Detection of organic amines using a ratiometric chromophoric fluorescent probe with a significant emission shift

Synthesis of the probe BI-CA-ID

As known, organic amines are nucleophilic that can react with α,β-unsaturated compounds. Thus, based on the nucleophilic addition reaction which can cause a rapid change in the fluorescence signal, the probe BI-CA-ID was synthesized. Through Knoevenagel reaction of an indole and carbazolyl, the reaction site was developed. To enlarge the conjugation of the probe, the biphenyl fragment was introduced by Suzuki reaction. The Synthetic route is shown in Scheme 1. The ¹H NMR, ¹³C NMR, and high-resolution mass spectrometry (HRMS) characterization of BI-CA-ID are shown in the supporting information.

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Scheme 1.

Synthetic route toward BI-CA-ID.

Optical response of BI-CA-ID toward the organic amine, n-BuNH₂

Absorption and fluorescence spectroscopy were used to examine the sensing behaviors of BI-CA-ID toward n-BuNH₂ (Figure 1(a)). Two distinct absorption bands at 292 and 501 nm occur with BI-CA-ID, with the first attributed to a π-π* transition and the second to intramolecular charge transfer (ICT) transition. An increase in the level of n-BuNH₂ (0–2.5 equiv.) added to BI-CA-ID (40 μM) led to a gradual decrease of the absorption peak at 501 nm and a progressive increase of the absorption band at around 292 nm. The result was a very noticeable color change from red to colorless, indicating the inhibition of both the π-conjugation and the ICT progress of BI-CA-ID on nucleophilic addition of -NH₂ to the α,β-unsaturated structure. Note that the occurrence of a well-defined isosbestic point at 346 nm implied the formation of only a single new species during the sensing process. The DL of the probe was 0.024 μM, calculated from the plot of the ratio (A₂₉₂/A₅₀₁) and concentration of n-BuNH₂.

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Figure 1.

(a) Absorption spectra of BI-CA-ID (40 μM) with increased n-BuNH₂ concentrations (0–2.5 equiv.) in solution (CH₃CN:water = 1:1). (b) The titration curve plotted from the absorbance ratio of BI-CA-ID (A₂₉₂/A₅₀₁). (c) The BI-CA-ID (40 μM) fluorescence spectra with increased n-BuNH₂ concentrations (0–2.5 equiv.). (d) The titration curve plotted from the BI-CA-ID fluorescence ratio (I₃₈₂/I₆₀₁). Excitation wavelength: 500 nm, excitation slit: 5 nm, and emission slit: 20.0 nm.

The above studies provided the basis for examining the changes in the fluorescence spectrum under the same conditions (Figure 1(c)). The excitation of the probe at 500 nm produced a dual emission with peaks at 382 and 601 nm. The band at 601 nm is attributed to the ICT emission band, and the band at 382 nm is ascribed to the biphenyl carbazole moiety. The addition of n-BuNH₂ gradually quenches the fluorescence intensity of the solution at 601 nm and gradually increases the intensity at 382 nm, yielding a bright blue emission that is clearly visible by the naked eye on UV-lamp irradiation at 365 nm. Simultaneously, a clear iso-emissive point at 523 nm indicated the formation of the BI-CA-ID-NH-adduct. The distinct wavelength difference between the two emission bands is almost 220 nm, which makes the probe favorable for ratiometric fluorescent detection of organic amines. A good linear relationship between the intensity ratio (I₃₈₂/I₆₀₁) and the concentration of n-BuNH₂ could be obtained in the range of 2.0 × 10⁻⁶ to 8.0 × 10⁻⁶ M (R² = 0.9924). This ratiometric fluorescence change is potentially useful for the quantitative determination of organic amines, with the DL of BI-CA-ID being 0.43 μM. The quantum yield of the probe was calculated to be 0.077.

Selectivity studies

To assess the specificity, BI-CA-ID (40 μM) was treated with the following anions and molecules (0.8 mM) under identical conditions: Cl⁻, Br⁻, I⁻, NO 2 − , NO 3 − , S²⁻, HSO 3 − , SO 4 2 − , PO 4 3 − , CO 3 2 − , Ac⁻, oxalic acid (OX), and amino acids: Cys, Gly, Glu, and His. Of the ratiometric absorption and fluorescence of the probe in the presence of different anions and molecules shown in Figure 2, only alkyl amines responded to BI-CA-ID with a large blue shift and a marked color change from red to colorless. Both primary and secondary amines were characterized by rapid color changes within 1 s, while tertiary amines displayed similar changes after 10 min; the unique steric hindrance of tertiary amines may be the cause of the different response times. Nevertheless, the weak nucleophilic ability prevents any reaction of aniline with BI-CA-ID, thus indicating negligible optional changes. At the same time, almost no change in optical properties occurred in the other anions and molecules. These results confirm the efficacy of BI-CA-ID for use as an efficient signaling probe for alkyl amines.

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Figure 2.

(a) Response of BI-CA-ID to various anions (EDA is ethylenediamine) and molecules. (b) and (c) Absorption and fluorescence response of BI-CA-ID (40 μM) in the presence of different anions and molecules (0.8 mM). Excited at 500 nm, excitation slit: 2.5 nm, and emission slit: 20.0 nm.

To further examine the high selectivity for organic amines of BI-CA-ID, a panel of related alkyl amine compounds was analyzed under identical conditions. The corresponding changes in the fluorescence and absorbance spectra before and after addition of different amines were recorded as shown in Figure 3. As expected, in the presence of alkyl amines, the F₃₈₂/F₆₀₁ ratio was enhanced significantly, and a decrease in absorbance at 501 nm occurred, indicating similar response and color changes as depicted in Figure 1. All the results further demonstrated the acquisition of a new ratiometric fluorescent probe for detecting organic amines.

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Figure 3.

(a) The color changes after adding different amines. (b) and (c) The fluorescence and absorbance response of BI-CA-ID in the presence of different organic amines (400 μM) after 10 min. (a) From left to right: blank solution (0), butylamine (1), isopropylamine (2), ethanediamine (3), dicyclohexylamine (4), diisopropylamine (5), diisopropylethylamine (6), and triethanolamine (7). Excited at 500 nm, excitation slit: 2.5 nm, and emission slit: 20.0 nm.

Effect of pH toward the probe

Next, to examine the influence of the pH value on the probe, we explored its response to different pH values. Initially, the pH titration was carried out in water. As shown in Figure 4, there is little influence upon BI-CA-ID when the Ph = 4–10, which indicates its suitability for near-neutral pH solutions.

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Figure 4.

(a) Plot of the absorbance ratio (A₂₉₂/A₅₀₁) of BI-CA-ID (40 μM) with increasing value of pH in water. (b) Plot of the intensity ratio (I₃₈₂/I₆₀₁) of BI-CA-ID (40 μM) with increasing value of pH in water, excited at 365 nm.

General and materials

All reagents were purchased from commercial suppliers and used without further purification unless otherwise specified. Qingdao Hai Yang silica gel (200–300 mesh) was used for flash chromatography and an F-4600 FL spectrophotometer with a 10 mm quartz cuvette was used to measure the fluorescence spectra and relative fluorescence intensity. A Specord Plus instrument was used to capture the ultraviolet–visible (UV-Vis) spectra. A Bruker spectrometer operating at 400 MHz, with tetramethylsilane (TMS) as the internal standard, was used to collect the ¹H and ¹³C NMR spectral data. A Bruker Compact TOF mass spectrometer was used to record the high-resolution mass spectra (HRMS).

Synthesis of the probe BI-CA-ID

9-Dodecyl-9H-carbazole (1): Compound 1 was synthesized via the report. ⁴⁰ A mixture of KOH (5.19 g, 62.7 mmol) and dimethylformamide (DMF; 25 mL) was stirred at room temperature for 30 min. Next, carbazole (3.00 g, 17.9 mmol) was added and the mixture reacted for another 4 h. DMF (5 mL) containing 1-bromododecane (5.0 mL, 20.9 mmol) was added dropwise to the mixture and reacted overnight. The solution was poured into icewater to form a precipitate which was then filtered. After recrystallization from ethanol, white needle crystals (4.8 g, 14.3 mmol) were obtained in 80% yield.

9-Dodecyl-9H-carbazole-3-carbaldehyde (2): Refer to the report, ⁴⁰ compound 2 was synthesized. Phosphorus oxychloride (6.9 mL, 74.50 mmol) was added dropwise to DMF (5.8 mL, 74.50 mmol) cooled in the icewater and stirred for about 1 h. After the addition of compound 1 (5.00 g, 14.9 mmol), the mixture was heated at 90 °C for 6 h. After cooling to room temperature, the above solution was poured into water and filtered. The crude product was purified through column chromatography (SiO₂, PE:CH₂Cl₂ = 10:1) to give 2 as light yellow solid (3.79 g, 10.4 mmol, 70%). ¹H NMR (400 MHz, CDCl₃): δ 10.10 (s, 1H), 8.61 (s, 1H), 8.16 (d, J = 5.2 Hz, 1H), 8.01 (dd, J = 5.6, 0.80 Hz, 1H), 7.53 (t, J = 5.0 Hz, 1H), 7.47 (t, J = 6.2 Hz, 2H), 7.32 (t, J = 4.8 Hz, 1H), 4.33 (t, J = 5.0 Hz, 2H), 1.94–1.81 (m, 2H), 1.41–1.20 (m, 18H), 0.87 (t, J = 4.6 Hz, 3H).

6-Bromo-9-dodecyl-9H-carbazole-3-carbaldehyde (3): As reported, ⁴¹ a solution of N-bromosuccinimide (NBS; 0.98 g, 5.50 mmol) in tetrahydrofuran (THF) was added dropwise to a solution of compound 2 (2.00 g, 5.50 mmol) in THF (40 mL) at ambient temperature in the absence of light. After 4 h, the reaction was quenched by the addition of icewater, and the resulting white precipitate was filtered. The white solid was recrystallized from ethanol to give product 3 (1.8 g, 4.07 mmol, 74%). ¹H NMR (400 MHz, CDCl₃): δ 10.08 (s, 1H), 8.54 (s, 1H), 8.26 (s, 1H), 8.03 (d, J = 8.8 Hz, 1H), 7.60 (dd, J = 8.6, 1.4 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H), 4.30 (t, J = 7.2 Hz, 2H), 1.94–1.78 (m, 2H), 1.38–1.18 (m, 18H), 0.86 (t, J = 6.8 Hz, 3H).

6-([1,1′-biphenyl)-4-yl)-9-dodecyl-9H-carbazole-3-carbaldehyde (4): DMF (35 mL) was added to a 100 mL three-necked flask and kept under N₂ for about 4 h. Compound 3 (0.50 g, 1.13 mmol), 2-((1,1′-biphenyl)-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.38 g, 1.36 mmol), K₃PO₄ (0.96 g, 4.52 mmol), and Pd(PPh₃)₄ (0.09 g, 0.079 mmol) were added sequentially, and the resulting mixture was heated to 85 °C for 4 h. The solvent was removed under reduced pressure, and the crude product was recrystallized to give 4 as a white solid (0.361 g, 0.70 mmol, 62%). ¹H NMR (400 MHz, CDCl₃): δ 10.12 (s, 1H), 8.68 (s, 1H), 8.43 (d, J = 1.2 Hz, 1H), 8.04 (d, J = 5.6 Hz, 1H), 7.83 (dd, J = 5.6, 1.2 Hz, 1H), 7.80 (d, J = 5.6 Hz, 2H), 7.74 (d, J = 5.2 Hz, 2H), 7.69 (d, J = 4.8 Hz, 2H), 7.56–7.46 (m, 4H), 7.38 (t, J = 5 Hz, 1H), 4.38 (t, J = 4.8 Hz, 2H), 1.98–1.87 (m, 2H), 1.47–1.17 (m, 18H), 0.87 (t, J = 4.6 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 190.71, 143.41, 139.66, 139.60, 139.40, 138.55, 132.20, 127.79, 127.53, 126.54, 126.26, 126.17, 125.97, 125.03, 123.15, 122.50, 122.10, 118.00, 108.67, 108.10, 42.52, 30.87, 28.67, 28.56, 28.51, 28.44, 28.32, 28.30, 27.94, 26.22, 21.66, 13.11. HRMS (ESI): m/z [M⁺] calcd for C₃₇H₄₂NO⁺: 516.3261; found: 516.3243. And m.p.: 142.4–143.4.

Synthesis of the probe BI-CA-ID

The novel product BI-CA-ID was synthesized based on an analogous procedure. ⁴² As shown in Scheme 1, under an N₂ atmosphere, a mixture of 1-butanol (5 mL) and toluene (5 mL) containing compound 4 (0.2000 g, 0.39 mmol) and 1,2,3,3-tetramethyl-3H-indol-1-iumiodide (0.1175 g, 0.39 mmol) was stirred at 90 °C overnight. The solvent was evaporated and column chromatography was used to purify the residue. The product was obtained as a red solid (0.202 g, 0.25 mmol, 65%). ¹H NMR (400 MHz, CDCl₃): δ 9.14 (s, 1H), 8.82 (d, J = 1.2 Hz, 1H), 8.41–8.29 (m, 2H), 7.97–7.85 (m, 3H), 7.82 (dd, J = 8.8, 1.6 Hz, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 7.2 Hz, 2H), 7.55–7.30 (m, 9H), 4.37 (s, 3H), 4.27 (t, J = 7.2 Hz, 2H), 2.01–1.73 (m, 8H), 1.40–1.22 (m, 18H), 0.88 (t, J = 6.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 181.11, 156.59, 144.94, 142.77, 141.76, 140.80, 140.76, 140.00, 139.46, 133.49, 131.15, 129.46, 129.02, 128.90, 127.74, 127.44, 127.06, 126.04, 125.77, 124.60, 124.08, 123.98, 122.50, 120.46, 114.10, 110.24, 110.08, 109.24, 51.96, 43.97, 37.00, 32.19, 29.90, 29.88, 29.86, 29.79, 29.68, 29.61, 29.29, 27.57, 27.52, 22.96, 14.40. HRMS (ESI): m/z [M⁺] calcd for C₄₉H₅₅N₂⁺: 671.4360; found: 671.4331. And m.p.: 221.3–223.4.

General procedure for the measurement of spectra

Stock solutions of BI-CA-ID (0.1 mmol) were prepared in CH₃CN and then diluted to derive the target solution (40 μM). The target solution (1.5 mL) and 1.5 mL of different concentrations of n-BuNH₂ in water were combined to obtain the test solution (3 mL), which was then maintained at room temperature (25 °C) for 30 s. The absorption and fluorescence spectra were recorded, and the fluorescence ratio of I₃₈₂/I₆₀₁ measured with the excitation wavelength set at 500 nm.

Determination of the DL

The DL was obtained by 3S_b/k, where S_b is the standard deviation of the blank measurements (10 times) and k is the slope of the fitted line.

Quantum yield

The fluorescence quantum yield can be calculated by means of equation (1)

where the subscripts s and r refer to the sample and the reference, respectively, and Ф, F, A and n stand for quantum yield, the integrated emission intensity, the absorbance and refractive index, respectively. Rhodamine B (Φ = 0.97) in ethanol was used as the standard for calculating the fluorescence quantum yields of the probes.