Recent Advances in Excimer-Based Fluorescence Probes for Biological Applications

The fluorescent probe is a powerful tool for biological sensing and optical imaging, which can directly display analytes at the molecular level. It provides not only direct visualization of biological structures and processes, but also the capability of drug delivery systems regarding the target therapy. Conventional fluorescent probes are mainly based on monomer emission which has two distinguishing shortcomings in practice: small Stokes shifts and short lifetimes. Compared with monomer-based emission, excimer-based fluorescent probes have large Stokes shifts and long lifetimes which benefit biological applications. Recent progress in excimer-based fluorescent sensors (organic small molecules only) for biological applications are highlighted in this review, including materials and mechanisms as well as their representative applications. The progress suggests that excimer-based fluorescent probes have advantages and potential for bioanalytical applications.


Introduction
Excimer is an excited state dimer produced by molecular photo-association. Excimers are classified into two different types: one is dynamic excimers, the other static excimers [1]. The former is produced from the collision between the excited-state and ground-state molecules, the latter obtained upon photoexcitation of a ground-state dimer generated by weak molecular interactions such as π-π stacking or H-bond. Excimers can also be divided into two categories: intermolecular excimer and intramolecular excimer [2]. The formation of excimers and their fluorescence can be modulated by the concentration of corresponding monomers and external stimuli such as pH, environmental temperature, solvent polarity and chemical species [3][4][5][6]. The exceptional sensitivity of excimers could be used to monitor fine conformation within molecules, which is particularly useful in bioanalysis of nucleic acids [7][8][9][10] and proteins [11][12][13][14].
Excimer-based fluorescent probes have many advantages over conventional monomerbased fluorescent probes. On the one hand, excimer-based fluorescent probes yield specific fluorescence emissions at much longer wavelengths than the corresponding monomer emissions. Consequently, large Stokes shifts are obtained, which can effectively avoid fluorescence self-absorption. On the other hand, excimer-based fluorescent probes exhibit longer fluorescent lifetimes than these of monomers, which benefit measurements especially when the auto-fluorescence of biological species is high. Furthermore, excimerbased fluorescent probes can display dual emissions which can be used for ratiometric signaling. In various strategies for constructing ratiometric probes [15][16][17][18], an elegant approach is to modulate the monomer and its excimer emission with a single fluorophore molecule. Therefore, researchers have made every effort to find excimer-based fluorescent probes for various purposes such as the detection of essential elements [19][20][21][22][23], chemical warfare agents and explosives [24][25][26] and biomolecules [27][28][29][30]. This review is a collection of recent research articles discussed in the context of excimer-based fluorescent probes organized according to their fluorescent core scaffold and with their representative biological applications.

Design Strategies for Excimer-Based Fluorescent Probes
A valuable fluorescent probe for biological application should have some excellent qualities including high selectivity, high sensitivity, accurate quantitative analysis and facile preparation. To this end, probes with some characteristics including "off-on" fluorescence, red or near-infrared (NIR) fluorescence and ratiometric measurement are desired. Offon fluorescence exhibits low background signal as compared to on-off fluorescence, and provides high detection sensitivity. Red and NIR fluorescence supplies less light scattering and minimized tissue auto-fluorescence background. Ratiometric measurement can provide reliable quantitative analysis because of built-in self-calibration for signal correction by measuring the ratio of the intensity profiles at different wavelengths. In this review, two kinds of fluorescent probes are involved: one is off-on probes, and the other ratiometric probes (Figure 1).

Pyrene-Based Probes
Pyrene is an aromatic compound with a strong emission in solution. The aggregation of monomers (λ em = 370-420 nm) can produce excimer (λ em = 450-550 nm) [31], and monomers can be restored through deaggregation [32]. Pyrene excimer emission provides large Stokes shifts (ca. 140 nm) and long fluorescence lifetimes (40-60 ns). These particular properties have been used for the design of many fluorescent probes.
Fischbach and co-workers [33] developed a class of pyrene excimer-based fluorescent probes for the detection of protease. They employed a pyrene derivative (1) and anthraquinone ( Figure 2) as fluorophore and quencher respectively. A peptide segment serving as the protease substrate was equipped with self-complementary peptide nucleic acid (PNA) arms. The formed PNA duplex results in a hairpin-type arrangement, in which both fluorophore and quencher are forced into proximity. The probe mechanism is that the pyrene excimer fluorescence is restored upon protease-catalyzed scission of a peptide substrate ( Figure 3). With incubation with protease MMP-7 [34], a 48-fold enhancement of fluorescence at 480 nm was obtained.  Kraskouskaya and co-workers [35] reported a pyrene excimer-based fluorescent probe for the selective detection of proximally diphosphorylated protein sites. Compound 2 used as probe ( Figure 4) was synthesized by coupling a pyrene to Zn(II)-cyclen macrocycle. The probe mechanism is as follows: Diphosphorylation on proximal residues is required for the activation of a subset of proteins, resulting in pYpY (pY: staining all tyrosine residues) and pTXpY motifs (pT: staining all threonine residues), respectively (X = any amino acid). The probe formed a 1:1 complex with a pX-containing peptide/protein site, and therefore no excimer signal. However, for proximally diphosphorylated peptides, each pX residue can coordinate a Zn(II)-cyclen unit, promoting pyrene interaction and resulting in excimer emission ( Figure 5).  The evaluation of probe 2 was performed in HEPES using mono-and diphosphorylated sites (AYpYAA and ApYpYAA) as models. With 350 nm excitation, the probe produced a distinct shift of fluorescence from 380 to 480 nm in response to diphosphorylated peptides. The intensity of fluorescence at 480 nm was much larger than that of the response to monophosphorylated pY peptides, which suggested that the strong excimer fluorescence is specific to a diphosphorylated motif. Furthermore, titration experiments showed that the maximum excimer signal was obtained from a 2-peptide (2:1) complexation, in which the detection limit is calculated to be 0.6 µM. In addition, the selective response by fluorescence imaging was also observed ( Figure 6), which may broaden the use of the probe. Human immunoglobulin E (lgE) plays a pivotal role in various allergic reactions. Bai and co-workers [36] used pyrene (3) as fluorophore ( Figure 7) for lgE sensing. A aptamer-based probe was constructed with four base pairs at the stem to which pyrene was labeled at the 3 and 5 end, respectively ( Figure 8). Upon binding to lgE, the pyrene moiety at each terminal of the aptamer came into proximity, creating an excimer with emission at 485 nm ( Figure 9). Under optimized conditions, the detection limit for lgE is calculated to be 1.6 nM.   Pak and co-workers [37] developed a chemical reaction-based ratiometric fluorescent probe 4 ( Figure 10) for detection of hypochlorite (OCl − ). Probe 4 was constructed by the conjugation of a pyrene unit as fluorophore and an imidazoline-2-thione as a reactive site. The probe aggregated to excimer in PBS (pH = 7.4) with fluorescence at 480 nm, and upon reaction with OCl − , the reactive site imidazoline-2-thione was oxidized to imidazoline, which exhibited monomer fluorescence at 378 nm. The probe exhibited good sensitivity and the detection limit is calculated to be 0.2 µM.
Ratiometric tracking of endogenous OCl − was conducted in stained Raw 264.7 live cells with probe 4 using two-photon excitation. Endogenous OCl − was produced with ROS inducers (LPS: 100 ng/mL, IFN-γ: 50 ng/mL, and PMA: 10 nM). Upon excitation with 730 nm, the average fluorescence intensity of the stained cells was reduced, and the ratio of F green /F blue is calculated to be 2.7 (Figure 11c,f). However, the ratio of F green /F blue increased to 3.9 when inhibitors such as 4-aminobenzoic acid hydrazide (4-ABAH) [38] or flufenamic acid (FFA) [39] were added (Figure 11d-f). The results suggest that 7 could be applicable to ratiometric fluorescence imaging of OCl − in live cells. Wu and co-workers [40] also reported a chemical reaction-based ratiometric fluorescent probe for the detection of OCl − . Probe 5 was constructed by the conjugation of pyrene and 1,2,3,3-Tetramethyl-3H-indolium. The double bond between pyrene unit and indolium moiety in probe 5 can be broken by OCl − to generate pyrenecarboxaldehyde, the latter forming excimer in PBS (pH 7.4) ( Figure 12). This process correspondingly led to fluorescence changes from 613 nm to 470 nm and thus affords the ratiometric fluorescent detection of OCl − . The detection limit is determined to be 0.35 µM.  Endogenous ClO − in RAW 264.7 live cells was obtained by PMA (phorbol myristate acetate) [41]. As shown in Figure 14, red fluorescence was observed when the RAW 264.7 cells were incubated only with probe 5. However, when incubated with 2 mg/mL PMA and 5, the cells displayed blue fluorescence.
Fluorescence resonance energy transfer (FRET) is a nonradiative process and occurs when the excited-state energy of the donor fluorophore is transferred to the acceptor fluorophore in close proximity (≤10 nm) through long-range dipole-dipole interactions [42,43]. Wu and co-workers [44] constructed a FRET-based fluorescent probe for ratiometric quantitative monitoring of pH changes in living cells. Probe 6 was composed of two pyrene units and one fluorescein moiety conjugated with a linker (Figure 15), in which a bispyrene moiety served as the energy donor and fluorescein as the energy acceptor. The fluorescence at 526 nm (F 526 ) increased upon increasing pH values, while the fluorescence at 459 nm (F 459 ) decreased concomitantly; the relative ratio of F 526 /F 459 increased from 0.26 to 5.82 over the pH range of 3.0-10.0.  The applicability of probe 6 to quantifying intracellular pH was carried out in HeLa cells with H + /K + ionophore nigericin [45]. The intensity of fluorescence from pyrenyl excimer ( Figure 16, Channel 1) decreased, whereas the fluorescence from fluorescein ( Figure 16, Channel 2) increased with the increase of the pH value. It is worth noting that the color of cell imaging displayed a significant change from blue to green when intracellular pH was changed from 6 to 7, which may satisfy the requirement for real-time observations. The relative ratio of intracellular fluorescence intensities (F channel2 /F channel1 ) increased from 0.27 to 2.25 when the pH value increased from 4.0 to 8.0 in HeLa cells.

Perylene-Based Probes
Perylene and its derivatives have attracted significant interest due to their outstanding optical and electronic properties [46][47][48]. They have low LUMO orbitals, prominent photo-/thermal stability and high fluorescence quantum yields. In addition, the π-stacked perylene derivatives have strong electronic communications among the individual chromophores in close proximity in both their ground and excited states [49,50], resulting in excimers/aggregates with distinct red fluorescence [51][52][53]. A growing number of reports have utilized perylene derivatives as fluorescence probes for tracking, biolabeling, base-mismatch detection, biomolecule sensing and bioimaging [54][55][56][57][58]. Niu and co-workers [59] developed a turn-on benzoperylene excimer-based probe for wash-free cell membrane fluorescence imaging. Probe 7 was constructed with a hydrophilic spermine chain and a hydrophobic benzoperylene fluorophore group ( Figure 17). Probe 7 showed a very weak emission in monomer, but strong emission (λ = 607 nm) in excimer, and the excimer emission exhibited sensitivity to the micro-environment. After MCF-7 live cells were incubated with probe 7 (10 µM) for 5 min, a remarkable fluorescence in the cell membrane was observed ( Figure 18). The fluorescence was derived from the aggregation of probe 7 in the membrane due to both electrostatic and hydrophobic interactions between the probe and the lipid membrane. Furthermore, probe 7 exhibited some advantages of excellent solubility, good selectivity and large Stokes shift. Molecular self-assembly is an important approach in the construction of functional materials for biological application. Perylene and its derivatives exhibit distinct fluorescence in solution and in the aggregation state, and the fluorescence can be fine-tuned by exoteric factors [60][61][62]. Jana and co-workers [63] reported a new strategy for enhancement of excimeric NIR fluorescence by morphology tuning of self-assembled perylene derivative from nanoparticles to colloidosomes. Perylene derivative 8 (Figure 19) was synthesized and used as fluorescent dye, which displayed entirely distinct fluorescence in a different morphology by self-aggregation. Probe 8 showed a green emission (545 nm) in a monomer state and red emission (670 nm) in the aggregation (excimer). With a biocompatible nonionic surfactant pluronic F127, two micelles MM-8@plu127 (emission at 575 nm) and EM-8@Plu127 (emission at 670 nm) were obtained. As a powerful tool to validate identity, fingerprint identification has been used in a variety of fields including access control, forensic investigation and medical diagnostics [64,65]. Based on perylene excimer fluorescence, Wang and co-workers [66] developed a probe for on-site detection of latent fingerprints. Probe 9 is composed of one perylene core and four positively charged quaternary ammonium groups ( Figure 21).  The fluorescence image of the latent fingerprint was observed when a substrate containing a latent fingerprint was submerged into the developer solution (0.2 mM 9 in 20 mM Tris-HCl buffer, pH 7.4) upon excitation with 365 nm light ( Figure 22). Study of the mechanism showed that probe 9 displayed monomer fluorescence at a low concentration but excimer fluorescence at a high concentration. The fluorescence in the latent fingerprint resulted from the excimer of probe 9 due to the aggregation induced by electrostatic and hydrophobic interactions between the probe and fatty acids and other organic components in the fingerprint residues. The control experiment confirmed that no developed fingerprint fluorescence image was observed when a negatively charged perylene probe was used instead of probe 9. DNA methylation is a biochemical process in which a methyl group is added to cytosine or adenine bases of DNA nucleotides; this process is catalyzed by DNA methyltransferase (MTase) in the presence of S-adenosylmethionine (SAM). Thus, sensing DNA MTase activity and its inhibitor screening are of great importance for fundamental biochemical research, drug discovery and diagnosis of genetic diseases [67]. Wang and co-workers [68] designed a perylene excimer-based probe 10 for selective sensing of DNA MTase activity with the following working principle: A strong excimer fluorescence of probe 10 was induced by a cationic polymer (polycation) via electrostatic interactions. Upon addition of single-stranded DNA (an anionic polymer), strong electrostatic interactions between the polycation and the DNA weakened the binding of probe 10 to the polycation, resulting in the excimer fluorescence decreasing and monomer fluorescence increasing ( Figure 23). DNA adenine methylation (dam) MTase and the restriction endonuclease DpnI were chosen as the model MTase and endonuclease, respectively. When the 3 -OH terminus (3 -ddC) of a duplex DNA is removed, the DNA strands are not elongated by terminal deoxynucleotidyl transferase (TdT); in this case, the monomer fluorescence is detected. In the presence of MTase and endonuclease, the DNA is specifically methylated and cleaved into single-stranded fragments with new 3 -OH termini; in this case, the excimer fluorescence is produced.
Probe 10 exhibited high sensitivity and good selectivity. Maximum F monomer /F excimer value was obtained after 2 h of enzymatic reaction with 80 U/mL dam MTase. The F monomer /F excimer value increased gradually with the increase of dam MTase concentration, and the detection limit for dam MTase is calculated to be 0.2 U/mL ( Figure 24). Control experiments demonstrated that other methyltransferases such as M.SssI MTase and HpaII MTase could not induce noticeable changes in the ratio of F monomer /F excimer . Acetylcholinesterase (AChE) plays an essential role in the catalysis of hydrolysis of the neurotransmitter acetylcholine [69]. The decrease in the level of acetylcholine boosts the assembly of amyloid β peptides into amyloid fibrils and generates Alzheimer's disease (AD) [70,71]. Thus, it has great practical significance to create selective and sensitive probes for the detection of AChE. He and co-workers [72] employed 10 to construct a sensitive ratiometric fluorescence probe for the assay of AChE activity based on the monomerexcimer transition of probe 10 ( Figure 25). The probe mechanism is as follows: probe 10 mainly existed in a monomer state in a buffer solution with two fluorescence peaks at 548 nm and 587 nm, respectively, aggregated to supramolecular assemblies by mixture with lauroylcholine (employed as the substrate of AChE) and lauric acid, resulting in a redshifted excimer fluorescence at 680 nm. Upon addition of AChE, the excimer transferred to the monomer by the hydrolysis of lauroylcholine to lauric acid and choline; as a result, the fluorescence change of the excimer-monomer transition was observed. The optical response of probe 10 to AChE is represented in Figure 26. In MOPS buffer solution (pH 7.5), the excimer fluorescence at 680 nm decreased gradually; meanwhile, the monomer fluorescence increased gradually with increased concentration of AChE (Figure 26a). The intensity ratios (F monomer /F excimer ) of probe 10 plotted against the concentrations of AChE display a linear relationship at AChE concentration range from 5-150 mU/mL (R 2 = 0.999). The limit of detection is calculated to be 5 mU/mL (Figure 26b). Heparinase involves in various biological processes including angiogenesis, inflammation, tumor invasion and metastasis [73,74]. Li and co-workers [75] used probe 10 and cationic silver nanoparticles (Ag NPs) to construct a ratiometric fluorescence probe for sensing heparin and heparinase ( Figure 27) with the following mechanism: A strong excimer fluorescence of probe 10 was induced by cationic Ag NPs which were stabilized by a ligand MUTAB via strong electrostatic interactions between them. These strong electrostatic interactions between the probe and MUTAB-Ag NPs were weakened when negative-charged heparin was adsorbed on the surface of MUTAB-Ag NPs, which blocked the formation of probe excimer, resulting in the decrease of F excimer /F monomer ( Figure 28A), whereas the degradation of heparin by heparinase facilitated the formation of the probe excimer, which resulted in the increase of F excimer /F monomer ( Figure 28B). The probe exhibited good sensitivity and excellent selectivity; the linear relationships for heparin and heparinase are in range from 0-75 nM and 0-0.25 U/mL, respectively. Metal-organic framework (MOF) [76] is of great interest because of its potential applications in many fields due to its porous structure and tunable pore size. Zhou and co-workers [77] reported a ratiometric fluorescent probe for adenosine triphosphate (ATP) detection by encapsulating 10 into zeolitic imidazolate framework-8 (ZIF-8) nanocrystals. It was found that the formed 10@ZIF-8 exhibited significant excimer fluorescence at 700 nm; the excimer fluorescence was, however, reduced upon the addition of ATP due to strong binding between ATP and Zn 2+ . The strong binding between ATP and Zn 2+ triggered the decomposition of 10@ZIF-8, which resulted in the monomer fluorescence at 549 nm ( Figure 29).  The fluorescence responses to ATP exhibited that the excimer fluorescence decreased gradually with the increase in the ATP concentration and a good linear relationship (R 2 = 0.997) between the F excimer /F monomer value and the ATP concentration (0-175 µM) was obtained. The detection limit for ATP is calculated to be 10 µM.
The application of the probe to quantify ATP in cell lysates was conducted. Cell lysates (5%) were spiked with ATP in different concentrations. The value of F excimer /F monomer was linearly related (R 2 = 0.992) to the ATP concentration in the range from 0-150 µM ( Figure 30). The concentration of ATP in the cell lysates was calculated to be 3.1 mM.

Benzothiazole-Based Probes
Recently, Kim's group and Bouffard's group [78] reported two benzothiazole-based excimers with red fluorescence and large Stokes shifts. They synthesized two benzothiazole derivatives A and B (Figure 31), and found that both A and B exhibited aggregation in aqueous solution (acetonitrile-water = 1:9, v/v) resulting in excimer fluorescence at 618 nm and 659 nm, respectively.  Probe 11 can also be used as a staining agent for the gel electrophoresis of DNA ( Figure 33). Plasmid DNA was separated on agarose gel before staining with a solution of 4 and rinsing with water. Red emission bands indicating electrophoretic DNA migration were observed. Caspases play essential roles in the initiation and execution of the apoptotic pathway as well as in inflammation. Accurate assessment of caspase activity can provide valuable information for biomedical research and drug development [79][80][81]. Kim and co-workers [82] developed a benzothiazole-based excimer fluorescent probe for the detection of caspase-3. Probe 12 was constructed by a fluorophore (CV-NH 2 ) and a caspase-3 recognition peptide (DEVD) through an amide bond. The probe displayed weak fluorescence in aqueous media. The cleavage of DEVD peptide by caspase-3 produced the CV-NH 2 residue, which aggregated in aqueous solution and resulted in excimer fluorescence ( Figure 34). The response to caspase-3 was explored by monitoring the fluorescence change of Ac-DEVD-NH-CV. The red fluorescence at 660 nm increased upon the addition of caspase-3. The probe exhibited high selectivity for the detection of caspase-3 and the quantitative analysis of caspase-3 with probe 12 was obtained by the ratio of F 660 /F 541 . The ratio (F 660 /F 541 ) showed 95-fold enhancement incubation with caspase-3 from 0 to 150 ng/mL. A linear relationship in the range from 0-3 ng/mL (R 2 = 0.9938) was obtained and the limit of detection for caspase-3 was determined to be 5.1 pg/mL (0.17 pM).
In vitro imaging of caspase-3 with probe 12 was performed in HeLa living cells. A protein delivery system loaded with 150 ng/mL caspase-3 onto AuNP-His-Apt-composite, and was used to deliver caspase-3 into cells. A red fluorescence was observed when the 12loaded cells treated with AuNP-His-Apt-caspase-3 complex [83] (Figure 35E). By contrast, no fluorescence was observed without the complex ( Figure 35B). Furthermore, significantly decreased fluorescence was detected when the 12-loaded cells were pretreated with the inhibitor Z-VAD-fmk [84] before incubation with the complex ( Figure 35H). Results showed that the probe can permeate efficiently into the cells and be activated by caspase-3. Costaining with the DNA indicated that the probe accumulated in the cytosolic compartment of the cells.

Conclusions and Outlook
In the past decade, considerable progress has been made in exploring excimer-based fluorescent probes for the detection of bioactive molecules and biological process due to convenient availability, high sensitivity and superior bioimaging capability. Many outstanding excimer-based fluorescent probes [85][86][87][88][89][90][91] are not involved herein due to the length of the article. In this review, fluorescent probes based on organic small-molecules are summarized (Table 1) including design strategies, reaction mechanism and applications to biological detection or imaging.
Although great progress has been made, however, some problems remain for practical applications: most pyrene-based probes suffer from short excitation and emission wavelength which limit their applications in biological system. Perylene-based probes exhibit poor solubility in both organic solvent and aqueous solution. In addition, the types of molecules with excimer emission are not rich enough, and more novel molecular structures with excimer emission need to be found and studied.
From a practical point of view, the new fluorescent probes based on excimer emission will be further explored in the future and several characteristics should be considered. First, highly sensitive, selective and accurate detection is required especially in early-stage diagnoses, which can reduce the mortality rate. Second, for the in vivo detection and imaging, NIR or multi-photon fluorescent probes are desired because of their low background signal and deep penetration depth. Third, quantitative detection is needed since bioactive molecules exhibit different levels of activity at different stages of disease development. In addition, solubility, stability, cell penetrability and cytotoxicity are important parameters in practical applications, which also need to be considered in the design of probes.