A rhodamine derivative probe for highly selective detection of Cu(II)

Background : Abnormal Cu(II) ions levels may affect many biological functions, and it is of great importance to detect Cu(II) ions in organisms. Methods : Herein, we report a near-infrared (NIR) fluorescent probe EtRh-N-NH 2 for the detection of Cu(II). In the probe structure, a rhodamine core was used, and a hydrazine group was employed as the responsive site. Results & Conlusions : EtRh-N-NH 2 displayed sensitive, specific and fast response upon Cu(II) with excellent linear relationship between the concentration and fluorescence emission intensity in 0–1 µ M range. The releasing EtRh-COOH exhibited 762 nm of emission wavelength with a 75 nm of Stokes shift.


Introduction
The copper element amount in the human body and other biological systems is only trace but essential and indispensable [1,2].In organisms, copper, existing as an oxidation state Cu(II), participates in many important redox reactions [3].The Cu(II) concentration should maintain at a certain range of about 40 µg L −1 to meet the normal metabolism of organisms in the human body [4].Excessive Cu(II) concentration can cause gastrointestinal dysfunction or liver and kidney damage.But on the other hand deficient Cu(II) concentration can cause a variety of neurodegenerative diseases [5,6].Therefore, it is significant to develop a reliable method to monitor Cu(II) in the complex organisms.
The traditional techniques based on inductively coupled plasma (ICP) method (including atomic absorption spectroscopy (AAS) [7], atomic emission spectroscopy (AES) [8], and mass spectrometry (MS) [9]) could detect Cu(II) ions concentration in high accuracy; however, it is difficult to monitor the Cu(II) compounds in realtime and in the biological system using these ICP methods [10].There are lots of methods for the detection of copper ions, mainly including fluorescent hybrid nanomaterials [11] and fluorescent chemical sensors [12].Emerging fluorescent hybrid nanomaterials includes quantum dots (QDs), metal nanoclusters (NCs), carbon dots (CDs), MXenes and polymer nanoparticles (NPs) [13,14].Fluorescence chemical sensors are mostly based on the following types: deoxyribonucleic acid [15], fluorophores [16,17], metal-organic framework (MOF) [18], copolymers [19], Schiff bases [20], and silica nanoparticles [21].Among them, chemical sensors containing fluorophores have attracted much attention.By introducing various ligands and fluorophores, such as fluorescein, rhodamine, naphthalimide and boron dipyrromethene (BODIPY), the selective de-tection of metal ions can be successfully achieved.The process involves different sensing mechanisms including photo-induced electron transfer (PET), fluorescence resonance energy transfer (FRET), intramolecular charge transfer (ICT), chelation enhanced fluorescence (CHEF), excited state intramolecular proton transfer (ESIPT) and Aggregation induced emission (AIE).Compared with nanomaterials, organic chemical probes are of superior biocompatibility, flexible structural modification and relatively low toxicity.In addition, fluorescence bioimaging technology allows us to detect a variety of metal ions including Cu(II) in vivo at a subcellular level [22,23], thus they were receiving much more attention.
Cu(II) fluorescent chemical sensors are mostly designed and synthesized based on inorganic [rhenium (I), ruthenium (II), iridium (III), zinc (II), and gold (I)] [24] and organic materials [25].And these probes can be classified into two categories based on their mechanism of the fluorescence production [26,27].The first ones produce fluorescence via the coordination with the Cu(II) ions; however, the fluorescence is generally weak due to the heavy atom effect and the low efficiency of photoinduced electron transfer (PET) process.Secondly, organic material chemical sensors are designed by the fluorescence technology of organic small molecules to sense metal ions.Through the complexation of ligands and metallic copper, it can produce spectral shifts, binding/association/dissociation constants, and color changes to achieve the sensing effect of Cu(II).The second category of Cu(II) detection probes produce fluorescence after the response with Cu(II) ions.Before the response, little fluorescence was emissive; thus, image with higher signal to background ratio can be achieved.Because of its instant response, high sensitivity and selectivity, easy measurement and real-time monitoring, fluorescent sensors based on organic materials have received extensive attention [28].Among the NIR dyes, rhodamine derivatives [29][30][31][32] are widely used as responsive fluorescent probes by their characteristic spirolactam ring-opening and -closing process (Supplementary Table 1).More importantly, the rhodamines are of high fluorescence quantum yield, large molar extinction coefficient, good stability upon photo irradiation, and easy structural modification.However, many reported rhodamines and their derivatives used for Cu(II) detection exhibited short fluorescence emission wavelength (mostly less than 600 nm), and probes with emission in the NIR range (600-900 nm) are more advantageous because of their better tissue penetration depth [33][34][35].Besides, the Stokes shifts of the reported dyes are usually small (less than 50 nm), leading to a severe autofluorescence issue in fluorescence imaging [36].Herein, we designed a new rhodamine-based near-infrared probe EtRh-N-NH 2 acting as a turn-on chemo-sensor for Cu(II) with a spirocyclic hydrazide group as the Cu(II) recognition group [37,38].The probe EtRh-N-NH 2 shows negligible fluorescence.However, upon the treatment with Cu(II) compound, the hydrazide group was hydrolyzed to generate a ring-opened dye EtRh-COOH with extremely strong fluorescence signals at 762 nm.Thus, the Cu(II) compound turns on the fluorescence (Scheme 1).Meanwhile, it is indicated from the results that the response is highly specific for Cu(II) compound.In the aspect of analytical chemistry, within the range of 0-1.0 µM, the response shows an excellent linear relationship with the R 2 = 0.997.Besides, the detection limit is only 6 nM.In addition, the Stokes shift of the probe is as large as 75 nm.Finally, the probe reveals its ability to detect various active substances in vitro.

Synthesis of EtRh-N-NH 2
As described, EtRh-N-NH 2 was synthesized via a three-step method from 2-(5-(diethylamino)-2hydroxybenzoyl) benzoic acid (Scheme 2).At first, the reaction of compound 1 and cyclohexanone generates the three-membered ring intermediate 2 in the presence of concentrated sulfuric acid.Then the afforded 2 was condensed with 4-diethylaminobenzaldehyde to give raise to the fluorophore EtRh-COOH.Finally, the carboxyl part of the fluorophore underwent a ring-closure reaction to obtain the desired aminohydrazide probe EtRh-N-NH 2 .

Mechanistic study of the response
As depicted in Scheme 3, we proposed the mechanism of the fluorescent turning-on process of the designed probe EtRh-N-NH 2 .The Cu(II) powerfully bonds and chelates with the amino and carbonyl groups of the probe, leading to the hydrolysis of the hydrazide and formation of the strong fluorescent EtRh-COOH.Eventually, the fluorescence is turned on.
To validate the mechanism, we performed the HPLC experiments.As shown in Fig. 1, the retention times for EtRh-COOH and EtRh-N-NH 2 were 3.20 min and 4.38 min, respectively.Upon adding Cu(II) compounds, the peak of EtRh-N-NH 2 disappeared gradually; meanwhile, the peak of EtRh-COOH raised proportionately, suggesting the generation of EtRh-COOH.In addition, the high-resolution detection of the mixed solution of the probe EtRh-N-NH 2 and Cu(II) compounds can simultaneously obtain the mass spectrum peaks of EtRh-N-NH 2 and EtRh-COOH (Supplementary Fig. 1).Therefore, we proved the reaction mechanism in Scheme 3.

Optical properties of EtRh-COOH and EtRh-N-NH 2
The absorbance spectra of EtRh-N-NH 2 (in PBS) shows a clear peak at approximately 350 nm, and emission intensity of EtRh-N-NH 2 from 690 nm to 900 nm is very weak.Besides, the solution is colorless; however, it turned blue after adding Cu(II).The 350 nm absorption peak decreased but a new band near 687 nm appeared and enhanced (Fig. 2A); meanwhile, a new band arose at 762 nm in the emission spectra (Fig. 2B).When the addition of Cu(II) with different concentrations (0-40 µM) is added to the probe(10 µM), the UV absorption of the probe at 687 nm and the fluorescence intensity at 762 nm gradually increase with the increase of Cu(II) concentration (Fig. 2C,D).The addition of 30 µM of Cu(II) triggered 235 fold enhancement of emission intensity in the range of 600-900 nm in 25 minutes (Supplementary Fig. 2).And in the range of 0-1.0 µM, the fluorescence emission intensity of the probe EtRh-N-NH 2 at 762 nm shows a good linear relationship (R 2 > 0.99) with a detection limit (LOD) of 6 nM (LOD = 3 δ/k), displaying a very good sensitivity (Fig. 2E).The results manifest that the quantification of Cu(II) concentration can be achieved by utilizing the fluorescent spectral change behavior of EtRh-N-NH 2 on Cu(II).

Influence of pH and temperature to the response
The effect of pH on the fluorescent change of probe EtRh-N-NH 2 on Cu(II) was studied (Fig. 3A).It was shown that the fluorescence spectrum of the EtRh-N-NH 2 solution remained unchanged at the pH range of 2.0 to 12.0 in the absence of Cu(II), which also indicated the excellent stability of the probe EtRh-N-NH 2 in PBS solution.However, after the addition of Cu(II) (30 µM), a significant increase of the fluorescence intensity was observed when the pH was in the range from 6.0 to 8.0, and the increasement was almost equal within the range, but relative fewer increase was achieved was when pH was less than 6.0 or greater than 8.0.When the pH is below 4 or above 12, the response became relatively weak, presumably due to the reduced chemical reaction activity between Cu(II) and spirocyclic hydrazide group in the probe structure under strong acidic or basic environment.Meanwhile, the temperature effect of the fluorescent response was also investigated (Fig. 3B).The experiment data showed that the response efficiency was almost the same in the range of 25-50 • C. Therefore, the probe EtRh-N-NH 2 can be used in both physiological pH (6.0-8.0) and temperature (37 • C).

Selectivity studies
The selectivity of the probe over Cu(II) was examined to check if the probe can be employed in complex biochemical environment.Firstly, the solution of the probe EtRh-N-NH 2 was treated with several common metal cations, including Al 3+ , Ba 2+ , Ca 2+ , Co 2+ , K + , Mg 2+ , Mn 2+ , Na + , Pb 2+ , Zn 2+ , Cd 2+ , Hg 2+ , Fe 3+ , Fe 2+ , Ni 2+ and the mixture of all metal ions.The results suggested that only little enhancement was achieved in both absorbance and fluorescence spectrum (Fig. 4A,B), and the enhancements of these ions were insignificant comparing to that of Cu(II), demonstrating the superior selectivity of the probe EtRh-N-NH 2 upon Cu(II) over other metals.
Human serum albumin (HSA) is the most abundant serum protein in the blood and can be used as a carrier for a variety of cargo molecules.Its apparent affinity for divalent copper has always been a concern, and it is considered to be the main Cu(II) binding ligand in blood and cerebrospinal fluid.We tested the binding ability of the probe to Cu(II) in the presence of different concentrations of HAS (Fig. 4C).The results proved that the affinity between HSA and Cu(II) was much high than the affinity between EtRh-N-NH 2 and Cu(II).Moreover, considering that the Cu(II) can participate in the intracellular redox reaction, we conducted the control experiments with different reducing and oxidizing reagents (Fig. 4D).GSH can function as a reducing reagent intracellularly and turn Cu(II) into Cu(I).And when both Cu(II) and excessive GSH was added, negligible fluorescence was generated, demonstrating that Cu(I) was not responsive over the probe.Besides, when the reactive oxidizing species (H 2 O 2 and ClO -) were reacted with the probe, little fluorescence was formed, proving that the probe was nonreactive with by ROS.
In view of the above HSA and GSH responsive results, and considering the high content of HSA in the organism and GSH in the cells, the imaging of Cu(II) in living cells with the probe EtRh-N-NH 2 would be indirect.Therefore, probes with less interference by HSA and GSH are needed to develop for the direct imaging of Cu(II) in living cells.

Reagents and instruments
The chemical reagents used are commercially available and purchased from J&K or Innochem chemical reagents.Commonly used solvents and chemical raw materials were all at analytically pure standard, and they were used without purification.The 1 H NMR and 13 C NMR spectrum were performed on a nuclear magnetic resonance spectrometer 400 (ASCEndTM400 (AVANCE HD III) with tetramethylsilane (TMS) as the internal standard.The high-resolution mass spectrum were tested on the American thermoelectric orbital ion trap mass spectrometer (LTQ-Orbitrap).The high-performance liquid chromatography was measured on the Agilent 1260 Infinity high performance liquid chromatograph.The ultraviolet were measured on the LS-45/55 fluorescence spectrophotometer (PerkinElmer, USA), and the fluorescence spectrum were measured on the UV-3200 ultraviolet-visible spectrophotometer.

Conclusions
In conclusion, we developed a rhodamine derivative EtRh-N-NH 2 as a fluorescent probe for the detection of Cu(II).The Cu(II) compounds were successfully recognized by the hydrazine moiety, releasing the product EtRh-COOH with high fluorescence and producing the fluorescence signal.The recognition was sensitive, specific, and rapid.The detection limit is only 6 nM.The product EtRh-COOH exhibited a NIR region of emission wavelength at 762 nm, and its Stokes shift is as large as 75 nm.The development of probes with the ability of Cu(II) imaging in living cells is underway.

Scheme 1 .
Scheme 1. Mechanistic proposal of the response of EtRh-N-NH2 upon treatment with Cu(II).

Fig. 3 .
Fig. 3.The investigation of the influence of pH and temperature to the response.(A) Fluorescence response of 10 µM EtRh-N-NH2 in the pH range of 2.0-12.0(black line) with 30 µM of Cu(II) (red line).(B) Fluorescence response under different temperature conditions (25-40 • C) of 10 µM EtRh-N-NH2 (black line) in the presence of 30 µM Cu(II) (red line).In the pH and temperature experiment, the probes are tested after incubating with Cu(II) for more than 30 minutes (λex = 690 nm).