A novel ratiometric pH probe for extreme acidity based on FRET and PET

Abstract

In this study, a novel ratiometric pH probe RC1 was successfully developed. RC1 was constructed by integrating a coumarin fluorophore as a fluorescence resonance energy transfer (FRET) donor into a rhodamine B fluorophore as a FRET acceptor, which is associated with rhodamine B dyes possessing spirocyclic (non-fluorescent) and ring-opening (fluorescent) forms with response to pH. At weak basic pH, the photo-induced electron transfer (PET) process of the N atom of aromatic imino in the rhodamine moiety partly quenches the coumarin emission. At acidic pH, the PET process is gradually inhibited upon acidification, enhancing the fluorescence intensity of coumarin remarkably; at the same time, the spirolactam form of rhodamine changes to a ring-opening form followed by the FRET process between coumarin and rhodamine. Hence, the emission intensities of coumarin and the rhodamine moiety simultaneously increase along with the pH decrease. The sensing mechanism is an integration of the PET and FRET processes. Based on the ratios of fluorescence intensity at 583 nm and 470 nm (I₅₈₃/I₄₇₀), RC1 with a pK_a of 3.21 could be used in the ratiometric detection of pH in the range 2.20–4.20 with high selectivity. Furthermore, it can be applied to visualize extreme acidity in bacteria. The results demonstrate that RC1 can serve as an ideal probe for extremely acidic pH levels with excellent biological significance.

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Introduction

The measurement of pH is of great importance since pH is a key parameter in a wide range of fields, such as food production, chemical process control, environmental analysis, medical diagnosis and life sciences.^1,2 Moreover, for all living organisms, the maintenance of pH homeostasis is highly required. The internal pH spans from the basic to extremely acidic range in various prokaryotic species as well as in diverse subcellular compartments of eukaryotic cells.^3,4 Despite the fact that most of the living species could hardly live in highly acidic conditions (pH < 4), a great number of microorganisms such as “acidophiles” and Helicobacter pylori particularly favour harsh environments.⁵ Another example is enteric pathogens, which may possibly lead to fatal infections by means of passing through the strongly acidic mammalian stomach and reaching the small intestine. The acidic pH plays a critical role in numerous organelles, particularly the apparatus along the secretory and endocytic pathways in eukaryotic cells.⁶

The potentiometric pH sensor is well-established for routine pH determination; however, the limitations of potentiometric pH sensor make it unsuitable in intracellular pH and microscopy studies.⁷ In recent years, the detection of pH by fluorescence-based techniques is well established for both imaging and sensing applications. Small fluorescent organic molecules, quantum dots have been widely used for accessing pH changes in solutions or living cells.^8–14 To the best of our knowledge, however, most of the pH probes reported in the literature worked in a pH region 4–7,^15–23 and the fluorescent probes for lower pH region (pH < 4) were paid less attention.^{1,6,7,24–28} Therefore, pH probes for extreme acidity are still in great demand.

Most reported fluorescent pH probes are based on measurement of the fluorescence intensity changes at a single wavelength. However, fluorescence signals are easily influenced by environmental factors, such as temperature, solvent polarity, and excitation intensity. To alleviate these problems, ratiometric measurements based on the ratios of intensity at two wavelengths were developed. Constructing a fluorescence resonance energy transfer (FRET) system, which could reduce self-quenching and fluorescence detection error, is an optimal strategy for the design of ratiometric biosensors,²⁹ such as pH probe and metal ion probe.^1,30–34

We have been focusing on the design and synthesis of pH probes. In a continuation of an ongoing program aiming at developing novel pH probes with high selectivity and sensitivity; herein, we report a coumarin–rhodamine FRET system RC1 as a ratiometric pH probe. Compared with the probes we previously reported,^{15–17,27,28,35} RC1 responds to pH variations via the self-calibration of two emission bands in a ratiometric manner, providing more accurate detection of pH even in complicated biological samples. Moreover, it is suitable for monitoring pH changes in extremely acidic conditions, whereas others function in a much higher pH region (pH 4–7).^{15–17,35,36} RC1 is also applied in bacteria with satisfying effects.

Results and discussion

Design and synthesis of RC1

The probe RC1 was designed as a ratiometric fluorescent pH probe based on several considerations. The framework of rhodamine derivatives is an ideal model to construct pH probes due to its excellent photophysical properties such as long absorption and emission wavelength, high fluorescence quantum yield and the change in structure between spirocyclic (non-fluorescent) and ring-opening (fluorescent) forms with response to pH. On the other hand, coumarin derivatives are excellent organic dyes with the emission profiles tunable from the blue to near-infrared region by simply changing at coumarin core structure. Coumarin was chosen as the energy donor since the emission spectrum of coumarin and the excitation spectrum of rhodamine have substantial overlap, giving effective fluorescence energy transfer with a single excitation wavelength.^37–41 Therefore, rhodamine B moiety and coumarin moiety are a good pair for FRET design. The probe RC1 was easily synthesized by a one-step reaction (Scheme 1) and the structure of RC1 was fully characterized by IR, ¹H NMR, ¹³C NMR, and HRMS.

Scheme 1 The synthetic route of probe RC1.

Spectra characteristics of RC1

The absorption spectra of RC1 in buffer solution at different pH values were displayed in Fig. 1. RC1 exhibited a maximal band at 428 nm when pH = 7.20. The absorbance was almost unchanged along with decreasing of the pH value from 7.20 to 2.00. Moreover, a new absorption band significantly appeared at 567 nm and the absorbance increased gradually when the pH decreased, which was attributed to the structure transformation of rhodamine moiety from non-fluorescent spirolactam form to fluorescent ring-opening form. The colour of the solution changed from pale green to pink simultaneously, so RC1 can act as a “naked-eye” indicator for acidic pH.

Fig. 1 Absorption spectra of RC1 (10 μM) in buffer at different pH values (7.20, 6.80, 6.40, 6.00, 5.40, 5.00, 4.80, 4.60, 4.40, 4.20, 4.00, 3.80, 3.60, 3.40, 3.20, 3.00, 2.80, 2.60, 2.40, 2.20, 2.00).

As shown in Fig. 2a, when excited at 420 nm, RC1 exhibited a maximal emission band at 470 nm at pH 7.20. As the pH value decreased from 7.20 to 2.00, the emission intensity of coumarin moiety centred at 470 nm enhanced gradually. Meanwhile, a new fluorescence emission band of rhodamine moiety evolved at 583 nm and increased along with the addition of H⁺. The observation that both of the fluorescence intensities of coumarin (470 nm) and rhodamine (583 nm) increased upon addition of H⁺ could be explained. At pH 7.20, the photo-induced electron transfer (PET) process of the N atom of aromatic imino in rhodamine moiety quenched the coumarin fluorescence to a certain extent.¹ When pH decreased from 7.20 to 2.00, the PET process was gradually prohibited due to the protonation of the N atom of aromatic imino, enhancing the emission intensity of coumarin moiety. Meanwhile, the addition of H⁺ induced the ring-opening process of rhodamine moiety and the resultant, ring-opening form of rhodamine was an effective energy acceptor. FRET process from coumarin moiety to rhodamine moiety occurred accordingly, so the fluorescence intensity of rhodamine increased concomitantly. The results demonstrated that the sensing mechanism of RC1 was an integration of PET and FRET processes (Scheme 2). The fluorescence quantum yield of the rhodamine moiety was determined to be 0.21 at pH 2.00 (rhodamine B as a standard). As displayed in Fig. 2b, ratios of fluorescence intensity at 583 nm and 470 nm (I₅₈₃/I₄₇₀) showed an obvious change from 0.02 to 0.22 when pH decreased from 7.20 to 2.00. According to the pH dependent ratios, typical application range of RC1 could be found to be in the pH region 2.20–4.20 with good linearity (R = 0.99524). The plot of I₅₈₃/I₄₇₀ against pH indicated the pK_a was 3.21, suggesting that RC1 was suitable for studying the extremely acidic environment (pH < 4). Based on the ratios of fluorescence intensity at 583 nm and 470 nm (I₅₈₃/I₄₇₀), RC1 could be applied to detect the pH changes in the range 2.20–4.20 in a ratiometric manner.

Fig. 2 (a) Fluorescence spectra of RC1 (10 μM) in buffer at different pH values. The inset shows the emission intensity at 470 nm (I₄₇₀) at different pH values; (b) the fluorescence emission ratios (I₅₈₃/I₄₇₀) by pH values according to the fluorescent pH titration (pH 2.00–7.20), λ_ex = 420 nm. The inset shows the linear relationship of the emission ratios (I₅₈₃/I₄₇₀) and pH values from 2.20 to 4.20 (R = 0.99524). Incubation time: 6 h.

Scheme 2 The proposed PET and FRET processes of probe RC1.

There is an interesting phenomenon that the fluorescence intensity at 470 nm increased along with the pH decrease from 7.20 to 2.00 (Fig. 2a), which is different from other FRET-based ratiometric probes.^42–44 According to the theory of FRET, energy donor (coumarin moiety) emission should decrease when energy transfer takes place. In order to further discuss the mechanism, we investigated the optical properties of ethyl 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylate (C1). The absorbance and fluorescence intensity of C1 remained unchanged upon addition of H⁺ (Fig. S1 and S2, ESI†), and it could be speculated that the coumarin moiety of RC1 was stable when pH decreased from 7.20 to 2.00. As shown in Fig. 3, at pH 2.40, the fluorescence intensity at 470 nm decreased along with the extension of the time, and the fluorescence intensity at 583 nm increased on the contrary, which was a manifestation of the occurrence of FRET process from coumarin moiety to rhodamine moiety. After 250 min, the fluorescence intensity remained stable. Therefore, the ring-opening process of rhodamine spirolactam was slow. What should be emphasized was that the blocking of the PET process was the main factor on the coumarin emission rather than the FRET process, which differed from other FRET-based ratiometric probes to a great extent. A reasonable mechanism of RC1 upon acidification was proposed in Scheme 2. Overall, the observation that the emission intensity of both coumarin moiety (470 nm) and rhodamine moiety (583 nm) increased simultaneously upon addition of H⁺ was a comprehensive effect of PET and FRET processes.

Fig. 3 The fluorescence spectra of RC1 (10 μM) at different time at pH 2.40. The inset shows the time courses of fluorescence intensity at 470 nm (I₄₇₀) and 583 nm (I₅₈₃).

The time courses of the fluorescence intensity ratios (I₅₈₃/I₄₇₀) in buffer at pH 2.40 and 7.20 were performed at room temperature (Fig. S3, ESI†). The results indicated that intensity ratios (I₅₈₃/I₄₇₀) peaked in 250 min at different pH values, and then remained stable. Therefore, all samples were incubated in buffer for 6 h before the measurement.

The ¹H NMR spectroscopy (Fig. 4), determined in CD₃OD–D₂O (9 : 1, v/v) under neutral and acidic conditions, showed the evident down-field shift of signals of the xanthene protons and the methylene protons (N(CH₂CH₃)₂) in rhodamine fragment, due to the protonation of the N atom of aromatic imino in rhodamine fragment under acidic condition. Five minutes after the addition of CF₃COOD, the signal of the methylene protons (N(CH₂CH₃)₂) in rhodamine fragment was stable, which indicated the protonation was a rapid process. In addition, some new signals were observed at δ = 6.60–7.40 ppm, which could be ascribed to the ring-opening process of the spirolactam.

Fig. 4 The ¹H NMR spectra of probe RC1 in CD₃OD–D₂O (9 : 1, v/v). (a) Neutral condition; (b) five minutes after the addition of CF₃COOD; (c) three hours after the addition of CF₃COOD.

As oxygen and nitrogen can bind to many metal ions in solution, it is important to determine whether metal ions were potential interferents. As shown in Fig. 5, the probe RC1 showed excellent selectivity to H⁺ over representative metal ions under acidic (pH 2.00) and neutral conditions (pH 7.20). The effect of such metal ions on pH measurement was negligible.

Fig. 5 The fluorescence emission ratios (I₅₈₃/I₄₇₀) response to RC1 (10 μM) in buffer in the presence of diverse ions at pH 2.00 (a) and pH 7.20 (b). (1) blank, (2) Al³⁺ (50 μM), (3) Ba²⁺ (50 μM), (4) Ca²⁺ (100 μM), (5) Cd²⁺ (50 μM), (6) Co²⁺ (50 μM), (7) Cr³⁺ (50 μM), (8) Cu²⁺ (50 μM), (9) Fe³⁺ (50 μM), (10) Hg²⁺ (50 μM), (11) K⁺ (1 mM), (12) Mg²⁺ (100 μM), (13) Na⁺ (1 mM), (14) Ni²⁺ (50 μM), (15) Pb²⁺ (50 μM), (16) Zn²⁺ (50 μM), (17) Ag⁺ (50 μM), (18) Fe²⁺ (50 μM). Incubation time: 6 h.

We also examined the reversibility of the probe, RC1 showed excellent reversibility between pH 2.40 and 7.20 (Fig. S4, ESI†).

Fluorescence imaging in bacteria

To verify the potential biological application of the probe RC1, we detected the extremely acidic conditions in bacteria. After incubation with the probe for 2 h, LB medium at different pH (2.00, 3.00, 4.00) was used to incubate E. Coli, respectively. From the images captured with the laser confocal microscopy (Fig. 6a), it was noted that the bacteria in the medium at pH 2.00 exhibited much brighter fluorescence than pH 3.00 and pH 4.00. Both the blue fluorescence of coumarin moiety and red fluorescence of rhodamine moiety decreased along with the pH increase. The phenomenon was more apparent in the fluorescence intensity quantitation data analyzed by the Image J (Fig. 6b). The results demonstrated that RC1 was able to visualize extremely low pH in bacteria with good effects.

Fig. 6 (a) Confocal fluorescence images of E. coli containing RC1 (2 μM) at pH 2.00 (e–h), pH 3.00 (i–l), pH 4.00 (m–p). (a–d) Control. First column: blue fluorescence of coumarin moiety, second column: red fluorescence of rhodamine moiety, third column: white light images, fourth column: overlap images of the three. (b) Fluorescence intensity quantitation was analysed by the Image J. The results were presented as means ± SE with replicates n = 3. *, p < 0.05; **, p < 0.01.

We also investigated the toxicity of RC1 (Fig. S5, ESI†). The SRB assay results showed that the probe RC1 was almost non-toxic to the living cells, which was vital for the future biological application.

Experimental

Materials and general methods

Thin-layer chromatography (TLC) involved silica gel 60 F₂₅₄ plates (Merck KGaA) and column chromatography involved silica gel (mesh 200–300). Melting points were determined on an XD-4 digital micro melting point apparatus. ¹H NMR (300 MHz) and ¹³C NMR (75 MHz) spectra were acquired on a Bruker Avance 300 spectrometer, using DMSO as solvent and tetramethylsilane (TMS) as internal standard. IR spectra were recorded with an IR spectrophotometer VERTEX 70 FT-IR (Bruker Optics). HRMS spectra were obtained on a Q-TOF6510 spectrograph (Agilent). The pH was measured on a PHS-3C digital pH-meter (YouKe, Shanghai). UV-vis spectra were measured on a U-4100 UV-vis-NIR Spectrometer (Hitachi). Fluorescent measurements were performed on a Perkin Elmer LS-55 luminescence spectrophotometer. The fluorescence images of cells were performed with laser confocal microscopy (Carl Zeiss LSM-700, Germany).

All reagents and solvents were purchased from commercial sources and used without further purification. The solutions of metal ions were prepared from nitrate salts dissolved in deionized water. Britton–Robinson (B–R) buffers were mixed by 40 mM phosphoric acid, acetic acid and boric acid. Sodium hydroxide was used for tuning pH values. All samples were incubated in buffers (1 : 1, B–R buffer/EtOH, v/v) for 6 h before measurement.

Synthesis of RC1

N-(rhodamine-B)lactam-ethanolamine (compound 1) and 7-diethylamino-2-oxo-2H-chromen-3-carboxylic chloride (compound 2) were synthesized according to the literature.^45,46 Compound 1 (485 mg, 1.0 mmol) was dissolved in 10 mL dry DMF, then sodium hydride (120 mg of 60% suspension in mineral oil, 3.0 mmol) was added to the solution at 0 °C. To this slurry, compound 2 (279 mg, 1.0 mmol) dissolved in 10 mL dry DMF was added over a period of 30 min, then the mixture was stirred under N₂ atmosphere and ice bath for 4 h. The solvent was then evaporated under reduced pressure, and the crude product was dissolved in 150 mL CH₂Cl₂. The organic layer was washed three times with water (3 × 100 mL), dried and concentrated. The residue was purified by column chromatography on silica gel using dichloromethane–methanol (15 : 1, v/v) as eluent to afford RC1 as a yellow solid in a yield of 67%. Mp: 135–137 °C. IR (KBr), ν/cm⁻¹: 3443, 2970, 2928, 1761, 1694, 1617, 1589, 1513, 1353, 1218, 1116, 790. ¹H NMR (DMSO, 300 MHz), δ: 1.04 (t, 12H, J = 6.9 Hz, NCH₂CH₃), 1.14 (t, 6H, J = 6.9 Hz, NCH₂CH₃), 3.24 (q, 8H, J = 6.9 Hz, NCH₂CH₃), 3.34 (t, 2H, J = 6.3 Hz, CONCH₂CH₂O), 3.48 (q, 4H, J = 6.9 Hz, NCH₂CH₃), 3.90 (t, 2H, J = 6.3 Hz, CONCH₂CH₂O), 6.28–6.39 (m, 6H, xanthene–H), 6.51 (d, 1H, J = 2.1 Hz, ArH), 6.77 (dd, 1H, J = 9.0, 2.4 Hz, ArH), 7.00 (m, 1H, ArH), 7.47–7.51 (m, 2H, ArH), 7.54 (d, 1H, J = 9.0 Hz, ArH), 7.79–7.82 (m, 1H, ArH), 8.35 (s, 1H, –CH=C); ¹³C NMR (DMSO, 75 MHz), δ: 12.28, 12.32, 43.53, 44.35, 61.03, 64.11, 95.81, 97.22, 104.62, 106.64, 106.89, 108.13, 109.77, 122.31, 123.60, 128.19, 129.84, 131.66, 132.81, 148.31, 149.31, 152.49, 152.79, 153.76, 156.71, 158.02, 162.35, 167.55; HRMS calcd for C₄₄H₄₉N₄O₆ [M + H]⁺: 729.3652, found: 729.3670.

Bacteria culture and confocal microscopy study with RC1

E. coli (Trans 5a) were incubated in Luria–Bertani (LB) medium at 37 °C in a table concentrator (ZHI CHENG ZHWY-2112B, China) at 180 rpm for 2 h. Then the culture was centrifuged at 5000 rpm for 5 min to collect E. coli. The sediment was resuspended with fresh LB medium containing 2 μM RC1 dissolved in DMSO (DMSO : LB medium ≤ 2 : 1000, v/v). Then the cells were incubated in the above mentioned table concentrator for 2 h. After centrifugation again, the sediment was resuspended in LB medium at different pH values (2.00, 3.00, 4.00) containing 10 μM nigericin, which could equilibrate the intracellular and extracellular pH. Cells were smeared on slides and observed by laser confocal microscopy at the wavelength of 405 nm.

Conclusions

In conclusion, we have developed an FRET-based ratiometric probe RC1 that could selectively monitor pH changes in the intensity ratio of the two emission bands of coumarin and rhodamine moiety. The ratiometric response of RC1 to pH was an integration of PET and FRET processes and the comprehensive effect resulted in the intensity enhancement of both coumarin (470 nm) and rhodamine (583 nm). Probe RC1 with a pK_a of 3.21 could provide ratiometric measurement of pH values in a range from 2.20 to 4.20 with good linearity based on the ratios of fluorescence intensity at 583 nm and 470 nm (I₅₈₃/I₄₇₀). Moreover, RC1 exhibited no response to various metal ions including Al³⁺, Ba²⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe³⁺, Hg²⁺, K⁺, Mg²⁺, Na⁺, Ni²⁺, Pb²⁺, Zn²⁺, Ag⁺ and Fe²⁺. Furthermore, it could be successfully applied in the fluorescence imaging in bacteria. We believe that RC1 could be a powerful tool for the detection of lower pH levels and it would provide essential information in medicinal analysis in other real biological systems under extremely acidic conditions.

Acknowledgements

This study was supported by the National Basic Research Program of China (2010CB933504) and the National Natural Science Foundation of China (91313303).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Supplementary Fig. S1–S5. See DOI: 10.1039/c4ra16398b

‡ Equal contribution.

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