Bimodal detection of carbon dioxide using fluorescent molecular aggregates

Abstract

Cyano-substituted p-phenylenevinylene (R-1) aggregates exhibiting fluorescence and Raman spectroscopic responses towards CO₂ are described. The aggregation-induced emission (AIE) as well as the aggregation-enhanced Raman scattering (AERS) of R-1 in aqueous conditions was reduced in the presence of a small amount of CO₂, which enabled its easy and fast bimodal detection in different analytical samples.

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The capture, storage, release and conversion of CO₂ are issues of high priority research.^1,2 In this context, the easy and fast detection of CO₂ is an important objective. Hence, researchers are looking for simple and effective sensors/receptors for CO₂.^3–5 For this purpose, optical detection based on self-assembled fluorescent molecules is considered to be viable owing to its high sensitivity, fast response time, low cost, visual signal transduction, real-time in situ responses, and compatibility for in vivo analysis.³ A possible approach to realize such sensors depends on the analyte-triggered self-assembly or disassembly of a given molecular probe, producing a turn-on/off fluorescence response.⁶ Moreover, the combination of more than one detection modality is preferred in order to increase the selectivity and sensitivity of the probe.^3,6 For instance, fluorescent probes that are Raman-active can be used as bimodal sensors.⁷ This is particularly relevant considering the fact that many of the reported CO₂ sensors rely on single detection modality and often utilize more than one receptor components.⁵

Herein, we report the fluorescence as well as Raman-assisted sensing of CO₂ using a cyano-substituted⁸p-phenylenevinylene⁹ molecule bearing terminal tertiary amine moieties (Scheme 1). The receptor R-1 was synthesized as per Scheme S1 (ESI†). All intermediates and final compounds obtained were characterized using FT-IR, ¹H, and ¹³C NMR techniques and mass spectrometry (Fig. S1–S8, ESI†).

Scheme 1 Receptor R-1 and its interaction with CO₂.

The receptor R-1 was highly soluble in THF and DMSO and formed aggregates (R-1_Agg) in an aqueous medium, as indicated by the observed Tyndall effect (Fig. 1a, inset). The UV/Vis spectrum of R-1_Agg (1 × 10⁻⁴ M) in the aqueous medium shows a narrow blue-shifted band (λ_max = 348 nm) in comparison to the broad spectrum in DMSO (λ_max = 375 nm), where it mainly exists as molecularly dissolved species (Fig. 1a). The aggregation of R-1 was also marked by the origin of an intense yellow emission (λ_em = 550 nm) owing to the AIE properties of the chromophoric unit (Fig. 1b, inset).¹⁰ On the contrary, the R-1 monomer in DMSO displayed a blue-shifted weak emission at 465 nm, τ = 5.84 ps (Fig. S9a and Table S1, ESI†). The red-shift and structureless features of emission in aqueous conditions indicated the formation of excimer-like species on aggregation, as evident from the longer average lifetime value (τ = 5.67 ns).¹¹ Systematic aggregation studies were carried out in a DMSO-water mixture using UV/Vis and fluorescence spectroscopies, revealing that R-1 starts to aggregate in a 70 : 30 water–DMSO mixture. The maximum fluorescence intensity was observed in a 99 : 1 water–DMSO mixture (Fig. S9 (ESI†) and Fig. 1b, inset). The scanning electron microscopy (SEM) images of a drop cast sample of R-1_Agg (1 × 10⁻⁴ M, water/DMSO, 99/1 v/v) over a silicon wafer show particle-like morphology (Fig. 1b).

Fig. 1 (a) Absorption spectra of R-1 (1 × 10⁻⁴ M) in DMSO and an aqueous medium (water/DMSO, 99/1 v/v). Inset shows the images of the Tyndall effect of the corresponding solutions, indicating the aggregation behaviour of R-1 in aqueous medium (i). (b) SEM image of R-1_agg (1 × 10⁻⁴ M, water/DMSO, 99/1 v/v) drop cast over a silicon wafer. Inset shows the change in the fluorescence of R-1 in DMSO (A) and in a 99 : 1 water/DMSO mixture (B). Images were taken under 365 nm UV light illumination.

Having these results in hand, we tested the CO₂ sensing ability of R-1_Agg (1 × 10⁻⁴ M, water/DMSO, 99/1 v/v) on purging CO₂ into R-1_Agg while monitoring the corresponding changes in its absorption and emission properties. It was observed that in the presence of CO₂, the sharp absorption band at 348 nm of R-1_Agg broadened and red-shifted to 364 nm (Fig. 2a). Interestingly, gradual quenching in the emission of R-1_Agg was observed on purging of CO₂ (Fig. 2b and Fig. S10, ESI†). Fluorescence titration experiments revealed that the yellow emission of the aggregates reduced by 25-fold with 0.33 mL of CO₂. Fig. 2c shows the fluorescence decay profiles of R-1_Agg (probed at 550 nm) on purging with CO₂. R-1_Agg exhibited a triexponential decay with an average lifetime of 5.67 ns, whereas in the presence of CO₂, a faster decay with an average lifetime of 0.33 ns was observed (Table S1, ESI†). The changes in absorption, steady state and time-resolved emission characteristics indicated the transformation of R-1_Agg into a new-type of species having monomer-like absorption and emission features.

Fig. 2 (a) Absorption spectra of R-1_Agg (1 × 10⁻⁴ M) in aqueous medium (water/DMSO, 99/1 v/v) after purging with excess amount of CO₂ (0.33 mL). (b) Emission spectra of R-1_Agg (1 × 10⁻⁴ M, λ_ex = 370 nm) in the presence of different concentrations of CO₂ (0, 0.016, 0.032, 0.048, 0.064, 0.080, 0.096, and 0.33 mL). Inset shows the fluorescence change of R-1_Agg under UV illumination (λ_ex = 365 nm) before and after purging with CO₂. (c) Fluorescence lifetime decay profile of R-1_Agg (λ_ex = 370 nm, λ_em = 550 nm) in the presence of increasing CO₂ concentrations (0, 0.016, 0.048 and 0.33 mL). IRF = instrument response function. (d) Fluorescence response (λ_ex = 370 nm) of R-1_Agg towards CO₂ after purging with different gases (3.0 mL) in aqueous medium; from left to right: R-1_Agg, CO₂, O₂, H₂, Ar, N₂ and air.

This observation was also supported by dynamic light scattering (DLS) studies. The average particle diameter of R-1_Agg (1 × 10⁻⁴ M) as measured by DLS was found to be 156 ± 10 nm. On purging a small amount of CO₂, the size of the aggregates gradually decreased; this was inferred from the reduction in the average particle diameter to 102 ± 10 nm, which was further reduced to 83 ± 5 nm with excess (0.33 mL) of CO₂ (Fig. S11, ESI†). Furthermore, a reduction in the Tyndall effect of R-1_Agg in the presence of CO₂ (0.33 mL) was also noticed (Fig. S11c, ESI†). The results of the fluorescence titration studies, size distribution, Tyndall effect and CO₂ chemistry with amine moieties in an aqueous medium suggested that R-1_Agg was protonated in the presence of CO₂ gas, leading to a change in the molecular character from hydrophobic to hydrophilic (Scheme 1). Thus, the large R-1_Agg was converted into a mixture of monomers and smaller aggregates of the positively charged R-1.¹² These processes were indicated by the simultaneous red shift of the absorption maximum and the reduction in the AIE property of the fluorophores; this was corroborated by the pH-dependent fluorescence titration studies (Table S2 and Fig. S12, ESI†), yielding a pK_a value for R-1 as 6.98 (±0.007).

To further evaluate the selectivity of R-1_Agg towards CO₂, we carried out fluorescence titration experiments with different gases in excess amounts (3.0 mL) including N₂, O₂, Ar, H₂, and air under identical conditions (Fig. 2d). It was clearly observed that CO₂ produced the most significant effect on the fluorescence properties of R-1_Agg. The experimental results also indicated that the detection of CO₂ using R-1_Agg experiences less interference from other common gases present in air. In addition, the binding of CO₂ to R-1_Agg was found to be reversible (Scheme 1). The less emissive R-1_Agg–CO₂ adduct was readily converted back into highly fluorescent R-1_Agg by purging N₂/Ar or by adding a small amount of K₂CO₃ at room temperature. As shown in Fig. S13 (ESI†), on repeated purging of CO₂ and addition of 0.1 mM K₂CO₃ solution, the emission of R-1_Agg exhibits multiple cycles of reversible on–off changes.

Fluorescence microscopy imaging was carried out to explore the capability of R-1_Agg as a probe for monitoring the variation of external CO₂ concentration inside living cells. For this purpose, lung adenocarcinoma cells A549 were incubated with 100 μM of R-1_Agg for 1 h under normal atmospheric conditions (0.038% CO₂) at 25 °C as we observed that the above-mentioned concentration gave optimum fluorescence intensity with minimal cytotoxicity around 3 h (Fig. S14, ESI†). Also, initial localization of R-1_Agg was observed inside the nucleus in 1 h (Fig. S15, ESI†), but it eventually spread throughout the cells. As shown in Fig. 3, a yellow fluorescence signal can be observed, confirming the internalization of the aggregates inside the intercellular milieu. Thereafter, the cells were kept in an incubation chamber with 5% CO₂ for 2 h, and the fluorescence was monitored against time. The emission intensity of the yellow fluorescence was reduced and almost no fluorescence was observed, while the sample cells kept under ambient conditions still showed the corresponding emission (Fig. S16, ESI†). These observations suggest that the increase in external CO₂ concentration causes a reduction in the fluorescence emission intensity, thus revealing the capability of R-1_Agg to detect CO₂ variations in cellular conditions. Even though R-1 showed slight toxicity after a higher incubation time, the probe could efficiently sense CO₂ inside the cellular environment.

Fig. 3 (a) Fluorescence and (b) bright field images of R-1_Agg in A549 lung cancer cells incubated with 100 μM R-1_Agg. (c) Fluorescence image of A549 cells further incubated for 2 h at 25 °C and 5% CO₂ and (d) the corresponding bright field image.

The aggregation-enhanced Raman scattering (AERS) of R-1_Agg was exploited as an additional modality for chemosensing purposes.^7,13 First, the Raman spectrum of R-1_Agg (4 × 10⁻⁴ M, water/THF, 99/1 v/v) was recorded with a Raman microscope using 633 nm laser excitation.¹³ The spectra were obtained using a CCD detector with an integration time of 0.5 s and 10 accumulations. A tiny droplet of R-1_Agg was placed on a glass slide to obtain the corresponding Raman fingerprint, which was observed with a high signal-to-noise ratio. The Raman spectrum of R-1_Agg shows two peaks at 1584 cm⁻¹ (strong, C–C aromatic ring chain vibrations) and 1180–1208 cm⁻¹ (weak, C–O–C asymmetric vibrations). Furthermore, the droplets examined after purging the aggregate solution with CO₂ showed a reduction in the peak intensity to almost baseline; thus, by monitoring the Raman-active peaks of R-1_Agg, it was possible to detect CO₂ gas (Fig. 4a).

Fig. 4 (a) Raman spectra of R-1_Agg (4 × 10⁻⁴ M) in aqueous medium (water/THF, 99/1 v/v) and after purging with CO₂. (b) SERS spectra of R-1_Agg in the presence of Au-NPs (1/9 v/v) and after purging with CO₂.

Along with the inherent AERS phenomenon, we also looked for surface-enhanced Raman scattering (SERS) due to its enhanced sensitivity compared to normal Raman scattering. The Raman spectrum of R-1_Agg was recorded using spherical gold nanoparticles (Au-NPs) as the SERS substrate. R-1_Agg was mixed with as-prepared Au-NPs (size ca. 40 nm) in 1 : 9 ratio (v/v), and the mixture was incubated for 10 min to obtain maximum adsorption. The SERS spectra were recorded as described above, and ca. 25-fold enhancement in the intensity was observed when compared with normal Raman intensity obtained for R-1_Agg (Fig. 4). In comparison to the spectrum obtained from AERS, the SERS-assisted spectrum showed well-resolved peaks along with a unique signal at 2213 cm⁻¹ resulting from the –CN group, which can be monitored easily for the CO₂ sensing experiment.

In conclusion, the cyano-substituted p-phenylenevinylene derivative R-1 reported in this study underwent aggregation in an aqueous medium, exhibiting AIE and AERS phenomena, which were used for the detection of CO₂ gas among different specimens of neutral gases. The AIE-based CO₂ assay described here is useful for detecting CO₂ in biological specimens. The novelty of the present system is that a combination of fluorescence and AERS or SERS can be used for sensing CO₂. However, for ratiometric sensing and in vivo applications, further improvement over the present design is required and such studies are in progress.

R. K. M. is thankful to DST, Govt. of India, for an INSPIRE Faculty Award (IFA-12 CH-50). V. K. P. and K. K. M. acknowledge CSIR mission mode project, nano-biosensor and microfluidics for healthcare (HCP-0012) and fast-track translation project, cellular sensor (MLP0027), Government of India for financial support. A. A. is grateful to the DST-SERB, Govt. of India, for a J. C. Bose National Fellowship (SB/S2/JCB-11/2014).

Conflicts of interest

There are no conflicts to declare.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental details, synthesis procedures and characterization data of the compounds, and additional figures. See DOI: 10.1039/c9cc01564g

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