A new carboxamide probe as On-Off fluorescent and colorimetric sensor for Fe3+ and application in detecting intracellular Fe3+ ion in living cells

Journal Pre-proof A new carboxamide probe as On-Off fluorescent and colorimetric sensor for Fe3+ and application in detecting intracellular Fe3+ ion in living cells Soraia Meghdadi, Niloofar Khodaverdian, Azadeh Amirnasr, Pim J. French, Martin E. van Royen, Erik A.C. Wiemer, Mehdi Amirnasr

PII:

S1010-6030(19)31412-1

DOI:

https://doi.org/10.1016/j.jphotochem.2019.112193

Reference:

JPC 112193

To appear in:

Journal of Photochemistry & Photobiology, A: Chemistry

Received Date:

17 August 2019

Revised Date:

18 October 2019

Accepted Date:

22 October 2019

Please cite this article as: Meghdadi S, Khodaverdian N, Amirnasr A, French PJ, van Royen ME, Wiemer EAC, Amirnasr M, A new carboxamide probe as On-Off fluorescent and colorimetric sensor for Fe3+ and application in detecting intracellular Fe3+ ion in living cells, Journal of Photochemistry and amp; Photobiology, A: Chemistry (2019), doi: https://doi.org/10.1016/j.jphotochem.2019.112193

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A new carboxamide probe as On-Off fluorescent and colorimetric sensor for Fe3+ and application in detecting intracellular Fe3+ ion in living cells Soraia Meghdadi a,⁎, Niloofar Khodaverdian a, Azadeh Amirnasr b,⁎, Pim J. French c, Martin E. van Royen d, Erik A.C. Wiemer b, Mehdi Amirnasr a

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Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran

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Erasmus Medical Center, Erasmus MC Cancer Institute, Dept. of Medical Oncology, Rotterdam, the Netherlands.

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Department of Neurology, Cancer Treatment Screening Facility (CTSF), University Medical Center Rotterdam, Erasmus MC, Rotterdam, The Netherlands. d

Corresponding authors: M. Amirnasr, S. Meghdadi [email protected]

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*E-mail addresses:

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Department of Pathology, Cancer Treatment Screening Facility (CTSF), Erasmus Optical Imaging Centre (OIC), University Medical Center Rotterdam, Erasmus MC, Rotterdam, The Netherlands.

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Graphical abstract

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[email protected]

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Highlights 

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Eco-friendly one-pot synthesis of a new indole quinoline-based chemosensor, H2IQ. Selective detection of Fe3+ by fluorometric (ON-OFF) and calorimetric methods. Remarkably low LOD (0.43 μM) and fast detection time (30 seconds). No interference from other common interfering M+, M2+ and M3+ metal ions. Membrane permeable; can readily detect intracellular Fe3+ via bioimaging.

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Abstract

A novel quinoline-functionalized carboxamide derivative, 1H-indole-2-carboxylic acid

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quinoline-8-ylamide (H2IQ), has been designed and synthesized via a benign method for detection of Fe3+. The On-Off H2IQ chemosensor is highly selective and sensitive toward Fe3+

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in the presence of other competing cations. This sensor displays rapid Fe3+ mediated decrease of florescence intensity at 445 nm, and also intense color change from colorless to bright yellow in DMSO-acetonitrile (1:9 v/v) solution. The 1:1 binding mode of the H2IQ with Fe3+ is confirmed by means of Job's plot and ESI-MS. The association constant (Ka) and limit of detection (LOD) for the resulting Fe3+ complex is 3.7 × 105 M-1 and 4.3 × 10-7 M respectively. Other interfering ions such as Na+, K+, Ca2+, Mg2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Mn2+, 2

Cr3+ and Al3+, show either no or slight change in the fluorescence intensity of H2IQ in the presence of Fe3+. The cytotoxicity of H2IQ was examined using the MTT assay. H2IQ is cell penetrant and does not overtly affect cell viability in various human cell lines. Importantly, using Opera PhenixTM HCS live cell imaging system, we have shown that H2IQ can be used to detect the intracellular presence of Fe3+ ions in live cells. Keywords: Indole quinoline carboxamide, Fe3+ On-Off fluorescent probe; Colorimetric sensor; Intracellular imaging

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1. Introduction

Among heavy and transition metals, iron accounts for over 5 percent of Earth’s crust making it the fourth most abundant element [1] and plays essential roles in many biochemical processes

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[2-5]. Iron is an indispensable element for human; it is involved in metabolic processes [6],

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DNA synthesis and repair [7], energy metabolism [8], and electron and oxygen transport [9,10]. The average adult contains ~3−5 g of iron while its average cellular concentration is ~50−100

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μM [11,12]. Both its deficiencies and overloads have noticeable effects on human health and are associated with diseases and ailments, such as anemia [13-17], liver damages [18],

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hemochromatosis [19], neurodegenerative disorders [20], weakness [21], infectious diseases [22,23], lethargy, loss of hair [24] and Alzheimer’s disease [25,26]. Moreover, autosomal

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dominant genetic disorders such as hereditary hemochromatosis are among the potential causes of excess in iron absorption [27,28]. Iron accumulation over time can result in various

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malignancies, of which hepatic and cardiac complications are two typical examples of severe iron overload.

It has long been investigated that lipid peroxidation and direct protein and/or DNA damages

are among the potential early stage carcinogenic causes of failure in iron homeostasis [29]. Less predominantly, excess iron has also been reported to be involved at a later stage of carcinogenesis acting as pivotal nutrition for tumor growth [30]. 3

The human body lacks a mechanism for the regulation of iron levels; therefore, iron homeostasis can only be mediated by regulation in its absorption [31]. Because of the importance of Fe3+ for cellular homeostasis, it is highly desirable to develop simple and facile method for sensitive and selective detection of Fe3+. Various techniques including atomic absorption spectroscopy (AAS) [32,33], Inductively Coupled Plasma Mass Spectrometry (ICPMS) [34], catalytic spectrophotometry [35], voltammetry [36] quantum-dot (QD)-based fluorescence [37,38] have been employed. Unfortunately, these techniques are expensive and

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need extensive sample preparation methods. In recent years however, the development of fluorescence and calorimetric sensors for detection of Fe3+ in both biological systems and the environment have received considerable attention [39-42]. These sensors have significant

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advantages including their less invasive character, simplicity, high sensitivity and selectivity, instantaneous response, and low cost.

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Among the chemosensors that are used in fluorometric detection of metal ions, fluorescent

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carboxamide derivatives have experienced increasing attention [43-46]. Indole as a natural product has a flowery smell, and is a constituent of many flower scents, such as orange blossoms, Jasmine, and wallflower. Interestingly, its lipophilicity and bicompatibiliy has made

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it suitable for being used in the fabrication of organic conjugated fluorescent materials with possible application in the live cell imaging and as chemosensors [47]. Indole–based

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chemosensors have been extensively used for fluorometric detection of anions such as F– and

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CN– [48-50]. Recently, the multi-step synthesis of an indole derivative and its application for colorimetric detection of Fe3+ ion in acetonitrile has been reported [51]. In the present work, we report the facile synthesis of a small fluorogenic molecule H2IQ,

1H-indole-2-carboxylic acid quinoline-8-ylamide, as a colorimetric and “On-Off” fluorescent chemosensor for the detection of Fe3+ in DMSO-acetonitrile (1:9 v/v). This sensor, bearing both the chelating sites for Fe3+ (ionophore) and signaling unit (fluorophore) together, is

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prepared readily by condensation of 1H-indole-2-carboxylic acid and 8-aminoquinoline in a one pot synthesis. Moreover, the toxicity of this probe against the available cell lines was evaluated by utilizing MTT assay. Upon exposing the cells to the sensor, we detected a profound feature of this probe as it exhibited good cell penetration. The results of exploiting H2IQ for detection of the intracellular iron in live cells by an Opera Phenix TM confocal HCS system is also reported and discussed. 2. Experimental section

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2.1. Materials and methods

All solvents and materials for the synthesis were purchased from Aldrich and Merck and used as received. Elemental analyses for carbon, nitrogen and hydrogen were carried out using

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a Perkin-Elmer 2400 CHN-O elemental analyzer. Infrared spectra (KBr pellets) were recorded

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on a FT-IR JASCO 680 plus spectrophotometer. UV-vis spectra were obtained on a JASCO V-570 spectrophotometer. The emission spectra were recorded on a RF-530 1PC fluorescence

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spectrophotometer. The 1H NMR spectra were obtained on a Bruker AVANCE 400 spectrometer. Proton chemical shifts are reported in ppm relative to Me4Si as an internal

spectrometer.

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standard. The mass spectrum was determined by ESI recorded on a Shimadzu 2010-A mass

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2.2. Synthesis of carboxamide ligand, H2IQ The H2IQ was synthesized via the condensation of indolic acid and 8-aminoquinoline by a

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benign method using tetrabutylammonium bromide (TBAB) as the reaction medium (Scheme1) [52]. A mixture of 1.55 g (5 mmol) triphenylphosphite (TPP), 1.61 g (5 mmol) tetrabutylammonium bromide (TBAB), 0.81 g (5 mmol) indole-2-carboxylic acid and 0.72 g (5 mmol) 8-aminoquinoline in a 25 mL round bottom flask was placed in an oil bath. The reaction mixture was heated and stirred for one hour at 120 °C. After cooling the reaction mixture to room temperature, the resulting solid mixture was treated with 10 mL cold methanol. 5

subsequently, the final white solid was filtered off and washed with cold methanol. Yield: 0.62 g (43%); m.p: 253-255 °C; (EtOAc/Hexan, 30/70, Rf = 0.5, Fig S1). Anal. Calcd. for C18H13N3O: C, 75.25; H, 4.56; N, 14.63. Found: C, 75.69; H, 4.48; N, 14.39. FT-IR: Fig. S2, ῡmax/ cm-1 (KBr): 3346 (N-Hindole), 3285 (N-Hamidic), 1663 (C=O), 1540 (C-N). UV-Vis λmax/ nm (ε, L mol−1 cm−1) (DMSO): 316 (21530), 334 (22340). 1H NMR (Fig. S3, 400 MHz, 298 K, DMSO-d6): δ (ppm): 7.18 (t, 1H11), 7.33 (t, 1H10), 7.41 (s, 1H13), 7.41 (d, 1H6), 7.70-7.83 (m, 4H2,4,5,12), 8.54 (dd, J = 8, 1.6, 1H3), 8.78 (d, J = 7.6, 1H9) 9.08 (dd, J = 4, 1.6, 1H1), 10.69

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(s, 1H, NHindole), 12.10 (s, 1H, NHamide). 13C NMR (Fig. S4, 100 MHz, 293.5 K, DMSO-d6): δ, 159.15, 149.22, 138.03, 137.19, 136.78, 133.89, 131.20, 127.87, 127.08, 124.16, 122.41,

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122.16, 121.93, 120.20, 116.51, 112.50, 103.31.

H N

C

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+

TBAB

OPh

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P OPh

NH2 N H N

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NH OPh OPh

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-PhOH

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O C

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OH

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H N

OPh

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N +

OH

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Scheme 1. One pot synthesis of the chemosensor H2IQ.

2.3. General procedure for metal ion binding studies Stock solutions of metal nitrates of K+, Zn2+, Mg2+, Ca2+, Al3+, Co2+, Cd2+, Ni 2+, Cu 2+ and HgCl2, FeCl3, MnCl2, CrCl3 and NaClO4 at a concentration of 10-2 M in acetonitrile were prepared for fluorescence spectral analysis. A 10-3 M stock solution of H2IQ was prepared in DMSO. The spectral measurements were carried out using 5 × 10-5 M DMSO-acetonitrile (1:9

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v/v) solution of H2IQ at room temperature. UV-visible spectra were recorded by adding 32 µL of 10-2 M different metal ions to 2 mL of H2IQ solution (5.0×10-5 M) in a quartz cell. All fluorescence data were recorded in a quartz cell of 1cm optical path length at λex = 376 nm,

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with the excitation and emission slit widths set at 5.0 nm. The fluorescence titration experiments were conducted by adding 0-32 µL of 10-2 M Fe3+ ion (0-16 equiv.) to 2 mL of

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H2IQ solution (5.0×10-5 M). The emission intensity changes of H2IQ in the presence of different metal ions was also measured by adding 32 µL of each metal ion solutions (1.0×10-2

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2.4. Human Cell Culture

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M, 16 equiv.) to 2 mL of the chemosensor solution (5.0×10-5 M).

sNF96.2 [53] a cell line derived from a neurofibromatosis type 1 associated malignant

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peripheral nerve sheath tumor (MPNST) was obtained from the ATCC. HepG2, Hep3b (both hepatocellular carcinoma) and HEK293 (human embryonic kidney cells) were kind gifts from

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the Department of Pathology and Department of Genetics (Erasmus MC Rotterdam, The Netherlands), respectively. All cell lines were cultured in DMEM (Gibco Life Technologies) supplemented with 10% fetal bovine serum and 100 IU/mL penicillin and 100 μg/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37C. The cell lines were regularly checked for mycoplasma infection. 2.5. In vitro cytotoxicity assay 7

The in vitro cytotoxicity of H2IQ was determined using a standard MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay essentially as described before [54]. In brief: Cells were seeded in 96 well plates and after 24 h exposed to various concentration of the H2IQ (5 µg/mL, 10 µg/mL, 20 µg/mL and 40 µg/mL prepared in DMSODMEM; DMSO content: 0.05-0.2%). The exposure to H2IQ was terminated after 24 or 48 h by the addition of 20 µL of MTT solution (5 mg/mL in PBS) to each well after which the cells were left to incubate for 4 h. Subsequently the medium was discarded and 200 µL DMSO was

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used to dissolve the formed purple formazan. The color intensity, a measure for cellular viability, was quantified using a spectrophotometer by measuring the absorbance at 540 nm. 2.6. Assessment of the intracellular uptake of H2IQ

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Hep3b cells were plated in 6-well plates and were exposed to 20 and 40 µg/mL H2IQ for 30 min. Cells were fixed with 4% PFA for 15 to 30 min and their membrane fluorescently

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labelled using PKH26 (Sigma-Aldrich Chemie N.V., Zwijndrecht, Netherlands). Cells were

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imaged in 3D using a Zeiss CLSM510 confocal microscope equipped with a 63x oil immersion objective (NA 1.4). H2IQ and cell membranes are excited at 405 and 543 nm and visualized

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using a 420-480 nm and 560-615 nm bandpass filters, respectively. 3D projections were generated in the Zeiss AIM image browser and Image J.

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2.7. H2IQ fluorescent measurements in living human cells HepG2, Hep3b, sNF96.2 and HEK293 cells were plated in 96-well CellCarrier TM-96 Ultra

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microplates (PerkinElmer, Groningen, The Netherlands) and were exposed to 20 and 40 µg/mL H2IQ. After 2 h of H2IQ exposure, live cells were incubated with DMSO-DMEM solutions of iron (FeCl3) or DMSO as a control (DMSO content: 0.05-0.2%) and imaged at 2 h intervals using an Opera Phenix

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confocal HCS system (PerkinElmer, Groningen, The Netherlands)

equipped with a 10x air objective (NA 0.3) over a period of 24 h. H2IQ was visualized with 405 nm excitation and using a 435-480 nm emission filter (in combination with a transmission 8

channel to visualize cells). Harmony High Content Imaging and Analysis Software (PerkinElmer, Groningen, The Netherlands) was used to further analyze the fluorescent intensity in cells as measure of H2IQ uptake. 3. Result and discussion 3.1. UV-visible studies The absorption spectrum of H2IQ sensor in DMSO-acetonitrile shows two peaks at 316 nm

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and 334 nm at room temperature (Fig. S5). The interaction of H2IQ with different cations and its selectivity toward Fe3+ ion was initially checked by following its UV-visible spectral changes in the presence of different metal ions DMSO-acetonitrile (1:9 v/v) solution. As

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evident from Fig. 1, H2IQ shows distinct spectral changes with Fe3+ relative to other metal ions accompanied by a bathochromic shift and increase in the absorption intensity at 318 and 352

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nm. Moreover, there is no overlapping band from Fe 3+ spectrum in the absence of H2IQ (Fig. S6). These changes could be also distinguished by the appearance of a yellow color, attributable

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to tailing of the absorption bands to the visible region. The color change could be identified by naked eye which provides a facile method for visual detection of Fe3+ ion (vide infra). Addition

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of other metal ions to a mixture of H2IQ and Fe3+ have either no or negligible effect on the absorption spectrum of H2IQ-Fe3+ complex, indicating the selectivity of the chemosensor for

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infra).

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Fe3+ ions. This selectivity is further supported by complementary emission experiments (vide

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Fig. 1. UV-vis spectral changes of H2IQ (10-5 M) in DMSO-acetonitrile (1:9 v/v) solution upon

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addition of 16 equiv. of various metal ions. 3.2. Fluorescence titration studies

In order to evaluate the utilization of the ligand as a selective Fe3+ fluorescent sensor, a

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fluorescence study was performed (λex = 376nm) and fluorescence titration of H2IQ with Fe3+

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ion was carried out by adding the FeCl3 (10-2 M) in 5 μL increments to H2IQ (5 × 10-5 M) in (DMSO-ACN 1:9 v/v) solution (Fig. 2). As the concentration of Fe3+ ion is gradually increased,

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the H2IQ fluorescence intensity at 445 nm is linearly decreased, reaching its minimum at 16 equiv (80 μL). Addition of extra Fe3+ solution does not have any noticeable effect on the

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fluorescence intensity. The association constant for H2IQ with Fe3+ (Ka = 3.7 × 105 M-1) was determined using Benesi-Hildebrand equation (Fig. S7). The limit of detection for the this

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chemosensor (LOD = 4.3 × 10-7 M) was calculated based on fluorescent titrations (Fig. 2), using 3σ/S (Fig. S8).

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The quenching process of the fluorophore-iron system was analyzed by using the emission

titration data and Stern-Volmer equation (1) (Fig. S9). The value of KSV of H2IQ-Fe3+ for Fe3+, was determined to be 9.6 × 103 M-1, indicating strong quenching of the sensor in the presence of Fe3+. I0/I= 1 + KSV [Q]

(1)

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To determine the coordination stoichiometry between H2IQ and Fe3+ ion, Job method of continuous variation was applied with changing the molar ratio of Fe3+, XFe3+, (XFe3+ = [Fe3+] /([H2IQ] + [Fe3+]) from 0 to 0.9. Based on the Job’s plot of the H2IQ-Fe3+ (Fig. S10), the

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stoichiometry ratio between the chemosensor and analyte in DMSO-ACN )1:9 v/v) is 1:1.

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Fig. 2. Emission spectral changes of H2IQ (5 × 10-5 M) (in DMSO-acetonitrile (1:9 v/v) solution by gradual increase in the concentration of Fe3+ ion (10-2 M) in 5 μL increments (0-16

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equiv.).

The formation of 1:1 complex between H2IQ and Fe3+ was further confirmed by ESI-MS,

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(Fig. 3). Mass spectrum of H2IQ in the presence of Fe3+ ion was obtained in DMSO-ACN (1:9 v/v) at room temperature (mobile phase was acetonitrile). The isotopic peak at m/z = 454.70

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corresponds to the [(H2IQ)(CH3CN)FeCl2]+ ion and agrees well with the calculated m/z =

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455.12. The results indicated a 1:1 stoichiometry of the chemosensor and Fe3+ in Fe-complex. Apparently, the iron ion satisfies its need for electroneutrality through expanding its coordination geometry by the addition of an acetonitrile molecule (Scheme 2).

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Fig. 3. ESI-MS spectrum for H2IQ-Fe3+ in DMSO-ACN (1:9 v/v) with the peak at m/z = 454.70

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corresponding to [Fe(H2IQ)(CH3CN)Cl2]+.

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Scheme 2. Fluorescence quenching mechanism and proposed structure of H2IQ-Fe3+ complex.

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3.3. Interference experiments

To check the possible interference of other metal ions with H2IQ-Fe3+ interaction, the effect

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of 14 interfering metal ions (Al3+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Hg2+, K+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Zn2+) on the fluorescence properties of this chemosensor was investigated under similar conditions. While the fluorescence of H2IQ is quenched by Fe3+, there is no noticeable change in the emission intensity of H2IQ by these ions (Fig. 4). Moreover, competition experiments were carried out by following the change in the emission intensity of H2IQ-Fe3+ (Fig. 7). As evident, there is no noticeable change in the emission intensity of H2IQ-Fe3+ 12

complex after addition of other metal ions. The bar diagram presented in Fig. 6. shows the competitive selectivity of H2IQ (5 × 10-5 M) toward Fe3+ (16 equiv.) in presence of other metal

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ions.

Fig. 4. Fluorescence spectral change of H2IQ (5 × 10-5 M) in DMSO-acetonitrile (1:9 v/v)

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solution upon addition of 16 equiv. of various metal ions (λex = 376 nm).

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Fig. 5. Fluorescence spectral changes of H2IQ (5 × 10-5 M) upon addition of Fe3+ (16 equiv.)

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and subsequent addition of other metal ions (16 equiv.). λex = 376 nm.

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Fig. 6. Emission spectral changes of competitive selectivity of H2IQ (5 × 10-5 M) toward Fe3+ (16 equiv.) in the presence of other metal ions (16 equiv.) with λex = 376 nm. 3.4. The time dependence of the response

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To test the ability of H2IQ for rapid Fe3+ detection, the time courses of the fluorescence intensity of the probe, H2IQ, in the presence of Fe3+ was investigated. As evident from Fig. 7,

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recognition interaction was almost complete immediately. The fluorescence intensity reaches

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the minimum value after 30 seconds, and then remains constant. Therefore, this fluorescent

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chemosensor could be applied in real-time tracking of Fe3+ in organisms.

Fig. 7. Time dependence of fluorescence intensity of H2IQ (5 × 10-5 M) sensor in the presence of Fe3+(10-2 M).

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3.5. Fe3+ ion sensing using colorimetric analysis While testing H2IQ for Fe3+ selectivity as a fluorescent sensor, we observed that it acts as a colorimetric sensor for Fe3+ too. The color of H2IQ changed from colorless to yellow when 16 equiv. of Fe3+ was added to its solution (Fig. 8). The yellow color of H2IQ-Fe3+ is stable in the presence of other metal ions (Fig. 9) and can be used for colorimetric detection of Fe3+ by

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“naked eye”.

Fig. 8. Color change of H2IQ solution after addition of different metal ions (16 equiv.) under

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visible light.

(16 equiv.) under visible light.

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Fig. 9. Stable yellow color of H2IQ-Fe3+ solution after addition of other interfering metal ions

3.6 Human cell lines display differential sensitivity to H2IQ.

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An in vitro cytotoxicity assay was conducted in order to evaluate the sensitivity of the human cell lines HEK293, sNF96.2 and HepG2 for H2IQ. To this end, exponentially growing

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cells were exposed to 5 µg/mL, 10 µg/mL, 20 µg/mL and 40 µg/mL to H2IQ for 24 and 48 h

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after which an MTT assay was performed to assess the viability of the cells. DMSO, the solvent of our fluorescent chemosensor (DMSO-DMEM solution with 0.2% DMSO), was included as a control. As shown in (Fig. 10), the compound was tolerated very well by HepG2 with cellular viability still well over 80% after 48 h exposure at 40 µg/mL. HEK293 cell were slightly more sensitive to H2IQ displaying 70% viability in the most extreme conditions (48 h exposure to 40 µg/mL). sNF96.2 cells proved to be most sensitive but still showed a viability of approximately

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60% at the most extreme conditions. Under the conditions of the experiment we could not

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determine IC50 values.

Fig. 10. In vitro cytotoxicity of H2IQ on HEK293, sNF96.2 and HepG2 cells. An MTT assay was carried out after 24 or 48 h of continuous exposure to 5, 10, 20 or 40 µg/mL of H2IQ. The viability of drug treated cells is depicted relative to the viability of untreated (normal) cells. Measurements were performed in triplicate, shown are averages ± standard deviation.

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The relative insensitivity to H2IQ was confirmed in proliferation experiments where HepG2 cells, exposed to 20 and 40 µg/mL, was monitored over the period of 24 h using a live cell imaging system (Opera PhenixTM HCS system, PerkinElmer). As shown in supplementary movie 1, proliferative behavior of HepG2 cells was still detectable. 3.7 Cell-permeability characteristics of H2IQ upon its cellular uptake Cell-permeability characteristics of the compound were investigated in 30 min and 24 h exposure period, using confocal microscopy and live-cell imaging. As shown in supplementary

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movie 2, H2IQ crystals (presented in green for visualization purposes) of the compound are visible in a 3D projection of fixed and PKH26 (red) fluorescently labeled cells upon 30 min induction. A live-cell imaging was conducted using the Opera Phenix TM HCS system

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(PerkinElmer) in order to monitor compound adsorption by the cells, over the period of 24 h

detectable through the entire period.

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(supplementary movie.1). The cells did not secrete the compound as the H2IQ fluorescence was

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3.8 Fluorescence intensity of H2IQ in living cells upon iron addition To assess the capability of H2IQ to indicate the presence of free Fe3+ inside cells, HepG2,

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Hep3b, sNF96.2 and HEK293 cells were exposed to 5 µg/mL of H2IQ in the absence or presence of 50 µg/mL Fe3+ (dissolved in DMSO) which was added two hours after the addition

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of H2IQ. We found that the addition of Fe3+ significantly and consistently reduced the H2IQ

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fluorescence signal observed in all four cell lines examined (Fig. 11). This difference was more pronounced in the presence of 40 g H2IQ. Over time the fluorescent signal of H2IQ gradually decreased both in the presence and absence of added Fe3+, possibly due to intracellular breakdown or modification to non-fluorescent derivatives, secretion or monitor bleaching during imaging. These observations clearly demonstrate that the fluorescent signal coming

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from H2IQ in living cells depends on the presence and concentration of Fe3+ ions indicating

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that indeed H2IQ may be used as sensor for detection of intracellular iron.

Fig. 11. Change in the fluorescence intensity of H2IQ upon iron-overload in living cells. The intensity values were measured per well using the live cell imaging system. Average of the duplicates (per condition) was calculated. A paired t-test was used for defining the significance of changes in the fluorescence intensity between different experimental conditions and error bars indicating standard deviation. 2 h time interval in the period of 24 h is indicated starting from point 1.

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4. Conclusion In summary, we have synthesized a highly selective and sensitive colorimetric and turn-off fluorescent carboxamide sensor H2IQ based on indole and quinolione, in a one-step reaction, using TBAB as the reaction medium. This chemosensor showed rapid detection of Fe3+ in DMSO-acetonitrile (1:9 v/v) with color change from colorless to yellow, and displayed fluorescence quenching response only towards Fe3+ with a remarkably low limit of detection (LOD = 0.43 μM) and fast detection time within 30 seconds. More importantly, the emission

without any interference from other common metal ions

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Declarations of interest

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intensity of this probe could be applied in real time tracking of Fe3+ ion in living organisms.

None.

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Acknowledgements

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We would like to acknowledge “Cancer Therapy Screening Facility” (CTSF) of Erasmus Medical Center and “Daniel den Hoed Foundation” for providing us with the cell imaging

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facilities. Moreover, helpful scientific support and discussions provided by Dr. Ya Gao and Behnam Tavakoli is gratefully acknowledged.

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Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at

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doi:https://doi.org/10.1016/j.jphotochem. References

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