Introduction

Coumarin is an aromatic heterocyclic compound made up of two fused six-member aromatic rings, between benzene and pyrone, to form as a benzopyrone. The academic literature contains an abundance of information regarding the synthesis and bioactivities of coumarin derivatives1,2,3. Research involving this ring system has been applied to a wide range of areas including pharmaceuticals4, optical brighteners5, fluorescents6,7,8,9,10,11,12,13,14 and laser dyes15. Recently, we developed a novel mixture of simple organic fluorescents, including furocoumarin, to generate high purity white light emission when applied as a coating to a commercial UV LED16. Furocoumarins are one of the coumarin derivatives that can be classified into two groups, i. furan fused benzene ring (psoralen and angelicin) and ii. furan fused lactone ring (furo[3,2-c]coumarin, furo[2,3-c]coumarin and furo[3,4-c]coumarins)17. Both psoralen and angelicin compounds are commonly studied because of their abundance in nature compared to the fused furan on the lactone ring17. In this study, furo[3,2-c]coumarin has been chosen as a suitable fluorescent heterocyclic candidate as it gives an excellent yield based on published reports18,19,20. Furthermore, the synthesis method for furo[3,2-c]coumarin is both efficient and straightforward (one-pot). It is found in natural products, for example, rhizome of Salvia miltiorrhiza Bunge and exhibits potent biological activity (antitumor, antioxidant, anticoagulant, antifungal, anticancer) with several therapeutic applications21. Nair and co-workers reported their preparative procedure which involves a [4 + 1] cycloaddition with in-situ generated heterocyclic coumarin methides and isocyanides18. Since coumarins typically show excellent spectroscopic properties, high stability and low toxicity22, we hypothesized that furo[3,2-c]coumarin derivatives could have potential as fluorescent sensor probes.

The study of fluorescent probes for metal ion detection is a vibrant research field, attracting great interest due to both the importance of detecting heavy metals but also because this sensing approach can offer high sensitivity and fast response times with relatively simple instrumentation requirements23,24,25. Due to the low concentrations at which metal ions are present, for example in biosystems and in the environment, high-sensitivity probes are essential for practical applications26,27. In recent years, a large number of fluorescent sensors from coumarin derivatives have been reported for metal ion detection28, such as Cu2+ 29,30,31,32, Zn2+ 33,34,35,36,37, Al3+ 38,39, Mg2+ 40,41,42 and Fe3+ 43,44,45,46,47. Reference48 gives an overview of some of the sensing materials used for Fe3+ detection.

Among the metal ions, iron is an essential trace element found in living organisms, and both its deficiency and excess are associated with various disorders, such as Alzheimer’s, Parkinson’s disease49,50,51 and anemia52. An excessive amount of iron in the human body can cause toxic damage to various organs including the heart and liver52, whilst a lack of iron is related to weakened cognitive growth and decreases the capacity for physical work53. In severe excess it is known to be lethal and death has occurred following human ingestion of ~40 mg/kg54. The major source of daily iron intake for humans is from food (e.g., green vegetables contain 20–150 mg/kg55) with drinking water (assuming an average concentration of 0.3 mg/L) accounting for ~0.6 mg of daily intake. Iron concentration in surface waters is usually <~1 mg/L but much higher concentrations are encountered in groundwater (e.g., >50 mg/L). Excess iron in the environment can also arise due to chemical treatment processes (e.g., coagulation) and from corrosion of ferrous materials. In the USA, the environmental protection agency (EPA) guidelines state that the maximum level of Fe3+ in drinking water is 5.37 µM56, and in the UK, the drinking water inspectorate (DWI) has set a maximum concentration limit for total iron at 200 µg/L57.

The analysis of Fe3+ is of great importance for various application areas including biomedical58, environmental59 and aquatic60. In previous work successful attempts have been reported for the detection of Fe3+ 43,44,45,46,47. However, in each case, selectivity is not demonstrated for some heavy metals (that exhibit properties similar to those of Fe3+) which could interfere with detection61. For example, we note that Ru3+, which amongst the variety of transition metal ions, theoretically, has the greatest similarity to Fe3+, is not tested for potential interference. Ruthenium is mainly used in the electronics62,63,64 and chemical industries65,66, but it also used for biomedical purposes such as anti-cancer drugs67,68. Therefore, for any Fe3+ fluorescent probe, it is important to extensively demonstrate selectivity, testing with other heavy metals including ruthenium, as it can be present in the environment69, biological systems70 and water71 samples.

Herein, for the first time, we perform a fluorescent study of furo[3,2-c]coumarin derivatives. In particular, we show that the derivative, 2-(cyclohexylamino)-3-phenyl-4H-furo[3,2-c]chromen-4-one (FH), is as an effective fluorescent sensor which exhibits high selectivity for Fe3+, tested against 13 other competing metal ions, including Ru3+ and Fe2+. Finally, we demonstrate the potential of this novel chemosensor for the rapid measurement of Fe3+ in real water samples.

Results and discussions

The structures of the furocoumarin derivatives (FH, FCl, and FNO2) were characterized by 1H NMR and FTIR. These results are in good agreement with the chemical structures for furocoumarin from the literature18,19. Table 1 summarizes the UV-Vis and fluorescence spectroscopy data of FH, FCl and FNO2. Fig. S1, shows the UV-Vis spectra of FH, FCl and FNO2 in ethanol. In Fig. 1, the fluorescence spectra of FH and FCl show higher intensity than FNO2. The main contributing factor responsible for the high fluorescence intensity of furocoumarin is related to its planar and rigid structure72. Fluorescence of FNO2 was severely quenched, contrary to the responses for FH and FCl. Chloro- in FCl is a weaker electron withdrawing group (EWG) than -NO2 in FNO2, however, the chloro- substituent can also donate through the aromatic ring, which has a high electron density, as the atom is enriched with non-bonding electrons. Therefore, it can be through a π-electron delocalization promoter rather than a nitro group, which acts as a relatively strong EWG as illustrated in Fig. 2. In this case, chlorophenyl would be a donor group to the furocoumarin moiety (an acceptor group). It has been reported that the EWG decreases electron density of the aromatic ring with the exception of the halogen substituent group73. The EWG of the nitro group in the benzene ring (nitroaromatic) has empty π∗ orbitals of low energy, which are good acceptors of electrons. Therefore electron-rich fluorescent molecules can potentially undergo strong quenching via a photoinduced electron transfer (PET)74, fluorescence resonance energy transfer (FRET) or electron exchange energy transfer with nitroaromatics75,76,77. Hence, we attribute the higher fluorescence intensity to the chloro- over the nitro- substituent.

Table 1 Concentration [M], Absorbance (Abs), fluorescence lifetimes (τ) and quantum yield (ФF) for fluorescence properties of furocoumarin derivatives in ethanol solution. nd = not determined.
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Figure 1
figure 1

Fluorescence Spectra of Furocoumarin derivatives (FC, FH, FNO2) in ethanol. Inset: Photograph image of furocoumarin in ethanol under UV lamp illumination.

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Figure 2
figure 2

Possible mechanisms whereby chloro- substituent (R = Cl) donates electron through aromatic ring compared with nitro- substituent (R = NO2).

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Fluorescence and UV–Vis titration studies of FH with other metal ions

The photophysical complexation studies of FH with an extensive series of metal salts including: Na+, K+, Mg2+, Ca2+, Mn2+, Fe2+, Fe3+, Al3+, Ni2+, Cu2+, Zn2+, Co2+, Pb2+ and Ru3+ in methanol, was performed using fluorescence spectroscopy. As shown in Fig. 3, the mixture of FH with Fe3+ was the only test sample that exhibited no fluorescence emission (i.e., turn-off) in the wavelength range from 430 to 700 nm. Remarkably, in the presence of 50 μM of various metal ions, fluorescence spectra of FH exhibited an appreciable fluorescence emission except in the case of Fe3+, which resulted in a noticeable turn-off fluorescence response. This fluorescence spectral change was also observed visually when examined with a UV transilluminator (380 nm) as illustrated in Fig. S2. The interaction of FH with Fe3+ leads to an immediate fluorescence turn-off, while for the other metal ions, a slight fluorescence quenching is observed by the naked eye. As mentioned, the planar and rigid structure of the FH molecule makes it a highly fluorescent compound. However, when chelation occurs, there is a transfer of charges within the fluorescent ligand-metal system which then causes fluorescence quenching78,79. Therefore, it can be inferred that the fluorescence quenching of FH in the presence of Fe3+ is due to a ligand-metal charge transfer (LMCT) mechanism. This suggestion is supported by considering the paramagnetic nature of Fe3+ with an unfilled d shell, this would take part in the energy and/or electron transfer processes leading to quenching of the fluorescence80,81. We suspect, when Fe3+ binds with FH, the fluorescent opens a non-radiative deactivation channel induced by the unfilled d shell, resulting in fluorescence quenching due to electron transfer82. Thus, the mechanism of LMCT could happen promptly due to the strong paramagnetic quenching property of Fe3+, leading to a severe fluorescence quenching effect (i.e., turn-off) to coordinate between FH and Fe3+.

Figure 3
figure 3

Fluorescence spectra of FH (0.5 μM) in the presence of different metal ions (100 equiv.) in methanol.

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To gain a quantitative evaluation of the relation between the change in emission intensity of FH and the amount of Fe3+ interaction, a fluorescence titration experiment was carried out with varying concentrations of Fe3+ (Fig. 4). The emission intensity of the peak at 511 nm was systematically quenched by increasing the concentration of Fe3+ from 5 to 50 μM. Moreover, the emission intensity at 511 nm was linearly proportional (correlation coefficient, R2 > 0.99) to the concentration of Fe3+ over the range of 0–30 μM, with a limit of detection of 1.93 µM (Fig. S3). These observations revealed that FH is suitable for use as a sensor for the quantitative measurement of Fe3+. To investigate the binding stoichiometry between FH and Fe3+, a Job’s plot experiment was carried out by keeping the total concentration of FH and Fe3+ ions at 20 μM and changing the molar ratio of Fe3+ from 0 to 1. As shown in Fig. S4 the result indicates a maximum molar fraction of 0.7, indicating the formation of 1:2 complex of FH and Fe3+. This agrees with complexes previously reported83,84. On the basis of changes in emission intensity at 511 nm, the stoichiometric ratio and apparent binding constant of FH with Fe3+ was determined using Benesi–Hildebrand (B-H) linear regression analysis. From the B − H plot, a 1:2 stoichiometry between FH with Fe3+ was confirmed with an association constant of 5.25 × 103 M−1 (Fig. S5).

Figure 4
figure 4

Fluorescence emission spectra of FH (0.5 μM) titrated with Fe3+ (0–100 equiv.) in methanol.

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Competition experiment using fluorescence spectroscopy

To further investigate the practical applicability of FH as a selective sensor for Fe3+, a competition experiment was carried out for FH in the presence of Fe3+ mixed with other metal ions (Na+, K+ Mg2+, Ca2+, Fe2+, Mn2+, Al3+, Ni2+, Cu2+, Zn2+, Co2+, Pb2+, Ru3+). Interestingly, the fluorescence emission intensity was quenched in every case after mixing Fe3+ with each of the candidate metal ions (Fig. 5). Thus, FH shows great promise as a highly selective and sensitive fluorescence turn-off sensor for the detection of Fe3+ even in the presence of other analogous ions (in particular, Fe2+ and Ru3+). Furthermore, based on the general trend in Fig. 5, it is apparent that 3+ cations tend to exhibit stronger binding that effects fluorescence quenching of FH. This may be due to stabilization of the binding with an anion (NO3−); 2 bonds at FH and one bond with anion. Consider, for example Al3+, where the cation can bind in a similar way. This tridentate binding is certainly more stable than the other 2+ cations with bidentate binding. It is also apparent that Fe3+ shows better binding with FH than Fe2+ which can be attributed to the cationic radii, since Fe3+ is much smaller than Fe2+ about half the size of the Fe3+ radius85. When considering 1+ cations it is interesting that Na+ also quenches FH but with K+ to a lesser extent. This is probably related to the single bond with FH that is not very stable. Moreover, Na+ has better electronegativity compared to K+, which one expects promotes better binding with FH.

Figure 5
figure 5

Competitive experiments in the FH + Fe3+ system with potential interfering metal ions. FH (0.5 μM), Fe3+ (50 μM), and other metals (50 μM). Excited at 374 nm and emission measured at 511 nm.

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Proposed sensing mechanism

To study the reasonable binding mode of FH and Fe3+, mass spectrometry analysis has been carried out and supports the formation of a 1:2 FH-Fe3+complex. As illustrated in Fig. S6, FH exhibits an intense protonated peak at m/z 360.21, while in the presence of Fe3+, a peak at m/z 595.55 is observed, which is attributed to the formation of a protonated FH:(Fe3+NO3)2 complex. For the mentioned results above, as well as the Job’s plot (Fig. S4), we suspect that the sensing mechanism for the 1:2 binding modes of the FH-Fe3+complex is as suggested in Fig. 6. IR spectroscopy was used to elucidate the coordination mode between FH and Fe3+ (Fig. S7), shows the FTIR spectra of FH before and after the addition of Fe3+. A shift in the characteristic absorption band in the FTIR spectra confirmed the coordination behavior for FH-Fe3+. Upon the introduction of Fe3+, an extremely broad peak appeared between 3665 and 3125 cm−1, which is attributed to the involvement of nitrogen from the primary amine (NH) and oxygen from furan in the binding of Fe3+. Furthermore, the stretching vibration frequency of the pyrone carbonyl (C=O) at 1720 cm−1 is shifted to 1605 cm−1.

Figure 6
figure 6

Proposed binding mode of FH with Fe3+.

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Fluorescence and UV–vis titration studies of FH with other metal ions (in water/methanol (9:1, v/v))

Fluorescence quenching in protic solvents is a common problem with previously reported fluorescence sensors86, In order to confirm FH is not susceptible to this issue and to demonstrate a real-world sample application, the photophysical properties of sensor FH were examined in a predominantly aqueous environment, water/methanol (9:1, v/v) at 5 µM. This composition of 9:1 v/v water/methanol was at the maximum solubility of FH in water. Changes to the fluorescence properties of FH caused by various metal ions are shown in Fig. 7. The result shows Fe3+ also produces significant quenching in the fluorescent emission of FH. The other tested metals only show relatively insignificant changes, except Co2+, Na+ and K+. So, it can be concluded that FH also has high selectivity for recognition of Fe3+ in a predominantly aqueous solution. The fluorescence spectra of FH (5 µM) in water/methanol (9:1, v/v), in the presence of various concentrations of Fe3+ ion (0.2–8 equiv.), are shown in Fig. 8, which shows quenching in the fluorescent emission of FH when the concentration of Fe3+ is increased. A Job’s plot of FH with Fe3+ also indicates the formation of a 1:2 complex (Fig. S8). A competitive assay (Fig. 9) confirms that FH can still detect Fe3+ even in the presence of other heavy metals. Thus, in a predominantly aqueous solution, FH exhibits high selectivity for Fe3+ over the other tested metal ions except Co2+, Na+ and K+.

Figure 7
figure 7

Fluorescence spectra of FH (5 μM) in the presence of different metal ions (10 equiv.) in water/methanol (9:1, v/v).

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Figure 8
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Fluorescence emission spectra of FH (5 μM) titrated with Fe3+ (0.2–8 equiv.) in water/methanol (9:1, v/v).

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Figure 9
figure 9

Competitive experiments in the FH + Fe3+ system with interfering metal ions. FH (5 μM), Fe3+ (50 μM) and other metals (50 μM) in water/methanol (9:1, v/v). Excited at 374 nm and emission measured at 511 nm.

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Determination of Fe3+ in real water samples

To investigate the applicability of the FH sensor in realistic environmental samples, recovery studies were carried out in mineral drinking water and tap water samples doped with Fe3+, using fluorescence emission spectroscopy. Testing on these water samples was performed without any sample pre-treatment except for the addition of FH, Fe3+ and allowing 1 minute for mixing. From Table 2, we can see that the recoveries of Fe3+ were from 91.5% to 125%. These data indicate that FH as a sensor has significant potential for the practical detection of Fe3+ in various aqueous samples where other potentially competing species are present.

Table 2 Analytical results of FH-Fe3+ in water samples.
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Conclusion

In summary, we have successfully synthesized and for the first time, characterized, the fluorescence properties of furocoumarin derivatives (FH, FCl and FNO2). These were synthesized by mixing 4-hydrocoumarin, benzaldehyde derivatives, and cyclohexyl isocyanide under reflux conditions within 24 h using singlestep high yielding chemistry (82–92% yield). All compounds are purified from recrystallisation preventing the need for time consuming column chromatography and showing that this chemistry is amenable to automated high throughput synthesis and screening technologies. Both FH and FCl produce strong fluorescence intensity whilst FNO2 does not, as a result of strong electron withdrawing from –NO2 causing fluorescence quenching of furocoumarin. Furthermore, the fluorescence study has led us towards a successful demonstration of a novel coumarin-based fluorescent (FH) ratiometric chemosensor, with an LMCT mechanism attributed to the recognition of Fe3+ in methanol and also in water/methanol (9:1, v/v). FH formed 1:2 complexes with Fe3+ and exhibited a fluorescence turn-off response to Fe3+. Extensive competitive selectivity experiments in methanol have been performed for Na+, K+, Mg2+, Ca2+, Mn2+, Fe2+, Al3+, Ni2+, Cu2+, Zn2+, Co2+, Pb2+ and Ru3+ demonstrating that FH has higher selectivity towards Fe3+ (fluorescence turn-off) than other analogous ions and other previously reported Fe3+ sensors (to the best of our knowledge). In an aqueous environment the probe selectivity reduces but the “turn off” effect is still operational confirming water does not fully quench fluorescence. The potential of this sensor has been further highlighted by testing with untreated mineral and tap water samples. This result sets the foundation for a second generation of sensors with improved sensing properties and water solubilizing groups with the real potential of developing a fully aqueous furocoumarin based sensor, which is the subject of future work.

Materials and Methods

Materials

All reagents were purchased from commercial suppliers and used without further purification. The salts used in stock solutions of metal ions were Al(NO3)3.9H2O, CaCl2, CoCl2.6H2O, Cu(NO3)2.4H2O, FeCl2.4H2O, Fe(NO3)3.9H2O, KOH, MgCl2, MnCl2, NaOH, NiCl2.6H2O, Pb(NO3)2, RuCl3 · H2O, Zn(NO3)2 · 6H2O.

Instrumentation

1H NMR (400 MHz) spectra were acquired on a Bruker AVANCE 400 MHz NMR Spectrometer using TMS (tetramethylsilane) as internal standard. All stock solutions of the samples for both UV-Vis and Fluorescence studies were prepared at 0.1 mM in different solvents (ethanol, chloroform and ethyl acetate) and diluted in 10 mL with appropriate concentrations. UV-vis absorption and fluorescence spectra of the furocoumarin derivatives (in solution) were recorded on a CARY 60 UV-Vis spectrophotometer and CARY Eclipse Fluorescence Spectrometer, respectively. Excitation and emission monochromator band pass were kept at 5 nm using a quartz cell cuvette (1 × 1 cm). The absolute quantum yields were calculated using quinine sulfate in 0.1 M H2SO4 as a standard. Fluorescence lifetime measurements were performed with the use of an FLS 1000 Spectrometer (Edinburgh Instruments, Livingston, UK) at room temperature. In these experiments the fluorescence lifetimes of the furocoumarin derivatives in methanol were measured using the photon counting technique (requiring at least 10,000 photons per second to be counted because the signal-to noise ratio becomes unsatisfactory at lower count rates87) with an excitation wavelength set to 374 nm in all the cases. UV-vis absorption and fluorescence spectra of FH and all metal ions were performed with the use of a Cary 5000 UV-Vis-NIR Spectrophotometer (Agilent Technologies) and FLS 1000 Spectrometer (Edinburgh Instruments), respectively. Paper spray ionization mass spectrometry (PSI-MS)88,89,90 was performed on a Waters Xevo TQ-MS (Waters, Wilmslow, UK).

Synthesis of furo [3,2-c] coumarin derivatives

Equimolar amounts of 4-hydroxycoumarin and benzaldehyde derivatives were dissolved in benzene (0.2 M) and heated under reflux (Fig. 10). After 30 minutes, cyclohexyl isocyanide (1 eq.) was added to the reaction mixture and further refluxed for 24 h. The pure compound was obtained by recrystallization from diethyl ether to produce up to 85% yield. These compounds have been reported and the characterization data agree with previous studies18,19.

Figure 10
figure 10

Synthesis of furo[3,2-c]coumarin derivatives.

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2-(Cyclohexylamino)-3-phenyl-4H-furo[3,2-c]chromen-4-one. FH, 92% yield, light yellow powder, m.p. = 120–122 °C, FTIR = 3250 (NH), 2925–2850 (cyclohexane), 1720 (C=O of pyrone), 1570 (C=C of pyrone), 1H NMR = 1.18–2.08(m, 10H), 3.55–3.58 (m, 1H), 4.29 (d, J = 8.32 Hz 1H), 7.27–7.31 (m, 2H), 7.39 (d, J = 4 Hz, 1H), 7.43 (t, J = 8H, 3H), 7.52 (d, J = 8 Hz, 2H), 7.77 (d, J = 8 Hz, 1H), 1H NMR spectrum of FH as shown in Fig. S9. UV-Vis = 375 nm (in ethanol).

2-(Cyclohexylamino)-3-(4-chlorophenyl)-4H-furo[3,2-c]chromen-4-one. FCl, 90% yield, bright crystalline yellow, m.p. = 110–112 °C, FTIR = 3289 (NH), 2930–2857 (cyclohexane), 1707 (C=O of pyrone),1593 (C = C of pyrone), 1H NMR = 1.16–2.07 (m, 10H), 3.57 (br, 1H), 4.21 (s, 1H), 7.33–7.28 (m, 1H), 7.41–7.39 (m, 4H), 7.47 (d, J = 6.4 Hz, 2H), 7.77 (d, J = 7.6 Hz, 1H), 1H NMR spectrum of FCl as shown in Fig. S10. UV-Vis = 375 nm (in ethanol).

2-(Cyclohexylamino)-3-(4-nitrophenyl)-4H-furo[3,2-c]chromen-4-one. FNO2, 85% yield, reddish orange powder, m.p. = 145–147 °C, 3389 (NH), 2929–2851 (cyclohexane), 1736 (C=O of pyrone), 1574 (C=C of pyrone), 1H NMR = 1.19–2.11 (m, 10H), 3.67 (m, 1H), 4.60 (d, J = 7.96 Hz 1H), 7.34 (t, J = 6.80 Hz, 1H), 7.45–7.40 (m, 2H) 7.69 (d, J = 8.72 Hz, 2H), 7.77 (d, J = 7.64 Hz, 1H), 8.22(d, J = 8.64 Hz, 2H), 1H NMR spectrum of FNO2 as shown in Fig. S11. UV-Vis = 380 nm (in ethanol).

Fluorescence spectral responses of FH to metal ions

The analysis was conducted for two different solvent systems: pure methanol and a water/methanol mixture (9:1, v/v). All stock solutions of the furocoumarin (FC) and various metal ions (Mg2+, Ca2+, Mn2+, Fe2+, Fe3+, Al3+, Ni2+, Cu2+, Zn2+, Co2+, Pb2+ and Ru3+) were analyzed at a concentration of 0.001 M, except Na+ and K+ at 0.2 M in methanol. Then, each of the metal ions were diluted to 50 μM, while FH was diluted to 0.5 μM in methanol. For the water/methanol solvent system, FH was diluted to 5 μM.

For testing, FH was mixed with each of the metal ions for up to 1 minute (by stirring until no layers could be visually observed) after which UV-Vis and fluorescence analysis were carried out. The fluorescence emission spectra were recorded from 430 to 700 nm with an excitation wavelength at 374 nm. Both excitation and emission slit widths were set at 1 nm. For the competing analysis, the fluorescence changes of FH in methanol were measured by the treatment of 50 μM Fe3+ ion in the presence of 50 μM other interfering metal ions. All of the background metal ions tested showed no interference with the detection of Fe3+ by competitive experiment.