Cellular fluorescence imaging based on resonance energy transfer

Journal Pre-proof Cellular fluorescence imaging based on resonance energy transfer Wei Zhang, Xiaolei Liu, Ping Li, Wen Zhang, Hui Wang, Bo Tang PII:

S0165-9936(19)30465-0

DOI:

https://doi.org/10.1016/j.trac.2019.115742

Reference:

TRAC 115742

To appear in:

Trends in Analytical Chemistry

Received Date: 19 August 2019 Revised Date:

28 October 2019

Accepted Date: 15 November 2019

Please cite this article as: W. Zhang, X. Liu, P. Li, W. Zhang, H. Wang, B. Tang, Cellular fluorescence imaging based on resonance energy transfer, Trends in Analytical Chemistry, https://doi.org/10.1016/ j.trac.2019.115742. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Cellular fluorescence imaging based on resonance energy transfer

Wei Zhang, Xiaolei Liu, Ping Li, Wen Zhang, Hui Wang, Bo Tang*

College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Institute of Biomedical Sciences, Shandong Normal University, Jinan 250014, P. R. China. E-mail: [email protected]

Abstract: Fluorescence imaging of intracellular active molecules plays an important role in understanding the occurrence and development of diseases. Resonance energy transfer (RET) has been recognized as a powerful technology in the design of fluorescence probes for biological imaging. Here, we summarize the novel approaches to fabrication of RET-based fluorescent probes systems such as the organic molecular fluorescent probe, nano fluorescent probe, protein probe and their recent applications in cellular fluorescence imaging.

Keywords: fluorescence imaging, cells, resonance energy transfer, active small 1

molecules, fluorescent probe

1. Introduction Cells are the fundamental units of life, cellular components maintain cellular homeostasis. The deviation of Intracellular homeostasis is closely related to the occurrence, development, transformation and treatment of common diseases such as cancer, diabetes and cardiovascular diseases [1,2]. Intracellular reactive molecules such as reactive oxygen species (ROS), microRNAs and proteins play important roles in the maintenance of cell homeostasis [3-5]. Thus, studies on changes in the concentration of active molecules that regulate cell homeostasis have drawn wide attention [6-8]. Fluorescence imaging has emerged as powerful technique to investigate spatiotemporal dynamics of reactive molecules or events in cell [9,10]. A unique feature of fluorescence imaging is its ability to afford high spatial resolution, which enables visualization of reactive molecules or events in cell. Resonance energy transfer (RET)，which involves energy transfer from excited donor to a suitable acceptor molecule, is a powerful technique for sensing changes in the distance between donor and acceptor[11-13]. RET technique can be an accurate measurement of molecular proximity at distance of 1 to 10 nm. Therefore, the RET technique has been widely applied in the field of biochemistry and structural biology [14-17]. In this review article, we present an overview of recent progress in RET-based systems for cellular fluorescence imaging such as the organic molecular fluorescent probe, nano fluorescent probe and protein probe. Among them, in the 2

molecular fluorescent probe section, we mainly review pH imaging, active small molecule imaging and metal ion imaging. The nanoprobe section is mainly introduced according to the material classification of the constructed nanoprobe. Then a brief review of protein probes based primarily on their application. Finally, we gave a short introduction for molecular beacons and aptamer-based probes. Altogether, in the review, we mainly focus on the construction mechanism of the probe for cellular fluorescence imaging, summarize key applications and discuss future prospects.

2. Cellular fluorescence imaging 2.1 Molecular probe 2.1.1 Cellular fluorescence imaging of pH Organic small molecule fluorescent probes have been rapidly developed in cell imaging research due to their design feasibility and ease of synthesis and controllability [6,7,9, 18]. In this section, we mainly review the synthesis principle of organic small molecule fluorescent probes and their application in pH, active small molecules and metal ion detection. As an important physiological indicator, intracellular pH play critical role in biological processes such as cell proliferation and apoptosis. A ratiometric dual-emission pH probe (SR1) including a pH responsive amino-naphthalimide unit and a pH-insensitive coumarin segment (Fig. 1) was designed by Tian et al. [19]. The probe was used to measurement of pH in the range from 4.0 to 10.0. Finally, the probe applied to monitor intracellular pH changes in mouse macrophage J774A.1 and human cervical cancer HeLa cell lines, respectively. 3

Fig. 1. Schematic illustration of the dual-emission sensing of pH with the chemosensor through FRET and PET turn-on fluorescence sensing mechanisms [19].

Lin et al. report a novel dual site controlled and lysosome-targeted intramolecular charge transfer-photoinduced electron transfer-FRET fluorescent probe (CN-pH), which was essentially the combination of a turn-on pH probe (CN-1) and a turn-off pH probe (CN-2) by a nonconjugated linker [20]. Coumarin and naphthalimide fluorophores were selected as donor and acceptor to construct the FRET platform (Fig. 2). Hydroxyl group and morpholine were simultaneously employed as the two pH sensing sites. Meanwhile, the morpholine also can serve as a lysosome-targeted group. Finally, the probe has been applied for imaging of the lysosomal pH values, as well as visualizing chloroquine-stimulated changes of intracellular pH in living cells. Sun et al. reported a naphthalimide-rhodamine based fluorescent probe (RBN) and applied for ratiometric sensing of cellular pH via FRET, which can respond to different pH precisely through ratiometric fluorescence intensity [21]. RBN can be employed to distinguish cancer cells from normal cells on the basis of different fluorescent response. Wang et al. designed and synthesized a novel fluorescence probe for visualizing acidification in fungal cells [22]. The probe was designed for pH through 4

the H+-triggered reversible closed-open process of spiroboronate. Owing to excellent cellular permeability and ignorable biotoxicity in fungi, the probe manifested precise pH change from 7.5 to 6.5 in amiodarone treated fungal cells.

Fig. 2. Molecular structure of the CN-1, CN-2 and CN-PH [20].

2.1.2 Intracellular small molecules Multiple active small molecule species in cells such as ROS and thiol compounds, widely mediate important processes including carcinogenesis, body inflammation, tissue peroxidation, protein cross-linking degeneration, DNA damage and cell signal transduction [6,7,9]. The concentration level and spatial-temporal distribution of active small molecules not only reflect the physiological and pathological state of the cells, but also play an important role in regulating cell function [10,15]. Superoxide anion is the source of other ROS, therefore, the development of methods for superoxide anion imaging is of great significance. Rochford and Tseng et al. reported BODIPY-luminol chemiluminescent resonance energy-transfer (CRET) cassette (Fig. 5

3) where the luminol chemiluminescent agent is covalently linked to the BODIPY energy-transfer acceptor in a molecular dyad [23]. The efficiency of intramolecular CRET investigated for the BODIPY-luminol dyad was 64%. Finally, the probe was used for imaging of superoxide anion in PMA activated splenocytes.

Fig. 3. Molecular structure of the BODIPY-luminol conjugate alongside the luminol and meso-(4-carboxyphenyl)BODIPY reference systems [23].

A red-emitting BODIPY-based FRET molecular probe (REB) for selective detection of cysteine and homocysteine was designed by James et al. [24]. The FRET energy donor is a BODIPY moiety and the energy acceptor is based on 4-hydroxylstyryl BODIPY moiety. The probe REB itself is non-fluorescent, while in the presence of cysteine or homocysteine a red emission at 590 nm is switched on, producing a pseudo-stokes shift of up to 77 nm. Finally, the probe was used for fluorescent imaging of cellular thiols. Chang et al. designed a red-emitting turn-on FRET-based molecular probe REF for selective detection of cysteine and homocysteine [25]. It is noteworthy that the probe REF is pH-independent at the physiological pH range. Finally, live cells imaging demonstrated the utility of probe 6

REF as a biosensor for thiols. Zhao and Mao et al. developed a ratiometric fluorescent probe BRT based on boron dipyrromethene (BODIPY) and rhodamine-thiohydrazide FRET platform (Fig. 4) for sensing hypochlorous acid (HOCl) [26]. Upon mixing with HOCl, the fluorescence colour of probe BRT changed from green to orange. Moreover, probe BRT was applied to monitor HOCl in living RAW 264.7 cells. A ratiometric fluorescent probe (RIL) for imaging lysosomal HOCl was prepared by Cao and Shen et al. based on the imidazo[1,5-a]pyridine-rhodamine platform [27]. The probe RIL performed well in fluorescence imaging of endogenous HOCl in RAW264.7 cells. Notably, the probe could target lysosomes and monitor lysosomal HOCl changes due to lysosomal targeting ability of morpholine moiety. Qian et al. reported a new ratiometric naphthalimide-rhodamine two-photon fluorescent turn-on probe RHSDN with a ‘naked-eye’ recognition capability for HOCl [28]. Besides, the chemodosimeter RHSDN works excellently within a wide pH range of 4–11.

Fig. 4. Synthesis of probe BRT and detection mechanism [26].

2.1.3 Fluorescent imaging of metal ion Metal ions are widely present in tissue cells and body fluids and play an important 7

role in the physiology and pathology of human body [10,11,14,15]. The concentration of metal ions must be precisely controlled within a certain range, and abnormal concentration change of ions is closely related to the occurrence and development of diseases. Lin et al. designed and synthesized coumarinrhodamine-based FRET ratiometric fluorescent chemodosimeters [29]. The probe exhibit several favorable merits, including a large variation in the emission ratio, well-resolved emission peaks, high sensitivity, high selectivity and good cell membrane permeability. Importantly, these excellent attributes enable us to demonstrate ratiometric imaging of Cu2+ in living cells by using these novel ratiometric fluorescent chemodosimeters. Qian et al. reported two novel intramolecular FRET dendritic compounds RhB-BODIPY and NPI-BODIPY have been synthesized utilizing click reaction and both two compounds were applied to imaging of Hg2+ in living cell [30]. Ye et al. designed and synthesized a novel reversibly naphthalimide-rhodamine based FRET fluorescent chemosensor for Hg2+ [31]. Rhodamine was selected as the FRET energy acceptor, owing to its non-fluorescent spirolactam to fluorescent open-ring amide equilibrium, and naphthalimide as the energy donor owing to its fluorescence spectrum can match well with the absorption spectrum of rhodamine. Notablely, naked-eye detection was realized through the changes of fluorescence color, and the sensor was applied for detecting Hg2+ in living cells. Qian et al. reported a novel intramolecular FRET and photo-induced electron transfer (PET) based dendritic compound PDI–BODIPY [32]. The probe exhibited excellent properties for rapid, selective, and sensitive determination of Hg2+ with high 8

energy transfer efficiency. Moreover, the imaging of Hg2+ in Hela cells further certified the potential value of the probe. Qian et al. reported a novel pyridyltriphenylamine-rhodamine dye PTRh and a pyridyltriphenylamine derivative PTO [33]. PTRh performed typical FRET signal from pyridyltriphenylamine to rhodamine along with notable color change from green to rose when interacting with Hg2+ (Fig. 5). In addition, Han and Liu et al. reported a novel BODIPY-RhB ratiometric fluorescent probe BRF for Hg2+ based on a FRET mechanism [34]. Three different probes, including RC-Am, RC-SB and RC-SBR were prepared by changing the linker between the rhodamine and coumarin units from amide to imine to reduced imine [35]. Moreover, FRET process starts only when the spirolactam ring of the rhodamine unit opens up. The binding performance of the probe with Al3+ remarkably changes, thereby affecting the FRET process.

Fig. 5. Possible binding mode of PTRh with Hg2+ ions [33].

Goswami et al. designed and synthesised a cell permeable small molecule fluorescent probe (RHD) for dual channel ‘naked-eye’ in vitro and in vivo detection of Al3+ and Zn2+, incorporating FRET and the ESIPT mechanism with a lower detection limit [36]. Zhu and Davidson et al. demonstrate a strategy to transfer the zinc(II) 9

sensitivity of a fluoroionophore with low photostability and a broad emission band to a bright and photostable fluorophore with a narrow emission band [37]. The two fluorophores are covalently connected to afford an intramolecular FRET conjugate. The FRET donor in the conjugate is a zinc(II)-sensitive arylvinylbipyridyl fluoroionophore, the absorption and emission of which undergo bathochromic shifts upon zinc(II) coordination. When the FRET donor is excited, efficient intramolecular energy transfer occurs to result in the emission of the acceptor boron dipyrromethene as a function of zinc(II) concentration. The broad emission band of the donor/zinc(II) complex is transformed into the strong, narrow emission band of the BODIPY acceptor in the FRET conjugates, which can be captured within the narrow emission window that is preferred for multicolor imaging experiments.

2.1.4 Visualizes Autophagosome–Lysosome Fusion Mitophagy occurs through the fusion of lysosomes with autophagosomes, which plays a pivotal role in regulating and maintaining cellular functions. Kim and Park et al. developed aupramolecular FRET pair based on the interaction between cyanine 3 conjugated cucurbit[7]uril (CB[7]-Cy3) and cyanine 5 conjugated adamantylamine (AdA-Cy5) (Fig. 6), which employed as a fluorescence tool for imaging cellular processes in live cells [38]. The results of the imaging revealed that CB[7]-Cy3 and AdA-Cy5 were intracellularly translocated and accumulated in lysosomes and mitochondria, respectively. FRET signal was reported through formed stable host– guest complex between the CB[7]-Cy3 and AdA-Cy5, thus, the fusion of lysosomes 10

and mitochondria was observed. Therefore, the FRET pair can visualize dynamic organelle fusion processes.

Fig. 6. a) Chemical structures of CB[7]-Cy3 and AdA-Cy5. b) Emergence of FRET after the fusion of two different organelles (lysosome and mitochondria) through the process of autophagy [38].

2.2 Nanoprobe 2.2.1 gold nanoparticles (AuNPs)-based probe Nanoprobes can achieve long in vivo retention time, multimodal imaging and multi-target detection through self-assembly. Gold nanoparticles have large specific surface area, unique optical properties and electrical properties [11,14,39]. Therefore, it can be used as a nano sensor and has wide value in the fields of food detection and pollution monitoring. A new nanoprobe was designed by Tang et al. and applied for 11

the fluorescence imaging of fluoride anion (F-) in living cells with high sensitivity and selectivity [39]. The design is based on the fluorescence resonance energy transfer (FRET) between CdTe quantum dots (CdTe QDs) and gold nanoparticles (Au NPs) through the formation of cyclic esters between phenylborinic acid and diol (Fig. 7). In the presence of F-, the boronate ester is converted to trifluoroborate, which causes the breakage of the linkage and disassembles CdTe QDs from AuNPs, and fluorescence recovery of the quenched CdTe QDs was achieved. Finally, the probe was applied for imaging F- in Fin macrophages cells. The present work provided a viable alternative to detection of F- in cells.

Fig. 7. Schematic illustration of (a) FRET formed between water-soluble CdTe QDs and AuNPs through cyclic ester bond. (b) FRET was broken by adding F-anions [39].

Based on a novel signal amplification strategy the carbon dots (CDs) functionalized with

aptamer

(CD-aptamer)

was

used

as

detection

probe

and

PAMAM-Dendrimers/AuNPs was used for covalent attachment of CA125-antibody and completing the sandwich assay method. Salimi et al. developed an ultrasensitive antibody-ssDNA aptamer sandwich-type fluorescence immunosensor for CA125 12

detection [40]. Xu et al. designed a probe termed FRET nanoflare to achieve ratiometric fluorescent detection of intracellular telomerase with higher specificity [41]. In the presence of telomerase, flares are displaced from the primer sequences and form hairpin structures, so that the donors and acceptors are brought into close proximity, resulting in high FRET efficiency. Cai and Sheng et al. designed gold nanoclusters-indocyanine green nanoprobes (Au NCs−INPs) by conjugation of Au NC assemblies with indocyanine green (ICG) for the therapeutic real-time monitoring based on FRET [42]. More importantly, a promising therapeutic monitoring strategy was performed based on FRET between Au NCs and ICG, suggesting that Au NCs-INPs could be utilized to evaluate the therapeutic response by real-time monitoring the change in Au NCs in fluorescence intensity together with ICG supersession.

2.2.2 ZrO2−Ln3+ NPs based probe Luminescent inorganic nanoparticles doped with trivalent lanthanide ions (Ln3+) have attracted growing attention owing to their potential applications in the flied of bioimaging and theranostic. Notably, zirconia (ZrO2) is considered as an ideal oxide host for Ln3+ doping to achieve intense long-lived luminescence of Ln3+. Based on such considerations, Chen et al. developed a new inorganic oxide biolabel (Fig. 8) based on amine-functionalized tetragonal ZrO2-Ln3+ NPs via a modified solvothermal method and ligand exchange procedure [43]. The bioconjugate method was used to achieve quantitative bioconjugation between amine-functionalized ZrO2−Ln3+ NPs 13

and various biomolecules including biotin and ATF. Based on the distinct optical properties and long-lived luminescence of Ln3+, the author demonstrated the application of biotinylated ZrO2 NPs as a sensitive TR-FRET bioprobe for avidin. In addition, ZrO2-Tb NPs bioconjugated with ATF of uPA exhibited specific recognition ability for cancer cells overexpressed with uPAR (a marker of tumor biology and metastasis), which have potential as fluorescent imaging tool for targeting various cancer cells.

Fig. 8. Schematic illustration showing the principle of TR-FRET detection of avidin. [43].

2.2.3. Silica nanoparticles Silica-based nanoparticles have caused great interest in the field of biomedicine due to their properties of easy surface modification, high specific surface area and good biocompatibility. Huang and Wang et al. reported a targeted cancer therapy imaging and sensing system (Fig. 9), which designed based on doxorubicin (DOX)-loaded green fluorescent mesoporous silica nanoparticles modified with folic acid (FA) [44]. An in situ formation method is used to prepare the systems, which act as the donor for 14

the FRET, owing to their emission (500 nm) overlaps with the absorption of the acceptor DOX (485 nm). Then the NH2-PEG-COOH and the folic acid is modified to the surface of the silica nanoparticles, so that the as-prepared mesoporous silica composite has the feature of nanostructure for drug loading, fluorescence imaging, targeting and real-time monitoring of intracellular drug release. When the as-prepared nanoparticles enter into the cells, green fluorescence of the nanoprobe gradually recovers along with the release of DOX, and thus achieved the aim of real-time monitoring of intracellular drug release.

Fig. 9. A diagram showing the synthesis of DOX–FMSN–PEG–FA, and sensing of the drug release process by FRET in a cancer cell [44].

2.2.4 Carbon dots based probe Carbon dots (CDs) is a new type of nanomaterials with low-cost, high fluorescence emission intensity, low toxicity and good biocompatibility [8]. Currently, the CDs have been widely used in the field of chemical analysis and biosensing. Wu and Zeng 15

et al. reported a multifunctional fluorescent nanoprobe is demonstrated for detecting mitochondrial H2O2 [45]. The nanoprobe is prepared by covalently linking a mitochondria-targeting unit (triphenylphosphonium, TPP) and a H2O2 recognition element (PFl) onto the surface of the CDs. In the presence of H2O2, the PFl moieties on CDs undergo structural and spectral conversion, the FRET-based ratiometric probe for H2O2 was thus achieved. In addition, the nanoprobe can specifically target the mitochondria, and it can detect the endogenously produced mitochondrial H2O2 in Raw 264.7 cells stimulated by PMA. Zhao and Zhang group developed a FRET-based ratiometric fluorescent nanoprobe for real-time monitoring of O2•− and •OH generation and also applied for in situ monitoring of the inflammation process in vivo [46]. The nanoprobe was composed of PEG functionalized GODs as donor connecting to hydroIR783, employing as both the O2•−/•OH recognizing unit and acceptor. The results demonstrated that the nanoprobe can monitor the changes of O2•− and •OH in living RAW 264.7 cells and realized in situ visual tracing of the change of O2•− and •OH in inflammatory mice model. Zhao group developed a dual-emission near-infrared pH response nanoprobe combined the advantages of porphyrin and CDs [47]. The nanoprobe has been applied to accurately detect intracellular pH values and monitor the change of intracellular pH related to oxidative stress.

2.2.5 Persistent luminescence nanoparticles Persistent luminescence nanoparticles can store the excitation energy and then 16

slowly release it by a photonic emission. The persistent luminescence can last several hours after removal of excitation resource, providing many conveniences in optical assay and imaging, which can reduce the shortlived background luminescence and auto-fluorescence interference from cells and tissues by exerting the delay time between pulsed excitation light and signal acquisition, provides a background-free approach in life science. Ju et al. used persistent luminescence nanoparticles (PLNPs) to design a time-resolved FRET (TR-FRET) system for biosensing, lifetime imaging of cell apoptosis and in situ lifetime recognition of intracellular caspase-3 [48]. Three kinds of PLNPs-based nanoprobes were prepared by linking dye-labeled peptides or DNA to carboxyl-functionalized PLNPs for the efficient detection of caspase-3, microRNA and protein, respectively. The nanoprobe was also applied for fluorescence lifetime imaging to monitor cell apoptosis, which exhibited a dependence of cellular fluorescence lifetime on caspase-3 activity.

2.2.6 Silver nanoparticles Silver nanoparticles are one of the most important precious metal nanoparticle materials. Due to their unique physicochemical properties such as quantum size effect, good electrical conductivity and superior permeability, they are widely used in biomedicine, catalysis and sensing [49, 50]. Xu et al. reported silver decahedral nanoparticles (Ag10NPs)-based FRET sensor for target cell imaging [49]. The sensor interacted with membrane protein tyrosine kinase-7 on the cell surface to attain fluorescence imaging of CCRF-CEM cells. The addition of CCRF-CEM cells resulted 17

in many sensors binding with cells membrane and the displacement of BHQ-1, thus disrupting the FRET effect and the enhanced fluorescence intensity of FITC. Liu et al. developed a method for imaging analysis of protein-specific sialylation on the cell surface via silver nanoparticle (AgNPs) plasmonic enhanced FRET [50]. In this strategy, the target monosaccharide was labelled with the FRET acceptor of Cy5 via bioorthogonal chemistry. In addition, aptamer linked AgNPs were combined with the Cy3 by DNA hybridization as the FRET donor, which could be recognize the target glycoprotein based on specific aptamer protein recognition (Fig. 10). The Cy5 fluorescence response was obtained under the Cy3 excitation wavelength via FRET. The results showed that the AgNP plasmonic enhanced FRET method has superior performance than that of the bare FRET method, therefore, to evaluate the expression of sialoglycoproteins in cell can be achieved.

Fig. 10. Schematic illustration of Ag nanoparticle enhanced FRET imaging for protein-specific sialylation on the cell surface [50].

18

2.2.7 Quantum dots Quantum dots (QDs) are semiconductor nanocrystals of zero-dimensional nanomaterials with particle sizes between 1 and 10 nm. Due to the quantum confinement effect, quantum dots have unique photoelectric properties [39]. Currently, it has been widely used in biological/chemical sensing, biomedical imaging. Sun et al. reported the design and application of a new ratiometric fluorescent probe (Fig. 11), which contains different-colored QDs as dual fluorophores, ultrathin silica shell as spacer, and meso-tetra(4-sulfonatophenyl)porphine dihydrochloride (TSPP) as receptor, for Zn2+ detection in aqueous solution and living cells [51]. Benefiting from the well-resolved dual emissions from different-colored QDs and the large range of emission ratios, the probe solution displays continuous color changes from yellow to green, which can be clearly observed by the naked eye. Feltz et al. report a FRET-based nanobiosensors for detection of intracellular Ca2+ and H+ transients [52]. Chen et al. reported a novel fluorescent turn-on biosensor based on FRET from GSH functionalized Mn-doped ZnS QDs to graphene oxide was constructed to determine glutathione S-transferases in cells and human urine [53]. Huang et al. report a QD-based FRET assembly and its utility for sensing Zn2+ [54]. The assembly on the QD scaffold is via first coating of poly(dA) homopolymer/double-stranded DNA, followed by loading of meso-tetra(4-sulfonatophenyl)porphine dihydrochloride (TSPP), both of which are electrostatic, offering the advantages of simplicity and cost-efficiency. Based on the Zn2+-chelation-induced spectral modulation, dual emission QD-poly(dA)-TSPP assemblies were developed as a ratiometric Zn2+ sensor 19

that was applied to visualize exogenous Zn2+ in cells.

Fig. 11. Schematic Illustration of the Preparation and Application of Dual-Colored QDs Based Ratiometric Fluorescent Probe for Detection of Zn2+ [51].

2.2.8 Fe3O4 Magnetic nanoparticles refer to magnetic, nanoscale metal oxide particles or composite nanoparticles. Owing to their sensitive magnetic responsiveness and good biocompatibility, magnetic nanoparticles have broad application prospects in biosensors, cell sorting, and biomedicine. Wang et al. presented a magnetic separation-assistant FRET inhibition strategy for highly sensitive detection of nucleolin using Cy5.5-AS1411 as the donor and Fe3O4-polypyrrole (Fe3O4@PPY) nanoparticles as the NIR quenching acceptor [55]. The superparamagnetism of Fe3O4@PPY enabled the separations and fluorescence measurements complete in the same vessel, and thus allowed the convenient but accurate comparison of the 20

sensitivity and fluorescence changes in the cases of separation or nonseparation.

2.2.9 Upconversion nanoparticles Upconversion luminescent nanomaterials (UCNPs) have significant advantages such as long fluorescence lifetime, low potential biotoxicity, large penetration depth, little damage to biological tissues, and almost no background light [11]. In recent years, it has been widely used in the fields of bio-imaging, bio-detection and photodynamic therapy. Chang et al. report a Zn2+ fluorescent nanoprobe by assembling

lanthanide-doped

upconversion

nanoparticles

(UCNPs)

with

chromophores [56]. Specifically, upconversion luminescence (UCL) can be effectively quenched by the chromophores on the surface of nanoparticles via a FRET process and subsequently recovered upon the addition of Zn2+, thus allowing for quantitative monitoring of Zn2+. Notablely, the fluorescent nanoprobe enables detection of Zn2+ in real biological samples.

2.2.13 MOFs Nanoscale MOFs have specific morphology and size, larger specific surface area and pore volume, which make potential applications of nanoscale MOFs in gas adsorption, sensing, drug delivery and catalysis [5]. Chu and Huo et al. adopt zeolitic imidazolate framework-8 (ZIF-8) as a nanocarrier for deliver a nucleic acid probe to living cells and developed a ratiometric fluorescence strategy based on DNAzyme for miRNA-21 imaging [57]. A Cy5-labeled 8-17 DNAzyme strand and a Cy3-labeled 21

substrate strand containing a segment complementary to the target miRNA-21 first formed a duplex probe, and thus FRET takes place. After adsorption on the ZIF-8 surface and cellular uptake, the probe/ZIF-8 nanocomposites degraded in acidic endosome and released duplex probes and Zn2+, and the latter can act as an effective cofactor for 8-17 DNAzyme. The intracellular miRNA-21 hybridized with the complementary segment of the substrate strand and results in dissociation from the DNAzyme−substrate duplex probe after DNAzyme cleaved the substrate into two fragments, accompanied by the change in the FRET signal. The present method has been applied to image levels of miRNA-21 expression in MCF-7, HeLa, and L02 cells, respectively.

2.2.18 Polymer nanoparticles An H2O2-activatable fluorescent unimolecular micelle-based nanoprobe using an aggregation-enhanced FRET strategy was developed by Zhu and Yan et al., which constructed

by

boronate

coupling

of

poly(fluorene-co-2,1,3-benzothiadiazole)

a

core

hydrophobic and

many

hyperbranched hydrophilic

poly(ethyleneglycol) arms (Fig. 12) [58]. The color-convertible, activatable nanoprobe exhibited obviously blue fluorescence in various normal cells, but becomes highly green-emissive in various cancer cells. After intravenous injection to tumor-bearing mice, green fluorescence signals was only detected in section of tumor tissue. Zhu et al. building up a ratiometric fluorescent H2S nanoprobe through encapsulated

a

semi-cyanine-BODIPY 22

hybrid

dye

(BODInD-Cl)

and

its

complementary energy donor (BODIPY1) into the hydrophobic interior of an amphiphilic copolymer (mPEGDSPE) [59]. A remarkable red-shift in the H2S response can efficiently switch off the FRET from BODIPY1 to BODInD-Cl, subsequently recovering fluorescence of the donor. Based on the unique self-assembled micellar aggregate Nano BODIPY achieved an extremely fast response, thus the ratiometric strategy allowed in situ imaging of endogenous H2S and monitoring its physiological and pathological consequences. Liang and co-worker developed a peptide-based four-color fluorescence polydopamine nanoprobe and applied for multiplexed sensing and imaging of tumor-related proteases in living cells [60]. Due to fast response and high selectivity, the proposed nanoprobe achieved simultaneous and high-contrast imaging of multiple proteases in living cells.

Fig. 12. Synthesis of HPFBT-star-PEG and illustration the mechanism of the color conversion from blue to green in response to H2O2 through an aggregation-enhanced 23

FRET effect [58].

2.3 Protein probe 2.3.1 Imaging of Src/Ca2+ signaling and glutathione redox potential Fluorescent proteins were increasingly being used for FRET analysis of targets in biological systems, and their techniques and materials were constantly being improved [16,17]. Gene-encoding the fluorophore to the target protein to produce a light chimera, which has the obvious advantage that the fluorescene group and the protein can be co-expressed in cell and precisely display the position and relative expression level of the target protein. Zhang et al. established a combination of FP biosensors for dual-parameter ratiometric imaging, consisting of a new FRET pair mVenus (yellow FP)/mKO(orange FP)-based (abbreviated as YO) biosensor and a single FP-based biosensor Grx1-roGFP2 [61]. By using this dual-parameter ratiometric imaging approach, they achieved simultaneous imaging of Src/Ca2+ signaling and glutathione (GSH) redox potential in cell. Moreover, the author provided direct evidence that epidermal growth factor (EGF)-induced Src signaling was negatively regulated by H2O2 via its effect on GSH-based redox system, suggesting the power of the dual parameter imaging strategy for elucidating new biological process between different molecular events that occur in a single cell.

2.3.2 Fluorescence imaging of protein glycoforms The glycosylation of proteins is closely related to the occurrence and development 24

of diseases. Therefore, monitoring the levels of glycosylation of specific protein in living cells is highly demanded. Suzuki et al. demonstrate the detection of glycoforms of a specific glycoprotein using the FRET technique [62]. Using model proteins, the author detect characteristic FRET signals from the specific glycoform-bearing target glycoprotein (Fig. 13). The analytical imaging tool allows studying the roles of specific glycan modifications of target protein. The strategy for visualization of a specific sugar on a target glycoprotein was shown. Glycans were metabolically labelled with azide sugars and click chemistry, and the target protein was conjugated with GFP. The specific glycoprotein can be visualized by FRET between GFP and the glycan-conjugated fluorophore.

Fig. 13. FRET-based imaging of the glycosylation pattern of a specific glycoprotein [62].

2.3.3 Simultaneous imaging of Srcand Ca2+ signaling Zhang et al. developed a new combination of spectrally distinguishable FRET pairs for dual-parameter molecular imaging: mTagBFP/sfGFP (blue and green FP, B/G) and mVenus/mKOk (yellow and orange FP, Y/O) [63]. Using the dual FRET pairs, they achieved imultaneous imaging of Src and Ca2+ signaling in single cells stimulated with epithelial growth factor (EGF). By converting traditional FRET 25

biosensors into B/Gand Y/O-based biosensors, additional applications were available to reveal the dynamic relationships of multiple molecular events with in cell.

2.3.4 FRET-based biosensor for imaging MMP-9 activity A nontoxic, genetically encoded FRET biosensor was reported by Wlodarczyk et al. [64]. The biosensor enables the research on the proteolytic activity of MMP-9 with high temporal and subcellular resolution at the precise region of MMP-9 action in cell. The application of the biosensor both in vitro and in cells was investigated by ratiometrically analyzing the cleavage of the biosensor by a purified auto-activating mutant of MMP-9 and endogenously secreted protease in tumor and neuronal cells.

2.3.5 Intraorganelle Zn2+ Imaging Merkx et al. reported a versatile alternative FRET sensor containing a de novo Cys2His2 binding pocket that was formed on the surface of the donor and acceptor fluorescent domains [65]. This eZinCh-2 sensor binds Zn2+ with a high affinity and displaying a substantially larger change in emission ratio (Fig. 14), which successfully used for measuring Zn2+ in the ER, mitochondria, and secretory vesicles, respectively. In addition, the organelle-targeted eZinCh-2 combined the previously reported redCALWY sensors can be used in multicolor imaging of intracellular Zn2+ simultaneously in the cytosol and the ER or mitochondria.

26

Fig. 14. The structure of eZinCh-2 sensor and its application in Cytosolic and Intraorganelle Zn2+ Imaging [65].

2.3.6 Monitoring and quantification of alcohols A genetically encoded nanosensor based on conformational changes in the human odorant binding protein (hOBPIIa) that converts the response into a ratiometric FRET signal was developed by Mohsin et al. and applied for quantification of alcohol [66]. The hOBPIIa was flanked by fluorescent proteins ECFP (Enhanced Cyan Fluorescent Protein) and Venus at the N- and C-terminus respectively. The formed nanosensor was named the fluorescent indicator protein for odorants (FLIPO). The FLIPO was able to determination of FRET response in a concentration-dependent manner. The developed nanosensor can selectively identify ethanol, not affected by pH and provides rapid response. The FLIPO-42 achieved non-invasively real-time measure ethanol dynamics in bacterial, yeast and mammalian cells, which provided useful tool in both prokaryotic and eukaryotic systems.

2.3.7 Reveals Fyn Kinase Regulation 27

A highly sensitive Fyn biosensor based on FRET was developed by Wang and Lu et al. and applied to monitor Fyn kinase activity in cells [67]. The results showed that Fyn kinase activity can be induced in both mouse embryonic fibroblasts (MEFs) and T cells by ligand engagement. Further experiments indicated that the membrane accessibility was necessary for Fyn activation, the localization of Fyn outside of its microdomains causes its hyperactivity, suggesting that membrane microdomains provide a suppressive microenvironment for Fyn regulation in MEFs. The present work provided a novel Fyn FRET biosensor for cell imaging and its application in revealing an intricate biological process of submembrane regulation of Fyn in live MEFs.

2.4 Other probes 2.4.1 Intracellular poly(A)+-RNA localization Molecular aptamer beacon is a molecular imaging and detection tool that allows visualization of small or large molecules combined the selectivity and sensitivity of molecular beacon and aptamer technologies [68]. Hah et al. described a method for time-dependent visualization of intracellular poly(A)+-RNA localization in living mammalian cells, and time-resolved intracellular binding dynamics of molecular beacons at single-molecule level by a FRET-based molecular beacon [69]. As the poly(A)-targeting probes, the FRET-based molecular beacons contained Cy5 and Cy3 fluorescent dyes at the strand ends and a poly(A)-targeting sequence inside the strand. Results indicated that the poly(A)-targeting probes can specifically recognize 28

poly(A)-RNA, which exhibited a distribution of the probe itself and localization of the target RNA sequence in cells.

2.4.2 FRET for Temperature Imaging Thermoresponsive double hydrophilic block copolymers (DHBCs)-based fluorescent thermometers were fabricated by Liu and Zhang et al. and investigated their application in intracellular temperature imaging [70]. Blue-emitting coumarin monomer (CMA), green-emitting 7-nitro-2,1,3-benzoxadiazole (NBD) monomer (NBDAE) and red-emitting rhodamine B monomer (RhBEA) were copolymerized separately

with

Nisopropylacrylamide

PEG-b-P(NIPAM-co-CMA),

(NIPAM)

to

obtain

dye-labeled

PEG-b-P(NIPAM-co-NBDAE),

and

PEG-b-P(NIPAM-co-RhBEA), respectively (Fig. 15). Due to the favorable FRET potentials between CMA and NBDAE, NBDAE and RhBEA, as well as the slight tendency between CMA and RhBEA fluorophore pairs, three polymeric thermometers based on traditional one-step FRET were fabricated through mixing two of these three fluorescent DHBCs, but exhibiting limited advantages. Thus, two-step cascade FRET among three polymeric fluorophores was further interrogated, in which the NBD selected as a bridging dye via transferring energy from CMA to RhBEA. Through optimizing conditions, a ∼8.4-fold ratio change occurred in the temperature range of 20-44 °C, and the detection sensitivity improved significantly, up to low as 0.4 °C, which exhibited superior performance than other one-step FRET thermometers in intracellular temperature imaging of living cells. 29

Fig. 15. (a) Fabrication of Polymeric Ratiometric Fluorescent Thermometers; (b) Schematic Illustration for the Intracellular Temperature Imaging and Temperature Readout of Living Cells upon Cellular Internalization of the Cascade FRET Thermometer [70].

2.4.4 Visualization of tumor-related mRNA Tumor-related mRNA as a disease marker, its high sensitivity and specific detection is of great significance for the diagnosis of disease. Wang et al. reported a method based on FRET and hybridization chain reactions (HCRs) for in situ sense tumor-related mRNA (TK1 mRNA) with high sensitivity in single cells and tissue sections [71]. Using the strategy, each copy of the target mRNA can propagate a chain reaction of hybridization events between two alternating hairpins to form a nicked duplex that contains repeated FRET units, amplifying the fluorescent signal. Notably, 30

based on the FRET strategy, complicated washing steps was not necessary and experimental time was sharply reduced. Tan and Zhang et al. reported an intracellular DNA nanoprobe, termed DNA tetrahedron nanotweezer (DTNT), was developed to image tumor-related mRNA in cells based on the FRET “off-on” signal readout mode [72]. The DTNT was self-assembled from four single-stranded DNAs. In the absence of target mRNA, the respectively labeled donor and acceptor fluorophores are separated, thus resulting in low FRET efficiency. However, in the presence of target mRNA, DTNT altered its structure from the open to closed state, thus bringing the dual fluorophores into close proximity obtained high FRET efficiency. In addition, the results showed that DTNT could effectively distinguish cancer cells from normal cells through distinguish changes of mRNA expression levels in cells.

2.4.5 Adenosine triphosphate sensing Exploiting the split aptamer and the FRET off-on sensing mechanism could efficiently avoid false-positive signals. Based on this consideration, a DNA nanoprobe based on the split aptamer and the DNA triangular prism (TP) for adenosine triphosphate (ATP) sensing in cells was developed by Huan et al. [73]. The ATP as a target induced the self-assembly of split aptamer fragments and thus make the dual fluorophores into close proximity obtained high FRET efficiency (Fig. 22). Importantly, the DNA TP-based nanoprobe could effectively detect ATP and distinguish among changes of ATP levels in living cells.

31

3. Conclusions and perspectives RET probes are valuable tools in monitoring molecular events in cells. In this review article, we described the progress of cellular fluorescence imaging based on RET, and summarized some key applications. These successful applications demonstrate that RET based systems is a good platform to design new fluorescence probes. Organic small molecule probes have high sensitivity, good selectivity, low damage and good biocompatibility. However, there are still some problems with molecular RET-based fluorescent probes such as low energy transfer efficiency, easy diffusion in cells, rapid metabolism and difficulty in achieving multi-target detection. By regulating electron transfer to further improve energy transfer efficiency; Enhance the localization ability of the probe by screening appropriate targeting groups to solve the problem of easy diffusion in cells; Combining the advantages of molecular probes and nanomaterials to solve multiple identification problems. The nanoprobes have features of long retention times in cell and easy to further functional modification, enable multimodal imaging and multi-target detection through self-assembly. Fluorescent proteins are increasingly being used for FRET analysis of targets in biological systems, while fluorescent proteins are less efficient in energy transfer and are susceptible to conformational changes of the proteins. For each type of probe problem, we can design an appropriate strategy to improve the performance of the probe. For cellular active molecules, it is expected to achieve real-time in situ fluorescence imaging of intracellular active molecules. We hope that future RET probes will be able to simultaneously image complex molecular events in signaling 32

pathways and provide technical support for the study of mechanism of disease.

Acknowledgments This work was supported by National Natural Science Foundation of China (21535004, 91753111 and 21874084), and Natural Science Foundation of Shandong Province (ZR2019JQ07). The Key Research andDevelopment Program of Shandong Province (2018YFJH0502).

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43

Highlights

1. An overview of RET-based fluorescent probes systems in analytical chemistry was presented 2. The construction mechanism of the probe for cellular fluorescence imaging and key applications was described. 3. Future advances and challenges of fluorescent probes systems was discussed.

The authors declare no competing financial interests.