Red-Emitting Fluorescence Sensors for Metal Cations: The Role of

Article Cite This: ACS Sens. 2019, 4, 1552−1559

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Red-Emitting Fluorescence Sensors for Metal Cations: The Role of Counteranions and Sensing of SCN− in Biological Materials Lukas Lochman,† Miloslav Machacek,† Miroslav Miletin,† Š tep̌ ań ka Uhlírǒ va,́ † Kamil Lang,‡ Kaplan Kirakci,‡ Petr Zimcik,† and Veronika Novakova*,† †

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Faculty of Pharmacy in Hradec Kralove, Charles University, Akademika Heyrovskeho 1203, 500 05, Hradec Kralove, Czech Republic ‡ Institute of Inorganic Chemistry of the Czech Academy of Sciences, 250 68 Husinec-Ř ež, Czech Republic S Supporting Information *

ABSTRACT: The spatiotemporal sensing of specific cationic and anionic species is crucial for understanding the processes occurring in living systems. Herein, we developed new fluorescence sensors derived from tetrapyrazinoporphyrazines (TPyzPzs) with a recognition moiety that consists of an azacrown and supporting substituents. Their sensitivity and selectivity were compared by fluorescence titration experiments with the properties of known TPyzPzs (with either one aza-crown moiety or two of these moieties in a tweezer arrangement). Method of standard addition was employed for analyte quantification in saliva. For K+ recognition, the new derivatives had comparable or larger association constants with larger fluorescence enhancement factors compared to that with one aza-crown. Their fluorescence quantum yields in the ON state were 18× higher than that of TPyzPzs with a tweezer arrangement. Importantly, the sensitivity toward cations was strongly dependent on counteranions and increased as follows: NO3− < Br− < CF3SO3− < ClO4− ≪ SCN−. This trend resembles the chaotropic ability expressed by the Hofmeister series. The high selectivity toward KSCN was explained by synergic association of both K+ and SCN− with TPyzPz sensors. The sensing of SCN− was further exploited in a proof of concept study to quantify SCN− levels in the saliva of a smoker and to demonstrate the sensing ability of TPyzPzs under in vitro conditions. KEYWORDS: phthalocyanine, aza-crown, fluorescence, counteranion, intramolecular-charge transfer

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etection of various cations,1 anions,2 or small biomolecules is an important issue in medicine, biotechnology, and environmental science.3 Many types of sensors have been described in the literature, including ion-selective electrodes/optodes,4,5 genetically encoded biosensors,6 nanoparticle-based sensors,7,8 or synthetic probes based on fluorophores.3 The latter modality provides many advantages, such as simple instrumentation, fast response, nondestructive monitoring, low-volume measurement, reversibility, and possibility of application in vivo.3 Fluorophores based on BODIPY, cyanine, rhodamine, or fluorescein have been employed in fluorescent sensors.3,9 These dyes may, however, suffer from weak stability, blue-shifted absorption and emission, or low brightness of a sensor (due to low extinction coefficients or low fluorescence quantum yields (ΦF) in ON state).9 Aza-analogues of phthalocyanines from the group of tetrapyrazinoporphyrazines (TPyzPzs) have been shown to possess optimal properties, because TPyzPzs are stable, absorb and emit light over 630 nm with molar absorption coefficients of more than 150 000 L mol−1 cm−1 in the most intensive absorption Q-band, and possess ΦF of approximately 0.30.10 In addition, the periphery of TPyzPzs can be easily decorated © 2019 American Chemical Society

with suitable substituents that alter the photophysical properties. These fluorophores have become a fundamental part of fluorescence sensors for pH11 or metal cation12−14 recognition, using intramolecular charge transfer (ICT) for switching on and off. A TPyzPz sensing device for CO2 monitoring has also been developed.15 Various recognition moieties that are specific for particular analytes are able to switch a sensor ON and OFF. Among the different moieties, aza-crowns have been widely used for metal cations. However, aza-crown recognition moieties still have many drawbacks. Despite numerous studies on the role of the size of the aza-crown,14,16 its type16,17 and rigidity,12,18 our understanding of the structure−activity relationships remained limited due to the use of different fluorophore types, different solvents, and different salts in experiments. Based on data from the literature,19−21 counteranions play an important role in Received: January 11, 2019 Accepted: May 16, 2019 Published: May 16, 2019 1552

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ACS Sensors Table 1. Data Determined from Titration of TPyzPz Sensors with KSCN in THF Cpd

1A

2A

3B

4B

5C

6C

7D

8D

ΦF ONa KA (M−1)b FEFc

0.094 1200 7.8

0.20 28 000 18.2

0.20 150 000 16.7

0.17 955 000 12.1

0.21 660 000 10.0

0.18 1.1 × 106 18.0

0.015 26 000 38.5

0.0020 33 000 2.0

a ΦF ON - fluorescence quantum yield in the ON state. bKA - apparent association constant. cFEF - fluorescence enhancement factor for complete binding.

Chart 1. Structures of TPyzPzs Involved in the Study Together with Types of Recognition Moieties (mono-azacrown (A), combination of aza-crown-lariat ether (B), aza-crown-additional crown (C), and tweezer of two aza-crowns (D))

ΦF(M+) is the ΦF value at complete saturation of the recognition moiety by an analyte. Sensing of SCN− in Saliva. A saliva sample (5−6 mL) from a smoking volunteer was collected in a conical centrifuge tubes (50 mL) and refrigerated at 4 °C for 30 min. Then, 3 × 1 mL of saliva was transferred into an Eppendorf tube (1.5 mL) and centrifuged at 3000 rpm for 10 min. 500 μL of the supernatant was transferred in a 10 mL volumetric flask and diluted to the mark with absolute EtOH (99.9%). The prepared solution was centrifuged at 3000 rpm for 10 min, 2.5 mL of the supernatant was used as follows: 25 μL of a 100 μM 4B stock solution in absolute EtOH (99.9%) was added (c ∼ 1 μM), and the fluorescence emission spectrum (λexc = 580 nm) was measured. Then, defined amounts (typically 5−50 μL) of a KSCN stock solution in 95% EtOH/water (v/v) (0.05 M, 0.1 M, 0.5 M) were added, and the emission spectra were recorded after each addition. The SCN− concentration was determined by the method of standard addition23 from the dependence of the fluorescence intensity on the concentration of the added SCN− by extrapolation of the linear dependence to zero (y = 0). The data in Figure S22 represent the mean of three independent experiments. The fluorescence of a blind sample (i.e., 4B in 95% EtOH/water (v/v)) was subtracted from each measured fluorescence intensity to correct the obtained dependence for the fluorescence of sensor in the OFF state. In Vitro Sensing. HeLa cells were seeded on Petri dishes suitable for confocal microscopy (WillCo Wells, The Netherlands) at approximately 7.5 × 104 cells per dish and allowed to grow for 24 h in a CO2 incubator at 37 °C in a humidified 5% CO2 atmosphere in cell culture medium (Dulbecco’s Modified Eagle’s Medium without phenol red (Lonza, Belgium) supplemented with 10% heatinactivated fetal bovine serum (Sigma, Germany), 1% penicillin/ streptomycin solution (Lonza), 10 mM HEPES buffer (Sigma), and 4 mM L-glutamine (Lonza)). The 12 h incubation with 5 μM TPyzPz 4B, 6C or always-ON control TPyzPz was conducted in the dark.

cation recognition, but this role has not been systematically studied. Therefore, we have focused on the following aims: (1) to design and prepare TPyzPz sensors with recognition moieties that overcome drawbacks of known recognition moieties, (2) to compare their sensing properties with published TPyzPz sensors under the same experimental conditions, (3) to study the role of a counteranion in metal cation recognition, and (4) to prepare a sensor that is suitable for use in aqueous medium and confirm its sensing ability in biological materials.

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EXPERIMENTAL SECTION

Syntheses are in Supporting Information (SI). Fluorescence Quantum Yields (ΦF). These were determined by the comparative method22 using zinc phthalocyanine in THF as a reference compound (ΦF = 0.32 in THF22). Determination of Association Constants. A dye stock solution in THF (15−20 μL) was added to a 2.45 mL of THF (HPLC grade, Br− > Cl− > F− (i.e., more polarizable and larger anions imparted higher stability to the complexes21). In general, the interaction strength between a cation and an anion differs substantially. To the best of our knowledge, the role of counteranions in aza-crown recognition in solutions is not well understood. Therefore, to gain insight into this phenomenon, appropriate sodium salts (i.e., CF3SO3−, ClO4−, SCN−, NO3−, and Br−) and TPyzPz sensors with a suitable sized aza-crown that is 1556

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ACS Sensors toward SCN− salts is a consequence of the synergic effects due to (i) cation size-fit into the specific aza-crown moiety; (ii) association of SCN− with central zinc cation of the TPyzPz macrocycle; and (iii) chaotropic ability of SCN− to reduce the local water density around to the recognition moiety. SCN− Sensing. The concentration of SCN− in human plasma varies between 10 and 140 μM, and the SCN− concentrations in saliva and milk are much higher (i.e., typically 0.5−1.6 mM31). The main biological functions of SCN− involve protection against microbes and the formation of an antioxidative environment. Its insufficiency may lead to deterioration of inflammatory diseases. However, increased levels of SCN− in saliva (e.g., due to smoking31) are toxic. Therefore, continuous and rapid monitoring of SCN− levels in human fluids and in vitro is important. From titration experiments of sensors with KSCN (Table 1, Figure S21), it is obvious that TPyzPzs bearing recognition moieties B and C are the most effective from the series for the sensing of SCN− (i.e., FEF are above 10, association constants up to 106 M−1, and ΦF ∼ 0.20 in the ON state). More specifically, sensors 4B and 6C appear to be the best candidates due to their good solubility in aqueous solutions. To establish a proper medium for SCN− sensing in biological samples, the functionality of 4B (1 μM) was investigated in EtOH/water mixtures containing 70% to 100% EtOH (Figure S22). 95% EtOH/water (v/v) with the highest FEF (52×) and association constant 870 M−1 (KA was substantially lower in comparison with THF due to presence of water) was selected as the best medium for sensing SCN− in biological samples. Limits of detection (LOD) for KSCN recognition in 95% EtOH determined on the bases of signal-to-noise ratio approach (S/N = 3) were 8 and 6 μM for 4B and 6C, respectively, which is a much lower concentration than the common SCN− level in human body. Noteworthy, sufficient FEF (6.2 a 1.8 pro 4B a 6C) was preserved in the presence of physiological concentration of biologically relevant cations (150 mM Na+ and 3 mM Ca2+) indicating the good selectivity of 4B and 6C toward KSCN. The method of standard addition,23 which involves the direct addition of KSCN to aliquots of the analyzed sample, was used to determine the concentration of SCN− in saliva (Figure S23). The initial linear part of the dependence was used for the analysis. Extrapolation of the linear regression yielded a value of 3.31 ± 0.70 mM, which is biologically relevant34 and indicates that 4B may be suitable for detection of SCN− levels in human fluids. A detailed study, which is beyond the scope of this project, is required to fully validate this application of TPyzPz sensors. In some cases, the SCN− anion has antioxidant and antimicrobial effect and is involved in innate defense of mucosal surfaces. Many researchers are currently investigating its transport and mode of action on the subcellular level to elucidate its biological properties and protective role in cystic fibrosis, inflammatory diseases, atherosclerosis, neurodegeneration, and certain cancers.35 Therefore, the development of a fluorescent “SCN− turn-on” sensor for live cell imaging with visible light fluorescence is in high demand because it can minimize sample damage and native autofluorescence events associated with ultraviolet excitation. In following experiments, we documented the response of 4B and 6C to the increased levels of SCN− inside cells. Our primary focus was evaluating the toxicity of the sensors using 4B as model compound. No changes in the HeLa cell viability were observed up to the limit

of solubility of 4B in the cell culture medium (5 μM, Figure S25), indicating high suitability for in vitro experiments. Fluorescence microscopy using costaining with different probes for subcellular compartments revealed that 4B localizes exclusively in lysosomes (Figure S24). Cellular uptake (Figure S28) disclosed slow entry of studied sensors into cells; however, concentration inside cells was sufficient for sensing studies (see below). The fluorescence intensity of both 4B and 6C inside the cells responded well to increased intracellular levels of SCN−. The red signals of the TPyzPz sensors approximately doubled upon treatment of the stained cells with 10 mM KSCN or NaSCN. On the other hand, no increase of fluorescence intensities was observed in control experiments in the presence of 10 mM KCl or KNO3 (Figure 3, Figure S26).

Figure 3. Photomicrographs of HeLa cells stained for nuclei (blue, Hoechst 33342) and incubated with 4B (5 μM) (red) before and after addition of appropriate salts (10 mM). Red fluorescence intensity profiles correspond to the emission along respective white bars before addition, yellow profiles to the emission at the same positions after addition of a salt.

Fluorescence intensity of TPyzPz bearing eight (4-(hydroxymethyl)-2,6-diisopropylphenoxy) groups serving as an alwaysON control, was not affected by the addition of different salts (Figure S27). This proof-of-concept study provides promising evidence that the deliberate design of TPyzPz sensors allows for sensitive sensing of SCN− in biological systems. The prospects of our approach will be investigated in follow-up studies.

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CONCLUSIONS In conclusion, we have designed and prepared TPyzPz sensors bearing structurally new recognition moieties based on an azacrown and a supporting substituent. Their improved sensing properties, essential for good brightness, were demonstrated by a detailed study of a series of TPyzPz sensors possessing 1557

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ACKNOWLEDGMENTS The work was financially supported by the Czech Science Foundation [No. 14-02165P] and by the project EFSA-CDN (No. CZ.02.1.01/0.0/0.0/16_019/0000841) cofunded by ERDF. Authors would like to thank Radim Kučera, Nela Váňová and Juraj Lenčo for MS measurements.

different types of recognition moieties under the same experimental conditions. The results indicated similar sensitivity for 3B and 5C compared to simple aza-crown (2A) with a 2-fold higher FEF. The values of ΦF of approximately 0.10 were achieved, which is almost 20 times higher than the values of ΦF of tweezer TPyzPz sensors. Importantly, hydrophilic 4B and 6C exhibited even 5−20× improved KA values compared to lipophilic 3B and 5C. The study of the role of the counteranion highlighted importance of their chaotropic ability expressed by the Hofmeister series. High selectivity of studied TPyzPzs toward KSCN was explained by a unique sensing mechanism combining size-fit recognition of cation, coordination of SCN− with central zinc cation, and chaotropic ability of SCN− allowing desolvation of cation. The high ΦF ON (∼0.2 in THF) and high molar absorption coefficients in the red region of UV−vis (∼150 000 L mol−1 cm−1) are good prerequisites for high brightness and, consequently, for good sensitivity of the sensors. Clear comparison of sensitivity with reported sensors is, however, difficult due to different conditions used for the analysis. In this respect, LOD may be considered as a suitable tool. From this point of view, TPyzPzs 4B and 6C showed good LODs (∼7 μM), much lower than the normal SCN− level in the human body. This value is comparable with the LODs of reported fluorescence sensors (0.01−600 μM)31,36 used for SCN− detection. The ability of 4B and 6C to detect SCN− in biological materials was further investigated in a proof of concept study based on two pilot approaches. The first experiment involved determination of the levels of SCN− in saliva of a smoker, and the second one tested the ability to switch on the fluorescence of 4B and 6C in HeLa cells. The biological relevance of the obtained value in the saliva (i.e., 3.31 ± 0.70 mM) along with the successful in vitro experiments confirmed the reasonability of a follow-up investigation of TPyzPz sensors in biological environments.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.9b00081. Syntheses, spectral properties, fluorescence titration experiments, stoichiometry assessment, characterization of prepared nanoparticles, sensing in vitro (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel. +420 495 067 394. ORCID

Kamil Lang: 0000-0002-4151-8805 Kaplan Kirakci: 0000-0002-1068-5133 Petr Zimcik: 0000-0002-3533-3601 Veronika Novakova: 0000-0002-2183-1220 Notes

The authors declare no competing financial interest. 1558

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