A ratiometric near-infrared naphthalimide-based fluorescent probe with high sensitivity for detecting Fe<sup>2+</sup> <i>in vivo</i>

A ratiometric near-infrared naphthalimide-based fluorescent probe with high sensitivity for detecting Fe²⁺ in vivo

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Lina Huang^a, Yu Chen^a, Yuqiang Zhao^b, Yumin Wang^a, Junwei Xiong^a, Junfeng Zhang^a^,^*, Xianghua Wu^a^,^*, Ying Zhou^b^,^*

Chinese Chemical Letters | 2020, 31(11) : 2941 - 2944

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Chinese Chemical Letters | 2020, 31(11): 2941-2944

• Communication •

A ratiometric near-infrared naphthalimide-based fluorescent probe with high sensitivity for detecting Fe²⁺ in vivo

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Lina Huang^a, Yu Chen^a, Yuqiang Zhao^b, Yumin Wang^a, Junwei Xiong^a, Junfeng Zhang^a^,^*, Xianghua Wu^a^,^*, Ying Zhou^b^,^*

Affiliations

^a College of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming 650500, China

^b College of Chemical Science and Technology, Yunnan University, Kunming 650091, China

Published: 2020-11-15 doi: 10.1016/j.cclet.2020.06.006

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Abstract

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Iron is one of the essential trace elements in the human body. It plays an important role in human biology and pathology. Deregulation of iron levels in cells is associated with disease development. In this work, we synthesized a novel near-infrared intramolecular charge transfer (ICT) based ratiometric fluorescent probe to detect Fe²⁺, by using naphthalimide and indole moieties as building blocks. Our work showed that the radiometric probe has excellent selectivity, sensitivity and rapid response. Moreover, we could successfully perform real-time monitoring of Fe²⁺ in HeLa cells and C. elegans.

Key words

Fluorescent probe / Ratiometric / Fe²⁺ / Naphthalimide / Near-infrared

Cite this Article

Lina Huang, Yu Chen, Yuqiang Zhao, Yumin Wang, Junwei Xiong, Junfeng Zhang, Xianghua Wu, Ying Zhou. A ratiometric near-infrared naphthalimide-based fluorescent probe with high sensitivity for detecting Fe²⁺ in vivo[J]. Chinese Chemical Letters, 2020 , 31 (11) : 2941 -2944 . DOI: 10.1016/j.cclet.2020.06.006

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As a vital element of life, iron can adopt multiple oxidation states, which makes it critical for catalytical conversion in biological systems. Its deregulation may trigger oxidative stress events, leading to dysregulation of iron levels and is associated with various aging-related diseases such as cardiovascular disease, neurodegenerative diseases, Alzheimer's disease and multiple cancers [1-5]. Iron binds to hemoglobin and promotes the oxygen transfer from the lungs to cells. At cellular level iron acts as part of cytochromes and respiratory enzymes to help electron transfer and participate in mitochondrial respiratory chain [6-8]. In many physiological processes, iron plays an indispensable role, mainly in the form of ferrous and iron ions. Therefore, it is of great interest to effectively monitor and detect ferrous ions.

Naphthalimide derivatives have gradually become one of the hot research subjects in the field of fluorescence probes, including pH probes [9-11], fluorescent probes for ions [12-15], biological small molecules [16-19] and macromolecules [20-23] probes, etc. In this study, we selected naphthalimide and indole as building blocks for a new ICT-based ratiometric fluorescence probe, based on the following considerations: (1) Naphthalimide dyes are classic intramolecular charge transfer (ICT) dyes, which have excellent two-photon characteristics. They are widely used to develop two-photon fluorescent probes [24-26]; (2) Due to the excited ICT state between the electron donor and the electron acceptor in the same dye molecule, the naphthalimide dye has a relatively large Stokes shift. This may be beneficial for probe design with long wavelengths, such as red or near-infrared emissions; (3) The indole moiety is a traditional structural unit of a cyanine derivative [27]. It is worth noting that due to the presence of the chemically active methylene of the indole unit, the structure of the innovative probe can be easily modified. In general, we combined the advantages of classic naphthalene dyes and traditional indole moieties to construct a near-infrared (NIR) ratiometric fluorescent Probe 1 with a novel structure for detecting Fe²⁺. From the structural perspective, Probe 1 extends the π system by introducing naphthalene and indole moieties to achieve adjustable emission from visible spectrum to NIR and has a large red shift (Scheme 1).

Fluorescent probes have the advantages of low radioactivity, low cost, simplicity and rapid application [28-36]. In recent years, several fluorescent probes for detecting Fe³⁺ have been developed [37, 38], but relatively few probes were developed for detecting Fe²⁺ [39-43]. Here we have developed a novel near-infrared ICT-based ratiometric fluorescence probe 1 to detect Fe²⁺. Probe 1 was made up by two parts: One is the NIR fluorophore of naphthalimide derivatives, the other is the Fe²⁺ detecting group of N-oxides.(Scheme S1 in Supporting information). Probe 1 showed fast response and a large red shift for Fe²⁺ detection. When the N-oxide was reduced by Fe²⁺, the NIR fluorescence emission (λ_em = 690 nm) increased significantly with the increase of ICT. In addition, Probe 1 exhibited high selectivity and sensitivity towards Fe²⁺ with low detection limit. Moreover, the novel Probe 1 was successfully utilized for imaging Fe²⁺ in cells and C. elegans.

All chemicals and solvents were purchased from commercial suppliers, and were used directly without further purification in this article. Flash chromatography was carried out on silica gel (200–400 mesh), ¹H NMR and ¹³C NMR spectra were recorded on Bruker 500 MHz and Bruker 126 MHz magnetic resonance spectrometers, respectively, mass spectrometry was recorded with Agilent 1100 LC-MSD/TOF mass spectrometer. The UV–vis spectra were recorded on UV-240 IPC spectrophotometer. The fluorescence spectra were recorded on F97XP FL spectrophotometer. The Olympus BX51 inverted fluorescence microscope and the Olympus IX71 inverted fluorescence microscope were used for biological imaging of cells and C. elegans, respectively. The fluorescence quantum yields were recorded with HORIBA Scientific Fluorolog.

Based on previous literatures [44, 45], we synthesized intermediate compounds 1-4. The structure of Probe 1 was confirmed by ¹H NMR, ¹³C NMR and HR-MS analysis. More details of synthetic processes and structure characterizations can be found in Supporting information.

To invest the responses of Probe 1 towards Fe²⁺, the spectral properties in PBS/DMSO = 2:8 (0.01 mol/L, pH 7.4) buffer solution were tested. We first investigate the UV–vis absorption of Probe 1 to the addition of different equivalents of Fe²⁺. As shown in Fig. 1a, the Probe 1 solution showed an intensive peak at 420 nm. Upon the addition of Fe²⁺ (45 μmol/L), the absorption of Probe 1 exhibited obvious decrease at 420 nm and strong increase at 510 nm. Then, the fluorescence spectra of Probe 1 with different equivalents of Fe²⁺ were investigated. As proposed, free Probe 1 (10 μmol/L) showed a fluorescence emission peak at 550 nm. With the addition of the Fe²⁺, the intensity of Probe 1 at 550 nm gradually decreased, while it enhanced at 690 nm, reaching the NIR region (Fig. 1b). It was observed that the color changed from yellow to pink. In PBS/DMSO = 2:8(0.01 mol/L, pH 7.4) buffer solution with the Probe 1 concentration of 2 × 10^-6 mol/L, after adding different concentration of Fe²⁺, the fluorescence intensity at 690 nm increased to a certain extent. The fluorescence intensity at 690 nm was excellent linearly associated with the concentration of Fe²⁺ (R² = 0.99287)(Fig. S1 in Supporting information). The calculated detection limit (LOD, 3σ /slope) of Fe²⁺ is 5.9 μmol/L, which also indicates a high sensitivity to Fe²⁺.

Highly selective response is necessary for efficiently detecting relevant analytes among complex biological samples. Thus, we investigated the UV–vis absorption and fluorescence selectivity of Probe 1 in PBS/DMSO = 2:8 (0.01 mol/L, pH 7.4) buffer solution. Upon treatment with various competitive species (Li⁺, Na⁺, K⁺, Ag⁺, Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Sn²⁺, Pb²⁺, Hg²⁺, Fe³⁺, Cr³⁺, F^-, Cl^-, Br^-, NO₂^-, NO₃^-, SO₄^2-, SO₃^2-, HCO₃^-, ClO^-, AcO^-, Cys, Hcy, GSH), there was a strong absorption at 420 nm, which was consistent with the characteristic absorption of the free Probe 1. In addition, there was almost no obvious change. After the interaction of Fe²⁺ (45 μmol/L) with Probe 1, there was obvious absorption at 510 nm (Fig. 1c, Fig. S2 in Supporting information). The color of the Probe 1 solution changed from yellow to pink. This result indicated that the Probe 1 is an effective Fe²⁺ sensor, which can be visually addressed. Similarly, we tested the fluorescence spectrum selectivity of various analytes for Probe 1. As shown in Fig. 1d, after Probe 1 interaction with Fe²⁺, the emission peak changed significantly, and the maximum emission peak appeared at 690 nm. In contrast, when other analytes, such as common RSS, ROS and anions, were added to the solutions of Probe 1, the fluorescence spectrum hardly changed, which showed the newly developed Probe 1 had an excellent selectivity in Fe²⁺ recognition (Fig. S3 in Supporting information). A histogram of the fluorescence intensity at the maximum emission of 690 nm is shown in Fig. 1e and Fig. S4 (Supporting information). More importantly, we also performed competition experiments. As shown in Fig. 1f and Fig. S5 (Supporting information), in the presence of other potentially interfering species, the fluorescence intensity of probe 1 at 690 nm response to Fe²⁺ in the presence of other potentially interfering species was approximately 5 times higher than that of the free Probe 1. The results showed that Probe 1 has a high selectivity for Fe²⁺ even in a complex biological environment, and the specific fluorescence response of Probe 1 towards Fe²⁺ was not affected by other biologically species analytes. In addition, the results confirming the Probe 1 had great potential for the specific detection of Fe²⁺.

It is worth noting that the fluorescence intensity increased at 690 nm in both different equivalent of Fe²⁺ (0, 15, 30, 45 equiv.) and time-dependent manner (Fig. 2a), and reached its maximum within 100 s. Furthermore, the fluorescence intensity of the free Probe 1 remains almost unchanged for a long time. These results indicated that the reaction between Probe 1 and Fe²⁺ is completed in a short time period, releasing the free compound 4 fluorophore. The presence of free compound 4 was verified by analyzing highresolution mass spectrometry (HR-MS), and this mass peak is shown at m/z 493.2729. The proposed reaction mechanism shown in Scheme 1 was supported by these above experimental results.

In order to investigate the applicability of Probe 1 in the physiological system in vivo, we evaluated the effect of Probe 1 on Fe²⁺ in the range of pH 3-11. As shown in Fig. 2b, the fluorescence intensity of the free probe hardly changed at pH 3-11. After treatment with Fe²⁺, a significant increase in fluorescence signal was observed over a wide pH range of 6–8, which indicates that Probe 1 has the potential to detect Fe²⁺ under physiological conditions. The fluorescence quantum yields of Probe 1 is 15.68% and after reaction with Fe²⁺ is 28.16%.

According to the reported literatures [46-48], Scheme 1 showed the potential response mechanism of the Probe 1 to Fe²⁺. Probe 1 was reduced to compound 4 with enhanced fluorescence from yellow to red. The products after the reaction between Probe 1 and Fe²⁺ were analyzed by HR-MS. As can be seen from Fig. S6 (Supporting information), a new peak was found at m/z 494.2802, which corresponds to the reduced compound 4(calculated value 493.2729). After the reaction between Probe 1 and Fe²⁺ (45 equiv.) in PBS/DMSO = 2:8 (0.01 mol/L, pH 7.4) buffer solution, the fluorescence intensity at 690 nm (Fig. S7 in Supporting information) was similar to 45 equiv. compound 4 in PBS/DMSO = 2:8 (0.01 mol/L, pH 7.4) buffer solution. Therefore, the process of the Fe²⁺ detection of Probe 1 was a typical ICT mechanism.

To demonstrate the potential of Probe 1 in vivo, Probe 1 was used for fluorescence imaging studies in HeLa cells under hypoxia and normal oxygen conditions. In Fig. 3, HeLa cells were treated with different concentrations of Fe²⁺ (0, 15, 30 and 45 μmol/L) for 2 h, followed by incubation with Probe 1 (20 μmol/L) at 37 ℃ for additional 2 h. No obvious change was observed only when HeLa cells were incubated with Probe 1 (Fig. 3c1). When Fe²⁺ was added to the cells, the fluorescence intensity increased with increasing amounts of Fe²⁺, and strong red fluorescence was observed throughout the cytoplasm. These results confirmed that Probe 1 penetrates the living cell membrane and effectively detects Fe²⁺.

Based on the fluorescence imaging performance of Probe 1 in cells, we also completed the in vivo imaging study of Probe 1 in C. elegans. As show in Fig. 4, the C. elegans were first treated with Fe²⁺(45 μmol/L) for 2 h under the N₂ atmosphere and later treated with Probe 1 for another 2 h at the room temperature (20 ℃), a strong red fluorescence in emitting from the entire C. elegans body was observed. In contrast, C. elegans treated with Probe 1 alone exhibited weaker fluorescence.

In summary, we have successfully designed and synthesized a near-infrared ratiometric fluorescent Probe 1 for Fe²⁺ detection based on novel naphthalimide and indole derivatives as fluorophore. Probe 1 showed excellent Fe²⁺ sensing performance including predominant selectivity and sensitivity, low detection limit, rapid response, high stability over a wide pH range and large red shift. Notably, Probe 1 was successfully employed to monitor Fe²⁺ in HeLa cells and C. elegans. Based on the above advantages of Probe 1, we believe that the Probe 1 is a promising research tool for Fe²⁺ detection in vitro and in vivo.

Declaration of competing interest

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

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We thank the National Natural Science Foundation of China (Nos. 21672185 and 21867019) and the "Youth Talent of WanRen Project" Foundation of Yunnan Province of China for the financial support. J.F. Zhang acknowledges the "LianDa Scholar Project" and "Graduate Research and Innovation" Foundation of Yunnan Normal University (No. Ysdyjs2019122).

Appendix A. Supplementary data

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Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.cclet.2020.06.006.

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Year 2020 volume 31 Issue 11

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doi: 10.1016/j.cclet.2020.06.006

Receive Date：2020-02-20
Online Date：2026-01-31
Published：2020-11-15

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Received：2020-02-20
Revised：2020-05-30
Accepted：2020-06-02

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^a College of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming 650500, China

^b College of Chemical Science and Technology, Yunnan University, Kunming 650091, China

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表12种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Scheme 1 The reaction mechanism of Probe 1 with Fe²⁺.

Fig. 1 The UV–vis absorption spectra (a) and fluorescence emission spectra (b) of probe 1 (2 ×10^-5 mol/L) in the presence of Fe²⁺ (0-45 equiv.) in PBS/DMSO = 2:8 (0.01 mol/L, pH 7.4); (c) The UV–vis absorption spectra for probe 1 (2 × 10^-5 mol/L) response to Fe²⁺ (45 equiv.) and other various metal ions (45 equiv.) in PBS/DMSO = 2:8 (0.01 mol/L, pH 7.4); (d) The fluorescence intensity of probe 1(2 ×10^-5 mol/L) response to Fe²⁺ (45 equiv.) and other various metal ions (45 equiv.) in PBS/DMSO = 2:8 (0.01 mol/L, pH 7.4); (e) The fluorescence intensity of probe 1 (2 × 10^-5 mol/L) with different metal ions (45 equiv.) in PBS/DMSO = 2:8 (0.01 mol/L, pH 7.4) at 690 nm. From 1 to 18: Blank, Fe²⁺, Na⁺, K⁺, Ag⁺, Mg²⁺, Mn²⁺, Li⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Sn²⁺, Pb²⁺, Hg²⁺, Fe³⁺, Cr³⁺; (f) The fluorescence intensity variety of probe 1(2 × 10^-5 mol/L) towards Fe²⁺ (45 equiv.) with other various metal ions (45 equiv.) addition in PBS/DMSO = 2:8 (0.01 mol/L, pH 7.4) at 690 nm. λ_ex = 500 nm, slit: 10 nm. From 1 to 17: Blank, Li⁺, Na⁺, K⁺, Ag⁺, Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Sn²⁺, Pb²⁺, Hg²⁺, Fe³⁺, Cr³⁺.

Fig. 2 (a) Fluorescence time response of probe 1 (2 × 10^-5 mol/L) in the presence of different concentration of Fe²⁺ (0, 15, 30, 45 equiv.) in PBS/DMSO = 2:8 (0.01 mol/L, pH 7.4). (λ_ex = 500 nm, slit: 10 nm); (b) Fluorescence emission intensities (690 nm) of probe 1 (2 × 10^-5 mol/L) at varied pH values (3-11) in the absence/presence of Fe²⁺ (45 equiv.).

Fig. 3 Fluorescence confocal imaging of probe 1 (2 × 10^-5 mol/L) in HeLa cells under normal oxygen and hypoxic conditions, incubated in the PBS buffer solutions at varying amount of Fe²⁺ for 2 h. (a1-d1): only with probe 1 (2 × 10^-5 mol/L); (a2- d2): with probe 1 (2 × 10^-5 mol/L) and Fe²⁺ (45 μmol/L); (a3-d3): with probe 1 (2 × 10^-5 mol/L) and Fe²⁺ (45 μmol/L). (a1-a3): bright-field image; (b1-b3): blue channel, stained with DAPI; (c1-c3): red channel; (d1-d3): merged images. Scale bar: 20 μm.

Fig. 4 Bright field images (a-d) and fluorescence images (e-h) in C. elegans. (a, e): probe 1 (2 × 10^-5 mol/L) only; (b, f): probe 1 (2 ×10^-5 mol/L) and Fe²⁺ (15 μmol/L); (c, g): probe 1 (2 × 10^-5 mol/L) and Fe²⁺ (30 μmol/L); (d, h): probe 1 (2 ×10^-5 mol/L) and Fe²⁺ (45 μmol/L). Fluorescence images were collected in red channels. Scale bar: 100 μm.

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