Fmoc-Tyr (15 mg mL −1 ) using bath sonication for 10 min (80 Hz, Elma P-30H), followed by direct tip sonication on ice for two times for 20 min (12 W, QSonica Q125). After sonication, the suspension was centrifuged for 3 h at 21,130 rcf (Eppendorf 5424 R) to separate the individually suspended SWCNTs from aggregates and impurities. Successful suspensions resulted in homogenous dark suspensions with sharp absorption peaks characterized by UV-vis-NIR spectroscopy (Shimadzu UV-3600 PLUS). The concentration of the SWCNT-FmocY suspension was determined using the extinction coefficient [39] ε 632 nm = 0.036 L mg −1 cm −1 to be between 100 and 150 µg mL −1 . Oxidative Near-Infrared (NIR) Fluorescence Spectroscopy: Fluorescence emission spectra were recorded in a 96-well-plate mounted on an inverted microscope (Olympus IX73). A super-continuum whitelight laser (NKT-photonics, Super-K Extreme) with a bandwidth filter (NKT-photonics, Super-K varia, Δλ = 20 nm) was coupled to the microscope as the excitation source at λ ex = 730 nm with a laser intensity of 20 mW. Fluorescence emission was spectrally resolved using a spectrograph (Spectra Pro HRS-300, Teledyne Princeton Instruments) with a slit-width of 500 µm and a grating (150 g mm −1 ). The fluorescence intensity spectrum was recorded by an InGaAs-detector (PylonIR, Teledyne Princeton Instruments) with an exposure time of 10 s. Excitation-emission maps were recorded using an excitation wavelength range of 500 -840 nm in 2 nm steps. The fluorescence emission peak intensity of the (9,4)-chirality was determined via a Lorentzian fit.
Inductively Coupled Plasma Mass Spectrometry (ICP-MS): Metal detection was performed on a 7800 single quadrupole ICP-MS equipped with a 4SPS Autosampler and "MicroMist" nebulizer (Agilent Technologies, Santa Clara, CA, USA) of the Adama Center for Novel Delivery Systems in Crop Protection, Tel Aviv University. The Mg 24 , Cu 63 , Zn 66 , and Pb 208 isotopes were quantified, respectively.
Nuclear Magnetic Resonance Measurements ( 1 H-NMR): FmocY Ox was filtered through a centrifuge filter (Amicon, MWCO 10 kDa) to remove the enzyme. The sample was lyophilized and dissolved in DMSO-d6. FmocY was dissolved in DMSO-d6. NMR-spectra were recorded on Bruker Avance I and Avance III 400 MHz ( 1 H) spectrometer.
Transmission Electron Microscopy (TEM): Ten microliters of the sample solutions (50 µg mL −1 SWCNT-FmocY Ox , 50 µg mL −1 SWCNT-FmocY, 15 mg mL −1 FmocY ox , 15 mg mL −1 FmocY, and 1.5 µg mL −1 SWCNT-SC) were applied to a carbon-coated grid and incubated for 2 min. The excess solution was removed by blotting the grid with a piece of filter paper, followed by staining with 10 µl of 2% (w/v) uranyl acetate solution for 2 min. After blotting with excess stain solution, the grid was left to air dry. The negatively stained samples were imaged using a JEM-1400Plus TEM operating at 80 kV. Images were recorded using SIS Megaview III camera, iTEM the TEM imaging platform (Olympus).

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.