Development of a colorimetric and NIR fluorescent dual probe for carbon monoxide

Abstract

Carbon monoxide (CO) has captivated great attention in part due to the discovery of the therapeutic and cell-signalling role that CO plays in biological systems. The ever-increasing interest in CO has resulted in the development of rapid and facile approaches for the sensitive and selective detection of CO, which still remains challenging. Herein, the first example of a colorimetric and near-infrared fluorescent dual probe for carbon monoxide (CO) has been developed by assembling an allyl chloroformate moiety with a naphthofluorescein fluorophore, which enables the label-free and visual detection of CO. The living cell imaging results indicate that the probe shows great potential in sensing intracellular CO.

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Introduction

Carbon monoxide (CO), a colorless and odorless gas, has long been primarily considered as a toxicant or hazardous material due to its strong affinity to hemoglobin.¹ As an environmental toxicant, high concentrations of CO (above 70 ppm) can induce both acute and chronic health hazards. On the other hand, recent studies have shown that CO, like nitric oxide (NO) and hydrogen sulfide (H₂S), serves as an essential second gasotransmitter in the body.^2,3 Everyone has a low level of carboxyhemoglobin in the blood stream, approximately 1–4%. During the past few years, CO has captivated considerable attention because of its biological significance and potential therapeutic applications.⁴ For example, CO modulates vasorelaxation at low concentrations and lowers the blood pressure. Although, CO plays a significant role in various physiological processes, many aspects of its physiological/pathological functions remain still unclear. The major obstacle in this area is the lack of real-time approaches to selectively track CO in biological systems. Some established approaches for CO detection include gas chromatography, chromogenic detection, laser sensor-infrared absorption, electrochemical assays, etc.,^5–8 which are usually unsuitable for real-time tracking of intracellular CO in a noninvasive manner.

Recently, fluorescence-based imaging techniques have emerged as powerful tools to monitor and sense various biomolecules in biological systems with high spatial and temporal resolution.^9–12 A variety of fluorescent probes for endogenous gasotransmitter, such as NO and H₂S, have been reported over the past decade.^13,14 However the development of fluorescent probes for CO is still at a very early stage. A very limited number of fluorescent probes for CO have been developed to date,^15–23 including our newly reported probe.²⁴ However, almost all of these probes display emissions in the visible region, which greatly restricts their applications in biological imaging, due to the strong autofluorescence and limited tissue penetration. By contrast, near-infrared (NIR) fluorescent probes are extremely favourable for bioimaging because of deep tissue penetration, minimum autofluorescence and photo-damage.²⁵ To this end, development of NIR fluorescent probes for selective and sensitive detection of intracellular CO is highly desired and urgent.

On this basis, we prepared a first example of colorimetric and NIR fluorescent dual probe (NF-APC). This dual probe consists of a naphthofluorescein fluorophore condensed with allyl chloroformate moieties and possesses the desirable properties described above (Scheme 1). Our overall strategy for this probe relies on CO-induced in situ generation of Pd⁰, which consequently mediate the Tsuji–Trost reaction.²⁶ Naphthofluorescein was chosen as a signaling fluorophore due to its long excitation and emission wavelengths (>600 nm), relatively large Stokes shift and easy availability.^27–29 Based on the literature and our previous work, allyl chloroformate was linked to the hydroxyl groups of naphthofluorescein as the response site to trap Pd⁰ according to the Tsuji–Trost reaction. We anticipated that the derivatization of the hydroxy groups of naphthofluorescein in position 4 and 9 with allyl chloroformates would lead to a colorless and non-fluorescent closed lactone form, and the free naphthofluorescein moiety would be released in the presence of CO and Pd²⁺, leading to the color change and the generation of strong NIR fluorescence emission.

Scheme 1 Proposed sensing mechanism of NF-APC for CO based on in situ generation of Pd⁰.

Results and discussion

The desired probe NF-APC was easily prepared in one step (Scheme S1†). With NF-APC in hand, we first test its colorimetric and fluorescent response to Pd²⁺ and Pd⁰. As expected, NF-APC exhibited extremely weak absorbance and fluorescence in DMSO–PBS buffer (4 : 6, v/v, Fig. S6†). Strikingly, NF-APC displayed remarkable enhancement in the absorption and fluorescence spectra after incubation with Pd(PPh₃)₄, and also excellent selectivity towards Pd⁰ over Pd²⁺. We expected that the addition of CO into the Pd²⁺-containing buffer would generate Pd⁰, which would consequently mediate the Tsuji–Trost reaction, thus leading to specific spectral changes.

To investigate the response of NF-APC to CO, UV-vis and fluorescence spectroscopic studies were conducted to monitor the time-dependent changes of the absorption and fluorescence spectra upon addition of CO into the Pd²⁺-containing buffer. In this study, the water-soluble complex [Ru(CO)₃Cl(glycinate)] (CORM-3) was used as the CO source. As shown in Fig. 1, NF-APC exhibits extremely weak absorption and emission in the visible region. After the addition of 10 equiv. of CORM-3, a new absorption peak appeared at around 620 nm, which increased gradually with incubation time and reached its maximum value after about 45 min of incubation (Fig. 1A and S7A†). The significant absorbance enhancement (15-fold) was accompanied by a marked and vivid color change from achromatic to blue in ambient light (insert of Fig. 1A), demonstrating that NF-APC can serve as an on-site and visual indicator for CO. In the fluorescence spectra, a new strong emission peak appeared at approximately 670 nm and its intensity increased gradually with incubation time, reaching a plateau in 45 min (Fig. 1B and S7B†). The turn-on fluorescence at 670 nm (35-fold increase) may be attributed to the elimination of the allyl chloroformate in NF-APC, and to the concomitant formation of the free naphthofluorescein moiety, which was confirmed by almost the same absorbance and emissive peak with naphthofluorescein from commercial source (Fig. S8†), and the HRMS result (Fig. S9†). Besides, the turn-on red fluorescence could also be seen with the naked eye under UV light (insertion of Fig. 1B). The effect of pH on the probe response and stability were also studied and the results suggested that NF-APC was stable in the pH range of 5–8.5, and displayed remarkable response from 7 to 8.5 (Fig. S10†). These results indicated that NF-APC could be used as an efficient naked-eye tool for colorimetric and NIR fluorescent turn-on probe for CO.

Fig. 1 UV-vis (A) and fluorescence spectral changes (B) of NF-APC against time in the presence of CORM-3 (10 equiv.) in PBS buffer at 25 °C. Conditions: [NF-APC] = 10 μM; [Na₂PdCl₄] = 60 μM; DMSO–PBS buffer (4 : 6, v/v), pH 7.4, λ_ex = 620 nm. Inset: color changes and emission changes of NF-APC upon addition of CORM-3.

Further UV-vis and fluorescence titration experiments were carried out to explore the sensing ability of NF-APC to CO. As shown in Fig. 2 and S11,† the new absorption band at 620 nm and the emission at 670 nm increased gradually with the increasing of CORM-3 concentrations. The plot of absorbance and fluorescence intensity versus CORM-3 concentration showed linear correlation in the range of 0–40 μM (Fig. S12†), and the detection limit for CO was deduced to be as low as 0.255 μM and 0.127 μM (3σ/k), respectively, indicating that our probe was highly sensitive to CO. Additionally, NF-APC displayed excellent photostability, which is very important for its application (Fig. S13†).

Fig. 2 UV-vis (A) and fluorescent (B) spectroscopic titration of NF-APC by stepwise addition of CORM-3 in PBS buffer at 25 °C. Conditions: [NF-APC] = 10 μM; [Na₂PdCl₄] = 60 μM; DMSO–PBS buffer (4 : 6, v/v), pH 7.4, λ_ex = 620 nm. The spectra were recorded at 45 min intervals.

We next assessed the selectivity of NF-APC towards CO and various biologically relevant chemical species. As shown in Fig. 3, only the addition of CORM-3 caused such strong signal changes in the absorption and fluorescence spectra of NF-APC. In contrast, nearly negligible signal interference was observed in the presence of other reactive biological species, such as ClO⁻, H₂O₂, H₂S, O₂⁻, NO, HSO₃⁻, SO₃²⁻, Cys, Hcy and GSH (Fig. 3), indicating that NF-APC exhibited excellent specificity for CO over other biologically relevant species. Taking all these results together, NF-APC displayed great potential for the sensitive and selective detection of CO with both colorimetric and fluorescent outputs.

Fig. 3 (A) UV-vis and (B) fluorescent spectral response of NF-APC towards CO and other reactive biological species (100 μM). Conditions: [NF-APC] = 10 μM; [Na₂PdCl₄] = 0 μM or 60 μM; DMSO–PBS buffer (4 : 6, v/v), pH 7.4, λ_ex = 620 nm. The spectra were recorded after incubation for 45 min.

Encouraged by the remarkable properties of this probe for the detection of CO in solution, we further investigated the intracellular applications of NF-APC to image CO in living cells by using confocal laser scanning microscopy. In this regard, a standard MTT assay was performed to evaluate the cytotoxicity of NF-APC, CORM-3 and Na₂PdCl₄ to HeLa and 293T cells. The results indicated that NF-APC, CORM-3 and Na₂PdCl₄ exhibited very low cytotoxicity to living cells for 48 h (Fig. S14†). As shown in Fig. 4A–H, co-staining living HeLa cells with Hochest 33342 nuclear stain and NF-APC revealed that NF-APC alone or in the presence of Pd²⁺ exhibited nearly no fluorescence emission, consistently with the results obtained in solution. In sharp contrast, after incubation with CORM-3 for 45 min, a distinct strong fluorescence emission was detected (Fig. 4I–L). These observations demonstrated that NF-APC is both cell-permeable and capable of sensing CO in living cells.

Fig. 4 Confocal fluorescence images of living HeLa cells using a 543 nm laser. HeLa cells were co-stained with Hochest 33342 (10 μM) and NF-APC (10 μM), Na₂PdCl₄ (20 μM), without (A–H) and with (I–L) incubation of CORM-3 (50 μM).

Conclusions

To conclude, we have successfully developed NF-APC, a tailor-made colorimetric and NIR-emitting fluorescent dual probe for CO detection, based on naphthofluorescein fluorophore. NF-APC displayed specific color change and significant turn-on fluorescence response towards CO over a variety of reactive species, which make it a facile and reliable sensing tool for CO. Moreover, living cell imaging results revealed that NF-APC is suitable for tracking intracellular CO species. All these properties make this probe a promising useful tool for CO detection in biomedical research.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (21502056), the Natural Science Foundation of Guangdong Province, China (2016A030310463) and the Pandeng project for undergraduate in Guangdong Province (j2tw-k315001-16 to L. F. Z.).

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Footnote

† Electronic supplementary information (ESI) available: Synthesis and characterization of NF-APC, experimental procedures, and supplemental spectra and graphs. See DOI: 10.1039/c6ra14409h

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