Rhodamine-based ratiometric fluorescent probes based on excitation energy transfer mechanisms: construction and applications in ratiometric sensing

Abstract

Ratiometric fluorescent probes allow the simultaneous measurement of two fluorescence signals at different wavelengths followed by calculation of their intensity ratio, which can provide more precise measurement results than intensity-based fluorescent probes. Excitation energy transfer is widely used in the design of ratiometric fluorescent probes. Rhodamine is a convenient platform for the construction of “OFF–ON” ratiometric chemosensors. Rhodamine-based ratiometric fluorescent probes based on the excitation energy transfer mechanism can be constructed by conjugated or non-conjugated connections with other chromophores. In this review, we summarized the recent advances regarding rhodamine-based ratiometric fluorescent probes based on excitation energy transfer. We reviewed these probes according to the classification of “through-space” and “through-bond” probes; we focused on the contributions of different donor fluorophores and the types of connections between the energy donors and acceptors.

---

Ruiqi Zhang

Ruiqi Zhang is currently a graduate student in the Environmental and Chemical Engineering College at Tianjin Polytechnic University (Tianjin, China), under the supervision of associate professor Fanyong Yan. His current research interest involves the design of ratiometric fluorescent probes.

Fanyong Yan

Fanyong Yan is an associate professor in the school of Environmental and Chemical Engineering at Tianjin Polytechnic University (PR China). He received his PhD degree from Tianjin University in 2007. His research interests focus on the design and synthesis of molecular probes and sensing materials for temperature-responsive and optical sensors.

Yicun Huang

Yicun Huang is currently a graduate student in the Environmental and Chemical Engineering College at Tianjin Polytechnic University (Tianjin, China). Her research interests include the design and synthesis of fluorescent probes and their applications.

Depeng Kong

Depeng Kong is currently a graduate student in the Environmental and Chemical Engineering College at Tianjin Polytechnic University (Tianjin, China), under the supervision of associate professor Fanyong Yan. His current research interest involves the design of fluorescent probes.

Qianghua Ye

Qianghua Ye is currently a graduate student in the Environmental and Chemical Engineering College at Tianjin Polytechnic University (Tianjin, China). Her research interests include the design and synthesis of carbon dots and their applications.

Jinxia Xu

Jinxia Xu is a lecturer in the school of Environmental and Chemical Engineering at Tianjin Polytechnic University (PR China). She received her PhD degree from Nankai University in 2010. Her research interests focus on the design and synthesis of molecular probes and functional complexes.

Li Chen

Li Chen received her PhD degree in 1999 from Hokkaido University, Japan. She is a professor in the college of Materials Science and Engineering at Tianjin Polytechnic University (PR China). Her main research fields include intelligent polymers, fluorescent chemosensors, biodegradable biopolymers, and nanotechnology.

---

1. Introduction

To date, many organic small molecule-based fluorescent probes for various targets have been described.¹ However, most of these probes only have one signal for detection, which results in major limitations, such as background interference. Ratiometric measurement utilizes the simultaneous measurement of two fluorescence signals at different wavelengths followed by calculation of their intensity ratio, which provides a built-in correction for environmental effects.² Intramolecular charge transfer (ICT) mechanism chemosensors exhibit an intramolecular push–pull electronic effect, which can afford simultaneous recording of the ratio of the signals of two emission intensities at different wavelengths.³ Moreover, these systems exhibit solvent-induced effects and are sensitive to the environment. Excitation energy transfer (EET) is a mechanism describing energy transfer between two chromophores.^4,5 Two fluorophores are linked by a conjugated or non-conjugated spacer that can reduce the influence of such factors. Normally, ratiometric fluorescent probes based on fluorescence resonance energy transfer (FRET) processes are linked by a non-conjugated spacer, and the energy transfer occurs through space (Scheme 1a); probes based on through-bond energy transfer (TBET) processes are linked by rigid conjugated linkers, and the energy transfer occurs through the electronically conjugated linker (Scheme 1b).^6,7 In 2012, Peng's group summarized organic cassettes of energy transfer based on traditional FRET and TBET.⁸ They classified the systems by their fluorophore skeletons and stressed the function of BODIPY, rhodamine, fluorescein, coumarin, and other fluorophores in the construction of the energy transfer systems. Yuan et al. reviewed the rational design and biological applications of synthetic rhodamine-based FRET probes.⁹ They focused on first-generation rhodamine-based FRET probes (where the energy donor is linked to the interaction site) and second-generation rhodamine-based FRET probes (where the energy donor is selectively incorporated into the 4/5-position carboxylic acid group through an appropriate linker to form a FRET platform). In recent years, ratiometric fluorescent probes have undergone rapid development. Furthermore, many novel rhodamine-based energy transfer probes have been successfully designed, synthesized and applied. Due to their outstanding properties, rhodamine-based ratiometric fluorescent probes have received much attention.

Scheme 1 (a) Fluorescence resonance energy transfer, (b) through-bond energy transfer.

Rhodamine fluorophores have excellent photophysical properties, such as large absorption extinction coefficients, sharp fluorescence emissions and high fluorescence quantum yields. Moreover, they can undergo great fluorescence enhancements via a structural change from the spirocyclic state (non-fluorescent) to the ring-open (fluorescent) state, induced by metal ions, inorganic anions,^10–13 etc. Their absorption and emission spectra appear at a longer wavelength (ca. 580 nm); thus, in the FRET system, the rhodamine fluorophore often acts as the energy acceptor.⁸ A conjugated or non-conjugated spacer moiety is chosen as the linker, and other fluorophores such as naphthalimide¹⁴ and coumarin¹⁵ are chosen as energy donors; rhodamine is a convenient platform to construct “OFF–ON” ratiometric chemosensors. Currently, many novel rhodamine-based energy transfer probes are being successfully designed, synthesized and applied.

In this review, we mainly summarized the recent advances in EET systems with rhodamine skeletons, including rhodamine–naphthalimide, rhodamine–coumarin, rhodamine–dansyl, rhodamine–quinoline, and rhodamine and other organic dyes; meanwhile, rhodamine nanoparticles, quantum dots, carbon dots and micelles have also been described.

2. Through-space energy transfer cassettes

2.1 FRET systems with naphthalimide as the energy donor

Naphthalimide dyes have been received much attention due to their outstanding properties, such as excellent chemical stability, a large Stokes shift, high fluorescence quantum yield and strong emission in the visible range. The absorption and emission spectra of naphthalimide can be adjusted by attaching different electron-donating groups via C-4 substitution, such as N-substituted groups, C-substituted groups, and O-substituted groups.¹⁶ At the same time, the emission wavelengths of naphthalimide derivatives are at 450 to 650 nm, which overlap with the absorption of rhodamine. Thus, in the FRET system, naphthalimide fluorophore can act as the energy donor.

Many of these probes are based on analyte promoted spiro-opening of rhodamine, while some other chemosensors exploit special reactions with the FRET process.

Using an ethylenediamine moiety as the linker, Luxami et al. synthesized a reversible naphthalimide–rhodamine-based fluorescent sensor, F1.¹⁷ F1 could act as a ratiometric sensor for Hg²⁺ based on FRET (Fig. 1). F1 displayed a blue fluorescence centered at 485 nm when excited at 410 nm in CH₃CN–HEPES buffer (1 : 1), pH = 7.12 ± 0.1. Upon addition of Hg²⁺ to the solution, the intensity of the fluorescent peak at 485 nm gradually decreased and that of a new fluorescent band centered at 585 nm gradually increased; thus, the FRET process occurred. The ratio of the fluorescence intensities at 585 nm and 485 nm (I₅₈₅/I₄₈₅) exhibited a drastic change from 0.11 to 15.6, a 141-fold variation in the emission ratio. Fang et al. synthesized a similar ratiometric Hg²⁺ probe, F2, based on FRET by connecting 4-propylamine-1,8-naphthalimide with rhodamine 6G ethylenediamine; the limit of detection was found to be 7.91 × 10⁻⁷ M (Fig. 2).¹⁸ Through connecting a pyrrolidine-1,8-naphthalimide directly with rhodamine B hydrazine, Mahato et al. synthesized a ratiometric Hg²⁺/Cr³⁺ probe, F3, based on FRET. The limits of detection for Hg²⁺ and Cr³⁺ were 0.35 and 0.14 ppb, respectively (Fig. 3).¹⁹

Fig. 1 FRET fluorescent probe F1 for Hg²⁺ detection.

Fig. 2 FRET fluorescent probe F2 for Hg²⁺ detection.

Fig. 3 FRET fluorescent probe F3 for detection of Hg²⁺ and Cd³⁺.

Through the introduction of different electron-donating groups by C-4 substitution, naphthalimide–rhodamine fluorescent sensors can show different properties.

Wang et al. synthesized a series of FRET-based fluorescent ratiometric chemosensors, F4a–f (Fig. 4).²⁰ In terms of the degree of separation of Fe³⁺ and Cr³⁺ (F_Fe³⁺/F_Cr³⁺), F4b had the best selectivity toward Fe³⁺. In ethanol/Tris–HCl buffer (9 : 1, v/v) solution; when excited at 420 nm, it only showed an emission band at 520 nm. In the presence of Fe³⁺ ions, F4b showed an emission band at 577 nm, which is the region of rhodamine. The fluorescence ratio at I₅₇₇/I₅₂₀ showed a 17-fold increase in the case of Fe³⁺ complexation, with a detection limit of 0.418 ppm.

Fig. 4 FRET chemosensors F4a–f for Fe³⁺ detection.

Li et al. synthesized a series of fluorescence probes, F5a–c, based on naphthalimide–rhodamine compounds (Fig. 5).²¹ For compound F1b, the small distance between two fluorophores and the lower number of binding sites made it difficult to bind Al³⁺. Meanwhile, Al³⁺ was difficult to bind to F5c; thus, the FRET process was forbidden. The diethylenetriamine linker offers a suitable distance between the energy donor (naphthalimide) and acceptor (rhodamine) and the binding sites. Therefore, the FRET process could occur in F1a. In the absence of Al³⁺ ions, the system of F5a was FRET-OFF; when it was excited with 420 nm in H₂O/EtOH (99 : 1, v/v) buffered (Tris–HNO₃, pH 7.0) solution, an emission at 530 nm appeared. Meanwhile, along with the addition of Al³⁺, a new emission of rhodamine at 580 nm increased; the steady decrease in emission intensity at 530 nm showed that the FRET process occurred. The ratio of the fluorescence intensities at 580 nm and 530 nm (I₅₈₀/I₅₃₀) gradually increased with increasing Al³⁺ concentration and varied from 0.42 to 4.88, an 11.5-fold variation in the emission ratios. The limit of detection was found to be 1.0 × 10⁻⁷ M. Furthermore, confocal laser microscopy studies confirmed that F5a could be used as an imaging probe for detecting Al³⁺ in HeLa cells.

Fig. 5 FRET chemosensors F5a, b and c for Al³⁺ detection.

Tang et al. synthesized a naphthalimide–rhodamine compound, F6, as a ratiometric fluorescent probe for adenosine triphosphate (ATP) detection (Fig. 6).²² The interaction of ATP with F6 through hydrogen bonds and unique π–π stacking interactions triggered the opening of the spirolactam of rhodamine. In the absence of ATP, the system F6 was FRET-OFF; when excited at 420 nm, an emission from naphthalimide at 530 nm appeared, while along with the addition of ATP (FRET-OPEN), a new emission of rhodamine at 580 nm increased and a steady decrease in emission intensity was observed at 530 nm. The emission intensity ratio, I₅₈₀/I₅₃₀, gradually increased with increasing ATP concentration and varied from 0.34 to 10.8, with the concentration of ATP ranging from 0 to 50 μM; the limit of detection was found to be 10 μM. Furthermore, confocal laser microscopy studies confirmed that the probe F6 could also be used as an imaging probe for detecting ATP in HeLa cells.

Fig. 6 Fluorescent probe F6 for ATP detection based on FRET.

Based on the pH dependent spirolactam to ring-opening amide equilibrium of the rhodamine core,^23–26 Georgiev et al. reported a pH probe (F7, Fig. 7).²⁷ Due to the photoinduced electron transfer (PET) quenching process in peripheral 1,8-naphthalimides and the non-fluorescent spirolactam form of rhodamine, in alkaline solution, F7 did not emit light. PET was not feasible at pH < 6.5, and the rhodamine was in the ring-opened form at pH 2 to 6. At pH 6.5, when excited with 400 nm in water/DMF (4 : 1, v/v) solution, an emission band at 520 nm was observed; at pH 2, the emission band at 520 nm decreased while the observed emission shifted to 561 nm, which could be attributed to the energy transfer from 1,8-naphthalimide to the ring opened form of the rhodamine. The energy transfer efficiency was 91%.

Fig. 7 Fluorescent probe F7 for H⁺ detection based on FRET and PET.

Fan et al. reported a naphthalimide–rhodamine FRET probe, F8, as a ratiometric and intracellular pH probe, in which 1,2,3-triazole was identified as an ideal bridge and biocompatible moiety (Fig. 8).²⁸ At pH > 6.2, F8 showed green fluorescence and a yellow color which was mainly ascribed to the optical properties of 1,8-naphthalimide. When the pH changed from 6.20 to 2.00, the intensity of the green fluorescent band at 538 nm gradually decreased and a new pink fluorescent band at 580 nm gradually increased (excitation wavelength at 430 nm). The FRET efficiency was 72%. Furthermore, F8 was highly membrane permeable and could localize in lysosomes.

Fig. 8 Fluorescent probe F8 for H⁺ detection based on FRET.

Yu et al. reported a FRET fluorescent probe, F9, based on the spirolactam of rhodamine through induced hydrazide and oxalaldehyde (Fig. 9).²⁹ In the absence of Cu²⁺, F9 exhibited emission at 525 nm, attributed to the 1,8-naphthalimide donor, when excited at 355 nm in ethanol/water (1 : 9, v/v, 50 mM HEPES, pH 7.4) solution (FRET-OFF). Upon addition of Cu²⁺ to the solution, the intensity of the fluorescent peak at 525 nm gradually decreased and a new fluorescent band at 580 nm gradually increased, indicating that the spirolactam ring was open and the FRET process occurred. The limit of detection was found to be 0.25 μM. At the same time, F9 could also be used as an imaging probe for detecting Cu²⁺ in RAW cells.

Fig. 9 FRET fluorescent probe F9 for Cu²⁺ detection.

Based on the Hg²⁺-promoted ring opening of the spirolactam of the rhodamine moiety, leading to a cyclization reaction of a thiourea moiety, Song et al. designed a FRET probe (F10, Fig. 10).³⁰ When excited in MeOH/water (7 : 3, v/v) solution with 420 nm, an emission peak at 543 nm was observed, which could be assigned to naphthalimide. Upon addition of Hg²⁺ to the solution, the emission peak at 543 nm decreased, and the emission peak of rhodamine at 589 nm appeared and increased remarkably, which showed that the FRET process occurred. The ratio of the fluorescence intensities at 589 nm and 543 nm (I₅₈₉/I₅₄₃) exhibited a great change, from 0.35 to 11.49. The detection limit of probe for Hg²⁺ was 2.11 × 10⁻⁸ M.

Fig. 10 FRET fluorescent probe F10 for detection of Hg²⁺.

F11 was synthesized as a ratiometric Au³⁺ probe. The reaction of rhodamine with N-(2-ethynlphenyl) increased steric strain around the lactam moiety, which increased the ground state energy of the probe and thus accelerated the gold ion-promoted ring-opening process (Fig. 11).³¹ When Au³⁺ was added to excited F11 at 420 nm, it showed an emission band at approximately 587 nm, which is the region of rhodamine. Selectivity and competition experiments showed that this system displayed high sensitivity and excellent selectivity over other metal ions. The detection limit for Au³⁺ was 0.5 ppb. Furthermore, F11 enables fluorescent imaging of gold species in N2A cells.

Fig. 11 Fluorescent probe F11 for Au³⁺ detection based on FRET.

2.2 FRET systems with coumarin as the energy donor

Coumarins, an important class of benzopyrones, are found in many plants and essential oils. Because of their strong light absorption, high fluorescence quantum yield, and large Stokes shift, coumarins have been widely investigated as fluorescent probes.³² Furthermore, many coumarins and their derivatives possess antimicrobial, anti-inflammatory, and anticancer properties;³³ they have been widely used in pharmacology and medicine. With the introduction of variable substituents to the coumarin fragment, the fluorescent properties of these derivatives can be tuned to meet different application requirements.³⁴ Meanwhile, the emission band of coumarin overlaps with the absorption of rhodamine; thus, in rhodamine–coumarin FRET systems, the coumarin fluorophore acts as the energy donor.

Cao et al. synthesized a ratiometric fluorescent chemosensor, F12, containing a coumarin moiety bound to rhodamine hydrazide (Fig. 12).^35,36 F12 can respond to Al³⁺ with two different sets of fluorescence signals. F12–Ca²⁺ complex gave a green emission with a peak centered at 490 nm. When excited at 377 nm, F12–Al³⁺ showed an emission band of rhodamine centred at 580 nm. Al³⁺ ions could displace the Ca²⁺ ions in the F12–Ca²⁺ complex. In the absence of Al³⁺ ions, the system of F12–Ca²⁺ was FRET-OFF, while upon excitation at 377 nm, an emission band from the donor at 490 nm appeared; meanwhile, upon addition of Al³⁺, the FRET process occurred, a new emission band of rhodamine centred at 580 nm increased, and a steady decrease in the emission intensity at 490 nm was observed. The detection limit of Al³⁺ was 9.4 × 10⁻⁹ M. At the same time, F12 could also be used as an imaging probe for detecting Al³⁺ in hepatoma cells.

Fig. 12 Fluorescent probe F12 for Al³⁺ detection based on FRET.

The measurement of pH values inside cells is of great significance. Shen et al. designed a ratiometric pH probe, F13, based on FRET and PET (Fig. 13).³⁷ The coumarin moiety served as a FRET donor with the rhodamine moiety as a FRET acceptor; a 2-aminoethanol moiety was chosen as the linker. The pK_a of F13 was 3.21, suggesting that F13 was suitable for studying acidic environments (pH < 4). At weakly basic pH, the PET process of the N atom of the rhodamine moiety partly quenched the coumarin emission. The PET process was gradually inhibited upon acidification; meanwhile, the rhodamine spirolactam changed to the ring-open form, followed by the FRET process between coumarin and rhodamine. Based on the ratio of the fluorescence intensities at 583 nm and 470 nm (I₅₈₃/I₄₇₀), F13 could be used for the ratiometric detection of pH in the range of 2.20 to 4.20 in buffer (1 : 1, Britton–Robinson buffer/EtOH, v/v) solution. Furthermore, confocal laser microscopy studies confirmed that F13 could also be used as an imaging probe for detecting pH in bacteria. Zhang et al. synthesized a coumarin–rhodamine based chemosensor as a ratiometric pH probe.³⁸ An N-ethylamino piperazine moiety was chosen as the linker. This probe (pK_a = 4.98) could be used in the ratiometric detection of pH in the range of 4.20 to 6.00 in Britton–Robinson buffer solution. At the same time, this probe has been successfully applied in fluorescence imaging in HeLa cells.

Fig. 13 Fluorescent probe F13 for H⁺ based on FRET and PET.

Wang et al. reported a coumarin–rhodamine based FRET fluorescent probe, F14, with a thiourea moiety as the receptor unit and m-phenylenediamine as a linker (Fig. 14).³⁹ Upon gradual addition of Hg²⁺, the emission peak at 478 nm decreased, while a new emission band centered at 587 nm appeared and gradually increased when excited at 440 nm. The ratio of the fluorescence intensities at 587 nm and 478 nm (I₅₈₇/I₄₇₈) exhibited a drastic change from 0.11 to 15.6, which represents a 994-fold variation in the emission ratios. The FRET efficiency was calculated to be 90%, while the detection limit of Hg²⁺ was 3.2 × 10⁻⁹ M. Furthermore, probe F14 was successfully used for recognition of Hg²⁺ in HeLa cells.

Fig. 14 FRET fluorescent probe F14 for detection of Hg²⁺.

Based on the fact that the energy difference of the HOMO and LUMO for the coumarin moiety is lower than that of an azido-capped rhodamine moiety, but significantly higher than that of the rhodamine moiety, Wei et al. took advantage of the unique reduction of an azide moiety with H₂S to synthesize a coumarin–rhodamine-based dyad, F15, for H₂S detection (Fig. 15).⁴⁰ In the absence of S²⁻ ions, the F15 system was FRET-OFF, while upon excitation at 400 nm, an emission from the donor at 465 nm appeared; meanwhile, upon the addition of S²⁻, a new emission of rhodamine at 525 nm increased and showed a steady decrease in emission intensity at 465 nm, indicating that the FRET process occurred. Selectivity and competition experiments showed that this system featured high sensitivity and excellent selectivity over other ions. Furthermore, the probe F15 could detect endogenous production of H₂S in living HEK293 cells.

Fig. 15 Fluorescent probe F15 for H₂S based on FRET.

Although partial success has been obtained in the design of first-generation and second-generation rhodamine-based FRET probes, further improvements in terms of versatility, sensitivity, and synthetic accessibility are required. To address these issues with first-generation and second-generation rhodamine-based FRET probes, third-generation rhodamine-based FRET probes have been designed and synthesized. The design of third-generation rhodamine-based FRET probes involves the utilization of 3-(piperazin-1-yl)-phenol as a raw material, because the piperazine can act as a linker. This approach only yields a single FRET probe; meanwhile, the energy donor is distant from the reaction site.

Guan et al. designed a series of rhodamine–coumarin-based ratiometric fluorescent probes, F16a–c, based on FRET (Fig. 16).⁴¹ F16a, whose energy donor is linked to the interaction site, was not suitable to detect the analyte. In F16b, the interaction site is far away from the donor, unlike F16a; thus, the sensing sensitivity should not be affected. However, F16b was very difficult to synthetize and separate. To solve this problem, F16c was designed by utilization of 3-(piperazin-1-yl)-phenol as a raw material; the piperazine acted as a linker, and the energy donor (coumarin) was distant from the reaction site of Cu²⁺. Thus, the coumarin donor would not affect the interactions between the reaction site and Cu²⁺. This strategy was suitable for the target analytes. In the presence of Cu²⁺ ions, a fluorescence emission band appeared from 481 nm to 581 nm when excited at 410 nm in HEPES buffer solution (pH 7.0, containing 20% CH₃CN as a cosolvent). The ratio of the fluorescence intensities at 581 nm and 481 nm (I₅₈₁/I₄₈₁) exhibited a drastic change from 0.059 to 1.63, a 27.6-fold variation in the emission ratio. At the same time, the chemosensor F16c could also be used as an imaging probe for detecting Cu²⁺ ions in MCF-7 cells. Furthermore, this new FRET technology has great potential for the development of a wide variety of ratiometric fluorescent probes.

Fig. 16 Fluorescent probes F16a, b and c and F16c for the detection of H₂S based on FRET.

Zhang et al. reported two ratiometric HOCl probes, F17a–b, based on FRET. The coumarin moiety acted as a FRET donor, the rhodamine moiety acted as a FRET acceptor, and a piperazine moiety was chosen as the linker (Fig. 17).⁴² F17a took advantage of diacylhydrazine as a detection group, while F17b utilized a rhodamine acid as a detection group. Probe F17a could respond to OCl⁻ under alkaline conditions. In a range of concentrations of OCl⁻, a new emission occurs at 580 nm, and the intensity increases with increasing hypochlorite dose; meanwhile, a steady decrease in emission intensity at 470 nm was observed, indicating that the FRET process occurred. Probe F17a showed an excellent linearity between the intensity ratio (I₅₈₀/I₄₇₀) and the concentration of OCl⁻ from 170 to 230 μM. Probe F17b could rapidly respond to HOCl at pH < 6.0. Probe F17b showed a trend of the change in fluorescence intensity that was opposite to that of probe F17a. Due to the chlorination-induced cyclization properties of rhodamine acid, the fluorescence intensity increased markedly at 470 nm, and a great decrease in emission intensity was observed at 580 nm with increasing hypochlorous acid dose (FRET-OFF). Probe F17b shows an excellent linearity between the intensity ratio (I₄₇₀/I₅₈₀) and the concentration of HOCl from 60 to 160 μM. Hou et al. introduced a triphenylphosphonium moiety to realize the OCl⁻ mitochondrial target ability.⁴³

Fig. 17 Fluorescent probe F17a, b for HOCl based on FRET.

2.3 FRET system with quinoline as energy donor

The emission of quinoline is at 440 nm, which overlaps with the absorption of rhodamine. Qin et al. developed a FRET ratiometric fluorescence probe, F18, which contained rhodamine spirolactam and 8-hydroxy quinoline moieties (Fig. 18).⁴⁴ The N atom of the quinoline ring, with an unshared electron pair, partly quenched the fluorescence emission of quinoline; upon excitation at 350 nm, a weak fluorescence emission at 425 nm was observed. Upon addition of Fe³⁺, the coordination of N atoms and Fe³⁺ resulted in efficient inhibition of the PET process; as a result, the fluorescence increased. At the same time, a new emission of rhodamine at 585 nm increased, which was attributed to FRET from quinoline to rhodamine. The detection limit of Fe³⁺ was 8.3 × 10⁻⁷ M. Similarly, Adhikari et al. took advantage of the ethanediamine moiety as a linker to connect quinoline-2-carboxaldehyde–rhodamine and reported a ratiometric fluorescent probe for Au³⁺ based on FRET.⁴⁵ The detection limit of Au³⁺ was 0.5 nM.

Fig. 18 Fluorescent probe F18 for Fe³⁺ detection based on FRET.

F19, a FRET ratiometric fluorescence probe for Cd²⁺ ions, was synthesized from a rhodamine spirolactam with a quinolone–benzothiazole conjugated dyad (Fig. 19).⁴⁶ In MeOH/H₂O (1/4, v/v, 1 mM HEPES buffer) solution, when excited with 360 nm, an emission from the quinoline dyad at 470 nm appeared; meanwhile, along with the addition of Cd²⁺, the FRET process occurred, a new emission of rhodamine at 585 nm increased, and a steady decrease in emission intensity was observed at 470 nm. The ratio of the emissions at the two wavelengths (I₅₈₅/I₄₇₀) increased from 0.054 to 2.07 with increasing Cd²⁺ concentration; the FRET efficiency was calculated to be 48%, while the detection limit of Cd²⁺ was 2.7 × 10⁻⁷ M.

Fig. 19 Fluorescent probe F19 for Cd²⁺ detection based on FRET.

Through connecting an 8-aminoquinoline derivative with rhodamine spirolactam, Zhou et al. reported a FRET-based ratiometric chemosensor, F20, for Hg²⁺ ion detection (Fig. 20).⁴⁷ An ethanediamine moiety was chosen as the linker. F20 could respond to Hg²⁺ with two different sets of fluorescence signals. In the presence of Hg²⁺ ions, a fluorescence resonance energy transfer phenomenon occurred in F20–Zn²⁺ by exciting the quinolone–zinc complex moiety at 420 nm; upon gradual addition of Hg²⁺ into F20–Zn²⁺ solution, the emission peak of the sensor at 497 nm decreased with concomitant formation of a new peak at 562 nm. Furthermore, the ratiometric change of the fluorescence spectra became evident with a clear isoemission point at 547 nm. The detection limit of Hg²⁺ was 2.95 × 10⁻⁸ M. Sen et al. synthesized a similar ratiometric chemosensor for Hg²⁺ bearing a rhodamine and 8-aminoquinoline derivative moiety; a hydrazine moiety was chosen as the linker.⁴⁸ The FRET efficiency was calculated to be 97.4%. The detection limit was determined and was found to be 90 ppb.

Fig. 20 Fluorescent probe F20 for Hg²⁺ detection based on FRET.

2.4 FRET systems with other organic fluorophores as energy donors

In addition to the several categories of organic fluorophores mentioned above, there are yet other fluorescent dyes with attractive photophysical properties which have been used in energy transfer systems. Dansyl, 1-dimethylamino-5-sulfonylnaphthalene, has been extensively used to fluorescently label proteins, peptides, and lipids due to the environmental sensitivity of its fluorescence and its favorable lifetime.⁴⁹ Furthermore, its broad emission (450 to 600 nm) overlaps perfectly with the absorption of rhodamine; thus, dansyl fluorophore can act as the energy donor in a Dansyl–rhodamine FRET system. The absorption and emission wavelengths for the classical BODIPY chromophore are centered at 470 to 530 nm; extension of the π conjugation could shift the BODIPY emission to a longer wavelength.⁸ Fluorescein has a high quantum yield and photostability; its emission band, at ca. 521 nm, matches well with the absorption spectrum of rhodamine.

With an ethylenediamine moiety as the linker, through connecting dansyl with rhodamine 101, F21 was synthesized as a ratiometric sensor for Fe³⁺ based on the resonance energy transfer process (Fig. 21).⁵⁰ The fluorescence intensity ratio at 605 nm and 515 nm (I₆₀₅/I₅₁₅) exhibited a great change from 0.29 to 62.2, about a 214-fold emission ratio increase due to FRET modulation. The efficiency of the energy transfer was calculated to be 84%; the detection limit of Fe³⁺ was 0.64 μM in CH₃CN–Tris (9 : 1, v/v) buffer solution. Choosing a diethylenetriamine moiety as the linker, Xie et al. synthesized a dansyl–rhodamine based probe for Hg²⁺ based on FRET.⁵¹ The efficiency of energy transfer was calculated to be 65%; the detection limit was 7.7 × 10⁻⁸ M.

Fig. 21 Fluorescent probe F21 for Fe³⁺ detection based on FRET.

Hu et al. connected the dansyl donor and rhodamine acceptor by the click reaction and introduced an ion ligand to report a FRET Cu²⁺ sensor F22 (Fig. 22).⁵² In CH₃CN–H₂O (1 : 1, v/v) buffered solution, a Cu²⁺ titration experiment showed that upon excitation at 420 nm, the fluorescence intensity around 540 nm decreases along with the incremental addition of Cu²⁺; simultaneously, a new emission band around 568 nm gradually increases. The ratio of the emission intensities at 568 and 540 nm (I₅₆₈/I₅₄₀) exhibited a drastic change from 0.6 to 16.8, a 28-fold variation in the case of Cu²⁺ complexation. The detection limit of the sensor was determined to be 0.12 μM. The F22 could also be used as an imaging probe for detecting Cu²⁺ ions in HeLa cells.

Fig. 22 FRET fluorescent probe F22 for detection of Cu²⁺.

The emission of BODIPY was at 510 nm in CH₃CN–H₂O (60 : 40) buffer solution, which overlaps with the absorption of rhodamine. Yu et al. employed an o-phenylenediamine as a NO trapper to synthesize a ratiometric NO sensor F23 (Fig. 23).⁵³ In the absence of NO, F23 exhibited an intense emission band with maxima at 510 nm, which was typical for BODIPY; upon addition of NO solution, the emission peak at 510 nm gradually decreased and a new emission peak at 590 nm of rhodamine gradually increased, which reflected that an efficient FRET process was turned on. The detection limit of NO was determined to be 26 nM. Furthermore, the probe F23 could detect endogenous production of NO in living MCF-7 cells.

Fig. 23 Fluorescent probe F23 for NO detection based on FRET.

Sui et al. reported a ratiometric chemosensor, F24, for Pd²⁺ ion detection (Fig. 24).⁵⁴ F24 was synthesized by connecting 3-amino-2-naphthoic acid directly with rhodamine B ethylenediamine. In EtOH/H₂O (1 : 1, v/v) solution, with increasing Pd²⁺ concentration, the emission of the naphthylamine moiety at 490 nm gradually decreased and a new emission peak of rhodamine appeared at 590 nm. The fluorescence intensity ratios (I₅₉₀/I₄₉₀) of F24 exhibited a linear relationship in the concentration range from 0 μM to 2 μM of Pd²⁺ ions. The detection limit of Pd²⁺ was 45.9 nM. In addition, this probe was successfully applied for detecting Pd²⁺ ions in live mice.

Fig. 24 Fluorescent probe F24 for Pd²⁺ detection based on FRET.

Using an imidazole derivative of the phenanthrene moiety as an energy donor and a rhodamine 6G moiety as an energy acceptor, Reddy et al. synthesized a ratiometric fluorescence sensor, F25, for pH detection based on FRET (Fig. 25).⁵⁵ In PBS buffer–DMSO (98 : 2, v/v) solution, at pH 7.0, F25 was present exclusively in the cyclic lactam form, FRET-OFF, while upon excitation at 400 nm, an emission from the donor at 493 nm appeared; meanwhile, the acyclic xanthene form of F26–H⁺ prevailed at pH 3.5 (FRET-OPEN), the emission peak at 493 nm decreased and the emission peak of rhodamine at 552 nm appeared. The energy transfer efficiency was calculated to be 42%. Furthermore, F25 could also be used as an imaging probe for detecting pH in Hct116 cells.

Fig. 25 Fluorescent probe F25 for pH detection based on FRET.

Kar et al. synthesized an indole functionalized rhodamine derivative, F26, which could specifically bind to Cu²⁺ in the presence of large excesses of other competing ions (Fig. 26).⁵⁶ In the presence of Cu²⁺ ions, a fluorescence emission band appeared from 490 nm to 587 nm upon excitation at 340 nm in CH₃CN/aqueous HEPES buffer (1 mM, pH 7.3; 1 : 4 v/v) medium. The FRET efficiency was calculated to be 51.5%. The detection limit was found to be 3.6 ppb. At the same time, this fluorescent probe could also be used as a fluorescence microscopic imaging probe for Cu²⁺ detection in HeLa cells.

Fig. 26 FRET fluorescent probe F26 for Cu²⁺ ion detection.

Liu et al. reported a FRET ratiometric fluorescence probe F27 for Hg²⁺ detection which comprised an indole–triazole conjugate moiety donor and a rhodamine acceptor, linked by a hydrazine moiety (Fig. 27).⁵⁷ Compound F27 displayed a large shift (from 490 to 590 nm) in its emission spectrum with increasing Hg²⁺ concentration in EtOH–MOPS mixed solvent. The detection limit of Hg²⁺ was 11 nM. Furthermore, F27 could also be used as an imaging probe for Hg²⁺ detection in MCF-7 cells.

Fig. 27 Fluorescent probe F27 for Hg²⁺ detection based on FRET.

F28 is a ratiometric fluorescent Hg²⁺ probe consisting of a phenothiazine donor and a rhodamine acceptor (Fig. 28).⁵⁸ In the presence of Hg²⁺ ions, a fluorescence emission band appears from 520 nm to 580 nm when excited at 415 nm in acetonitrile. The intramolecular energy transfer efficiency from the phenothiazine donor to the rhodamine acceptor was calculated to be 76.8%. The detection limit of Hg²⁺ was 9.2 × 10⁻⁹ M.

Fig. 28 Fluorescent chemosensor F28 for Hg²⁺ detection based on FRET.

F29 is a FRET ratiometric fluorescence mercury probe with a 7-nitrobenz-2-oxa-1,3-diazole (NBD) fluorophore as the energy donor and rhodamine B as an energy acceptor; a 1-(2-aminoethyl) piperazine moiety was chosen as the linker (Fig. 29).⁵⁹ In the presence of Hg²⁺ ions, a fluorescence emission peak appeared from 525 nm to 580 nm upon excitation at 420 nm in CH₃CN–H₂O (9 : 1 v/v) mixture medium. The FRET efficiency was calculated to be 86.5%. The detection limit was found to be 5.7 × 10⁻⁹ M.

Fig. 29 Fluorescent probe F29 for Hg²⁺ detection based on FRET.

Using per-acetyl glycosyl as an energy donor and rhodamine B as an energy acceptor, F30 was synthesized through a click reaction. Fluorescent probe F30 could detect Hg²⁺ based on FRET (Fig. 30).⁶⁰ In the absence of Hg²⁺ ions, the F30 system was FRET-OFF, while upon excitation at 315 nm, an emission from the donor at 470 nm appeared; meanwhile, along with the addition of Hg²⁺, the FRET process occurred, a new emission of rhodamine at 584 nm increased, and a steady decrease in emission intensity at 470 nm was observed in aqueous solution (H₂O/CH₃CN = 4 : 1, v/v). The detection limit of Hg²⁺ was 7.6 × 10⁻⁸ M.

Fig. 30 FRET Fluorescent probe F30 for Hg²⁺ ion detection.

In addition, double ion detection probes and two-photon technology have opened up new prospects for traditional energy transfer systems.

Fan et al. reported the design of ratiometric chemosensor F31 for Al³⁺ ions by connecting a 6-ethoxychromone-3-carbaldehyde chromone with rhodamine 6G hydrazide. Based on the spiro-ring of rhodamine, F31 could respond to Al³⁺ with two different sets of fluorescence signals (Fig. 31).⁶¹ The prepared zinc complex of the sensor could detect Al³⁺ on the basis of the fluorescence resonance energy transfer mechanism, and the detection limit for Al³⁺ was 1.83 × 10⁻⁷ M.

Fig. 31 Fluorescent probe F31 for Al³⁺ and Zn²⁺ ion detection.

The emission peak of fluorescein is at 523 nm, which overlaps perfectly with the absorption of rhodamine B; at the same time, based on the spiro-ring of fluorescein and rhodamine, a fluorescein–rhodamine dyad probe could detect two different ions. Based on Hg²⁺-promoted desulfation followed by a cyclization reaction of thiourea and fluoride ion-selective cleavage of the Si–O linkage, Chereddy et al. reported a fluorescein–rhodamine dyad probe, F32 (Fig. 32).⁶² F32 permitted ratiometric detection of Hg²⁺ and F⁻ ions, individually and collectively, with different fluorescence outputs.

Fig. 32 Fluorescent probe F32 for Hg²⁺ and F⁻ ion detection.

Wang et al. developed a bichromophoric two-photon excited FRET-based ratiometric fluorescent probe, F33 (Fig. 33).⁶³ F33 had a large two-photon absorption cross-section in the 735.8 nm region (359.9 GM). F33 was capable of undergoing efficient FRET following either two-photon (IR laser) or single-photon (UV-visible) excitation. For probe F33, using a 735.8 nm laser as the two-photon (TP) excitation light, the probe was excited and the fluorescence emitted from the energy donor was observed (FRET-OFF); meanwhile, along with the addition of Cu²⁺, the spirolactam ring of rhodamine opened (FRET-OPEN), and the fluorescence emission band of the acceptor could be observed. The energy transfer efficiency was nearly perfect (>99%). Furthermore, F33 could produce a larger Stokes shift and prevent photodamage to biological samples.

Fig. 33 Fluorescent chemosensor F33 for Cu²⁺ detection based on FRET.

2.5 FRET systems with nanoparticles acting as vehicles

To date, many FRET-based chemosensors have been designed with small molecules which contain two fluorophores linked by a non-conjugated spacer.⁶⁴ These small organic molecule sensors usually have high detection sensitivity to analytes; however, most of them are hydrophobic and require organic solvent as a cosolvent to detect analytes in the organic phase. Recently, fluorescent particles have received increasing attention. Fluorescent particles as three-dimensional scaffolds for the development of new tunable and versatile sensing devices have been widely used in biological and environmental fields.^65,66 Notably, FRET systems could also be built into nanoparticles, such as silica nanoparticles, quantum dots, organic particles and inorganic particles.^67–71 These water-dispersible particles with different dyes exhibit high fluorescence brightness, selectivity, and photostability in the aqueous phase.

Mesoporous silica is an excellent host material with good optical transparency in the visible-light region and good biocompatibility. Wu et al. reported a FRET ratiometric fluorescence sensor, F34, through thioRB-esters connected with mesoporous silica nanoparticles (MSN) functionalized with fluorescein isothiocyanate (FITC) (Fig. 34).⁷² In the absence of HOCl, F34 was FRET-OFF, while upon excitation at 488 nm in Na₂HPO₄–citrate buffer solution, an emission from the donor at 525 nm appeared; upon the addition of HOCl, the analyte triggered oxidative opening of the intramolecular thioether, a new emission of rhodamine at 590 nm increased, and a steady decrease in the emission intensity at 525 nm showed that the FRET process occurred. The detection limit was 5 μM. At the same time, F34 could also be used as a fluorescence image probe for detecting HOCl in L929 cells.

Fig. 34 Fluorescent chemosensor F34 for HOCl detection based on FRET.

Liu et al. developed a FRET ratiometric silica core–shell nanoparticle fluorescent probe F35 based on 7-nitro-benzo-furazan (NBD) and a rhodamine B derivative for Hg²⁺ detection (Fig. 35).⁶⁴ The fluorophores were linked to silica core–shell nanoparticles by γ-aminopropyl triethoxysilane. With increasing concentration of Hg²⁺, the ratio of the emission intensity at 578 nm to that at 526 nm (I₅₇₈/I₅₂₆) increased steadily upon excitation at 420 nm in water. The detection limit was 100 nM.

Fig. 35 Fluorescent chemosensor F35 for Hg²⁺ detection based on FRET.

Lu et al. developed a FRET fluorescent probe, F36, for ratiometric sensing of Hg²⁺ in water with periodic mesoporous organosilica (PMO) nanoparticles (NPs) as the scaffold (Fig. 36).⁷³ Silylated rhodamine 6G with a spiro-ring as the energy acceptor was covalently attached on the pore walls of anthracene encapsulated PMO NPs, which played the role of energy donor. In the absence of Hg²⁺ ion, the system of F38 was FRET-OFF; upon excitation at 370 nm in HEPES buffer solution, an emission from anthracene at 450 nm appeared, and a significant increase of the I₅₅₀/I₄₃₀ value was observed upon the addition of Hg²⁺ ion. The detection limit was 6.0 × 10⁻⁹ M.

Fig. 36 Fluorescent chemosensor F36 for Hg²⁺ detection based on FRET.

Graphene quantum dots exhibit an excitation-dependent photoluminescence emission; the emission peak shifts from ca. 440 nm to ca. 550 nm, while the excitation wavelength changes from 280 to 460 nm. Graphene quantum dots could be prepared from graphite powder by a modified Hummers' method. Liu et al. synthesized a FRET ratiometric probe, F37, based on graphene quantum dots (GQDs) and vulcanized rhodamine B derivative moiety (Fig. 37).⁷⁴ An ethanediamine moiety was chosen as the rigid linker. In the absence of Hg²⁺ ions, F37 was FRET-OFF, while upon excitation at 400 nm in HEPES buffered (pH 7.4) water–ethanol (8 : 1, v/v), an emission from graphene quantum dots at 500 nm appeared; meanwhile, upon the addition of Hg²⁺, a new emission of rhodamine at 585 nm increased, and a steady decrease in emission intensity at 500 nm was observed, which suggested that FRET process occurred. The detection limit was 0.23 μM. Furthermore, F37 could be used as a fluorescence microscopic image probe for detecting Hg²⁺ in HeLa cells.

Fig. 37 Fluorescent chemosensor F37 for Hg²⁺ detection based on FRET.

Choosing CdSe (CdZnS) quantum dots as the energy donor, Moquin et al. described a nanosensor, F38, for caspase-1 enzymatic activity ratiometric measurements. F38 consisted of a rhodamine-labeled, caspase-1 cleavable peptide and quantum dots (QDs) (Fig. 38).⁷⁵ In the absence of caspase-1, when excited at 355 nm, they transfer their energy to the rhodamine moiety, and emission was observed at 590 nm. After enzymatic cleavage of the peptide molecules, the acceptor molecules were detached from the QDs, FRET could no longer occur, and the emission of the QDs at 550 nm appeared. Furthermore, F38 could function as a sensitive assay to measure caspase-1 enzymatic activity in microglia cells.

Fig. 38 Fluorescent chemosensor F38 for caspase-1 enzymatic activity detection based on FRET.

Carbon dots (Cds), a new species of fluorescent material, possess great dispersibility in water and good biocompatibility and have received much attention. Kim et al. developed Cds by microwave irradiation of acrylic acid and 1,2-ethylenediamine. The carbon dots exhibited an excitation-dependent photoluminescence emission; when excited at 350 nm, a fluorescence emission band at 410 nm appeared. Kim et al. developed a FRET ratiometric fluorescent probe, F39, with rhodamine 6G and carbon dots (Fig. 39).⁷⁶ In the presence of Al³⁺ ions, the fluorescence emission band shifted from 410 nm to 560 nm when excited at 350 nm in aqueous solution. The detection limit was determined and was found to be 3.8 × 10⁻⁵ M. Furthermore, F39 could be embedded to fabricate a paper-based sensor strip, with which the detection of Al³⁺ was performed in aqueous solution.

Fig. 39 Fluorescent chemosensor F39 for Al³⁺ detection based on FRET.

Polymers with well-designed fluorescent probes have been widely explored in recent decades. Georgiev et al. reported a pH sensitive probe, F40, based on highly water-soluble fluorescent micelles (Fig. 40).⁷⁷ F40 was synthesized by grafting a 1,8-naphthalimide–rhodamine bichromophoric FRET system (RNI) to the PMMA block of a well-defined amphiphilic diblock copolymer, poly(methyl methacrylate)-b-poly(methacrylic acid) (PMMA48-b-PMAA27). The RNI-PMMA48-b-PMAA27 adduct was capable of self-assembling into micelles with a hydrophobic PMMA core, containing the anchored fluorescent probe and a hydrophilic shell composed of PMAA blocks. F40 had greater photostability in comparison with the pure organic dye label and could serve as a highly sensitive pH probe in water. At the same time, this fluorescent probe could be used as a fluorescence image probe for detecting pH in HeLa cells and HEK cells.

Fig. 40 Fluorescent chemosensor F40 for H⁺ detection based on FRET.

Due to its excellent solubility in pure aqueous solution and the good match of its fluorescence emission spectrum with the absorption spectrum of rhodamine 6G, poly[p(phenylene ethynylene)-alt-(thienylene ethyn-ylene)] (PPETE), whose fluorescence is insensitive to environmental factors, has been chosen as an energy donor. Wu et al. reported a Fe³⁺ sensitive FRET-based ratiometric fluorescent probe, F41 (Fig. 41).⁷⁸ In the presence of Fe³⁺ ions, the fluorescence emission band shifted from 442 nm to 538 nm when excited at 400 nm in aqueous solution; the ratio of the emissions at the two wavelengths (I₅₃₈/I₄₄₂) increased from 0.18 to 3.7, and the FRET efficiency was calculated to be 61.8%. The detection limit was determined and was found to be 3.0 × 10⁻⁷ M. Furthermore, confocal laser microscopy studies confirmed that the reagent F41 could also be used as an imaging probe for detecting Fe³⁺ in Hela cells.

Fig. 41 Fluorescent chemosensor F41 for Fe³⁺ detection based on FRET.

3. Through-bond energy transfer cassettes

The fluorescence resonance energy transfer strategy has been widely applied in designing ratiometric probes for bioimaging fields. Unfortunately, for FRET systems, sufficiently large spectral overlap is necessary between the donor emission and the acceptor absorption, which limits the resolution of double-channel images. For ratiometric probes based on TBET, the donor is linked directly by an electronically conjugated bond with the acceptor, and energy transfer occurs through a conjugated bond without the need for spectral overlap,⁸ which could afford a large wavelength difference between the two emissions with improved imaging resolution and higher energy transfer efficiency than that of the classical FRET system.

Based on the Hg²⁺-promoted ring opening of the spirolactam of a rhodamine moiety, leading to a cyclization reaction of a thiourea moiety, Gong et al. synthesized a TBET probe, T1. T1 used vulcanized rhodamine B derivative as the signaling moiety, with a spirolactam structure (nonfluorescent); coumarin moiety acted as the energy donor (Fig. 42).⁷⁹ On excitation of T1 at 420 nm in a buffered (0.01 M HEPES, pH 7.2) H₂O/THF (1 : 1, v/v) solution, an emission band at 470 nm was observed, which could be assigned to the coumarin moiety. Upon gradual addition of Hg²⁺ to the solution of T1, the emission at 470 nm decreased and the emission of rhodamine at 580 nm appeared and increased remarkably, which showed that the TBET process occurred. The ratio of the emission intensities at 589 nm and 543 nm (I₅₈₉/I₅₄₃) exhibited a great change from 0.0133 to 9.280, a 697.7-fold increase. The TBET efficiency was calculated to be 97.4%, and the detection limit of Hg²⁺ was 7 nM. Furthermore, T1 has been preliminarily used for ratiometric imaging of Hg²⁺ in Hela cells.

Fig. 42 Fluorescent chemosensor T1 for Hg²⁺ detection based on TBET.

Bhalla et al. synthesized a ratiometric fluorescence probe, T2, which contained a hexaphenylbenzene derivative as the energy donor and rhodamine B as the energy acceptor (Fig. 43).⁸⁰ T2 was the first report where a donor–acceptor system displayed solvent dependent switching of its energy transfer mechanism in the presence of Hg²⁺ ions. The FRET mechanism was operative in THF and CH₃CN, whereas in protic solvents such as MeOH, TBET was operative. Chopra et al. developed a similar TBET fluorescence probe which could sense picric acid in pure methanol.⁸¹

Fig. 43 Fluorescent probe T2 for Hg²⁺ detection based on TBET and FRET.

Through the conjugated connection of a naphthalimine derivative with rhodamine spirolactam, T3 was synthesized as a ratiometric sensor for Cu²⁺ detection based on TBET (Fig. 44).⁸² Cu²⁺ acts as not only a selective recognizing guest but also as a hydrolytic promoter. In the absence of Cu²⁺, upon excitation at 420 nm, a emission peak of naphthalimine derivative appeared at 537 nm; there is no TBET in the free T3. The addition of Cu²⁺ caused a significant decrease in the emission intensity at 535 nm, while simultaneously, a new emission band at around 577 nm gradually increased; thus, the TBET process occurred. The detection limit of T3 was 3.88 × 10⁻⁷ M. Furthermore, confocal laser microscopy studies confirmed that the reagent T3 could also be used as an imaging probe for detecting Cu²⁺ in MCF-7 cells.

Fig. 44 Fluorescent probe T3 for Cu²⁺ detection based on TBET.

Wang et al. developed a TBET ratiometric fluorescence probe, T4, using a naphthalimine derivative moiety as the energy donor and rhodamine B as the energy acceptor (Fig. 45).⁸³ A p-phenylenediamine moiety was chosen as the rigid linker. In the presence of Fe³⁺ ions, the fluorescence emission band shifted from 535 nm to 586 nm; when excited at 420 nm in a CH₃OH–H₂O (4 : 6, v/v) solution, the ratio of the emissions at the two wavelengths (I₅₈₆/I₅₃₅) exhibited a 30-fold enhancement. The detection limit was found to be 0.105 μM.

Fig. 45 Fluorescent probe T4 for Fe³⁺ detection based on TBET.

Based on the fact that cerium(IV) ammonium nitrate (CAN) specially promotes an irreversible oxidative cyclization of N-acylhydrazones into 1,3,4-oxadiazoles under mild conditions, Goswami et al. developed a TBET ratiometric fluorescence probe (T5) which uses carbazole moiety as the energy donor and rhodamine B as the energy acceptor (Fig. 46).⁸⁴ On excitation of T5 at 450 nm in CH₃–CN : H₂O (7 : 3, v/v), a weak emission band at 483 nm was observed, which could be assigned to the carbazole moiety. Upon gradual addition of Ce⁴⁺, the emission at 483 nm decreased, while the emission of rhodamine at 585 nm appeared and increased remarkably, which showed that the TBET process occurred. The TBET efficiency was calculated to be 98%, and the detection limit was 0.685 μM. Yang et al. developed a similar TBET fluorescence probe which could detect Hg²⁺.⁸⁵ The energy transfer efficiency reaches 99.6%; the detection limit was determined to be 3 ppb.

Fig. 46 Fluorescent probe T5 for CAN detection based on TBET.

The donor fluorophore was connected directly to the rhodamine by a conjugated linker. Zhou et al. synthesized naphthalene derivatives with an appended rhodamine based fluorescent chemosensor, T6 (Fig. 47).⁸⁶ The fluorescence spectrum of compound T6 exhibited an emission at 475 nm, attributed to the naphthalene moiety, when excited at 420 nm in Tris–HCl/CH₃CH₂OH (9 : 1, v/v, 10 mM) aqueous solution. After addition of Cu²⁺, fluorescence appeared at 575 nm. The efficiency of energy transfer was calculated to be 93.7%; the detection limit of Cu²⁺ was 3.0 × 10⁻⁷ M. Furthermore, T6 is a two-photon probe which had a two-photon active absorption cross-section of 115 GM at 475 nm upon excitation at 780 nm. At the same time, T6 could be used as an imaging probe for detecting Cu²⁺ ions in HeLa cells. Furthermore, Zhou et al. synthesized a similar naphthalene derivative appended rhodamine based TBET fluorescent chemosensor for Pd²⁺ with an anthracene appended rhodamine spirolactam as the signaling moiety.⁸⁷ The detection limit was estimated to be 2.3 × 10⁻⁷ M, and the energy transfer efficiency was calculated to be 90%.

Fig. 47 Fluorescent probe T6 for Cu²⁺ detection based on TBET.

4. Conclusion and outlook

In this review, we focused on the recent major contributions to the field of rhodamine-based FRET probes since 2012. These included rhodamine–naphthalimide, rhodamine–coumarin, rhodamine–dansyl, rhodamine–quinoline, and other systems. The ingenious design of the probes endowed them with high detection sensitivity and practicability. Notably, FRET systems can also be built in nanoparticles, such as rhodamine–mesoporous silica, rhodamine–quantum dots, rhodamine–Cds, and rhodamine–organic micelles. Excitation energy transfer cassettes between rhodamine with nanoparticles might provide an alternative approach for constructing FRET-based detection systems for sensing a variety of analytes in aqueous media. These water-dispersible particles with fluorescent probes exhibited high fluorescence brightness, high selectivity, biocompatibility and photostability in the aqueous phase. At the same time, rhodamine-based TBET probes underwent rapid development; many new TBET fluorescence probes have been successfully designed and synthesized, and appropriate connections between the donor and acceptor improved the energy transfer efficiency. Actually, the above-mentioned sensor molecules indicated that rhodamine-based fluorescent probes are feasible for practical use. With the rapid development of fluorescent probes, rhodamine-based EET fluorescent probes will be widely used in ion detection, protein labelling, organelle localization and other applications.

Conflict of interest

The authors confirm that this article content has no conflict of interest.

Acknowledgements

The work described in this manuscript was supported by the National Natural Science Foundation of China (No. 21374078, 51303132), the Tianjin Research Program of Application Foundation and Advanced Technology (No. 15JCYBJC18100, 15JCTPJC59200), and the State Key Laboratory of Separation Membranes and Membrane Processes (No. Z1-201507).

Notes and references

1. L. D. Lavis and R. T. Raines, ACS Chem. Biol., 2008, 3, 142–155.
2. R. Y. Tsien and A. T. Harootunian, Cell Calcium, 1990, 11, 93–109.
3. X. Peng, J. Du, J. Fan, J. Wang, Y. Wu, J. Zhao, S. Sun and T. Xu, J. Am. Chem. Soc., 2007, 129, 1500–1501.
4. J. Y. Liu, H. S. Yeung, W. Xu, X. Li and D. K. P. Ng, Org. Lett., 2008, 10, 5421–5424.
5. M. Kumar, N. Kumar, V. Bhalla, H. Singh, P. R. Sharma and T. Kaur, Org. Lett., 2011, 13, 1422–1425.
6. L. Stryer and R. P. Haugland, Proc. Natl. Acad. Sci. U. S. A., 1967, 58, 719–726.
7. G. S. Jiao, L. H. Thoresen and K. Burgess, J. Am. Chem. Soc., 2003, 125, 14668–14669.
8. J. Fan, M. Hu, P. Zhan and X. Peng, Chem. Soc. Rev., 2013, 42, 29–43.
9. L. Yuan, W. Lin, K. Zheng and S. Zhu, Acc. Chem. Res., 2013, 46, 1462–1473.
10. J. Park, B. A. Rao and Y. A. Son, Fibers Polym., 2015, 16, 953–960.
11. M. Wang, F. Yan, Y. Zou, L. Chen, N. Yang and X. Zhou, Sens. Actuators, B, 2014, 192, 512–521.
12. F. Yan, T. Zheng, D. Shi, Y. Zou, Y. Wang, M. Fu, L. Chen and W. Fu, Sens. Actuators, B, 2015, 215, 598–606.
13. Z. Zhang, C. Deng, L. Meng, Y. Zheng and X. Yan, Anal. Methods, 2015, 7, 107–114.
14. C. Wang, D. Zhang, X. Huang, P. Ding, Z. Wang, Y. Zhao and Y. Ye, Sens. Actuators, B, 2014, 198, 33–40.
15. X. Hu, X. Zhang, G. He, C. He and C. Duan, Tetrahedron, 2011, 67, 1091–1095.
16. R. Jin, Mol. Phys., 2013, 111, 3793–3800.
17. V. Luxami, M. Verma, R. Rani, K. Paul and S. Kumar, Org. Biomol. Chem., 2012, 10, 8076–8081.
18. Y. Fang, Y. Zhou, J. Y. Li, Q. Q. Rui and C. Yao, Sens. Actuators, B, 2015, 215, 350–359.
19. P. Mahato, S. Saha, E. Suresh, R. Di Liddo, P. P. Parnigotto, M. T. Conconi, M. K. Kesharwani, B. Ganguly and A. Das, Inorg. Chem., 2012, 51, 1769–1777.
20. C. Wang, Y. Liu, J. Cheng, J. Song, Y. Zhao and Y. Ye, J. Lumin., 2015, 157, 143–148.
21. C. Y. Li, Y. Zhou, Y. F. Li, C. X. Zou and X. F. Kong, Sens. Actuators, B, 2013, 186, 360–366.
22. J. L. Tang, C. Y. Li, Y. F. Li and C. X. Zou, Chem. Commun., 2014, 50, 15411–15414.
23. X. F. Zhang, T. Zhang, S. L. Shen, J. Y. Miao and B. X. Zhao, J. Mater. Chem. B, 2015, 3, 3260–3266.
24. K. A. Alamry, N. I. Georgiev, S. A. El-Daly, L. A. Taib and V. B. Bojinov, J. Lumin., 2015, 158, 50–59.
25. N. I. Georgiev, M. D. Dimitrova, A. M. Asiri, K. A. Alamry and V. B. Bojinov, Dyes Pigm., 2015, 115, 172–180.
26. N. I. Georgiev, A. M. Asiri, K. A. Alamry, A. Y. Obaid and V. B. Bojinov, J. Photochem. Photobiol., A, 2014, 277, 62–74.
27. N. I. Georgiev, A. M. Asiri, A. H. Qusti, K. A. Alamry and V. B. Bojinov, Dyes Pigm., 2014, 102, 35–45.
28. J. Fan, C. Lin, H. Li, P. Zhan, J. Wang, S. Cui, M. Hu, G. Cheng and X. Peng, Dyes Pigm., 2013, 99, 620–626.
29. C. Yu, Y. Wen, X. Qin and J. Zhang, Anal. Methods, 2014, 6, 9825–9830.
30. J. Song, M. Huai, C. Wang, Z. Xu, Y. Zhao and Y. Ye, Spectrochim. Acta, Part A, 2015, 139, 549–554.
31. H. Seo, M. E. Jun, K. Ranganathan, K.-H. Lee, K.-T. Kim, W. Lim, Y. M. Rhee and K. H. Ahn, Org. Lett., 2014, 16, 1374–1377.
32. M. Tasior, Y. M. Poronik, O. Vakuliuk, B. Sadowski, M. Karczewski and D. T. Gryko, J. Org. Chem., 2014, 79, 8723–8732.
33. S. Z. Ferreira, H. C. Carneiro, H. A. Lara, R. B. Alves, J. M. Resende, H. M. Oliveira, L. M. Silva, D. A. Santos and R. P. Freitas, ACS Med. Chem. Lett., 2015, 6, 271–275.
34. X. Liu, J. M. Cole, P. G. Waddell, T.-C. Lin and S. McKechnie, ACS Med. Chem. Lett., 2013, 117, 14130–14141.
35. L. Cao, C. Jia, Y. Huang, Q. Zhang, N. Wang, Y. Xue and D. Du, Tetrahedron Lett., 2014, 55, 4062–4066.
36. L. Cao, C. Jia, Q. Zhang, D. Chen, C. Zhang and Y. Qian, Chem. Res. Chin. Univ., 2014, 30, 362–367.
37. S. L. Shen, X. F. Zhang, S. Y. Bai, J. Y. Miao and B. X. Zhao, RSC Adv., 2015, 5, 13341–13346.
38. X. F. Zhang, T. Zhang, S. L. Shen, J. Y. Miao and B. X. Zhao, RSC Adv., 2015, 5, 49115–49121.
39. M. Wang, J. Wen, Z. Qin and H. Wang, Dyes Pigm., 2015, 120, 208–212.
40. L. Wei, L. Yi, F. Song, C. Wei, B.-f. Wang and Z. Xi, Sci. Rep., 2014, 4, 4521.
41. X. Guan, W. Lin and W. Huang, Org. Biomol. Chem., 2014, 12, 3944–3949.
42. Y. R. Zhang, X. P. Chen, S. Jing, J. Y. Zhang, Q. Yuan, J. Y. Miao and B. X. Zhao, Chem. Commun., 2014, 50, 14241–14244.
43. J. T. Hou, K. Li, J. Yang, K. K. Yu, Y. X. Liao, Y. Z. Ran, Y. H. Liu, X. D. Zhou and X. Q. Yu, Chem. Commun., 2015, 51, 6781–6784.
44. J. C. Qin, Z. Y. Yang and G. Q. Wang, J. Photochem. Photobiol., A, 2015, 310, 122–127.
45. S. Adhikari, S. Mandal, A. Ghosh, P. Das and D. Das, J. Org. Chem., 2015, 80, 8530–8538.
46. K. Aich, S. Goswami, S. Das, C. Das Mukhopadhyay, C. K. Quah and H. K. Fun, Inorg. Chem., 2015, 54, 7309–7315.
47. X. Zhou, W. Yan, T. Zhao, Z. Tian and X. Wu, Tetrahedron, 2013, 69, 9535–9539.
48. B. Sen, M. Mukherjee, S. Pal, K. Dhara, S. K. Mandal, A. R. Khuda Bukhsh and P. Chattopadhyay, RSC Adv., 2014, 4, 14919–14927.
49. S. Haldar, K. Raghuraman and A. Chattopadhyay, J. Phys. Chem. B, 2008, 112, 14075–14082.
50. P. Xie, F. Guo, R. Xia, Y. Wang, D. Yao, G. Yang and L. Xie, J. Lumin., 2014, 145, 849–854.
51. P. Xie, F. Guo, L. Wang, S. Yang, D. Yao and G. Yang, J. Fluoresc., 2015, 25, 319–325.
52. Z. Hu, J. Hu, Y. Cui, G. Wang, X. Zhang, K. Uvdal and H.-W. Gao, J. Mater. Chem. B, 2014, 2, 4467–4472.
53. H. Yu, L. Jin, Y. Dai, H. Li and Y. Xiao, New J. Chem., 2013, 37, 1688–1691.
54. S. Sun, B. Qiao, N. Jiang, J. Wang, S. Zhang and X. Peng, Org. Lett., 2014, 16, 1132–1135.
55. U. G. Reddy, H. A. Anila, F. Ali, N. Taye, S. Chattopadhyay and A. Das, Org. Lett., 2015, 17, 5532–5535.
56. C. Kar, M. D. Adhikari, A. Ramesh and G. Das, Inorg. Chem., 2013, 52, 743–752.
57. H. Liu, H. Ding, L. Zhu, Y. Wang, Z. Chen and Z. Tian, J. Fluoresc., 2015, 25, 1259–1266.
58. W. Zhao, X. Liu, H. Lv, H. Fu, Y. Yang, Z. Huang and A. Han, Tetrahedron Lett., 2015, 56, 4293–4298.
59. B. Biswal and B. Bag, J. Photochem. Photobiol., A, 2015, 311, 127–136.
60. K. B. Li, H. L. Zhang, B. Zhu, X. P. He, J. Xie and G. R. Chen, Dyes Pigm., 2014, 102, 273–277.
61. L. Fan, J. C. Qin, T. R. Li, B. D. Wang and Z. y. Yang, Sens. Actuators, B, 2014, 203, 550–556.
62. N. R. Chereddy, P. Nagaraju, M. V. N. Raju, K. Saranraj, S. Thennarasu and V. J. Rao, Dyes Pigm., 2015, 112, 201–209.
63. D. Wang, A.-M. Ren, J. F. Guo, L. Y. Zou and S. Huang, RSC Adv., 2015, 5, 98144–98153.
64. B. Liu, F. Zeng, S. Wu, J. Wang and F. Tang, Mikrochim. Acta, 2013, 180, 845–853.
65. I. L. Medintz, H. T. Uyeda, E. R. Goldman and H. Mattoussi, Nat. Mater., 2005, 4, 435–446.
66. X. H. Wang, H. S. Peng, Z. Chang, L. L. Hou, F. T. You, F. Teng, H. W. Song and B. Dong, Mikrochim. Acta, 2012, 178, 147–152.
67. K. Susumu, H. T. Uyeda, I. L. Medintz, T. Pons, J. B. Delehanty and H. Mattoussi, J. Am. Chem. Soc., 2007, 129, 13987–13996.
68. L. Zhu, W. Wu, M. Q. Zhu, J. J. Han, J. K. Hurst and A. D. Q. Li, J. Am. Chem. Soc., 2007, 129, 3524–3526.
69. M. Frigoli, K. Ouadahi and C. Larpent, Chem.–Eur. J., 2009, 15, 8319–8330.
70. J. Chen, F. Zeng, S. Wu, J. Zhao, Q. Chen and Z. Tong, Chem. Commun., 2008, 5580–5582.
71. F. Luo, J. Yin, F. Gao and L. Wang, Mikrochim. Acta, 2009, 165, 23–28.
72. X. Wu, Z. Li, L. Yang, J. Han and S. Han, Chem. Sci., 2013, 4, 460–467.
73. D. Lu, H. Chen, X. Yan, L. Wang and J. Zhang, J. Photochem. Photobiol., A, 2015, 299, 125–130.
74. M. Liu, T. Liu, Y. Li, H. Xu, B. Zheng, D. Wang, J. Du and D. Xiao, Talanta, 2015, 143, 442–449.
75. A. Moquin, E. Hutter, A. O. Choi, A. Khatchadourian, A. Castonguay, F. M. Winnik and D. Maysinger, ACS Nano, 2013, 7, 9585–9598.
76. Y. Kim, G. Jang and T. S. Lee, ACS. Appl. Mater. Interfaces, 2015, 7, 15649–15657.
77. N. I. Georgiev, R. Bryaskova, R. Tzoneva, I. Ugrinova, C. Detrembleur, S. Miloshev, A. M. Asiri, A. H. Qusti and V. B. Bojinov, Bioorg. Med. Chem., 2013, 21, 6292–6302.
78. Y. X. Wu, J. B. Li, L. H. Liang, D. Q. Lu, J. Zhang, G. J. Mao, L. Y. Zhou, X. B. Zhang, W. Tan, G. L. Shen and R. Q. Yu, Chem. Commun., 2014, 50, 2040–2042.
79. Y. J. Gong, X. B. Zhang, C. C. Zhang, A. L. Luo, T. Fu, W. Tan, G. L. Shen and R. Q. Yu, Anal. Chem., 2012, 84, 10777–10784.
80. V. Bhalla, V. Vij, R. Tejpal, G. Singh and M. Kumar, Dalton Trans., 2013, 42, 4456–4463.
81. R. Chopra, V. Bhalla, M. Kumar and S. Kaur, RSC Adv., 2015, 5, 24336–24341.
82. J. Fan, P. Zhan, M. Hu, W. Sun, J. Tang, J. Wang, S. Sun, F. Song and X. Peng, Org. Lett., 2013, 15, 492–495.
83. C. Wang, D. Zhang, X. Huang, P. Ding, Z. Wang, Y. Zhao and Y. Ye, Talanta, 2014, 128, 69–74.
84. S. Goswami, S. Paul and A. Manna, RSC Adv., 2014, 4, 43778–43784.
85. B. Yang and W. Wu, Anal. Methods, 2013, 5, 4716–4722.
86. L. Zhou, X. Zhang, Q. Wang, Y. Lv, G. Mao, A. Luo, Y. Wu, Y. Wu, J. Zhang and W. Tan, J. Am. Chem. Soc., 2014, 136, 9838–9841.
87. L. Zhou, Q. Wang, X. B. Zhang and W. Tan, Anal. Chem., 2015, 87, 4503–4507.

---