Photoinduced electron transfer is the most popular principle in the design of fluorescence molecular sensors. The characteristic structure of PET sensors includes three parts as shown in Figure 1:

Figure 1: The general view of the principle of PET fluorescence molecular sensor.

In the PET sensors, photoinduced electron transfer makes the transfer of recognition information to fluorescence signal between receptor and fluorophore come true. Figure 1 shows the detailed process of how PET works in the fluorescence molecular sensor. The receptor could provide the electron to the vacated electoral orbital of the excited fluorophore. The excited electron in the fluorophore could not come back the original orbital, resulting in the quenching of fluorescence emission. The coordination of receptor and guest decreased the electron donor ability of receptor reduced or even disrupted the PET process, then leading to the enhancement of intensity of fluorescence emission. Therefore, the sensors had weak or no fluorescence emission before the coordination. However, the intensity of fluorescence emission would increase rapidly after the coordination of receptor and gust.

Intramolecular charge transfer (ICT) is also named photoinduced charge transfer. The characteristic structure of ICT sensors includes only the fluorophore and recognition group, but no spacer. The recognition group directly binds to the fluorophore. The electron withdrawing or electron donating substituents on the recognition group plays an important role in the recognition. When the recognition happens, the coordination between the recognition group and guest affects the electron density in the fluorophore, resulting in the change of fluorescence emission in the form of blue shift or red shift.

When the two fluorophores are in the proper distance, an intermolecular excimer can be formed between the excited state and ground state. The fluorescence emission of the excimer is different with the monomer and mainly in the form of new, broad, strong, and long wavelength emission without fine structures. The proper distance determines the formation of excimer, therefore modulation of the distance between the two fluorophores becomes crucial in the design of the sensors based on this mechanism. The fluorophores have long lifetime in the singlet state to be easily forming the excimers. They are often used in such sensors.

FRET is a popular principle in the design of the fluorescence molecular sensor. In one system, there are two different fluorophores, in which one acts as a donor of excited state energy to the receptor of the other. As shown in Figure 2, the receptor accepts the energy from the excited state of the donor and gives the fluorescence emission, while the donor will return back to the electronic ground state. There are three factors affecting the performance of FRET. They are the distance between the donor and the acceptor, the proper orientation between the donor emission dipole moment and acceptor absorption moment, and the extent of spectral overlap between the donor emission and acceptor absorption spectrum (Figure 3).

Figure 2: A schematic fluorescence resonance energy transfer system.

Figure 3: Diagram showing the spectral overlap for fluorescence resonance energy transfer system.

Fluorescence is a process involving the emission of light from any substance in the excited states. Generally speaking, fluorescence is the emission of electromagnetic radiation (light) by the substance absorbed the different wavelength radiation. Its absorption and emission is illustrated in the Jablonski diagram (Figure 4), a fluorophore is excited to higher electronic and vibrational state from ground state after excitation. The excited molecules can relax to lower vibrational state due to the vibrational relaxation and, then further retune to the ground state in the form of fluorescence emission.

Figure 4: Jablonski diagram of fluorescence.

Most spectrofluorometers can record both excitation and emission spectra. They mainly consists of four parts: light sources, monochromators, optical filters and detector (Figure 5).

Figure 5: Schematic representation of a fluorescence spectrometer.

As a PET sensor 2-{5-[(2-{[bis-(2-ethylsulfanyl-ethyl)-amino]-methyl}-phenylamino)-methyl]-2-chloro-6-hydroxy-3-oxo-3H-xanthen-9-yl}-benzoic acid (MS1) (Figure 6) shows good selectivity for mercury ions in buffer solution (pH = 7, 50 mM PIPES, 100 mM KCl). From Figure 7, it is clear that, upon the increase of the concentration of Hg²⁺ ions, the coordination between the sensor and Hg²⁺ ions disrupted the PET process, leading to the increase of the intensity of fluorescence emission with slight red shift to 528 nm. Sensor MS1 also showed good selectivity for Hg²⁺ ions over other cations of interest as shown in the right bars in Figure 8; moreover, it had good resistance to the interference from other cations when detected Hg²⁺ ions in the mixture solution excluding Cu²⁺ ions as shown in the dark bars in the Figure 8.

Figure 6: Structure of the PET fluorescence sensor 2-{5-[(2-{[bis-(2-ethylsulfanyl-ethyl)-amino]-methyl}-phenylamino)-methyl]-2-chloro-6-hydroxy-3-oxo-3H-xanthen-9-yl}-benzoic acid.

Figure 7: Fluorescence spectra of sensor MS1 (1 µM) upon addition of Hg²⁺ (0 - 3 µM) in buffer solution (pH = 7, 50 mM PIPES, 100 mM KCl) with an excitation of 500 nm.

Figure 8: The selectivity of MS1 for Hg²⁺ ions in the presence of other cations of interest. The light bars represent the emission of MS1 in the presence of 67 equiv of the interested cations. The dark bars represent the change in integrated emission that occurs upon subsequent addition of 67 equiv of Hg²⁺ to the mixed solution.

2,2',2'',2'''-(3-(benzo[d]thiazol-2-yl)-2-oxo-2-H-chromene-6,7-diyl) bis(azanetriyl)tetrakis(N-(2-hydroxyethyl)acetamide) (RMS) (Figure 9) has been shown to be an ICT fluorescence sensor. From Figure 10, it is clear that, with the gradual increase of the concentration of Hg²⁺ ions, fluorescence emission spectra revealed a significant blue shift, which was about 100-nm emission band shift from 567 to 475 nm in the presence of 40 equiv of Hg²⁺ ions. The fluorescence change came from the coexistence of two electron-rich aniline nitrogen atoms in the electron-donating receptor moiety, which prevented Hg²⁺ ions ejection from them simultaneously in the excited ICT fluorophore. Sensor RMS also showed good selectivity over other cations of interest. As shown in Figure 11, it is easy to find that only Hg²⁺ ions can modulate the fluorescence of RMS in a neutral buffered water solution.

Figure 9: Structure of the ICT fluorescence sensor 2,2',2'',2'''-(3-(benzo[d]thiazol-2-yl)-2-oxo-2-H-chromene-6,7-diyl)bis(azanetriyl) tetrakis(N-(2-hydroxyethyl)acetamide) (RMS).

Figure 10: Fluorescence spectra of RMS (5 µM) upon addition of Hg²⁺ (0 µM to 200 µM) in 0.05 M phosphate-buffered water solution (pH 7.5) with an excitation of 390 nm.

Figure 11: Fluorescence response of 10 µM RMS in the presence of 20 equiv of different cations of interest at the same condition: control (0), Cd²⁺ (1), Hg²⁺ (2), Fe³⁺ (3), Zn²⁺ (4), Ag⁺ (5), Co²⁺ (6), Cu²⁺ (7), Ni²⁺ (8), and Pb²⁺ (9).

The (NE,N'E)-2,2'-(ethane-1,2-diyl-bis(oxy))bis(N-(pyren-4-ylmethylene)aniline) (BA) (Figure 12) is the excimer fluorescence sensor. As shown in Figure 13, when BA existed without mercury ions in the mixture of HEPES-CH₃CN (80:20, v/v, pH 7.2), it only had the weak monomer fluorescence emission. Upon the increase of the concentration of mercury ions in the solution of BA, a strong excimer fluorescence emission at 462 nm appeared and increased with the change of the concentration of mercury ions. From Figure 14, it is clear that BA showed good selectivity for mercury ions. Moreover, it had good resistance to the interference when detecting mercury ions in the mixture solution.

Figure 12: Structure of the excimer fluorescence sensor (NE,N'E)-2,2'-(ethane-1,2-diyl-bis(oxy))bis(N-(pyren-4-ylmethylene) aniline) (BA).

Figure 13: Fluorescence spectra of BA (1 µM) upon addition of Hg²⁺ (0 µM to 10 µM) in the mixture of HEPES-CH₃CN (80:20, v/v, pH 7.2) with an excitation of 365 nm.

Figure 14: Fluorescence response of BA (1 µM) with 10 equiv of other cations of interest in the same condition. Bars represent the final (F) over the initial (F₀) integrated emission. The red bars represent the addition of the competing metal ion to a 1 µM solution of BA. The blue bars represent the change of the emission that occurs upon the subsequent addition of 10 µM Hg²⁺ to the above solution.

The calix[4]arene derivative bearing two pyrene and rhodamine fluorophores (CPR) (Figure 15) is a characteristic FRET fluorescence sensor. Fluorescence titration experiment of CPR (10.0 μM) with Hg²⁺ ions was carried out in CHCl₃/CH₃CN (50:50, v/v) with an excitation of 343 nm. As shown in Figure 16, upon gradual increase the concentration of Hg²⁺ ions in the solution of CPR, the increased fluorescence emission of the ring-opened rhodamine at 576 nm was observed with a concomitantly declining excimer emission of pyrene at 470 nm. Moreover, an isosbestic point centered at 550 nm appeared. This change in the fluorescence emission demonstrated that an energy from the pyrene excimer transferred to rhodamine, resulting from the trigger of Hg²⁺ ions. Figure 17 showed that CPR had good resistance to other cations of interest when detected Hg²⁺ ions, though Pb²⁺ ions had little interference in this process.

Figure 15: Structure of the FRET fluorescence sensor calix[4]arene derivative (CPR) bearing two pyrene and rhodamine fluorophores.

Figure 16: Fluorescence spectra of CPR (10.0 μM) in CHCl₃/CH₃CN (50:50, v/v) upon addition of different concentrations of Hg(ClO₄)₂ (0 μM to 30 μM).

Figure 17: Competition experiment of 10.0 μM CPR at 576 nm with 10 equiv of other cations of interest in the presence of Hg²⁺ (3 equiv) in the same condition. F₀ and F denote the fluorescence intensity of CPR and Hg²⁺ ions and the interested metal ions in the presence of CPR and Hg²⁺ ions.