Melanin is a ubiquitous biomacromolecule that is widely distributed in the animal and plant kingdoms. They are not only function as the pigment, but also involved in various biological activities, such as sequestering metal ions [1-3], free radicals scavenging [4-6], photocatalysts [7, 8], and photoprotection [9, 10]. Eumelanin is the most intensively studied melanin that produced from the 3, 4-dihydroxyphenylalanine precursor. The large number of studies have confirmed the structure of eumelanin as essentially an amorphous heterogeneous biopolymer composed primarily of random assembles of 5, 6-dihydroxyindoles (DHI) and 5, 6-dihydroxyindoles-2-carboxylic acid (DHICA) [11].

Biomacromolecules, includes nucleic acids and proteins are made from building blocks of nucleobase and amino acid, of which the sequence and composition variety leads to diverse structures and functions that are vital to life [12, 13]. Researchers construct synthetic materials made of these building blocks in a range of fields including in biotechnology, programmable structural synthons, and catalysis [14-16]. In each application, the function of material is highly dependent on the chemistry and assembly of the building blocks [17]. The structure complexity of melanin hindered the efforts in exploring its building blocks and use for constructive purpose. Recent advances in construction of melanin-like materials by oxidation polymerization of dopamine showed great potential in revealing fundamental structure information and related applications [18, 19]. However, the use of building blocks, 5, 6-dihydroxyindole and its derivatives, originated from melanin to construct functional materials is relatively unexplored.

Recently, the development of chemical probes for detection of Cu²⁺ has attracted considerable interest worldwide [20-25]. Note that Cu²⁺ plays an important role in human homeostasis, but also toxic to living organisms at high concentration [26-29]. Therefore, it is imperative to seek rapid, sensitive and efficient methods for detecting Cu²⁺. In the past, a large number of organic fluorescent probe, including cyanines [30], phthalocyanines [31], rhodamines [32] and coumarins derivatives [33], with excellent sensitivity and selectivity has been developed for Cu²⁺ detection [34-36]. However, the efforts of using biological building blocks to build fluorescence sensor is rare [37-39]. Herein, we report a new class of melanin-inspired dihydroxyindoles oligomers-based reversible fluorescence sensor for Cu²⁺ detection. We chose 5, 6-dihydroxyindoles (DHI), 5, 6-dihydroxyindoles-2-carboxylic acid (DHICA) and its derivatives 5, 6-dihydroxyindoles-2-carboxylic acid methyl ester (DHICMe) and 5, 6-dihydrocy indole-2-carboxylic acid ethyl ester (DHICEt) as the building blocks for constructing fluorescence probes. Notably, we find that these building blocks can be oxidized and further polymerized under alkaline condition to form fluorescent oligomers (Scheme 1). Though the structure complexity of melanin-based material hindered the efforts in revealing their structural information, the polymerization process can be monitored via the fluorescence spectrum.

Moreover, we found these fluorophores were quenched specifically by Cu²⁺ association over other metal ions, including Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Co²⁺, Cr³⁺, Fe³⁺, Mn²⁺, Sr²⁺ and Zn²⁺. In addition, fluorescence intensity can be switched between ON and OFF states by the Cu²⁺ association and disassociation process. This work suggests a new approach for constructing fluorescent probe originated from nature, which helps to reveal the fundamental information of melanin and potentials in biosensing.

DHICA were synthesized via ferricyanide oxidation of L-3, 4- dihydroxyphenylalanine (L-dopa), followed by rearrangement of the resulting dopachrome under anaerobic conditions [40]. DHICMe and DHICEt were prepared by the esterification DHICA and alcohols (Scheme S1 in Supporting information). The resulting products were confirmed by ¹H, ¹³C NMR spectra (Figs. S1–S3 in Supporting information), and the electrospray ionization mass spectrometry (ESI-MS) (Fig. S4 in Supporting information). The oxidative polymerization of DHI, DHICA, DHICMe and DHICEt under alkaline conditions yield corresponding oligomers namely, P-DHI, P-DHICA, P-DHICMe and P-DHICEt (Scheme 1). The successful polymerization was evidenced by the disappearance and shift of peaks from aromatic rings in ¹H NMR spectra, indicating the coupling between dihydroxyindoles units (Fig. 1a). To further characterize the extent of polymerization, ESI-MS was applied to detect the molecular weight of the possible oligomers. As shown in Fig. 1b, P-DHICA consisted of several oligomers, including dimer (compound D), trimers (compounds E, F and G) and tetramers (compounds H and I) with m/z of 388.2 [D+3OH+H]⁺, 488.2 [E+H]⁺, 588.3 [F+OH+K]⁺, 593.0 [G + OH+H]⁺, 672.9 [H + K]⁺, and 783.9 [I + OH+H]⁺, respectively. Meanwhile, P-DHI, P-DHICMe and P-DHICEt also consisted of serval dimers, trimers and tetramers, as suggested by ESI-MS (Figs. S5–S7 in Supporting information).

The fluorescence properties of P-DHI, P-DHICA, P-DHICMe and P-DHICEt were first evaluated in water by two-dimensional contour maps to estimate the optimal excitation-emission wavelength. As shown in Fig. S8 (Supporting information), the thickest contour regions of the spectrum can be clearly seen by the contour maps, that corresponded to the maximum fluorescence emission intensity of the fluorophores. Therefore, the optimal fluorescence excitation-emission wavelength can be obtained accordingly. In addition, the darker color in the spectrum indicated the higher fluorescence intensity. All of the two-dimensional spectrum showed a broad color distribution, indicating that all samples exhibited an excitation-dependent emission wavelength behaviour [41], due to the presences of multiple oligomers. With the increase of excitation wavelength, the maximum emission wavelength of fluorophore shifted to the longer wavelength. The emission intensity increased with increasing excitation wavelengths and then attenuated gradually, showing a maximum fluorescence emission intensity in the excitation wavelengths range of 330 nm and 370 nm, set as the optimal excitation wavelength. Therefore, the optimal excitation wavelength was determined to be 365 nm, 359 nm, 360 nm and 340 nm for P-DHI, P-DHICA, P-DHICMe and P-DHICEt, respectively.

The polymerization process was analyzed by its fluorescence change over time. DHICA has two active sites (positions a and c in Fig. 1) for polymerization, that resulted in the formation of linear oligomer P-DHICA. As shown in Fig. S9a (Supporting information) and Fig. 2a, the fluorescence intensity increased with polymerization time at first 10 min and then attenuated gradually, suggesting the oxidative degradation of hydroxyl radical at late stage [42, 43]. The trend stayed true for DHICMe and DHICEt, both formed linear oligomer during polymerization (Figs. S9b and c in Supporting information and Figs. 2b and c). In contrast, for DHI, its oxidative polymerization process is a little bit different from other dihydroxyindoles derivatives, probably due to an extra active site at position b of DHI (Fig. S5). The first 30 min of oxidative polymerization, the P-DHI emission wavelength showed a blue shift and the fluorescent intensity gradually decrease (Fig. S9d in Supporting information), then the emission wavelength kept constant and the fluorescence intensity increased continuously during the polymerization (Fig. 2d). We hypothesized that oxidative polymerization at the initial stage tends to form oligomer with a high degree of π conjugation, which will contribute to the delocalization of electrons. As the increase of polymerization degree of oligomers and the introduction of hydroxyl groups results in a decrease in the degree of conjugation [44]. Therefore, the fluorescence blue-shift phenomenon occurs at the initial stage of oxidation [45]. With these data in hand, we can selectively prepare the dihydroxyindoles-based fluorescent oligomers with desirable emission wavelength and intensity.

The design of fluorescent sensors for metal ion detection is based on the phenomena of fluorescence response on the metal chelation [46, 47]. Dihydroxydoles derivatives possess serval active site, such as catechol, carboxylate acid and quinone imine groups, for Cu²⁺ binding (Scheme S2 in Supporting information) [48, 49]. We examined the fluorescence quenching behaviour of P-DHI, P-DHICA, P-DHICMe and P-DHICEt at different concentration of Cu²⁺. As shown in Fig. 3a, the fluorescent intensity of the P-DHICA decreased continuously with the increase of Cu²⁺ concentration from 0 to 50 μmol/L. A plot of the ((I_F0-_IF)/I_F0) value versus the Cu²⁺ concentration also showed a positive correlation with the concentration of Cu²⁺, where I_F0 and I_F are the fluorescence intensities in the absence and presence of Cu²⁺ (Fig. 3b). Notably, the ((I_F0-I_F)/I_F0) value showed a linear increase with the concentration of Cu²⁺ (0–20 μmol/L) (R > 0.997, Fig. 3b). The detection limit for Cu²⁺ was estimated to be about 57 nmol/L according to the 3σ per slope, which is lower than maximum contamination level (20 μmol/L) of Cu²⁺ in drinking water permitted by the U.S. Environmental Protection Agency (EPA) [50]. In contrast, the change of fluorescence intensity becomes slower at higher Cu²⁺ concentration, suggesting the saturation of Cu²⁺ binding. The similar trend was also observed for P-DHI (Figs. S10a and b in Supporting information), P-DHICMe (Figs. S11a and b in Supporting information) and P-DHICEt (Figs. S12a and b in Supporting information). More interestingly, the significant fluorescence quenching of P-DHICA was exclusive to Cu²⁺ (Fig. 3c). Other metal ions, including Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Co²⁺, Cr³⁺, Fe³⁺, Mn²⁺, Sr²⁺ and Zn²⁺ showed little changes in the fluorescence intensity at the same concentration of 50 μmol/L. It is noted that the structural complexity of P-DHICA hindered the detailed investigation of the quenching mechanism. But we reasoned that Cu²⁺ is an effective fluorescent quenchers due to its paramagnetic nature by electron or energy transfer [51, 52]. Another two paramagnetic ion, Co²⁺ and Fe³⁺, exhibited much less ability towards fluorescence quenching. So it was suggested that carboxylic acid group of P-DHICA has high affinity for Cu²⁺ over other metal ions [53], which caused a more dramatic fluorescence quenching. This conclusion was in accordance with the affinity study of nature melanin [54]. Other metal ions showed a fluorescence enhancement effect towards P-DHICA, possibly via blocking of photoelectron transfer (PET) process [55, 56]. In comparison, P-DHI, P-DHICMe and P-DHICEt presented a similar fluorescence quenching behaviour, but the selectivity for Cu²⁺ was less significant (Figs. S13a–c in Supporting information). Meanwhile, the effect of pH on fluorescence intensity was evaluated at a wide range of pH. The result revealed that the fluorescence intensity of P-DHICA was stable in a wide range of pH from 2 to 10 (Fig. 3d). Compared to P-DHICA, the fluorescence intensity of other oligomer fluorophore has some fluctuations, suggesting the weaker association with Cu²⁺ (Figs. S14a–c in Supporting information). Lastly, we gave a quantitate evaluation of this selectivity by calculating the specific selectivity parameter W, defined as relative fluorescence intensity difference, of P-DHI, P-DHICA, P-DHICMe and P-DHICEt towards Cu²⁺. As showed in Table S1 (Supporting information), P-DHICA has the highest W values over all the metal ions, and therefore the best candidate for Cu²⁺ sensing. Again, this emphasize the importance of carboxylic group for the selective quenching of fluorescence.

In the above discussion, we attributed the fluorescent quenching phenomena to the binding of Cu²⁺. To validate this, we performed a Cu²⁺ binding and dissociation experiment, in which the fluorescence intensity of the P-DHICA can be switched between ON and OFF states by alternating addition of Cu²⁺ and pyrophosphate as the competition agent for Cu²⁺ (Fig. 4). The results indicating an excellent recyclability and reusability for the detection of Cu²⁺.

In summary, we successfully prepared fluorescent oligomers (P-DHI, P-DHICA, P-DHICMe and P-DHICEt) through oxidative polymerization of nature derived building blocks. The fluorescent properties were confirmed by two-dimensional fluorescence spectroscopy and useful for revealing the structural information. More importantly, we found P-DHICA has the highest selectivity to the Cu²⁺ detection, which worked over a wide range of pH values and had a good cyclability. We believed that our fluorescence sensors have great potential for use in medical testing.

This work was supported by the National Natural Science Foundation of China (No. 21774079) and State Key Laboratory of Polymer Materials Engineering, Sichuan University (No. sklpme2018-2-04).

Supplementary material related to this article canbefound, in the online version, at doi: https://doi.org/10.1016/j.cclet.2019.05.021.