An Easy-to-use Colorimetric Cyanine Probe for the Detection of Cu in

Department of Chemistry & State Key Laboratory of Molecular Engineering of Polymers & ..... intensity at 670 nm from 1.0 to 0.6 under nitrogen protect...
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Biological and Medical Applications of Materials and Interfaces

An Easy-to-use Colorimetric Cyanine Probe for the Detection of Cu in Wilson’s Disease 2+

Yibing Shi, Roumin Wang, Wei Yuan, Qingyun Liu, Mei Shi, Wei Feng, Zhiying Wu, Ke Hu, and Fuyou Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07081 • Publication Date (Web): 31 May 2018 Downloaded from http://pubs.acs.org on May 31, 2018

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An Easy-to-use Colorimetric Cyanine Probe for the Detection of Cu2+ in Wilson’s Disease Yibing Shia, Roumin Wangb, Wei Yuana, Qingyun Liua, Mei Shia,Wei Fenga, Zhiying Wub, Ke Hu*a, and Fuyou Li*a a

Department of Chemistry & State Key Laboratory of Molecular Engineering of Polymers &

Institute of Biomedicine Sciences & Collaborative Innovation Center of Chemistry for Energy Materials, Fudan University, 220 Handan Road, Shanghai 200433, P. R. China; bDepartment of Neurology and Research Center of Neurology, Second Affiliated Hospital, Zhejiang University School of medicine, 88 Jiefang Road, Hangzhou, Zhejiang, 310009, P. R. China

KEYWORDS: Wilson’s disease, colorimetric probe, cyanine dye, dopamine, urinary copper detection

Abstract: Copper (II) is one of the essential metal elements in human body, which can accumulate in many organs and finally excrete in urine. Excessive load of Cu2+ can cause liver cirrhosis, kidney dysfunction and many neurological symptoms in the case of Wilson’s disease (WD). Therefore, the selective and efficient detection of Cu2+ is of great importance. Although various fluorescent probes for Cu2+ have been reported, an efficient and capable probe is still rare for patients’ self-use on a routine basis. In this study, we developed an easy-to-use probe CY1 based on UV-Vis-NIR absorption changes with excellent sensitivity and selectivity for Cu2+. The

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mechanism of the oxidation of CY1 by Cu2+ was first explored. We demonstrated the role of the probe in quantitative detection of Cu2+ in the urine from WD patients and showed that it has great potential for clinical applications.

INTRODUCTION Though moderate uptake of copper is necessary, excess amount of that will eventually cause damage in liver cirrhosis and neurological symptoms1,2. Hepatolenticular degeneration (HLD), also named Wilson’s disease (WD)3,4, is an autosomal-recessive disease resulting in copper excessive accumulation in tissues and increased excretion in urinary systems5,6. In consequence, urinary copper detection is part of routine examination for WD patients. Conventional techniques for copper detection are limited to inductively coupled plasma (ICP) methods7,8, such as atomic absorption spectroscopy9 and atomic emission spectroscopy10. These methods need enormous space to accommodate the instrument (usually a whole room), complex procedures for examination (around one hour for preparation), and high expenditure for routine maintenance (argon and filter membrane needed). All these deficiencies keep them from household uses for WD patients. In contrast, portable UV-Vis spectrophotometer has been welcomed for small size, easy operations, and low cost11-13. Exploration of colorimetric probes for Cu2+ detection with high sensitivity and selectivity would benefit WD patients greatly for monitoring patients’ daily dosage and health conditions. One of the most widely used fluorescent probes for copper are cyanine-based sensors, especially conventional near-infrared fluorescent probes which have gained increasing popularity in imaging due to low autofluorescence and deep penetration depth14-17. Other fluorescent probes like quantum dot based ratiometric probe for copper detection with high sensitivity have also been developed 18. However, fluorescence-

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based technology commonly relies on invisible and unstable NIR emission as the detection signal and therefore unsuitable for home use. In contrast, UV-Vis-NIR absorption shift provides intuitive color changes and further quantitative analysis by a compact UV-Vis-NIR spectrometer is fast and easy. Herein, we explored the possibility of quantitative detection of copper based on the UV-Vis-NIR absorption spectrometry and implied them in clinical sample detection of Cu2+, which would contribute to the popularity of portable UV-Vis-NIR spectrometer as a method for routine detection of urinary copper. We select dopamine as the sensitive moiety for copper. Dopamine (DA, (1-amino-2-(3, 4)-dihydroxyphenyl ethane)) is an important substance in the primary metabolic pathway19. Dopamine oxidation generates ortho-quinones and semiquinones via intermolecular or intramolecular Michael addition20. Previous research shows that copper ions could oxidize dopamine anaerobically at the physiological pH condition (pH 7.4), with the complex of catechol (1, 2-dihyroxybenzene) and Cu2+ as the intermediate yielding Cu+ and a semiquinone20,21. Dopamine oxidation could also be accelerated by reactive oxygen species (ROS) and metal ions22-26. As a result, it is common to select dopamine as the reactive moiety when designing a small organic molecule probe. What is more, we know that WD results in overload of Cu2+ in liver tissue and in nervous system, and that the toxic intermediates in dopamine oxidation contribute to the neurological disorders4. Selecting dopamine as the reactive moiety for copper would help for further investigation of the decomposition mechanisms of dopamine at the same time25. In this study, we explored the mechanism of oxidation of CY1 by Cu2+ and developed dopamine-conjugated CY1 as the Cu2+ colorimetric probe for WD patients as routine household examination (Scheme 1). CY1 presented high sensitivity and selectivity for copper in vitro and in

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vivo, provided quantitative statistics about urinary copper from WD patients, and supplied information about pH values of the urine, which exhibited potential for further biomedical applications. RESULTS AND DISCUSSION Synthesis and characterization of CY1: Dopamine was chosen as the reactive moiety because of the high reaction sensitivity to Cu2+ 20. The preparation of CY1 followed previous literatures27,28. We selected 3-ethyl-1, 2, 2trimethyl-benz[e]indolium iodide (compound 1) as the heterocyclic base containing an activated methyl group and the unsaturated bisaldehyde 2-chloro-3-oxocyclohex-1-ene carbaldehyde (compound 2) as the Schiff base. Condensation reaction was carried out with the sodium acetate as the catalyst in acetic anhydride29. Further integration of dopamine unit to compound 3 was adopted from the literature of Keli Han group in 201227. The procedure to yield CY1 was simple and straight forward. With triethylamine as the catalyst, and DMF as the solvent, the overall yield was 48%. Detailed synthetic procedure was summarized in supporting information (Figure S1). Mechanisms of CY1 transforming to CY2 /CY3: Prior research showed that dopamine could rapidly react with Cu2+ to form a cyclic structure20,30. We demonstrated that this reactivity was still preserved even after dopamine being covalently linked to compound 3. Dopamine contained both an electron-deficient ring and an electron-donating amine group31. As a result, it could be attacked by nucleophiles easily. Different pH conditions of the environment might influence the UV-Vis-NIR absorption and fluorescent spectrum of CY1. Solution pH variation from 5.3 to 10.2 resulted in no significant

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absorption changes of CY1, demonstrating that there was no protonation for the amine group of dopamine moiety. The characteristic absorption at 553 nm and 823 nm didn’t rise up, indicating no formation of CY2 or CY3 (Figure 1a). Thermostability of our probe was also tested at 4 ◦C and 25 ◦C in three relevant pH conditions over 48 hours. Only basic solution at 25 ◦C would cause slight oxidation of our probe (Figure S9), suggesting the need of refrigeration for long time storage. Photostability was also tested with minimal changes (Figure S10). To investigate the reaction of CY1 to CY2/CY3, changes of UV-Vis-NIR absorption spectra of CY1 for Cu2+ were explored in EtOH/H2O (1/1; v/v) within different pH conditions (pH 6.8, pH 7.1, pH 8.0) (Figure 1). Addition of Cu2+ to CY1 solution induced significant changes in the absorption spectra. The aqueous solution was mildly acidic (pH 6.8), the absorbance at 670 nm decreased while the absorbance at 553 nm (ε=1.09×105 L•mol-1•cm-1) grew in, accompanied by color changes of the solution from blue to red. These phenomena indicated the formation of CY2 (Figure 1b). In alkaline solutions (pH 8.0), the absorbance at 670 nm (ε=2.14×105 L•mol-1•cm-1) decreased gradually while absorbance at 823 nm (ε=0.22×105 L•mol-1•cm-1) increased with an isosbestic point at λ =793 nm, indicating the formation of CY3 (Figure 1d). Color of the solution changed from blue to greyish blue as a result. The absorbance at 823 nm went through up and down with increasing concentration of Cu2+ when pH was 7.1; while the absorbance at 553 nm arose monotonically (Figure 1c). Calibration curve was obtained by plotting absorption change at 670 nm as a function of concentrations of added Cu2+ (Inset: Figure 1b, 1c, 1d). The slopes of every calibration curve were -0.0374 with slight differences in the intercepts, which remained the same with pH values in the range of 6.3-8.0. Here we selected the UV-Vis-NIR absorption spectra of CY1 upon addition of 2.0 equiv. Cu2+ to show characteristic absorption changes of the anaerobic oxidation of CY1 in acidic and

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alkaline solutions (Figure 2a and 2d). In Figure 2a, the anaerobic oxidation of CY1 by Cu2+ caused the increase of absorbance at 553 nm and decrease of absorbance at 670 nm in mildly acidic solution, indicating the generation of CY2. Dopamine oxidation occurred in alkaline solution too. There was characteristic absorption at 823 nm rising up, representing the generation of CY3. We monitored changes of the absorption intensity at 670 nm upon addition of Cu2+ at different concentrations in mildly (pH 6.8) and alkaline (pH 8.0) solutions to measure the oxidation kinetics, as shown in Figure 2b and 2e. The absorption intensity at 670 nm stopped decreasing at around 0.6 and 0.4 respectively upon addition of 2.0 equiv. and 5.0 equiv. of Cu2+, but showed complete reaction of CY1 upon addition of 10 equiv. Cu2+. It was unknown that why 2 and 5 equiv. of Cu2+ could not oxidize CY1 to completion. Nevertheless, we were able to conclude that Cu2+ performed as the oxidant during the anaerobic oxidation of CY1. Detailed statistics giving Figure 2b and 2e were presented in the supporting information (Figure S4). As we could observe from Figure 2c and 2f, concentration of Cu2+ as low as 5 µmol/L could react with CY1 (10 µmol/L) to completion within three hours. Along with the amount of Cu2+ increased to 10 µmol/L (1.0 equiv.) and 15 µmol/L (1.5 equiv.), time for the oxidation to completion shrank to around 100 min and 40 min respectively no matter in the alkaline or acidic solutions. The UV-Vis-NIR absorption intensity decreased from 1.0 to 0.1 within 2 minutes upon addition of 20 µmol/L Cu2+ when the solutions were exposed to air. In other words, aerobic oxidation of CY1 upon addition of Cu2+ was faster than anaerobic process. As exhibited in Figure 3a and 3b, the anaerobic absorption intensity at 670 nm of CY1 kept unchanged throughout the duration of the measurement (5 hour). The absorption intensity at 670 nm decreased from 1.0 to 0.9 when solution was exposed to air and decreased even more (from 1.0 to 0.7) when solution was bubbled with oxygen. Oxidation of CY1 was significantly

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accelerated in the presence of both Cu2+ and oxygen. Merely bubbling oxygen into the solution could cause decrease of UV-Vis-NIR absorption intensity of CY1 at 670 nm, which decreased from 1.0 to 0.2 in 13 hours. The reduction time would be prolonged to 108 hour when oxygen was replaced by air (Supporting information, Figure S5). Oxygen alone was involved in the oxidation of CY1, while oxygen and Cu2+ had synergistic effect in the oxidation. To one extreme, CY1 reacted to the completion with 2 equiv. of Cu2+ rapidly within 2 minutes when exposed to air atmosphere. Conversely, the same amount of Cu2+ only caused the decrease of the absorption intensity at 670 nm from 1.0 to 0.6 under nitrogen protection no matter in alkaline or acidic condition. It meant that oxidation could be carried out with Cu2+ or oxygen as the oxidant respectively, and Cu2+-assisted oxidation was much faster. To further confirm that Cu2+ accelerated the oxidation of CY1, we examined the absorption spectra under air, nitrogen, or oxygen (Figure S6, supporting information). Results of the addition of Cu2+ were collected when exposed to air and nitrogen respectively. UV-Vis-NIR absorption spectra almost kept the same no matter bubbled with nitrogen, air, or oxygen. The absorption at 670 nm decreased from 1.0 to 0.9 when the solution was under nitrogen protection with 2 equiv. Cu2+ (20 µmol/L) added and reacted for another 2 minutes. UV-Vis-NIR absorption intensity of CY1 decreased from 1.0 to 0.1 upon addition of 2.0 equiv. Cu2+ within 2 minutes. These indicated that reaction of CY1 by Cu2+ in air atmosphere was indeed a synergistic process. Products from oxidation of CY1 in acidic and basic solutions were confirmed through Maldi-TOF-MS (Figure 4). CY1 in neat acidic solution was in the protonated catechol form as evidenced by the mass at 729.865 at the very beginning (Figure 4a). Mass of intermediate1 shifted to 728.606 [M+1] after the addition of Cu2+, implying the mass loss of one from the catechol. Mass of the final product CY2 was 591.647 [M-1], which fitted nicely with the

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detachment of the dopamine moiety from CY1. The alkaline condition gave significantly different results as shown in Figure 4b. It started with the same mass of CY1 (m/z [M+1]: C50H54N3O2+) at 729.865, but the oxidation of CY1 by Cu2+ yielded various products. Intermediate 2 with the mass of 728.372 [M+1] and CY3 (m/z: C50H52N3O2+) with the mass of 725.126 [M+1] were two of them captured through Maldi-TOF-MS. According to the calculated mass change of CY1 and its oxidation products, we could deduct that the oxidation was limited in the dopamine moiety. The collective results from UV-Vis-NIR absorption spectra, Maldi-TOF-MS, and the 1H NMR (see Supporting Information) were able to decipher the mechanism of the oxidation of CY1 by Cu2+ in the acidic and alkaline conditions as discussed below. The blue shift of UV-VisNIR absorption peak of CY1 from 670 nm to 553 nm in acidic solutions indicated the shortened π-electron system and the weaker electron donor, which coincided with the generation of CY232. CY2 was believed to be the final product for oxidation of CY1 for the stability and absorbance at 553 nm27. We further confirmed its existence through Maldi-TOF-MS. Referring to alkaline oxidation of CY1 by Cu2+, there was the red shift of UV-Vis-NIR absorption spectra of CY1 from 670 nm to 823 nm, corresponding to the generation of cyclized structure of CY1. Absorption intensity at 823 nm of CY3 was lowered compared to that of CY1 at 670 nm in alkaline solutions, indicating that the cyclic structure was unstable. Compound 3 (Figure S1, supporting information) had the UV-Vis-NIR absorption at 815 nm, which was similar to the UV-Vis-NIR absorption of the final products CY3 at 823 nm in alkaline solution. Compound 3 contained electropositive nitrogen of benzoheterocycle as the electron acceptor and the other nitrogen of benzoheterocycle as the electron donor, so did the CY3. During the alkaline oxidation of CY1 by Cu2+, the ability of the nitrogen in dopamine moiety as the electron donor

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was attenuated and replaced by the other nitrogen of benzoheterocyclic compound. In this way, the π-electron system was extended. All this deduction was further demonstrated by the characterization of Maldi-TOF-MS and 1H NMR (Figure S7, supporting information). Results of Maldi-TOF-MS demonstrated that the alkaline oxidation of CY1 by Cu2+ was limited in the dopamine moiety. The subtraction of the mass of CY1 and CY3 was 4.73, corresponding to four diminished protons. The deducted structure of CY3 was further confirmed in this way. Also, the 1

H NMR indicated the disappearance of hydroxyl hydrogen in catechol and the amino hydrogen.

All the information demonstrated the cyclized structure of CY3. The oxidation mechanisms of CY1 at different pH conditions were summarized in Figure 4c. The dopamine moiety of CY1 kept unchanged at the very beginning when the solution was acidic. Further oxidation generated low concentration of CY2 with the cleavage of the C-N bond that led to the hypothesized byproduct of leucodopaminochrome. Leucodopaminochrome was detected in prior research where anaerobic oxidation of dopamine by Fe3+ was studied22,23. This intermediate was unstable in acidic aqueous solutions and could be further oxidized to generate dopaminochrome and CY2. In the alkaline condition, the catechol moiety was the predominant reactive moiety and would exist with one of the phenolic hydroxyl hydrogen off within the pH range of 8.0 – 13.731. The catechol moiety would chelate with Cu2+ to generate complexes of catechol with Cu2+. Further intramolecular electron transfer would yield corresponding semiquinone moiety, in which both of the phenolic hydrogen diminished. The semiquinone moiety was unstable and would be further oxidized by Cu2+ to generate the quinine moiety, which would undergo intramolecular 1, 4-Michael addition to form CY320. Specificity examination of CY1 to Cu2+:

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Because of the complex composition of human urine with multiple metal ions existing as potential source of interference, it was essential to test the redox specificity of CY1 to Cu2+. Specificity of CY1 to Cu2+ was examined through the absorbance spectra of CY1 (10 µmol/L) in EtOH/H2O (1/1, v/v) treated with various metal ions (Al3+, Ba2+, Ca2+, Cd2+, Co2+, Er3+, K+, Mg2+, Mn2+, Na+, Ni2+, Sm3+, Tm3+, Yb3+, Y3+, Zn2+, Ag+, Fe2+, Fe3+, Hg2+) successively at the concentration of 100 µmol/L when pH values were 6.8 (Figure 5a) and 8.0 respectively (Figure 5b). Neither the characteristic absorption at 823 nm or at 553 nm nor significant changes of absorption intensity at 670 nm was observed. The mechanism we summarized was specific for Cu2+-assisted oxidation of CY1. The concentrations of the added metal ions were quintuple that of Cu2+, which indicated significant sensitivity and selectivity of CY1 to Cu2+. CY1 as the probe for H2O2 have been reported and applied to in-vivo and in-vitro experiments27,28. Compared to Cu2+, oxidation of CY1 by H2O2 was slower and in higher concentration. We collected pictures of small bottles containing CY1 upon addition of Cu2+ in different pH conditions (pH 6.8, pH 7.1, and pH 8.0). Pictures of small bottles containing CY1 upon addition of multiple metal ions and H2O2 when pH value was 7.1 were specially taken to provide evidence for later clinical experiments, which was carried out when pH value was 7.1. As shown in Figure 5c, CY1 upon addition of Cu2+ presented intuitive color changes at different pH value, demonstrating remarkable convenience of CY1 as a colorimetric probe for copper. Test of other possible reactive anions or compounds such as sulfide (S2-), cysteine (Cys), glutathinone (GSH), hypochlorite (ClO-), and bisulfate (HSO3-), were conducted and presented in the supporting information (Figure S8), which showed negligible interference. Detection of Cu2+ in Urine Samples: We confirmed CY1 as an efficient probe for intracellular Cu2+ detection through confocal

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luminescence microscopy (CLM) experiments (Figure S13) and in-vivo Cu2+ through bioimaging (Figure S14). Clinical samples, such as urine from WD patients, were also tested to demonstrate the wide prospect of CY1 as a colorimetric probe for Cu2+ in biological applications. Ahead of detection of urinary copper from WD patients, we mimicked the calibration curve with the concentrations of Cu2+ added in urine as the x axis and the absorption intensity at 670 nm as the y axis (Figure S15, Supporting information). The slope was calculated to be 0.0374 (Inset: Figure 6b), which was the same with the slopes calculated in different pH conditions (Inset: Figure 1b-d). That was, slopes of the fitted line kept unchanged with different concentration of CY1 when pH value was in 6.3-8.0. Further calculation was based on this phenomenon. Concentrations of Cu2+ contained in these samples were detected by ICP method and by UV-Vis-NIR spectrometer respectively. Twelve samples were collected from four female patients and eight male patients with the age range from 13 to 36. Three samples were collected from normal people. Different diet they took and different health condition they suffered finally resulted in different physiological conditions. In addition, manual measurement error was also unavoidable for quantitative detection of Cu2+ in urine. All of those resulted in different absorption spectra of CY1 at the very beginning. Here we adopted UV-Vis-NIR absorption intensity at 670 nm of CY1 tested immediately after addition to the solution as the Istart. Spectra of CY1 kept unchanged in Urine/EtOH/HEPES (10/10/1; v/v/v) when immediately collected within 30 seconds. As shown in Figure 6b, the calculated concentrations of Cu2+ were accorded with the detected concentrations by ICP within the range of 0.3-20.0 µmol/L (Figure S16, S17, Supporting information). Pearson correlation (R) was calculated to be 0.996, indicating that the data obtained from our cyanine absorption probe method was in good agreement with ICP

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measurement (Figure 6b). Note that some reactive oxygen species such as H2O2 could potentially act as the interference for some dopamine containing fluorescent probes reported in the literature27,33,34. However, our colorimetric probe is not subject to this issue as was demonstrated in the specificity examination. CY1 also showed good repeatability. We observed the uprising of the absorption peak at 553 nm in mild acidic condition and that at 823 nm in alkaline condition upon addition of urine of WD patient (Figure 6c, 6e). In other words, CY1 also supplied information about urinary pH values in addition to provide quantitative concentration of Cu2+. That was the information we failed to obtain from fluorescent spectra of CY1 (Figure 6d, 6f), demonstrating that CY1 was a promising colorimetric probe rather than fluorescent probe. ICP is a demanding technique that only hospitals and research institution could afford to own. In contrast, portable UV-Vis-NIR spectrometer has been widely available, the volume of which could be as small as a suitcase and only reusable quartz cells are needed for examination. Popularity of household UV-Vis-NIR spectrometer would help a lot in routine detection of urinary copper for further supervision of daily dosage and physical conditions of WD patients. CONCLUSION We adopted CY1 as the colorimetric probe for Cu2+ and explored mechanisms of anaerobic and aerobic oxidation of CY1 by Cu2+. CY1 was oxidized to produce CY2 (λex=553 nm) in mild acidic solution and CY3 (λex= 823 nm) in alkaline solution through intramolecular 1, 4-Michael addition reaction. As a cyanine-based dye with the UV-Vis-NIR absorption at 670 nm and fluorescent emission at 800 nm, CY1 exhibited good sensitivity and selectivity, and was capable to detect Cu2+ in vitro and in vivo. We further applied CY1 to quantitative detection of Cu2+ in urines of

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WD patients. Concentrations of Cu2+ in these clinical samples were obtained through the calculation method we developed, which were in accordance with the concentrations detected through ICP method. Though sample size was limited, further evaluation of CY1 as a universal UV-Vis-NIR absorption probe for urinary copper would carry on. In addition, CY1 also provided different absorption spectra in acidic and alkaline solutions upon addition of urine of WD patients, which would provide more information about one’s diet, kidney and urinary system. In a nutshell, CY1 is an efficient and promising colorimetric probe for detection of Cu2+ and is believed to have further applications as a household probe to provide convenience for WD patients. EXPERIMENTAL SECTION Chemicals and Instruments: All reagents and chemicals were purchased from commercial sources and used without further purification. 2, 3, 3-Trimethylbenzoindolenine, iodoethane, dopamine hydrochloride (DA 98%), dimethyl formamide (DMF), phosphoryl chloride (POCl3), dichloromethane (DCM), cyclohexanone, ethanol, n-butanol, triethylamine (TFA), and toluene were obtained from Alfa Aesar Ltd. RECl3 (RE3+ = Y3+, Yb3+, Er3+, and Tm3+) were prepared according to the literatures35,36. Ethanol and deionized water were used to prepare all aqueous solutions. Solutions of Cu2+, Zn2+, Tm3+, Sm3+, Er3+, Yb3+, Y3+, Ba2+, Ca2+, K+, Fe2+, Na+ ions were prepared from their chlorate salts, and solutions of Ag+, Ni2+, Cd2+, Co2+, Fe3+, Al3+ ions were prepared from their nitrate salts. Solutions of HSO3-, ClO-, S2- were prepared from sodium salts 17,37. Characterization: 1H NMR and 13C NMR were measured on a Bruker AV-400 spectrometer with chemical shifts reported in ppm (in MeOD or DMSO-d6; TMS as internal standard).

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Electrospray ionization mass spectra (ESI-MS) were measured on a Micromass LCTTM system. UV-Vis-NIR absorption spectra were recorded on a shimaduzu 3000 spectrophotometer. Emission spectra were measured on an Edinburgh FLS 920 luminescence spectrometer. Inductively coupled plasma atomic emission spectra (ICP) was chosen to provide standard concentration of Cu2+ contained in the clinical samples, model for which was ICAP 7400 produced by Thermo Fisher Scientific. Synthesis of compound 138: 2,3,3-Trimethylbenzoindolenine (4.2 g, 20.0 mmoL) and iodoethane (3.1 g, 20.0 mmoL) were dissolved in toluene (20.0 mL). The mixture was stirred at 100 ̊C for 24 h leading to needle-like crystals.35 The reaction was cooled to room temperature, after filtered, washed with ether and dried, and to give 5.30 g (70%) of compound 1 as a solid. 1H NMR (400 MHz, CDCl3) δ 8.09 (t, J = 9.60 Hz, 2H), 8.04 (d, J = 8.20 Hz, 1H), 7.86 (d, J = 8.90 Hz, 1H), 7.73 (t, J = 7.30 Hz, 1H), 7.65 (t, J = 7.40 Hz, 1H), 4.84 (q, J = 7.10 Hz, 2H), 3.21 (s, 3H), 1.86 (s, 6H), 1.67 (t, J = 7.20 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 194.99, 137.90, 137.20, 133.70, 131.56, 130.09, 128.68, 127.85, 127.64, 122.88, 112.54, 55.92, 45.74, 22.68, 16.81, 13.84. M/Z (C17H20N+) = 237.219 [M+1]. Synthesis of compound 239: To a chilled solution of DMF (40.0 mL, 515.0 mmoL) in 20.0 mL DCM under N2 atmosphere, 37.0 mL of POCl3 (37.0 mL, 397.0 mmoL) in dichloromethane were added dropwise with an ice bath. After stirred for 30 min, cyclohexanone was added (10.0 g, 100.0 mmoL), and the resulting mixture was refluxed with vigorous stirring for 2 h at 80 ̊C, poured into ice cold water and kept it overnight to obtain compound 2 as a yellow solid. 1H NMR (400 MHz, CDCl3): δ2.46 (t, J = 6.20 Hz, 4H), 1.75 – 1.68 (m, 2H); MS (MALDI-TOF-MS): 172.164 [M].

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Synthesis of Compound 335: 5 mL acetic anhydride was added into the round-bottom flask containing compound 2 (365.3, 10.0 mmoL), compound 1 (172.6, 5.0 mmoL) and potassium acetate (98.1, 10.0 mmoL). Then the mixture was heated to 70 ̊C for 1 hour under the protection of N2, resulting in a green solvent. After chilled down, the mixture was poured into saturated sodium bicarbonate solution to separate green solid, which could be collected through filtration and further purified through column chromatography on silica gel. The chromatographic solution was DCM/MeOH (20/1). 1H NMR (400 MHz, CDCl3): δ 8.48 (d, J = 14.20 Hz, 2H), 8.15 (d, J = 8.50 Hz, 2H), 8.04 – 7.95 (m, 4H), 7.64 (t, J = 7.40 Hz, 2H), 7.49 (dd, J = 12.0, 7.20 Hz, 4H), 6.29 (d, J = 14.10 Hz, 2H), 4.38 (dd, J = 14.10, 6.90 Hz, 4H), 2.78 (t, J = 5.60 Hz, 3H), 2.12 (s, 3H), 2.04 (s, 12H), 1.53 (t, J = 7.00 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ 173.20, 149.71, 143.38, 139.26, 133.96, 131.99, 130.90, 130.21, 128.18, 127.77, 127.29, 125.13, 122.05, 110.64, 100.59, 51.14, 39.95, 27.59, 26.55, 20.82, 12.71. M/Z (C42H44ClN2+) = 611.265 [M-1]. Synthesis of CY127: A mixture of dopamine hydrochloride (0.5 g, 2.5 mmoL), compound 3 (0.4 g, 0.5 mmoL), and TFA (70.0 uL, 0.5 mmoL) with anhydrous DMF (10.0 mL) as solvent were reacted under N2 atmosphere at the temperature of 80 ̊C for 6 h. Then the solvent was removed through rotary evaporation, and then, after purified by flash chromatography with DCM/MeOH (v/v; 40/1) as eluent to give a deep blue solid (0.30 g, 48%). 1H NMR (400 MHz, DMSO): δ 8.82 (d, J = 5.60 Hz, 2H), 8.41 (t, J = 5.80 Hz, 1H), 8.17 (d, J = 8.60 Hz, 2H), 7.98 (d, J = 8.50 Hz, 4H), 7.68 (d, J = 13.20 Hz, 2H), 7.58 (dd, J = 14.10, 8.20 Hz, 4H), 7.40 (t, J = 7.50 Hz, 2H), 6.69 (d, J = 8.40 Hz, 2H), 6.53 (d, J = 8.50 Hz, 1H), 5.80 (d, J = 12.90 Hz, 2H), 4.12 (dd, J = 13.60, 7.10 Hz, 4H), 3.99 (dd, J = 12.80, 7.20 Hz, 2H), 2.97 (t, J = 6.60 Hz, 2H), 1.84 (s, 12H), 1.75 – 1.67 (m, 2H), 1.27 (dd, J = 15.30, 8.40 Hz, 10H). 13C NMR (101 MHz, CDCl3): δ 173.20, 149.71, 143.38, 139.26, 133.96, 131.99, 130.90, 130.21, 128.18, 127.77, 127.29, 125.13, 122.05, 110.64,

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100.59, 51.14, 39.95, 27.59, 26.55, 20.82, 12.71. ESI-MS m/z (C78H54N3O2): 729.526 [M+1]. Titration of CY1 by Cu2+: We added 10 μl CY1 (2 mmol) in EtOH to prepared 2 mL solution EtOH/Buffer (1/1; v/v) to acquire solutions of CY1 at the concentration of 10 μmol/L. UV-Vis absorption spectrum of CY1 at the very beginning was tested. We then collected UV-Vis absorption spectra of CY1 after addition of 10 μl Cu2+ (0.2 mmol) and stirred for 5 minutes with a magneton per time. Experiments were stopped until addition of Cu2+ would not cause changes to the UV-Vis absorption spectra. All the buffers (10 mM) and solutions of CY1 and Cu2+ were freshly prepared for the experiments. Calculation of Detection Limits: The detection limit was calculated according to the function: 3σ Detection Limit = k In this function, σ represented the standard deviation calculated based on the intensities of UVVis-NIR absorption peaks of CY1 (10 µmol/L) at 670 nm repeated for ten times, which was 0.001. The k could be calculated by the fitted lines of CY1 with the UV-Vis-NIR absorption intensity at 670 nm as the y axis and the concentration of Cu2+ as the x axis. The k represented the slope shown in Figure 1d, 1f and 1h, which was -0.0374. The value kept the same with pH ca. 6.3-8.0. The detection limit was calculated to be 80 nmol/L. Evaluation of pH Interference: We evaluated the influence of pH by measuring UV-Vis-NIR absorption spectra and fluorescent spectra of CY1 (10 µmol/L) in different pH (5.3, 6.3, 7.0, 8.0, 9.2 and 10.2) at the same time. Solutions were prepared with ethanol and buffer in one-to-one volume ratio, and buffers were prepared by triethanolamine and HEPES rather than PBS to avoid the interference of copper interacting with phosphate. All the parameters of UV-Vis-NIR

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absorption spectrophotometer and FLS920 luminescence spectrometer were kept the same all through the experiments. Aerobic and anaerobic oxidation: In order to explore mechanism of aerobic oxidation of CY1 by Cu2+, we prepared quartz cell equipped with rubber stopper. All the buffer solutions with different pH value were prepared by triethanolamine and HEPES rather than commonly used PBS to avoid interference from Cu2+ interacting with phosphate. This fact was demonstrated in supporting information (Figure S3). The calibration curves we obtained from titrations of CY1 in solutions free of buffer were the same with those with buffer. Prior to the injection into the quartz cell with newly unwrapped syringe, the buffer solutions were bubbled with nitrogen for 1 hour to remove oxygen dissolved in the solution as much as possible. Solutions of CY1 (10 µmol/L) would be protected with nitrogen throughout the experiment, so did the solution of Cu2+. We then collected the changes of absorption spectra of CY1 (10 µmol/L) after different amounts of Cu2+ (0 µmol/L, 20 µmol/L, 50 µmol/L, 100 µmol/L) were injected into the sealed cell respectively through a newly unwrapped syringe. The UV-Vis-NIR absorption and fluorescent emission spectra were collected every 5 minutes. Anaerobic oxidation of CY1 (10 µmol/L) by oxygen was carried on in quartz cells equipped with rubber stopper. Oxygen was bubbled into the solution all throughout the experiments. Oxidation by air went through exposure to atmosphere. Solutions for test were prepared by EtOH and buffer in one-to-one ratio, and the buffer solutions with different pH value were prepared by triethanolamine and HEPES. All the UV-Vis-NIR absorption spectra were collected every 5 minutes. Observation of oxidation of CY1 by 1H NMR Spectra: We then examined the oxidation of CY1 by Cu2+ through 1H NMR. 7.4 mg CY1 was dissolved in deuterated methanol and the pH value of this solution was 8.0. Thus the alkaline oxidation of CY1 was examined utilizing Cu2+

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dissolved in HEPES buffer solution to guarantee basic environment all through the experiment. 1

H NMR spectra were collected after the addition of 2.0 equiv Cu2+ and reacted for another 10

minutes with the help of ultrasonic apparatus. Acidic oxidation mechanism of CY1 was examined by addition of Cu2+ in deionized water considering Cu2+ itself was a weak acidic. We then collected the products of 100 mg CY1 and 1.0 equiv Cu2+ interacted for 10 minutes, purified them through column chromatography, confirmed CY2 through UV-Vis-NIR absorption spectrum and finally characterized CY2 through 1H NMR Spectra. pH stability and thermostability: We prepared solutions of CY1 (10 µmol/L) in EtOH/buffer (1/1; v/v) when pH values were 6.8, 7.1, and 8.0. Then absorption spectra of these solutions were collected per 1 hour for two days (48 hours in total) at 4 ̊C and 25 ̊C. CY1 presented good pH stability. It was more suitable for CY1 to be preserved at 4 ̊C. Cell Culture: The cell lines Raw630 were purchased from the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). Raw630 cells were grown in modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 ̊C and 5% CO2. Raw630 cells were planted on 14 mm glass coverslips and allowed to adhere for 24 h. Cytotoxicity of CY1: Vitro cytotoxicity was measured by performing MTT assays on the Raw630 cells. Cells were seeded into a 96-well cell culture plate at 5 × 104/well, with 100% humidity, and were cultured at 37 ̊C and 5% CO2 for 24 h; different concentrations of CY1 (0, 5, 10, 25, 50, 100, 200, 400, 800 µmol/L, diluted in RPMI 1640) were then added to the wells incubating for 24 h at 37 ̊C under 5% CO2. Then MTT (10 µL, 5 mg/mL) was added to each well for further incubation of four hours under same conditions. The optical density OD570 Value (Abs) of each well with background subtraction at 690 nm was measured on a Tecan Infinite M200 monochromator-based multifunction microplate reader. The following formula was used to

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calculate the inhibition of cell growth:

cell viability% =

mean Abs value of treatment group × 100 mean Abs value of control

Confocal Luminescent Imaging: The ability of CY1 for detecting intracellular Cu2+ was confirmed through confocal luminescence imaging experiments (Figure S13, Supporting information). There were two groups of Raw 630 cells incubated with CY1 (10 µM) for 30 min at 37 ̊C. Then both of them were washed softly to clear out materials unable to enter the cell. One group of Raw 630 cells would be observed immediately, as the contrast group. The other would be further treated with Cu2+ of 20 µmol/L and respectively observed after 10 minutes, 30 minutes, and 60 minutes. Every group contained several coverslips planted with Raw 630 cells to guarantee efficient cells for test. Detection of Cu2+ in vivo: 50 µmol/L CY1 intravenously injected into the Bab/c mouse and the signal were immediately through the 800 nm ∓ 12 nm filter under the emission of 660 nm filter. Then the same amount of CY1 was intravenously injected to a new mouse in the same size. This experimental mouse was further injected with (0.1 mMol/L) Cu2+ and chloral hydrate. The imaging signal was collected after 35 min, 1 hour, and 2 hours, the results were presented in Figure S14. All animal experiments were performed in compliance with the guidelines of National Institute for Food and Drug Control, China, and were approved by the Institutional Animal Care and Use Committee, School of Pharmacy, Fudan University. Detection of Cu2+ in Urine: According to the literature3,10, urinary copper excretion was 0.6 µmol/day for healthy people, and >1.6 µmol/day for WD patients. To make sure that the fitted line worked well in urine, we firstly detected the additional Cu2+ in the solution of

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Urine/EtOH/HEPES (10/10/1; v/v/v). The concentrations of additional Cu2+ were 0 µmol/L, 0.5 µmol/L, 1.0 µmol/L, 1.5 µmol/L, 2 µmol/L, 2.5 µmol/L, 3.0 µmol/L, 3.5 µmol/L, 4.0 µmol/L, 4.5 µmol/L, and 10 µmol/L. UV-Vis-NIR absorption spectra of CY1 for additional Cu2+ were presented in Figure S15 along with the fitted line. We then detected Cu2+ in urines of WD patients. Every sample was tested in the cuvette (10 mm × 10 mm) to acquire UV-Vis-NIR absorption spectra of Urine/EtOH/HEPES (10/10/1; v/v/v) alone, CY1 (10 µmol/L) dissolved in Urine/EtOH/HEPES (10/10/1; v/v/v) immediately, and CY1 (10 µmol/L) dissolved in Urine/EtOH/HEPES (10/10/1; v/v/v) after interacted with the Cu2+ contained. Volume of the urine for detection every time was 1 mL. We calculated the deduction between the UV-Vis-NIR absorption intensity at 670 nm of CY1 tested immediately (Istart) and after interaction (Iend). Concentration of Cu2+ was calculated according to the function: C$%&' =

I)*+ − I-./0. −0.0374

We then tested the concentration of Cu2+ of those samples through inductively coupled plasma atomic emission spectra (ICP). We summarized the concentrations of Cu2+ tested by ICP and by UV-Vis-NIR absorption spectra of CY1 (Figure 6b). Detailed absorption spectra of the samples were presented in supporting information (Figure S16, Figure S17).

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Figure 1: (a) Absorption and (Inset: a) fluorescent emission spectra of CY1 (10 µmol/L) in EtOH/H2O (1/1; v/v) in different pH conditions (pH 5.3, pH 6.3, pH 7.0, pH 8.0, pH 9.2 and pH 10.2). (b-d) UV-Vis-NIR absorption spectra of CY1 (10 µmol/L) dissolved in EtOH/H2O (1/1; v/v) upon titration of Cu2+ (20 µmol/L in total) when pH value was 6.8 (b), 7.1 (c) and 8.0 (d).(Inset: b-d) The fitted line was based on the absorption of CY1 at 670 nm and the corresponding concentration of Cu2+ added when pH value was 6.8 (Inset: b), 7.1 (Inset: c), 8.0 (Inset: d).

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Figure 2: (a, d) Absorption spectra of CY1 (10 µmol/L) measured at pH 6.8 (a) or pH 8.0 (d) upon the addition of 20 µmol/L Cu2+ under anaerobic conditions. (b, e) The absorbance changes at 670 nm in different pH conditions (b: pH 6.8; e: pH 8.0) upon the addition of different amount of Cu2+ (0 µmol/L, 20 µmol/L, 50 µmol/L, 100 µmol/L) were plotted as a function of time up till 5 hours. (c, f) Aerated absorbance changes at 670 nm of CY1 (10 µmol/L) in different pH conditions (c: pH 6.8; f: pH 8.0) upon addition of different amount of Cu2+ (0 µmol/L, 5 µmol/L, 10 µmol/L, 15 µmol/L, 20 µmol/L) was measured too. Solutions with different pH value were prepared by triethanolamine and HEPES, which were dissolved in EtOH/H2O (1/1; v/v).

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Figure 3: Changes of absorption intensity at 670 nm of CY1 (10 µmol/L): (a) in acidic solutions (pH 6.8) exposed to nitrogen, oxygen and air, and to nitrogen, and oxygen upon addition of Cu2+ (20 µmol/L); (b) in alkaline solutions exposed to nitrogen, oxygen and air, and to nitrogen, and oxygen upon addition of Cu2+ (20 µmol/L).

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Figure 4: (a-b) MalDI-TOF-MS of products resulted from CY1 reacting with Cu2+ when the pH of solution was 6.8 (a) and 8.0 (b). (c) The oxidation mechanism of CY1 prompted by of Cu2+ yielding CY2 and dopaminochrome in mild acidic solution (pH 6.8) and CY3 in alkaline solution (pH 8.0) was presented. The blue, red and green color coding indicates extension degree of πconjugation. The table inset shows the peak extinction coefficients for the colorimetric probe before the reaction (CY1) and after the reaction in acidic (CY2) or basic (CY3) conditions.

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Figure 5: (a-b) Absorption spectra of CY1 (10 µmol/L) in EtOH/H2O (1/1, v/v) treated with various metal ions (100 µmol/L, added successively), H2O2 and Cu2+ (20 µmol/L) when pH values were 6.8 (a) and 8.0 (b) respectively. (c) Pictures of CY1 (10 µmol/L) in EtOH/H2O (1/1; v/v) treated with Cu2+ (20 µmol/L) when pH values were 8.0, 7.1, and 6.8, and pictures of CY1 (10 µmol/L) in EtOH/H2O (1/1; v/v) treated with various metal ions.

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Figure 6: (a) Illustration of detection of Cu2+ in urine of WD patients by ICP and by spectroscopy with CY1 as the probe; (b) Concentrations of Cu2+ of WD patients by ICP method and by UV-Vis-NIR spectrometer with CY1 (10 µmol/L) in Urine/EtOH/HEPES (10/10/1; v/v/v) as the probe, and (Inset: b) fitted line of absorption changes at 670 nm of CY1 upon addition of Cu2+. (c-d) UV-Vis-NIR absorption (c) and fluorescent emission (d) spectra of CY1 (10 µmol/L) when pH value was 6.8, and (e-f) UV-Vis-NIR absorption (e) and fluorescent emission (f) spectra of CY1 (10 µmol/L) when pH value was 8.01 were measured in Urine/EtOH/HEPES (10/10/1; v/v/v). Urine was from sample 6 (S6).

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Scheme 1: (a) Oxidation of CY1 by Cu2+ in acidic solution (pH 6.8) and alkaline solution (pH 8.0) respectively and (b) Schematic illustration of CY1 as a copper ions detection probe in urine from WD patients.

ASSOCIATED CONTENT Supporting Information Additional syntheses and NMR spectra details, fluorescence characterizations, additional UVvis-NIR absorption spectra data for time based measurements, additional probe specificity test, probe stability test, in-vitro and in-vivo imaging, UV-vis-NIR absorption spectra for clinical sample test and statistics AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENT The authors thank the National Key R&D Program of China (2016YFC1303100), the National Nanotechnology Major Program (2017YFA0205100), and the National "1000 Youth Talents" Plan for financial support. REFERENCES

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