Advances in the development of fluorescence probes for cell plasma membrane imaging

Journal Pre-proof Advances in the Development of Fluorescence Probes for Cell Plasma Membrane Imaging Chaolong Liu, Xiaona Gao, Jingli Yuan, Run Zhang PII:

S0165-9936(20)30321-6

DOI:

https://doi.org/10.1016/j.trac.2020.116092

Reference:

TRAC 116092

To appear in:

Trends in Analytical Chemistry

Received Date: 2 July 2020 Revised Date:

28 September 2020

Accepted Date: 20 October 2020

Please cite this article as: C. Liu, X. Gao, J. Yuan, R. Zhang, Advances in the Development of Fluorescence Probes for Cell Plasma Membrane Imaging, Trends in Analytical Chemistry, https:// doi.org/10.1016/j.trac.2020.116092. 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. © 2020 Elsevier B.V. All rights reserved.

Advances in the Development of Fluorescence Probes for Cell Plasma Membrane Imaging Chaolong Liu a, Xiaona Gao a, Jingli Yuan a, Run Zhang b,* a

State Key Laboratory of Fine Chemicals, Department of Chemistry, Dalian University of

Technology, Dalian 116024, China b

Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St.

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Lucia, Queensland 4072, Australia. Email: [email protected]

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Abstract Cell plasma membrane, the boundary between an individual live cell and its microenvironment, plays important roles in cellular communication and signaling, and associates with many physiological and pathological processes. For better understanding the membrane-related biological processes, a number of fluorescent probes have been developed in the past few years. According to their different functions and targeting mechanisms, we herein provide an overview of the advances in the development of fluorescent probes for cell plasma membrane biology and biophysics research. Fluorescent probes specific for cell plasma membrane labelling are initially discussed based on their

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different targeting mechanisms, which is followed by the summary of membrane-targeting

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responsive fluorescence probes for various biomolecules detection. Challenges and future research

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dynamics investigations are outlined in the end.

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directions for the development of new generation fluorescent probes for cell plasma membrane

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Keywords

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Cell plasma membrane, Fluorescent imaging, Labelling, Responsive probe

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1. Introduction The cell is known as the “building blocks of life”. As the smallest biological unit of living organisms, the basic structure of the cell is consisted of cytoplasm and many organelles, such as ribosome, lysosome, mitochondrion, endoplasmic reticulum, Golgi and nucleus that are protected from surrounding environment by cell plasma membrane (Fig. 1A). This membrane is a biological glycerophospholipid bilayer in the periphery of the cell that plays import roles in cell biology, such as signal transduction and biomolecular transport [1, 2]. The bilayer structure of cell plasma membrane is primarily composed of a mix of lipids and proteins. Phospholipid molecule, a major

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lipid component of cell plasma membrane, has hydrophobic alkyl chains and a hydrophilic polar

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group. These molecules are spontaneously assembled to be a continuous membrane in an aqueous medium to form the basic backbone of cell plasma membrane. Cholesterol, interspersed in the

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glycerophospholipid bilayer [3], is an important component of bilayer to regulate cell plasma

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membrane stiffness [4, 5]. In addition to lipids, proteins are also loaded on the membrane and

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account for ~50% mass ratio of most cell plasma membrane [6]. The proteins have a variety of functions related to material transport, receptors, enzymes and immunity [7]. Building on the

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backbone of lipids and proteins, plasma membranes serve as barriers and gatekeepers that possess

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crucial functions in live cells, such as compartmentalizing the interior of cell from the environment, cell signaling and solute transporting [8]. Revealing biological function of cell plasma membrane is

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thus critical for various applications, such as drug screening, disease early diagnosis and treatment, and signal transduction control [9-11].

< Fig. 1 is here >

To this end, several methods have been developed for the cell plasma membrane analysis [12-16], and among these methods, fluorescence analysis using optical probes has been widely used in monitoring of cellular processes in situ in real-time due to its unique advantages, such as high sensitivity and simplicity, rapidity, and efficiency [17-24]. More specifically, for the analysis of cell plasma membrane dynamics, it is highly desirable to track the physiological processes on cell 3

surfaces in situ and in real-time. While the cell plasma membranes are too thin (5-8 nm) to be directly visualized by the light microscope, these structures are thus hardly to be distinguished with other structures in cells. Staining the cell plasma membrane with fluorescent probes, the boundaries can then be clearly observed under fluorescent microscopes. With this technology, monitoring cell activities, metabolism and cell-to-cell communication on cell surface can be achieved with “naked eyes”. Therefore, a numbers of bioanalytical probes and/or imaging agents have been recently developed for visualizing and tracking the behaviors of cell plasma membrane [25, 26], and such investigations have contributed and will continue to contribute to future investigating membrane

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biophysics and cell biology.

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Fluorescence probes for cell plasma membrane labelling are normally designed by incorporating cell plasma membrane targeting unit to fluorophore [27-33]. Selection an effective targeting unit is

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one of the most important steps for the development of membrane fluorescent probes. Generally, the

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design of membrane targetable unit relies on two key components of cell plasma membrane, that is,

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the phospholipid bilayer structure and cell plasma membrane proteins (Fig. 1B, C). The unique feature of hydrophilic outer membrane layer and lipophilic inner membrane layer makes some

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specially designed lipophilic ligands cell plasma membrane targetable, and the membrane proteins

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allow the developed fluorescent tags for cell plasma membrane targeting [8, 34, 35]. In addition to the fluorescent probes for cell plasma membrane labelling, recent research has also focused on the

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design of responsive fluorescence probes for biomolecules detection on cell plasma membrane. This kind of probe not only accumulates in the cell plasma membrane for labelling, but also responds to specific biomolecule and gives the changes of fluorescence signal for this biomolecule detection. In this review, the advances of fluorescent probes for the visualization of cell plasma membrane are summarized. The principles of probes’ design, targeting mechanisms and biological applications of these fluorescence probes are comprehensively overviewed. As shown in Fig. 2, the progresses of cell plasma membrane imaging agents (probes) are first summarized according to their different targeting mechanisms. This is followed by the discussion of responsive fluorescence probes for specific biomolecules detection on the membranes. Finally, the current challenges and the potential future directions of fluorescent probes for the visualization of cell plasma membrane are proposed.

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< Fig. 2 is here >

2. Fluorescence probes for cell plasma membrane labelling Through coupling cell plasma membrane targeting unit to well-documented fluorophores, the fluorescence probes can be readily designed for investigating the cell plasma membrane biology. Most of the fluorescence probes for cell plasma membrane labelling are developed by using lipophilic groups as the targetable units of phospholipid bilayer (Fig. 1B), such as lipophilic ligands,

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cholesterol, and tocopherol (Fig. 2). The cell plasma membrane proteins labelling approach (Fig. 1C) mainly relies on fusion protein tags, antibodies, small molecular ligands, and aptamers (Fig. 2). In

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this section, we will provide an overview of the advances in the development of fluorescent probes

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for cell plasma membrane labelling according to the different membrane targeting mechanisms.

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2.1 Phospholipid bilayer as the targeting sites for the development of cell plasma membrane

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probes

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2.1.1 Lipophilic ligands as the targeting units

Owing to the glycerophospholipid bilayer structure of cell plasma membrane, strongly lipophilic

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moieties of the fluorescent probes are capable of driving the probe molecules to the membrane’s

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lipid bilayer through the lipophilic-lipophilic interaction. After anchoring, the lipophilic probe molecules are easy to be internalized into the cells, resulting in a short retention time on cell plasma membrane [36, 37]. Inspired by the structure of cell plasma membrane lipid bilayer, amphiphilic structure-based fluorescent probes were then developed by incorporating membrane-targetable lipophilic moieties to hydrophilic groups-modified fluorophores [38-43]. These probes exhibit longer retention time on cell plasma membrane because the cellular internalization process is slowed down by the poor permeability of hydrophilic groups.

< Fig. 3 is here >

Quaternary ammonium group has been employed as hydrophilic moiety to improve the retention 5

capacity of cell plasma membrane fluorescent probes. Using 4’-(diethylamino)-3-hydroxyflavone as the fluorophore, Shynkar et al. reported a probe F2N12S (Fig. 3), for dual-color fluorescence imaging of cell plasma membrane [44]. F2N12S has a zwitterionic group for lipid interaction and a long (dodecyl) hydrophobic tail for the membrane anchoring. The fluorophore of F2N12S exhibits typical excited-state intramolecular proton transfer (ESIPT), resulting in two-band emission that the intensities are highly sensitive to the lipid composition of the cell plasma membrane. As a result, F2N12S could be used for dual-color ratiometric fluorescence imaging the loss of the plasma membrane asymmetry during the early steps of apoptosis. Using similar hydroxyflavone

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fluorophore, Kreder et al. synthesized two probes (F2N12SM, FC12SM) (Fig. 3) for fluorescence imaging of cell plasma membrane [40]. F2N12SM and FC12SM showed almost non-fluorescent in

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water but highly fluorescent in lipid membranes (>100-fold enhancement in fluorescence at 537 and

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fluorescent imaging of cell plasma membrane.

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621 nm for F2N12SM, FC12SM, respectively). The probe F2N12SM was then used as a tool for

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Recently, Collot et al. developed a family of six fluorescent membrane probes, MemBright, for advanced cellular imaging and neuroscience [41]. As shown in Fig. 4, the probes were designed by using the click chemistry of azide-modified clickable amphiphilic zwitterion (CAZ) with alkyne-modified cyanine fluorophores (Cyanine 3, 3.5, 5, 5.5, 7, and 7.5). The produced probes, including MemBright C3, C3.5, C5, C5.5, C7, and C7.5, formed soluble aggregates (nanoparticles with diameter of 22-32 nm) in aqueous media. Fluorescence of the MemBright probes were thus quenched in water due to the aggregation induced quenching (AIQ) process. Highly fluorescent molecular species could be obtained after these aggregates disassembled in the cell membrane bilayer. These probes are compatible with various microscopy techniques for long-term live-cell imaging and immunostaining by live video microscopy, multiphoton, and super-resolution microscopies. Moreover, MemBright probes were found to be particularly suitable in neuroscience and cell biology membrane imaging.

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Using a modified BODIPY as fluorophore, a dipolar fluorescence probe, Mem-SQAC (Fig. 3), was presented by Zhang et al. for long time tracking cell plasma membrane [38]. The probe Mem-SQAC was developed by coupling hydrophilic quaternary ammonium to the lipophilic BODIPY fluorophore. The bipolarity balanced structure of Mem-SQAC promotes it to be successfully used for rapid binding of cell plasma membrane for long time membrane analysis. Similar to probe F2N12SM, Mem-SQAC is also non-fluorescent in water (φ = 0.01), while intense emission was obtained in lipophilic environment. Such a turn “ON” fluorescence response provided an excellent approach to increase the signal-to-noise (S/N) ratio for cell plasma membrane imaging.

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Mem-SQAC was then used as the probe for fluorescence imaging of morphological changes of cell plasma membranes in the processes of apoptosis and endocytosis. Using BODIPY as the

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fluorophore and CAZ as the membrane targeting unit, Collot et al. recently reported the

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development of another probe, B-2AZ (Fig. 3), for cell plasma membrane staining [42]. Similar to

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MemBright, B-2AZ showed AIQ-mediated quenching of fluorescence in aqueous medium, while

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intense emission was obtained after a quick deaggregation process in the presence of plasma membrane. B-2AZ was successfully used in 3D-imaging to reveal fine intercellular tunneling

< Fig. 5 is here >

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nanotubes in KB cells and to stain the plasma membrane in glioblastoma cells in spheroids.

Amphiphilic dendritic polyglycerol perylene imido dialkylester (PIDE) in water forms micelles, where its fluorescence is quenched but can be switched “ON” after incorporating into a lipophilic environment, such as biological membranes. Based on this mechanism, Heek et al. developed a fluorescence turn “ON” probe ([G3]-PIDE-C10) (Fig. 5) for cell plasma membrane imaging [43]. [G3]-PIDE-C10 has two alkyl chains (C10) that can be used as cell plasma membrane targeting group. Due to the formation of micellar self-aggregates, [G3]-PIDE-C10 exhibited weak fluorescence in aqueous solution, while bright green fluorescence was obtained upon monomerization after incorporated into cell plasma membrane. Similar aggregation-mediated fluorescence response approach has been used by Peng et al. for the development of fluorescent-Gemini surfactant probe (8-TBT-8) (Fig. 5) for cell plasma membrane imaging [45]. 7

This quaternary-ammonium probe was designed by using thiophene-benzene-thiophene as fluorophore and two alkane tails as targeting moieties. 8-TBT-8 forms J aggregates in an aqueous solution to give green fluorescence, while it transfers to H aggregates with blue emission in an organic solvent. Fluorescent cell images studies showed membrane accumulation of 8-TBT-8, suggesting 8-TBT-8 is able to be used as the reagent for cell plasma membrane labelling. Matsuda et al. explored the use of lipophilic alkyl chains modified peptide anchors for the development of cell surface staining reagent [46]. Six types of peptide composed of five residues peptides were used for the design of membrane probes. Among them, a peptide with a sequence of

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NBD-Lys-Lys(X)-Lys-Lys-Lys(X)-NH2 (NBD: fluorophore, Lys(X): N-ε-palmitoyl-lysine) (XX2)

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(Fig. 5) provided the best performance for cell plasma membrane staining. The poor membrane permeability of the probe makes it to be modified on the cell surface with high efficiency (60-70%).

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More importantly, XX2 is capable of staining cell plasma membrane for a long time because the

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peptide can be represented after its endocytotic disappearance from the cell surface.

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Luminescent transition metal complexes have recently attracted enormous interests in the development of probes for biosensing and imaging due to their abundant photo-physical/-chemical properties and controllable cytotoxicity [47-53]. Among various metal complexes, luminescent platinum(II) (Pt(II)), ruthenium (II) (Ru(II)), rhenium(I) (Re(I)), and iridium(III) (Ir(III)) are contributing significantly to this field [54-59]. Through incorporating a hydrophobic C18 chain onto one of the ligands of cyclometalated Pt(II) complex, a luminescent Pt(II) complex (2) (Fig. 6) was synthesized by Koo et al. for plasma membrane imaging [39]. In addition to this membrane lipid anchoring ligand, the coordinated p-trisulfonated triphenylphosphine ([PPh3-3SO3]3-) ligand contributed to retain the probe 2 on cell plasma membrane for imaging. Probe 2 is able to be excited at both 400 (one-photon) and 720 nm (two-photon), allowing for the demonstrations of one-/two-photon luminescence imaging of HeLa cells’ membrane.

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< Fig. 7 is here >

Through coordinating with phenylpyridine ligands modified by lipophilic carbon chains of different lengths, Zhang et al. designed a series of cyclometalated Ir(III) complex luminescence probe (1-4) (Fig. 6) for cell plasma membrane staining [60]. Luminescent cell images revealed that the retention capacities of these complexes on cell plasma membrane is increasing progressively with the length of the carbon chains. Utilizing the different retention capacities of these iridium(III) complexes on cell plasma membrane, sensing and distinguishing between exogenous and

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endogenous analytes were then performed (Fig. 7). Similarly, Kim et al. also investigated the effect

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of lipophilic carbon chain length for membrane imaging using three 6-acyl-2-aminonaphthalene

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fluorophore-based TP probes (CH, CL, and CS) (Fig. 6) [61]. Cell imaging results found that CH is

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not suitable for membrane staining due to its highly water solubility, while CL and CS are excellent two-photon fluorescence probes for the cell plasma membrane imaging. Moreover, CS has a greater

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tendency to be located in the plasma membrane due to the poor water solubility.

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Aggregation-induced emission (AIE)-based luminescent materials are increasingly contributing to the field of bioassay [62-64]. For visualization of cell plasma membrane, several AIE-based fluorescent probes have recently been developed through exploring lipophilic-lipophilic interaction approach. Using 1,8-naphthalimide platform as the fluorophore, Li et al. prepared an AIE probe (FD-9) (Fig. 8) specific for tracking the cell plasma membrane [65]. FD-9 showed weak fluorescence in large polar solvents (e.g. methanol). When H2O fraction was >30% in CH3OH mixed solvent and >50% in THF mixed solvent, the twisted intramolecular charge transfer (TICT) activity-dominated emission of FD-9 was observed. The enhancement of FD-9’s emission in water was attributed to the AIE process that FD-9 aggregated in these mixed solvents. Cell imaging studies revealed that FD-9 could specifically track the cell plasma membrane for 4 days. Two triphenylamine (TPA)-based AIE probes (AS2CP-TPA, TTVP) (Fig. 8) were developed by 9

Zhang et al. and Wang et al. for near infrared (NIR) fluorescent imaging of cell plasma membrane [66, 67]. AS2CP-TPA showed good solubility in polar solvents such as DMSO and poor solubility in non-polar organic solvents such as toluene. In PBS buffer, AS2CP-TPA displayed only weak fluorescence, while distinct fluorescence was obtained after addition of lipid vesicles. The results of fluorescence response imply that AS2CP-TPA could be a good probe for plasma membrane imaging. For another AIE probe TTVP, its emission intensity in water was increased upon addition of THF (highest intensity was observed at 90% fraction of THF). The TTVP was then demonstrated to be able to fast staining cell plasma membrane by an easy-to-operate staining protocol (i.e., simply

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shaking cells with probe at R.T. for a few seconds). More interestingly, TTVP was found to be able to generate reactive oxygen species (ROS) upon visible light irradiation, providing a potential

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photosensitiser for photodynamic therapy (PDT) of cancer cells.

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Using tetraphenylethylene (TPE) as the AIE luminophore, cell plasma membrane probes TR4

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and TPE-MEM (Fig. 8) were developed [68, 69]. Among these two probes, TPE-MEM features

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alkyl chain for membrane anchoring and hydrophilic pyridinium salt moiety to contribute to retain the probe on cell plasma membrane. In addition to the unique AIE property, TPE-MEM is good water solubility, low cytotoxicity, and cell plasma membrane specificity, allowing for fluorescent membrane imaging (Fig. 9). Similar to TTVP, TPE-MEM is able to produce ROS under room-light irradiation. PDT effectiveness was then evaluated by cancer cell and tumor ablation, demonstrating the potential of TPE-MEM as a cell plasma membrane-selective photosensitizer for cancer treatment.

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Recently, another AIE-Based probe (Pent-TMP) (Fig. 10) was developed by Shi et al. for rapid 10

and sensitive imaging of plasma membranes [70]. Pent-TMP has a pentyl group for targeting cell plasma membrane through hydrophobic-hydrophobic interaction. Similar to probe TTVP, a ((trimethylammonium)propyl) pyridine group was also incorporated to Pent-TMP for labelling the cell

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interaction.

Pent-TMP

showed

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non-fluorescence in DMSO. When toluene fraction was >30% in DMSO mixed solvent, strong emission of the aggregates of Pent-TMP was observed. Pent-TMP was found to be able to visualize the plasma membrane morphology of neuronal cells, real tissue imaging, and labeling of

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the epidermal plasma membrane of live zebrafish.

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Polymers containing alkyl side chains were also explored for the development of fluorescence

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probes for cell plasma membrane staining. Modification of oleic acid chain with polyethylene glycol (PEG), Kato et al. reported two cell plasma membrane anchoring reagents (Fig. 11) [71]. In

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these two reagents, oleyl chain was introduced as lipid anchoring group at one end of polyethylene

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glycol (PEG). In comparison with single oleyl chain derivative, double oleyl chain derivative was

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found to be a useful anchoring reagent due to its high water insolubility, fast anchoring ability, and membrane retaining ability. The NHS-ester allowed facile modification with signaling unit, such as fluorescein, streptavidin-fluorescein, for fluorescent cell plasma membrane imaging. Similarly, PEG-conjugated phospholidpid (PEG-lipid) and poly(vinyl alcohol) carrying alkyl side chains (PVA-alkyl) (Fig. 11) were explored by Teramura and co-workers as the membrane anchoring moieties for cell plasma membrane studies [72-77]. Using FITC as the fluorescent reporter, cell plasma membrane dynamic and stability investigations revealed that the PVA-alkyl anchor presented longer retention time on the cell surface than PEG-lipid anchor. This was attributed to the multiple points of attachment by hydrophobic interaction between the PVA-alkyl polymer and the lipid bilayer. However, these synthetic polymers were gradually taken into the cytoplasm and excluded from the cell surface within 24 h. In order to retain the probes on the cell surface for a longer period, Kamitani et al. reported a cell surface modification method using the 11

synthetic polymers 5 (Fig. 11) [78]. With FITC as fluorophore and oleylamine chain as membrane anchoring moiety, 5 was found to be able to retain on the cell surface without rapid internalization. Further investigations of cell imaging showed over 12 h retention of 5 on the cell surface that could be attributed to the contributions of secondary amines in the polymer.

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In 2015, Liu et al. reported two red emitting polymers (S2, M2) (Fig. 12) for two-photon

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excitation fluorescence imaging of cell plasma membrane [79]. The preferential location of S2 and M2 at the cell plasma membrane was ascribed to: i) hydrophilic interactions between the carboxylic

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acid moiety of poly(styrene-co-maleic anhydride) (PSMA) and the water molecules near the

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membrane lipid head groups, ii) the hydrophobic interactions between the multiple aliphatic chains

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of the polymers and lipid molecules in the cell plasma membrane. These polymers had a considerable TP absorption cross section, facilitating TP fluorescence imaging of membrane in three

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different cell lines.

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DNA fragments contain highly charged natural sequences that cannot incorporate and penetrate

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the plasma membrane. This property of DNA allows for the development of membrane-anchorable nucleic acid-based cell plasma membrane probes [80, 81]. In recent years, a series of oligonucleotide-based membrane probes, termed as aptamers, have been developed using stearic acids as the membrane anchor ligand for studying the cell plasma membranes associated physiological processes.

< Fig. 13 is here >

Taking advantages of the aptamers’ long retention time and the alkyl chains’ strong interaction with lipid bilayer, Tan and coworkers reported several diacyl lipid-DNA aptamers for the visualization of physiological processes on cell plasma membrane [82-84]. For example, diacyl 12

lipid-DNA aptamer probe (lipid-DNA) was prepared as an anchor for cell plasma membrane targeting in 2013 [84]. A synthetic diacyllipid tail with two stearic acids was conjugated to the 5’ end of the aptamer for cell plasma membrane anchoring through a PEG linker. This PEG linker also contributed to minimize nonspecific and steric interactions between the cell-surface molecules and the aptamer. The diacyl lipid tail of the probe could effectively insert into the immune cell plasma membrane by its hydrophobic nature. These aptamer-modified immune cells were able to be recognized by recording the fluorescent signals (TAMRA conjugated to the 3’ end of the oligonucleotides as reporter), demonstrating a powerful approach in developing cell plasma

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membrane-targetable nucleic acid-based probe. Based on the success of lipid-DNA probe, a phosphorylated lipid-conjugated oligonucleotide (DNA-lipid-P) (Fig. 13) was recently developed to

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distinguish different cell plasma membranes [83]. Interestingly, the membrane-targetable lipid

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terminus of DNA-lipid-P was blocked by negatively charged phosphate groups. In the presence of

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alkaline phosphatase (ALP), DNA-lipid was formed and thus the cell plasma membrane adhesion

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ability was improved. DNA-lipid-P was then successfully used for anchoring on ALP-elevated cell plasma membranes, demonstrating its potential for disease diagnostics at the molecular level.

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By covalently conjugating hydrophobic diacyllipid tail with chemically stabilized RNA or DNA

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oligonucleotides, Liu et al. developed some cell plasma membrane anchored CpG ODNs probes for in vivo cell modification [85]. These conjugated oligonucleotides were synthesized by coupling

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20-mer oligonucleotides with cholesterol, single chain hydrocarbon or diacyllipids at the 5’ end, respectively. Imaging results suggested that diacyllipid tails provided the highest affinity for ODN insertion into cell plasma membranes over the probes with cholesterol and single chain hydrocarbon. In addition to these hydrophilic oligonucleotide-hydrophobic tails-based probes, highly stable, well‐ defined oligonucleotide micelles have also been investigated for cell plasma membrane by Liu et al. in 2010 [86]. The oligonucleotides were linked to hydrophobic diacyllipid tails to form amphiphilic oligonucleotide molecules that can be spontaneously self-assembled to 3D micellar nanostructures with hydrophilic DNA corona. Cell experiments investigations revealed that the internalization process of these micelles is depend on the nature of the hydrophobic tails as well as the DNA length.

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2.1.2 Cholesterol as the targeting unit Cholesterol is an essential lipid component of cell plasma membrane that interacts with the alkyl tails of phospholipids to regulate the rigidity of cell plasma membranes. Based on the relative in vitro affinities of their phospholipids, cholesterol is found to be associated strongly with the outer than the inner leaflet of cell plasma membrane bilayers [87, 88]. By virtue of unique functions of

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through coupling cholesterol with the fluorescent reporters.

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cholesterol, several probes have been reported for efficient cell plasma membrane anchoring

Using cholesterol as the cell plasma membrane anchoring group, Wu and coworkers reported

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several fluorescent probes for long-time and stable cell plasma membranes imaging [89-91]. In

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2015, a multisite membrane tracker (Chito–Chol–FITC) (Fig. 14) for the imaging of cell plasma

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membranes was developed by modifying cholesterol scaffold [89]. In Chito–Chol–FITC, PEG-cholesterol conjugated through amide groups served as membrane anchoring and FITC

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conjugated to amine groups served as the fluorescent reporter. Cellular imaging studies revealed

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that Chito–Chol–FITC could rapidly stain the cell plasma membrane within 5 min. More

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important, no obvious internalization was observed after staining the cell plasma membrane for 6 h. Crosslinking (fixation) the cells with 4% paraformaldehyde prevented the detachment of Chito– Chol–FITC from the cell surface by washing with alcohols and 0.2% Triton X-10 permeabilization, implying probe Chito–Chol–FITC could be used as a membrane labeling reagent that can tolerate lipid removal in immunostaining [91]. Instead of direct linking of FITC fluorophore, modification of chitosan with biotin produced a two-step synergistic cell surface modification strategy, where the cell plasma membrane labeling reagents (GC-Chol-Biotin and avidin-FITC) (Fig. 14) were developed for long time and stable cell imaging [90]. This strategy allowed for membrane anchoring through two steps procedure: i) anchoring GC-Chol-Biotin onto the plasma membrane to decorate the cell surface with biotin moieties; ii) linking the biotin moieties with FITC conjugated avidin through biotin-avidin recognition. These reagents were capable of visualizing cell plasma membrane for up to 8 h without 14

substantial internalization of the dyes. The application of this probe was then demonstrated for tracking cell plasma membrane behaviors, including shrinkage, membrane blebbing and vesiculation under continuous laser irradiation of Chlorine e6 (Ce6) for 5 h.

< Fig. 15 is here >

Similar to DNA-lipid-P [83], an ALP activatable probe (1P) (Fig. 15) for high spatial and

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temporal resolution imaging of membrane dynamics of live cells were reported by Wang et al. [92].

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1P consisted a cholesterol as the cell plasma membrane anchoring, a 4-nitro-2,1,3-benzoxadiazole (NBD) as the fluorescence reporter, and a phosphotyrosine that can be activated by cell surface

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phosphatase. This phosphotyrosine of 1P was able to be cleaved through phosphatase induced

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reaction. The application of 1P was then involved in monitoring of nanoscale heterogeneity in

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membranes of live cells, the release of exosomes, and the membrane dynamics of live cells.

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Using cholesterol as membrane anchoring group, Jia et al. reported a red-emitting fluorescence probe (Chol-PEG-Cy5) (Fig. 15) for cell plasma membrane labelling in vitro and in vivo [93].

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Chol-PEG-Cy5 was designed by linking cholesterol and Cy5 fluorophore through a PEG linker.

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Due to the intermolecular Förster resonance energy transfer (FRET), the fluorescence of Chol-PEG-Cy5 was quenched in water by the neighboring Cy5 fluorophore. Upon binding to cell plasma membrane, the FRET was disappeared and intense fluorescence at cell plasma membrane was obtained. With this Chol-PEG-Cy5 probe, fluorescence imaging applications were demonstrated to track the cell plasma membrane dynamics of cells and the epidermal cell surfaces of live zebrafish. Using 3D amphiphilic pyramidal DNA as the scaffold, effective membrane-anchored nanoprobe was developed by Li et al. [94]. These amphiphilic tetrahedrons DNA probes were self-assembled by four DNA oligonucleotides with a pendant DNA probe at one vertex and cholesterol tags at the other vertices. These probes could rapidly and efficiently accumulate onto the cell plasma membrane based on hydrophobic insertion between the cholesterol and the cellular phospholipid layer, providing a powerful approach for the investigations of cell plasma membrane-related communication network. 15

2.1.3 Tocopherol as the targeting unit

< Fig. 16 is here >

Tocopherol, known as vitamin E, is another lipid component of animal cell plasma membrane, where the lipophilic side chain intercalates in cell lipid monolayers. Using tocopherol as membrane

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anchoring groups, some of fluorescence probes have recently been developed for investigating the

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cell plasma membrane dynamics. For example, Tokunaga et al. reported a fluorescent aptamer

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sensor (toc-fApt) (Fig. 16) for the analysis of extracellular chemical transmitter dynamics [95]. The

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tocopherol-conjugated aptamer (toc-fApt) directly drive the fluorescent aptamer to the cell surface. Applications of toc-fApt was then demonstrated by fluorescent imaging of astrocytes. Together

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with the advantages of fluorescence imaging, the use of toc-fApt enabled the real-time monitoring

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of adenine compounds release. In another work, You et al. reported three DNA-based probes for the monitoring of the dynamic and transient molecular encounters on live cell plasma membranes [82].

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Three natural membrane components, including phospholipid, cholesterol and tocopherol were used

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to anchor the DNA probe system onto cell plasma membrane. These DNA probes were utilized to monitor rapid encounter events of membrane lipid domains.

< Fig. 17 is here >

2.1.4 others In addition to lipophilic alkyl chains, cholesterol and tocopherol, ligands having a special structure that binds specifically to lipids have also been used in the development of fluorescent probes for membrane analysis and imaging. Phosphatidylserine (PS), an anionic phospholipid, is an important component of the membrane of all eukaryotic cells [96, 97]. Based on the high affinity of cationic dipicolylamine-zinc (DPA-Zn) complexes to phosphatidylserine, several fluorescent probes 16

for imaging cell surface phosphatidylserine have been developed in the past few years [98-100]. For example, through exploiting an intramolecular indicator displacement sensing mechanism, Zwicker et al. reported the development of a fluorogenic probe (P-IID) (Fig. 17) for imaging of cell surface phosphatidylserine [99]. PIID showed weak fluorescence due to the coordination of the 6,7-dihydroxycoumarin with one of the DPA-Zn moieties. In the presence of externalized PS, this coordination was displaced by the interaction of DPA-Zn site with anionic PS headgroup. As a result, restoration of the coumarin fluorescence was obtained for fluorescence analysis and imaging of membrane. Recently, a BODIPY-based fluorogenic probe (BPDPA-Zn) (Fig. 17) was designed

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by Ashokkumar et al. for highly selective imaging of phosphatidylserine [100]. BPDPA-Zn has a BODIPY-based molecular rotor for sensing viscosity, two DPA-Zn moieties for recognition of PS,

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and a PEG linker to prevent non-specific interactions with cell membrane. BPDPA-Zn showed a

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turn-on fluorescence response upon binding to lipid membranes containing anionic phospholipid PS.

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Imaging results suggested that BPDPA-Zn could bind PS of early apoptotic selectively, and

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distinguish early and late apoptotic cells based on fluorescence intensity.

probes

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2.2 Cell plasma membrane proteins as the targeting sites for the development of fluorescent

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In addition to lipids, proteins are another type of important component of cell plasma membranes.

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Many proteins with important biological functions are embedded in cells membranes of live organisms. These membrane proteins are significantly contributing to the cell plasma membrane biology and biophysics [101, 102]. For instance, membrane proteins serve as molecular channels for transporting metabolites and nutrients, and as sensor proteins for probing the cellular environment [103, 104]. Therefore, fluorescent labelling of cell plasma membrane proteins has been reported as another important approach for understanding the physiological processes of cell plasma membranes. Until now, five approaches have been successfully explored in the development of protein labelling based fluorescent probes, including (i) tag-mediated fusion protein labelling methods; (ii) chemistry (small molecular ligands)-based protein labelling methods; (iii) antibody-based membrane protein labelling methods; (iv) aptamer-based labelling methods; and (v) others. In this section, we will discuss the advances of cell plasma membrane protein labelling techniques and their applications. 17

< Fig. 18 is here >

2.2.1 Fusion protein tags based fluorescent probes for membrane proteins Development of specific fusion protein fluorescent probes for cell surface proteins labelling relies on i) expressing target proteins as a fusion tag of peptide or protein that endows the protein of interest (POI) with new functionality, ii) fluorescent probes are coupled to the target protein. In

of

recent years, several peptide and protein fusion tags have been developed, including PYP-tag,

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SNAP-tag, Halo-tag, CLIP-tag, ACP-tag, b-lactamase-tag, etc. [105-110], and some of them have been successfully used in cell plasma membrane protein labelling. For example, Johnsson and

-p

coworkers reported a general approach (Fig. 18) for the covalent labeling of cell surface fusion

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proteins with chemically diverse compounds, such as fluorophores, affinity ligands, or quantum

lP

dots [106, 111, 112]. With this general approach, the POI was first fused to an acyl carrier protein (ACP). This fusion protein was then specifically labeled with coenzyme A (CoA) derivatives (as

na

substrates) through a post-translational modification that was catalyzed by phosphopantetheine

ur

transferase (PPTase). The membrane impermeability of PPTases and CoA derivatives make this

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method feasible for labeling of cell-surface proteins. This method was then used for multicolor imaging of cell surface proteins by screening the appropriate type of ACP and CoA derivatives, respectively.

< Fig. 19 is here >

On the basis of photoactive yellow protein (PYP)-tag labelling approach, Hori et al. developed a protein-labeling probe (FCTP) (Fig. 19) for specific staining of PYP tag [107]. FCTP showed weak fluorescence due to the intramolecular association between the fluorescein and coumarin moieties. After labeling of PYP, FCTP exhibited obvious “OFF-ON” fluorescence response. Despite the advantages of this approach, long time incubation (more than 24 h) is required to 18

complete the labelling reaction. To address this issue, improved fluorogenic probes, FCATP and FCANB (Fig. 19), were then developed for rapid protein labeling in 2012 [113]. Of these probes, FCANB can bind to the PYP tag with 110 times faster than that of FCTP. FCANB consisted of fluorescein as fluorophore, cinnamic acid thioester as a PYP-tag ligand, and nitrobenzene as a fluorescence quencher. The fluorescence of a probe is quenched by nitrobenzene, while this nitrobenzene quencher is dissociated after PYP labeling, accompanied with the “OFF-ON” fluorescence response. FCANB was cell plasma membrane impermeable and was able to specifically label the PYP-EGFR fusion protein on the cell surface. More important, the “OFF-ON”

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fluorescence response feature of FCANB with rapid labeling kinetics make it favorable for live cell

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plasma membrane imaging because tedious washing procedures are not required. Employing SNAP-tag technology, several fluorescent probes for cell plasma membrane protein

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labelling have been developed in the past few years. In 2011, Sun et al. reported the design and

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application of fluorescent benzylguanine probes for labelling of SNAP-tag fusion proteins on the

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cell surface [105]. To obtain optimal intramolecularly quenched substrates, different combinations of fluorophore-quencher pairs with the emission across the visible spectrum were investigated in

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this work. These fluorescence probes, carrying negatively charged groups, cannot passively cross

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cell plasma membranes, allowing for the long-term visualization of targets on cell plasma membrane. Of these probes, CBG-549-QSY7 (Fig. 19) was able to imaging the epidermal growth

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factor on cell plasma membrane with high sensitivity. Combing the time-resolved fluorescence resonance energy transfer (FRET) with SNAP-tag technology, Maurel et al. described a method for quantitative analysis of protein-protein interactions at the living cells surfaces [114]. In this time-resolved FRET (TR-FRET) scaffold, the long-lived europium cryptate was selected as the FRET donor, and Alexa Fluor 647, DY-647 or d2 were employed as FRET acceptors. By introducing a SNAP tag at the N-terminal end of GABAB1 and GABAB2 subunits, labelling cell-surface receptors with this TR-FRET fluorophore were achieved.

< Fig. 20 is here >

19

Johnsson and coworkers also reported several fluorescent probes for visualizing membrane protein-associated physiological processes on cell surface [115-117]. In 2011, a class of semisynthetic fluorescent sensor proteins, Snifits (Fig. 20), was developed for measuring metabolite concentrations on the cell surface [115]. In the absence of analyte, the intramolecular ligand of Snifits binds to the binding protein, which leads to a high FRET efficiency due to the short distance between donor and acceptor fluorophores. In the presence of analyte, the intramolecular ligand is able to be displaced. As a result, the distance between donor and acceptor fluorophores are increased, leading to inefficient FRET. With human carbonic anhydrase II (HCA) as the binding

of

protein, a cell surface targeted Snifit probe protein was then developed to demonstrate the general properties and applications of Snifit probe proteins for the investigations of the cell surface

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metabolite.

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< Fig. 21 is here >

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Using the solvatochromic molecule Nile Red as the fluorophore, Johnsson and coworkers

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developed an “OFF-ON” fluorescence response probe for “no-wash” labelling of SNAP-tagged

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plasma membrane proteins (Fig. 21) [116]. The probe showed weak fluorescence in a polar aqueous environment. After binding to the SNAP-tag, Nile Red is localized to the plasma membrane, allowing for its membrane insertion. The fluorescence of Nile Red is thus switched “ON” for the membrane imaging. Different SNAP-tag fusion proteins, including the human insulin receptor were then

employed

to

demonstrate

the

fluorescence

character

of

the

probe.

Using

4,5-dimethoxy-2-nitrobenzyl derivatives (DMNB) as the photocleavable linker, photoactivatable and photoconvertible fluorescent probes were then designed for selectively coupling to SNAP-tag fusion proteins on the cell surface [117]. Photoactivatable fluorescein and Cy3 (Q-Fl and Q-Cy3) and a photoconvertible Cy5-Cy3 probe were synthesized in this work for the visualization of SNAP-tag fusion proteins on the cell surface. With the photoactivatable Cy3 probe, characterisation of the mobility of a lipid-anchored cell surface protein and of a G protein coupled receptor (GPCR) were successfully demonstrated.

20

2.2.2 Small molecule ligands based fluorescent probes for membrane proteins The interaction between cell plasma membrane protein and specific molecular ligands, such as folic acid, plerixafor, methotrexate, and lapatinib has also been explored as another approach for the development of targetable cell plasma membrane probes for surface protein labelling. In comparison with aforementioned tag-mediated fusion proteins fluorescent probes, the small molecular ligands-based membrane protein labelling approach allow for directly labelling of natural

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proteins (proteins of interest) without genetic manipulation.

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< Fig. 22 is here >

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Lapatinib, a tyrosine kinase inhibitor (TKI) of human epidermal growth factor receptor 2 (HER2), can insert into the intracellular domain of transmembrane ER2 family proteins [118]. Based on this

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unique interaction mechanism, Wang et al. reported a lapatinib conjugated water-soluble polymer probe (PTL) for cell plasma membrane imaging [119]. This polymer consisted of polythiophene as

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the main skeleton and fluorophore, oligo-ethylene glycol (OEG) as the side chains and the lapatinib

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as intracellular transmembrane proteins targeting group to drive the PTL’s location in the cell plasma membrane region (Fig. 22). Fluorescence images of SK-BR-3 and MCF-7 cells demonstrated that PTL could locate in the cell plasma membrane region for targeted cell plasma membrane imaging. Taking advantages of the specific binding between plerixafor (PLE) and CXCR4 protein [120], another fluorescent polymer-based nanoprobe (PFPNP-PLE) was developed by Li et al. for cell plasma membrane imaging [34]. PFPNP-PLE has excellent photophysical properties (blue fluorescence), low cytotoxicity, and cell plasma membrane target ability, which enabled this probe to be used for cell plasma membrane labeling.

< Fig. 23 is here >

21

Based on the high affinity of methotrexate (MTX) to the folate receptor (FR) and benzenesulfonamide (SA) to the carbonic anhydrases (CA) [121-123], Hamachi and coworkers demonstrated two approaches for fluorescent labelling of endogenous membrane-bound proteins [124, 125]. In one of the ligand-directed acyl imidazole (LDAI) approach (Fig. 23) [124], MTX-conjugated fluorescein was used as a fluorescent ligand for labelling FR on the cell plasma membrane. After anchoring of this ligand to the target protein, the nucleophilic acyl substitution reaction occurred to release the ligand moiety and to yield labeled active protein with a vacant binding pocket. This LDAI approach allowed for the selective labelling of endogenous

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membrane-bound protein, such as FR on the live cell surface. Based on the mechanism of recognition-driven disassembly of ligand-tethered fluorophore, fluorescent nanoprobe was also

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fabricated by the same group for cell surface protein labelling and imaging [125]. The

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self-assembled nanoprobe showed weak fluorescence due to the aggregation-induced quenching

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(AIQ). Intense fluorescence was observed after response to the target protein, which was attributed

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to the recognition-induced disassembly of the probe. Using fluorescein or rhodamine as fluorophores, a series of probes were then synthesized for specific visualization of overexpressed

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FR and hypoxia-inducible CA on cancer cells.

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Huang et al. reported a versatile terminal protection assay strategy for the development of FA-linked DNA probe for wash-free quantification and imaging of cell surface FR [126]. FA was

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conjugated at 3’ end for specific recognition of the cell surface FR and the Ag nanoclusters (AgNCs) was linked at 5’ terminal for delivering detectable responses. The prepared AgNCs/DNA probe was able to be excited at 635 nm, and the emission maximum was 670 nm, thus providing a near infrared fluorescence imaging probe for FR on the cell surface. Binding of the FA to FR protected the DNA probe from degradation by exonuclease I, which allowed the probe for wash-free detection and imaging of cell surface protein. Cell imaging results indicated that this probe is capable of distinguishing FR-positive cells and FR-negative cells. Based on the interaction of the peptide ligand carbetocin with a G protein-coupled receptor (oxytocin receptor), Klymchenko and co-workers developed several turn-on fluorescent ligands for imaging G protein-coupled receptors in living cells [127-129]. For example, in 2015, three fluorogenic squaraine dimers-based far-red probes were prepared for background-free bioimaging 22

[128]. In aqueous media, these probes showed weak fluorescence due to the intramolecular H-aggregates, whereas in organic solvents, distinct fluorescence was obtained due to the unfolding of the dimer, i.e. disruption of the H-aggregate. Among the three probes, the one derived from the core-PEGylated squaraine showed the best performance in sensing OTR at the cell surface. To demonstrate the application of fluorescent probes for imaging G protein-coupled receptors in whole animal, a near-infrared fluorogenic dimer was designed recently based on the similar design concept [127]. Cyanine derivative (Cy5.5) decorated with a PEG8 chain was coupled with the OTR ligand to avoid non-specific interactions. Imaging results indicated that this probe could be used as a tool

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of

for background-free and target-specific imaging of the naturally expressed receptor in living mice.

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< Fig. 24 is here >

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2.2.3 Antibody-based membrane proteins fluorescent probes

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Based the highly specific binding of antigen-antibody, several antibody-based membrane proteins fluorescent probes have been delivered by simply labelling the antibody with fluorophores. For

ur

example, Chiu and coworkers reported several fluorescent semiconducting polymer dots (Pdots)

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probes for imaging cell surface Epithelial cell adhesion molecule (EpCAM) (Fig. 24) [130-132]. The Pdots with surface carboxyl-functionalization were first synthesized and then were used for biological conjugation with IgG or streptavidin through EDC-NHS cross-linking approach to obtain Pdot-IgG and Pdot-streptavidin probes. Imaging results revealed that Pdot-IgG conjugates could be directly used for cell surface EpCAM labelling. The Pdot-streptavidin probes, together with the primary anti-EpCAM antibody and biotinylated goat anti-mouse-IgG secondary antibody, also effectively labelled EpCAM on the surface of live MCF-7 cells. Taking advantage of the specific binding between trastuzumab (Herceptin) and the human epidermal growth factor receptor 2 (HER2), Li et al. described polymer particles for HER2-positive cancer cell detection [133]. The amine-functionalized polymer nanoparticles were conjugated to trastuzumab through EDC-Sulfo-NHS crosslinking reaction. As the overexpression of HER2 receptor on the SK-BR-3 breast cancer cell plasma membrane, these nanoparticles were then 23

employed for discriminating SK-BR-3 breast cancer cells from MCF-7 breast cancer cells and NIH/3T3 fibroblast cells. Similarly, Chiu and coworkers demonstrated streptavidin functionalized Pdots for specific labelling HER2 receptors on cell surface [134]. The nanoprobe was prepared by conjugating carboxyl-functionalized Pdots with streptavidin by carbodiimide-catalyzed coupling reaction. Together with primary anti-HER2 antibody and biotinylated goat antimouse IgG secondary antibody, the probe was effectively used for labelling HER2 receptors on SKBR3 cell surface. 2.2.4 Aptamer-based fluorescent probes for membrane proteins

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On the basis of the unique secondary or tertiary structures formed by single-stranded

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oligonucleotides, aptamers have potential as binding molecules for cell surface proteins labelling

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[84, 135, 136]. Nucleic acid aptamer approach has several advantages, including easy and

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reproducible synthesis, controllable modification, high stability, and ability to sustain reversible denaturation [137-142]. These properties allow for the development of aptamers-based fluorescence

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probes for the cell plasma membrane proteins targeting.

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The aptamer sgc8 has been identified to be able to effectively interact with the cell plasma membrane protein tyrosine kinase-7 (PTK7). In 2011, Shi et al. designed an activatable aptamer

ur

probe (AAP) for targeting membrane proteins of living cancer cells and in vivo imaging of cancer

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[143]. AAP is a single-stranded oligonucleotide consisting of three fragments: i) A-strand, the aptamer sgc8 for targeting PTK7; ii) T-strand, and iii) C-strand complementary to a part of the A-strand. The Cy5 fluorophore and BHQ2 quencher were covalently attached at either terminus of AAP. Due to the effective FRET-mediated quenching by BHQ2, AAP showed weak fluorescence in its free state. Upon binding to the cell plasma membrane protein PTK7, AAP underwent a conformational alteration, which led to a fluorescent turn-on response. Considering the close association of PTK7 with cancers, AAP was then successfully used for fluorescence imaging of the CCRF-CEM tumor site. Similar sgc8 aptamer-mediated membrane PKT-7 protein labelling method has been utilized by Zhao et al. for imaging analysis of protein-specific sialylation on the cell surface [144]. The Cy5 was conjugated to target monosaccharide through bioorthogonal chemistry, and Cy3 was linked to the AgNPs-sgc8 conjugate through DNA hybridization. The involvement of AgNPs in this system enhanced the FRET process from Cy3 to Cy5 for better performance in 24

protein-specific sialylation imaging. Recently, Li et al. reported sgc8 aptamer equipped micelles X-Sgc8-LM with improved stability and specificity for targeting cancer cells [145]. In this work, methacrylamide units was employed as the linker of sgc8 aptamer and lipid segments to help stabilize the micelle structure and mitigate nonspecific binding of the probe. Imaging results suggested that X-Sgc8-LM could selectively identified PTK7-positive cancer cells and significantly mitigate nonspecific binding.

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< Fig. 25 is here >

In 2013, a SYL3C DNA aptamer was identified by Song et al. for cancer cell targeted

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fluorescence imaging and circulating tumor cell (CTC) capture (Fig. 25) [146]. This aptamer is

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capable of recognizing the human cell expressed epithelial cell adhesion molecule (EpCAM)

lP

protein on the cancer cell surface. The SYL3C aptamer probe was obtained by labeled with FITC at the 5’ end. Using this aptamer probe, cell imaging and flow cytometric analysis were conducted,

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and the results demonstrated the feasibility of this probe for selective recognition a variety of cancer

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cells expressing EpCAM from the EpCAM-negative cells.

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A silver cluster-aptamer probe for marking and quantitatively counting membrane immunoglobulin M (mIgM) in live cells was developed by Liu et al. in 2014 [147]. The probe was constructed by modifying the silver cluster with the TDC05.1. sequence aptamer that is capable of targeting the mIgM epitope on the Burkitt’s lymphoma cell surface. The probe was successfully utilized for fluorescence imaging the mIgM of the B-cell receptor (BCR). Furthermore, the silver cluster of the probe also could provide mass quantification of mIgM by inductively coupled plasma-mass spectrometry (ICP-MS).

< Fig. 26 is here >

2.2.5 Others fluorescent probes for membrane protein 25

Proteins can interact with small peptides in a variety of ways. These protein-peptide interactions are part of many physiological processes. Taking advantages of the protein-peptide interaction, Gao et al. demonstrated a nanoprobe for specific targeting integrin GPIIb/IIIa on cell plasma membrane [148]. The probe consisted of AuNPs as fluorophore and peptide (H2N-CCYKKKKQAGDV-COOH) as integrin GPIIb/IIIa targeting moiety [149]. The probe has the ability of TP adsorption, allowing it to be used for TP fluorescence imaging of integrin GPIIb/IIIa on cell plasma membrane. Utilizing intrinsic enzyme-like catalysis property of the nanoprobe, the levels of integrin expression on human erythroleukemia cells were quantitatively counted. In another study, a multifunctional probe was designed and synthesized by Zhang et al. for targeting the αvβ3 integrin on cancer cell surface

surface,

and

a

5-amino-fluorescein

moiety

for

fluorescence

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cell

of

(Fig. 26) [150]. The probe consisted of a cyclic RGD peptide unit for targeting the αvβ3 integrin on imaging

and

a

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2-aminoethylmonoamide-DOTA group for loading stable europium ion and subsequent ICPMS

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quantification of the cancer cells. The feasibility of this probe for the detection and imaging of the

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αvβ3-positive cancer cells was then validated.

Based on the click reaction and bioorthogonal non-canonical amino-acid tagging (BONCAT)

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techniques, Wu et al. reported the development of functional semiconducting polymer dots for

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fluorescence membrane protein imaging [151]. The synthesized alkyne-/azido-functionalized proteins were obtained by incubating MCF-7 cells with azidohomoalanine (AHA) or

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homopropargylglycine (HPG). Then, these proteins on the MCF-7 surface were selectively recognition by azido-/alkyne-functionalized Pdots. N-azidoacetylgalactosamine (GalNAz) and Pdot-alkyne were also used for selectively target membrane glycoproteins by a click reaction. 3. Membrane-targeting responsive fluorescence probes In comparison with fluorescent cell plasma membrane labelling, the probes that are capable of both visualising and detecting one specific biomolecules on the cell plasma membrane are particularly desirable. In this context, several fluorescence probes have been successfully developed for the detection of biomolecules on cell plasma membrane [152-157]. In this section, we will outline recent advances in the development of responsive fluorescence probes for the detection and visualisation of various ions and molecules on cell plasma membrane. These species include microenvironment pH, metal ions, small reactive biomolecules, macromolecules, and membrane 26

tension. 3.1 Membrane-targeting responsive fluorescence probes for pH Cellular microenvironment pH plays an important role in biological processes, such as adhesion, migration and drug resistance [158-161]. Abnormal pH level is known to be associated with various pathological states, such as tumors, ischemic stroke, infection, and inflammation [162-164]. For example, extracellular pH is around neutral (pHe = 7.4) in normal tissues, while the extracellular pH in tumor microenvironment is around 6.7-7.1, which is much lower than that of normal tissues. As a result, changes of cellular microenvironment pH have been identified as a hallmark for tumors.

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Therefore, tracking the changes of intercellular pH and cellular microenvironment are very

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important for better understanding the biological processes of various diseases.

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< Fig. 27 is here >

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To track the extracellular pH, Ke et al. prepared a lipid-DNA-based probe for ratiometric fluorescence sensing of extracellular pH (Fig. 27) [152]. The probe has a hydrophobic diacyllipid

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tail for anchoring membrane lipid and DNA structure for retaining the probe on the cell surface. For

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ratiometric detection of extracellular pH, the probe was labelled with pH-sensitive FAM (carboxyfluorescein derivative) and pH-insensitive dye (TAMRA). As a result, the emission at 520 nm was increased with the pH changes from 6.0 to 8.0, while emission at 580 nm showed pH independent. The ratiometric fluorescence (F520/580) changes allowed for the detection and imaging of extracellular pH of HeLa cells. Using pH-insensitive gold nanoclusters (AuNCs) as the reference dye and FITC (fluorescein derivative) as the pH-sensing unit, Yang et al. developed another ratiometric fluorescent probe for monitoring the dynamic changes of extracellular pH in real time [153]. The probe showed ratiometric fluorescence response (F521/635) for pH from 5.0 to 9.5 with a pKa of 7.2. The probe carrying large amount of unlabeled cationic Fpen peptides to target cell plasma membranes, facilitating its fluorescence imaging of extracellular pH and monitoring cell surface acidification in normal and high glucose metabolism.

27

< Fig. 28 is here >

Based on the pH-sensitive i-motif sequence, several cell-surface-anchored DNA probes have been developed for pH detection [165-168]. For example, on the basis of FRET mechanism, Ying et al. engineered a cell-surface-anchored probe for ratiometric fluorescence detection of extracellular pH [169]. Two pH-insensitive dyes (rhodamine green and rhodamine red) were selected as the FRET donor and acceptor to be accommodated on the pH-sensitive i-motif structure. For cell surface anchoring, membrane amines were modified with NHS-biotin. This biotin modification

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allowed the i-motif probe staining on the cell surface through strong interaction of

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biotin-streptavidin. This interaction enabled the probe’s localization on the cell surface for the

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detection of the changes of extracellular pH. Similar FRET between rhodamine green and

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rhodamine red has also been explored by Zeng et al. for the construction of an i-motif DNA tweezer to dynamically monitor pH changes of extracellular microenvironments [170]. This probe consisted

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of a triple-stranded ssDNA framework that is labeled with cholesterol for cell plasma membrane

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targeting. The i-motif structure is pH sensitive, allowing for modulating FRET process for ratiometric fluorescence detection of pH. The application of this probe was then demonstrated to

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map the apoplastic-pH change during the alkalization process of plant roots caused by

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rapid-alkalinization factor (RALF1). Liu et al. reported a cell surface-anchored DNA NT for dynamically tunable sensing of extracellular pH on living cells (Fig. 28) [171]. The DNA NT could be efficiently anchored on the cell surface via multisite cholesterol moieties. Fluorophore TAMRA and FRET acceptor and quencher BHQ2 were conjugated to the DNA strands for fluorescence response to pH via pH-responsive triplex-duplex conformational transition. This probe was then used for sensing and imaging of pH changes in extracellular microenvironments. 3.2 Membrane-targeting responsive fluorescence probes for metal ions

< Fig. 29 is here >

Potassium (K+), calcium (Ca2+), and magnesium (Mg2+) are essential elements of the human body 28

that play important roles in many physiological events, such as hyperpolarization in signal transduction of neurons, regulating cell osmotic pressure, apoptosis, muscle contraction, exocytosis, and humoral secretion [172-176]. For the detection of these metal ions, a number of bioanalytical methods, particular the responsive fluorescence probes have been developed recently [177-183], and some of them are capable of detecting and visualizing these metal ions in cell plasma membrane. For Ca2+ detection, cell plasma membrane targetable probes, such as C18-Fura-2 and Calcium Green C18 probes (Fig. 29) have been developed by coupling of hydrophobic alkyl chain to Ca2+-responsive fluorophores [184-186]. These membrane probes were successfully used to

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monitor the changes of Ca2+ concentration in near-membrane and the translocation of Ca2+ across the plasma membrane. Taking advantages of two-photon (TP) excitation, Mohan et al. developed a

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TP fluorescent probe (ACaL) (Fig. 29) for near-membrane Ca2+ detection and imaging deeply

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inside live tissues [187]. ACaL is designed by coupling dodecanoyl to a Ca2+-responsive

re

fluorophore, which allowed for the plasma membrane anchoring and Ca2+ sensing. Under excitation +

in living cells and fresh rat

lP

at 780 nm, TP fluorescence imaging of near-membrane Ca2

< Fig. 30 is here >

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ur

na

hippocampal slice were demonstrated.

Based on the tag protein technology, Hirata et al. developed a protein-coupled fluorescent probe (TLSHalo) for selectively visualization of K+ at the cells surface (Fig. 30) [154]. TLSHalo consisted of TAC (2-triazacryptand [2,2,3]-1-(2-methoxyethoxy) benzene) chelator as the recognition moiety and HaloTag ligand as HaloTag protein targeting moiety. The probe is responsive to K+ in the physiological range, and retains its K+-sensing properties after covalent conjugation with HaloTag protein. TLSHalo was further employed to monitor extracellular K+ change induced by K+ ionophores or by activation of a native Ca2+-dependent K+ channel. In another work, Xiong et al. reported a diacyllipid-aptamer conjugate-based fluorescent probe for the monitoring of K+ in the cell microenvironment [175]. This probe consisted of a thrombin-binding aptamer (TBA) sequence as the K+-recognition unit, a C18 diacyllipid as the cell-membrane anchoring unit. The reversible coordination between K+ and the probe make it available for 29

real-time and reversible monitoring of K+ in the cell microenvironment. A universal diacyllipid-DNAzyme probe for cell plasma membrane-anchored detection of target metal ions in the cell microenvironment was deleloped by Qiu et al. in 2014 [188]. The probe consisted of a diacyllipid tail, a PEG linker and a DNAzyme sequence. In this system, the DNAzyme-fluorophore conjugates hybridized with the substrate that was linked to the quencher. The DNAzyme was activated after the binding of probe to the target metal ion. The activation of DNAzyme led to the cleavage of substrate and dissociation from the DNAzyme strand. As a result, the fluorescence signal was switched “ON” for this target metal ion detection. More importantly, the

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diacyllipid-DNAzyme probe can be designed for the detection of multiple metal ions through

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simply replacing the DNAzymes. The reliability and versatility of this diacyllipid-DNAzyme strategy based probe were then evaluated by using Mg-DNAzyme, Pb-DNAzyme, and

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Zn-DNAzyme sequences for Mg2+, Pb2+, and Zn2+ detection on the cell plasma membrane,

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respectively.

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na

< Fig. 31 is here >

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Iron, copper, zinc and other metal ions are essential trace elements within body that play vital roles in various biological processes of fundamental physiology [189-191]. For example, it has been reported that the disruption of iron homeostasis can lead to the oxidative stress and cell damage [192]. Nevertheless, the redox states of iron within body remain unclear due to the lack of bioanalytical tools for effective and fast detecting the redox states of iron ions in-situ in real-time. To tackle this issue, Niwa et al. developed a fluorescent probe (Mem-RhoNox) (Fig. 31) specific for the detection of Fe2+ on the plasma membrane [155]. The probe was designed by exploring dialkylarylamine N-oxide as the Fe2+-reactive site and palmitoyl group as the membrane-anchoring group. Cleavage of N-oxide by Fe2+-triggered reaction, the fluorescence of rhodamine (Em = 575 nm) was increased, allowing for the detection of Fe2+ at the surface of the plasma membrane of live HpeG2 cells and hippocampal neurons (DIV15). Imbalances of Cu2+ has been reported to be a potential hallmark for the various diseases, such as 30

Wilson’s and Alzheimer’s diseases. Therefore, development of an effective method for the detection and imaging of free copper ions near or across the plasma membrane will be helpful to better understand the biological roles of Cu2+ transport in these diseases [182, 183, 193-195]. In this context, Chen et al. developed a photocontrollable fluorescence probes (Mem-5, Mem-6) (Fig. 31) for the monitoring of Cu2+ on the cell plasma membrane [196]. The probes were designed by incorporating a cholesterol or 12-carbon aliphatic chain as membrane targeting group into a Cu2+-responsive fluorescein reporter. Under the UV-light irradiation, the nitrobenzyl group was cleaved, affording a fluorescein-based probe for Cu2+ ion detection. Fluorescence imaging was performed to demonstrate the capability of Mem-5 in photocontrolled detection and monitoring of

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Cu2+ in the cell surface in live cells. By modifying tetraphenylethene-pyridine (TPE-Py)-based

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AIE-fluorophore with a peptide (EEGTIGYG), Liu et al. synthesized a fluorescent probe,

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TPE-Py-EEGTIGYG (Fig. 31), for selective detection of Cu2+ on the cell plasma membrane [197].

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Cell imaging results revealed that the probe was largely distributed on the cell plasma membrane and the emission was significantly increased after cell plasma membrane anchoring. Further

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incubation of the cells with Cu2+, the fluorescence on the cell surface was quenched due to the

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interaction between TPE-Py-EEGTIGYG and Cu2+.

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To understand the biological functions of Zn2+ within body [178, 189, 198], Deng et al. reported fluorescence probes (ZTRS-alkyl) (Fig. 31) for the detection of Zn2+ on the cell plasma membrane

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[199]. The ZTRS-alkyl was designed by incorporating alkyl chain with different lengths to a naphthalimide-based Zn2+ chemosensor (ZTRS) [200]. The ZTRS-alkyl and the product binding with Zn2+ were found to be weakly fluorescent due to the AIQ. After the interaction between alkyl chain and cell surface lipid, dissociation of the aggregation led to the liberation of ZTRS-alkyl for Zn2+ detection with obvious enhancement of fluorescence. It was found that ZTRS-C12 has better performance on Zn2+ detection on cell surface, allowing it to be used as a tool for Zn2+ imaging in live HT-29 cells.

< Fig. 32 is here >

31

Mercury ion (Hg2+) is one of the most toxic heavy metals that causes several problems to human health and the environment [201-204]. This ion can be widely presented in the ambient air, water, soil and biota, and then be taken up and accumulated in the body, resulting in various biological issues, such as leukemia, cardiovascular disease and nervous system damage. The cell plasma membrane is believed to be a barrier that prevents harmful Hg2+ ions from entering the cell freely. Therefore, the development of fluorescent probes specific for the detection of Hg2+ on the cell plasma membrane is of great significance [201, 202]. Towards this end, Bai et al. reported a membrane-targetable probe (HGMem-3) (Fig. 32) for Hg2+ detection in 2018 [205]. Cholesterol tag was equipped on one phenol of the fluorescein for cell plasma membrane targeting. Similar to the

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design of Mem-5 and Mem-6 [196], HGMem-3 was caged by a photolabile group (2‐ nitrobenzyl

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group) for photocontrolled Hg2+ detection. After binding to the cell plasma membrane, the UV light

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irradiation allowed for cleavage of the cage and then recognition of extracellular Hg2+. HGMem-3

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showed a 1900-fold enhancement of the fluorescence intensity at 520 nm upon addition of Hg2+, and the detection limit was determined to be 4.9 × 10-7 M. HGMem-3 was then used for imaging of

< Fig. 33 is here >

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Hg2+ near cell plasma membrane of live HepG2 cells.

3.3 Membrane-targeting responsive fluorescent probes for reactive small molecule Reactive oxygen species (ROS), reactive sulfur species (RSS), reactive carbonyl species (RCS) and reactive nitrogen species (RNS) are a series of active small molecules that play important roles in many physiological processes such as immune regulation, cell metabolism, nerve damage, and signaling [19, 206-213]. As one of highly reactive ROS, hypochlorous acid (HOCl) plays essential roles in the immune system for rapid and effective killing and digestion the microbes. Imbalanced levels of HOCl is causal to or can exacerbate pathogenesis of various diseases such as arthritis, vascular disease, kidney disease, cancer and lung injury [206, 214-218]. More importantly, the elevated level of HOCl on the surface will change the cell plasma membrane structure. This will affect the deformability and permeability of the cell plasma membrane, resulting in cell lysis and 32

death [219-221]. To detect the HOCl on the cell surface, Peng et al. designed and synthesized a membrane-specific fluorescence probe (HOCMem) (Fig. 33) for the detection of near-membrane HOCl [156]. HOCMem was designed by integrating a photolabile group (2‐ nitrobenzyl group) and membrane-anchoring unit (cholesterol) with HOCl-sensitive fluorescein scaffold. Similar to aforementioned Mem-5, Mem-6 [196], and HGMem-3 [205], the analyte (HOCl) was able to be detected after UV-light mediated cleavage of 2‐ nitrobenzyl group. The cholesterol unit of HOCMem allowed it to be distributed on the cell surface for the detection and imaging of HOCl of live HepG2 cells. Using alkyl chain as the membrane anchoring group, Zhang et al. reported a

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series of Ir(III) complex-based phosphorescence probe for HOCl detection and imaging and distinguishing exogenous/endogenous HOCl [60]. Complex 3 with seven-carbon chain was

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employed as a fluorophore for the development of HOCl probe that was obtained by modifying the

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complex with a HOCl-responsive aldoxime group. The probe 3a (Fig. 33) showed weak

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fluorescence due to the quenching by the isomerisation of the aldoxime group. The product of the

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reaction between 3a and HOCl, 3b, exhibited intense luminescence. Then, the application of probe

< Fig. 34 is here >

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3a for the detection and distinguishing exo-/endo-genous HOCl were then demonstrated.

Nitric oxide (NO) has been well known as one of the three major messenger molecules (NO, carbon monoxide (CO) and hydrogen sulfide (H2S)). This molecule plays important roles in inflammation, cell apoptosis, etc. and is associated with various diseases, such as stroke, heart disease, hypertension, and neurodegeneration [54, 222-227]. NO can be produced within live cells and then quickly transferred to other tissues or cells. Therefore, detection of NO on cell surface could contribute to better understanding the biological process of NO-associated diseases. Previous fluorescence probes are mainly focused on the NO detection in the live cells and tissues, while the one that can detect cell surface NO is rare. The probe DSDMHDAB (Fig. 33) [228], developed by Yao et al. is capable of detecting and imaging of extracellular NO of live cells. The probe DSDMHDAB was designed by incorporating o-phenylenediamine (NO-recognition moiety) and C16 alkyl chain (cell plasma membrane anchor) into a BODIPY fluorophore. In PBS buffer of pH 33

7.4, rapid fluorescence “OFF-ON” response was obtained in the presence of NO. DSDMHDAB could be accumulated on the outer surface of the plasma membrane for visualizing the diffusion of NO across the plasma membrane. Similarly, using C18 alkyl chain as the membrane anchor and o-phenylenediamine as NO-responsive moiety, a naphthalimide fluorophore-based probe, Mem-NO (Fig. 34), for NO detection was reported by Zhang et al. in 2018 [229]. This probe showed almost non-fluorescent, and displayed substantial fluorescence enhancement (16-fold) upon reacting with NO. Moreover, Mem-NO exhibited strong TP excitation fluorescence activity, allowing for TP fluorescence imaging of endogenous NO in live neurons and HUVECs and exogenous NO in mouse

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brain tissues were performed (Ex = 810 nm).

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In addition to the probes for HOCl and NO detection in cell plasma membrane, recent researches have also witnessed the development of responsive probes for other membrane-related

incorporating

an

C16

aliphatic

chain

and

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Through

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biomolecules, such as biothiols, sulfur dioxide (SO2), and carbon monoxide (CO) [230-232]. a

maleimide

group

onto

the

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[1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) fluorophore, a membrane fluorescence turn “ON” probe 1 (Fig. 33) was developed by Mertens et al. for SH-containing molecules (e.g. Cys) detection

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[233]. Probe 1 exhibited weak emission due to the PET quenching mechanisms, while the emission

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was switched “ON” after reacting of maleimide with biothiols. Using cholesterol as membrane targeting group, a DNA-based fluorescence probe was recently reported by Feng et al. for the

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detection of signaling molecules in the cell plasma membrane microenvironment [234]. The DNA fluorescence probe is generated by self-assembly of a fluorophore coupled G4 DNA motif as the sensing cofactor. After the reaction with signaling molecules, the fluorescence emission of the cofactors was altered, accompanied by a change of FRET ratio for this signaling molecule detection. Using SO2 and NO sensing cofactors, two DNA sensors was successfully used for ratiometric fluorescence detection of SO2 and NO in the cell plasma membrane, respectively.

< Fig. 35 is here >

CO is a small fat-soluble gas molecule that can freely pass through cell plasma membranes. This 34

biological gas has also been known as one of the gasotransmitter that plays an important role in regulating various physiological processes [222, 235, 236]. Using a long and linear hydrophobic Nile red molecule as the targeting group, Xu et al. developed a cell plasma membrane-anchored fluorescent probe (ANRP) (Fig. 35) for the monitoring of CO release from living cells [237]. ANRP showed very weak fluorescence in the absence of CO, while intense fluorescence emission at 650 nm was observed in the presence of CO. Probe ANRP was then used as a tool for the detection of CO in buffer, tracking the CO release behavior in cells, and evaluating the

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over-expression of CO in cancer cells.

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3.4 Membrane-targeting responsive fluorescent probes for biological macromolecules

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Proteolysis catalyzed by cell plasma membrane proteases is associated with several biological

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processes such as cell metabolism, proliferation, and death [238-242]. Detection of cell plasma

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membrane surface protease activity is thus an interesting topic for the study their relationships with various human diseases. As one of proteases on cell plasma membrane, thioredoxin (Trx) is a

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redox-active protein that is overexpressed in many diseased cells [243]. In order to study the biological roles of Trx during inflammation, Lee et al. developed a Trx-specific probe (1) (Fig. 36)

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for monitoring of the Trx activity at (or around) the membrane site [244]. Probe 1 consisted of

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naphthalimide as fluorophore, a dodecyl alkyl chain as membrane anchoring group, and four carboxylic acid groups to increase its retention capacity on the membrane. After reaction with Trx, the emission of the probe at 472 nm is red-shifted to 540 nm, accompanied with 9-fold enhancement in fluorescence at 540 nm. Visualizations of membrane associated Trx were then demonstrated using 1 as a probe.

< Fig. 36 is here >

Matrix metalloproteinases (MMPs) are a class of secretory endopeptidases that have hydrolytic activity for a variety of extracellular proteins. This kind of enzyme has been reported to be closely related to the invasion and metastasis of cancer cells [245-248]. For the detection of MMPs on the 35

cell plasma membrane, Yang et al. designed a genetically encoded fluorescent protein probe to monitor the MMP-mediated hydrolysis of peptide and to analyze MMP inhibitor effects both in vitro and in living cells [249]. The probe was designed by linking a cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) through a 12 amino acid peptides LEGGIPVSLRPV with a MMP substrate site (MSS). The probe was immobilized on the surface of the cell plasma membrane through a peptide structure of 12 amino acids. Upon reaction with MMP, the MSS site was cleaved and the FRET from CFP to YFP was disrupted, resulting in ratiometric fluorescence (F528/476)

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< Fig. 37 is here >

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response for MMP detection and imaging on the cell surface.

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Furin is a membrane-localized proteolytic processing enzyme that contributes significantly to

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cell secretion and endocytosis. The malformed expression of furin leads to imbalance in physiological homeostasis, and thus is associated with various diseases [250-253]. By combining

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fluorescein (FITC) with Dabcyl (4-(dimethylaminoazo)benzene-4-carboxamide) quencher through a

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furin-responsive peptide, Mu et al. designed a fluorescent probe (MFP) (Fig. 37) for the detection

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of ferin on cell plasma membrane [157]. The dodecanoyl group was employed as the membrane targeting group to anchor the MFP on the plasma membrane. The MFP showed weak fluorescence due to the FRET from fluorescein to Dabcyl, while it exhibited a 12-fold fluorescence enhancement at 525 nm upon reaction with furin for 2 hours. The increase of fluorescence facilitated MFP for one-photon and two-photon fluorescence imaging of ferin on cell surface. Recently, by combining a membrane inserting peptide (MIP), green fluorescent protein (GFP) (ET donor), and a peptide with tetramethylrhodamine (TAMRA) (ET acceptor), Sun et al. reported a semisynthetic GFP assembly-based probe (sFPAP) for real-time detecting cell plasma membrane furin activity [254]. In the presence of furin, the specific substrate site was identified and hydrolyzed, resulting in inefficient of FRET from GFP to TAMRA. The fluorescence signal ratio at F507/580 was increased, allowing for ratiometric fluorescence detection of ferin on the cell surface. β-secretase (BACE) is a membrane-associated aspartic protease that cleaves amyloid precursor 36

protein (APP) in the extracellular domain [255-258]. For BACE detection, Folk et al. designed a membrane-anchored FRET probes (β β -MAP) (Fig. 36) to monitor BACE activity in living cells [259]. The fluorescent 7‐ dimethylaminocoumarin‐ 4‐ acetic acid (DMACA, FRET donor) was linked to a Dabcyl quencher through a BACE substrate linker (peptide). The cell plasma membrane anchoring moiety (cholesterol) was linked to the aspartic acid of the probe through a PEG linker. After reacting with BACE, the peptide linker is cleaved, resulting in the increase of DMACA fluorescence. The application of this probe was then demonstrated by imaging of BACE and inhibitor screening.

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Membrane-associated receptor-like protein tyrosine phosphatase (RPTP) is one subclasses of

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protein tyrosine phosphatase (PTP) that involves in most protein phosphatase activity near the plasma membrane [260]. In 2013, Li et al. developed a fluorescent probe (Flu7/Q12) (Fig. 36) for

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the detection and imaging of membrane-associated protein tyrosine phosphatase activity [261]. The

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hydrophobic 6-alkyl chains of Flu7 make the probe to be able to anchor the cell plasma membrane.

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The Q12 with a phosphate was then linked to the positively charged quaternary ammonium group of Flu7 through electrostatic interactions. Moreover, the 2‐ nitrobenzyl group on the Q12 makes the

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detection photo-controllable, i.e., the RPTP detection is available after UV irradiation-mediated

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cleavage of this photoactivatable cage. Using Flu7/Q12 as the probe, TP fluorescence imaging of

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RPTP activity on the cell surface and brain of Drosophila was demonstrated.

< Fig. 38 is here >

3.5 Fluorescent probes for lipid microdomains Membrane microdomains (also known as lipid raft) are specific subdomains in cell plasma membrane that are enriched in sphingolipids and cholesterol. The microdomains play a vital role in diverse cellular functions, such as membrane transport, signal transduction, metabolism, motility, polarized secretion, and adhesion [262-264]. Therefore, it is necessary to develop fluorescent probes as the imaging tools for unraveling the functions of lipid raft. The majority of currently available fluorescent probes for lipid raft are: (i) lipophilic dyes-based probes, which selectively and 37

directly stain specific membrane microdomains; (ii) environment-sensitive dyes-based probes, which partition into both membrane microdomains, while present different emission properties for discriminating the membrane microdomains. Baumgart et al. and Sezgin et al. systematically studied the distribution of the lipophilic dyes-based probes in liquid ordered (Lo) and disordered (Ld) phases [265, 266]. The results showed that most of the lipophilic probes partitioned strongly out of Lo and into Ld phases. This is mainly because the highly packed lipids in Lo phase usually exclude the exogenous molecules. The partition of these lipophilic probes between Ld and Lo phase was found to be dependent on the

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characteristics of the fluorophore, the chain length, the degree of unsaturation, the size, and

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molecular structure. In addition, the system of the host membrane lipids may also affect the lipid

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probes partitioning between Ld and Lo phase.

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To develop the environment-sensitive probes for lipid microdomains, environment-sensitive dyes are normally selected as the parent fluorophore. These fluorophores can partition into both Ld and

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Lo phases, and present different fluorescence color, intensity, or lifetime after the staining. The

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fluorescence differences of these probes in Ld and Lo phases are generally attributed to the intrinsic properties of the probes at different conditions, such as polarity, viscosity, and membrane tension.

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Laurdan (Fig. 38) is a typical solvatochromic dye that composes of a hydrophobic alkyl chain with

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a naphthalene fluorophore. The Laurdan distributes evenly in Lo and Ld phases, while shows strongly blue-shifted emission in Lo phase of model membranes. Polarity-sensitive emission feature of Laurdan has been widely used for imaging Lo and Ld phases in model membranes [267-269]. To improve the water solubility and sensitivity of Laurdan, Cho and co-workers developed a two-photon (TP) polarity-sensitive membrane probe, C-Laurdan, for lipid raft imaging (Fig. 38) [270]. Due to the coupling of the carboxylic group to Laurdan parent probe, C-Laurdan showed greater sensitivity to the solvent polarity. Using C-Laurdan as the probe, TP fluorescence imaging of lipid rafts on the cell surface was successfully demonstrated. Nevertheless, the function of this polarity probe heavily relied on the generalized polarization (GP) value, which may lead to a significant error for lipid rafts analysis because the ambiguity in determining the range of emission band and the calculation of the G factor [270, 271]. To address this issue, the same research group developed a second-generation probe (CL2) (Fig. 38) for the lipid rafts in living cells and tissues 38

[272]. Under two-photon excitation, this probe emits much brighter fluorescence in lipid rafts than in non-raft domains. The CL2 has also been demonstrated to be able to visualize the lipid rafts in the live cells and pyramidal neuron layer of the CA1 region in live tissues using two-photon microscopy. However, CL2 was found to be easily internalized into the cytoplasm, causing a blurred image. Towards this issue, Cho and co-workers then developed another TP fluorescence probe (SL2) (Fig. 38) by modifying head group of CL2’s carboxyl with sulfonate. This SL2 probe was then used for visualizing the lipid rafts in live cells and intact tissues by two-photon microscopy [273]. In comparison with CL2, SL2 has higher photostability and greater tendency to

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be located in the plasma membrane, which allows for direct visualization of the lipid rafts.

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Styryl derivative dyes that can be excited by visible light at one-photon excitation model were introduced for staining membrane microdomains [274, 275]. In 2011, Jin et al. developed an

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environmentally sensitive probe (Di-4-ANEPPDHQ) (Fig. 38) for the imaging of membrane lipid

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domains [274]. Upon excitation at 488 nm, Di-4-ANEPPDHQ showed significant emission

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spectrum shifts (60 nm) in Lo relative to Ld phase domains. Imaging results revealed that Di-4-ANEPPDHQ can stain the plasma membrane rapidly and can be used to measure the

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transmembrane potential changes in live cells. In 2013, Kwiatek et al. presented a new series of

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order-sensitive optical probes, PY3304, PY3174 and PY3184 (Fig. 38) for the determination of membrane order in artificial membranes, live cells, and live, intact vertebrate organisms [275]. For

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all three dyes, fluorescence spectra were red-shifted and the lifetimes were decreased in the disordered phase. The changes of fluorescence spectra and lifetimes enabled the probes to be used as the tools for ratiometric fluorescence and lifetime imaging of membrane order.

< Fig. 39 is here >

Using solvatochromic dye Nile Red as the fluorophore, Kucherak et al. developed a probe (NR12S) (Fig. 39) for investigating the cholesterol and lipid order in biomembranes [276]. Upon increase the solvent polarity, the emission spectra of NR12S were remarkably red shifted. NR12S is able to exclusively stain the outer leaflet of lipid vesicles and cells, exhibit large emission difference 39

in Lo and Ld phases, varied cholesterol content, and in living cells. Recently, Danylchuk et al. designed a switchable solvatochromic probe (NR4A) (Fig. 39) for imaging of plasma membrane organization in living cells using super-resolution microscopy [277]. Cell imaging results revealed that NR4A is able to be used as an imaging agent for visualizing lipid order in biomembranes at nanoscale resolution. Solvatochromic 3-hydroxyflavone derivatives with characteristic excited-state intramolecular proton transfer (ESIPT) emission have also been used for the design of lipid rafts probes [278-280]. For example, using 3-hydroxyflavone derivatives as fluorophore, Klymchenko et al. reported a

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probe (F2N8) (Fig. 39) for simultaneous investigating the hydration and polarity of lipid bilayers

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[278]. F2N8 is sensitive to membrane hydration Lo phase from Ld phase in large unilamellar vesicles (LUVs), accompanied by a dramatic change in the ratio of its two ESIPT-based emission

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bands. Fluorescence microscopy imaging studies revealed that F2N8 partitions exclusively into the

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Ld phase, which restricted its application in lipid domain imaging. Then, 3-hydroxyflavone-based

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probe (F2N12S) was developed by the same research group in 2009 for visualization of lipid domains in giant unilamellar vesicles [279]. F2N12S showed high selectivity to cell plasma

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membrane, which enabled its application in imaging the loss of the plasma membrane asymmetry

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during the early steps of apoptosis [44]. Moreover, F2N12S was found to be able to bind both Ld and Lo phases in model systems, allowing efficient visualization of these phases by ratiometric

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fluorescence imaging.

< Fig. 40 is here >

3.6 Fluorescent probes for membrane viscosity Membrane viscosity (or membrane fluidity) is a cell plasma membrane physical property that describes the movement of molecules within the phospholipid bilayer. Variations in membrane viscosity are associated with a variety of diseases, such as atherosclerosis, cell malignancy, hypercholesterolemia and diabetes [280, 281]. To better understand the importance of membrane viscosity in cellular biology and physiology, several fluorescent probes have been developed for its 40

analysis. In 2001, Haidekker et al. reported a series of fluorescent probes (2a-2g) (Fig. 40A) for the measurement of cell plasma membrane viscosity [282]. Probes 2b-2g have similar viscosity-dependent fluorescence properties with commercial 9-(dicyanovinyl)-julolidine, while improved membrane localization of probes 2b-2g was obtained. Of these probes, the farnesyl-containing probe 2g (farnesyl-(2-carboxy-2-cyanovinyl)-julolidine, FCVJ) displayed 20-times higher sensitivity than that of 9-(dicyanovinyl)-julolidine for probing membrane viscosity changes. For meso-substituted boron-dipyrrin (BODIPY) dye, the fluorescence lifetimes are changed upon

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the alterations of the viscosity due to the decreasing/increasing rotation of the meso-phenyl group.

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Based on this mechanism, several fluorescent probes have been developed for fluorescence lifetime imaging (FLIM) of viscosity [283, 284]. These membrane-soluble molecular rotors have been

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successfully used for viscosity probing in model lipid bilayers. However, the imaging of the plasma

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membrane in live cells was not realized due to the rapid and effective internalization of the dyes

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into the live cells. Using the same mechanism of BODIPY’s meso-phenyl group rotation, Lopez-Duarte et al. developed a viscosity sensitive molecular rotor 2 (Fig. 40B) for selectively

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stains the plasma membranes of live cells [285]. The double positive charge located on its

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hydrocarbon tail of probe 2 prevented the probe molecules from endocytosis. Efficient rotation opens access to a dark non-emissive state in which the emission is quenched. Both the fluorescence

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quantum yield and the lifetime of the molecular rotor 2 were increased dramatically with increasing viscosity. Imaging results revealed that the molecular rotor 2 could be successfully used for measuring viscosity in plasma membranes of live cells.

< Fig. 41 is here >

3.7 Membrane-targeting fluorescent probes for membrane tension The membrane tension is a common cellular phenomenon that is generated as a response to the applications of an external force on the cell plasma membrane and the internal changes of cell processes [286-288]. For the changes of membrane tension generated by cellular processes, the 41

membrane tension is closely linked to the cell migration, spreading, and phagocytosis. Despite the importance of membrane tension in many cell processes, bioanalytical methods, particularly the fluorescence probes for measuring membrane tension are limited. Taking advantages of environment-sensitive emission feature of Laurdan [289], Zhang et al. developed a probe for the investigations of cell plasma membrane tension [290]. The investigations showed that the emission of Laurdan was regulated by the mechanical strain of the lipid bilayer membrane. The generalized emission polarization value (GP) decreased when more water molecules penetration into the bilayer. The decrease of GP was attributed to the red shift of Laurdan’s emission spectra caused by

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dipole-dipole interactions and reorientation of water molecules near to the Laurdan probe in the bilayer. The changes of emission spectra and GP were then used to study osmotic tension-induced

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strain and changes in the hydration depth of lipid bilayer membrane under stress. In addition,

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further studies of molecular dynamics (MD) simulation and experimental validations revealed that

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the membrane tension is depend on the dipole potential of the membrane [291].

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Recently, a series of mechanosensitive membrane probes were described by Matile and colleagues for fluorescent detection of membrane tension [292-297]. Dithienothiophenes and their S,

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S-dioxides represents the first fluorescent molecular flippers (defined as “monomers in twisted

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push-pull probes with high surface area and fluorescence”) for mechanosensitive membrane probe [292]. These fluorescent flippers have high fluorescence (f > 80%) with lifetime over 4 ns. The

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excitation spectra changes of probes to cell plasma membrane tension were recorded. The planarization of these flippers in liquid-/solid-ordered (Lo/So) membranes led to obviously red shifts of excitation spectra (+80 nm), which were transcribed into the red shifts of emission spectra (+140 nm). In 2016, two mechanosensitive membrane probes with similar chemical structure were designed and synthesized by the same group (C-1, C-2) (Fig. 41) [294]. Due to the prevention of rotational quenching after membrane tension mediated planarization, the fluorescence intensity of the probes was increased with the increase of the planarization of the membrane. Cell imaging evaluation found that probe C-2 with high chemical stability can stain the cell plasma membrane exclusively within in a few minutes. More importantly, washing after staining is not necessary because it fluoresce only after inserting within membrane. On the basis of these research, a planarizable push-pull structure-based fluorescent probe (FliptR) 42

(Fig. 41) was then developed for measuring of membrane tension in 2018 [295]. FliptR consisted two large dithiothiophene thiophenes that are planarizable under high pressure (tension). The fluorescence lifetime of FliptR was increased with the level of planarization, i.e., lifetime was membrane tension dependent. Through modifying the FlipR with different organelle-targeting groups, probes were then developed for measuring the membrane tension of the organelle [296]. Further investigation involved in modifying the FliptR with biotin to form the biotinylated targets [297]. Through the specific biotin-streptavidin interaction, this probe provided a general strategy for measuring membrane tension at any place in any living cell. Recently, based on the tag protein

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technology, HaloFlippers were developed for imaging of localized membrane tension changes in living cells [298, 299]. HaloFlippers retains the mechanosensitivity of the parent Flipper and can

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report the changes of membrane tension at the subcellular level. By expressing HaloTag fusion

addition

to

aforementioned

twisting

based

fluorescence

probes,

bending

of

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In

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membranes expressing HaloTag protein fusions.

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proteins on that membrane of interest (MOI), HaloFlippers was able to highly specific label

N,N’-diphenyl-dihydrodibenzo[a,c]phenazine amphiphiles has also been explored by Matile group

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as the mechanosensitive probes for measuring of membrane tension [300]. The probes feature dual

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emission from bent and planarized excited states that could be used to distinguish micelles in water, monomers in solid-ordered (So), liquid-disordered (Ld) and bulk membranes. Moreover, it has been

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demonstrated that such discriminations could be achieved by fluorescence microscopy, ratiometric TP excitation fluorescence microscopy, and naked eyes.

4. Concluding remarks This review summarized the advances in the development of fluorescent probes for the visualisation of cell plasma membrane. We discussed the membrane fluorescence probes according to their different characters and functions, including structure, photophysical properties, targeting mechanism, and their biological applications. The strategies for the development of bioanalytical probes for cell plasma membrane labelling were initially summarized, followed by the discussions on the progresses of responsive fluorescence probes for various biological species detections, including pH, metal ions, reactive molecules, enzyme, and membrane microdomains, viscosity and 43

tension. Through careful surveying the advances of fluorescence probes for cell plasma membrane biology investigations, we proposed the following directions for the future research on the development of cell plasma membrane probes: (1) Currently reported fluorescence probes were developed commonly by integrating fluorophore with cell plasma membrane anchoring moieties, including alkyl chain, cholesterol, and tocopherol, cell plasma membrane protein tags, antibodies, and aptamers. To develop next generation fluorescent probes for cell plasma membrane studies, new targeting moieties with better membrane anchoring performance are demanded. (2) The probes with

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long retention time on the cell plasma membrane is desirable, but most of the reported membrane

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fluorescence probes are easily to be internalized. This makes long-time tracking the dynamics of cell plasma membrane in a longer period is not possible. Therefore, development of new

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fluorescence probes that are able to retain on the cell surface for a long time could be another

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direction. (3) Development of fluorescence probes that are capable of specifically targeting special

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cells (e.g. cancer cells, inflamed cells, etc.) will be an interesting research direction. This kind of probes could be potentially used to distinguish normal and diseased cells, contributing to diseases

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diagnostics and treatment monitoring. (4) Considering key roles of cell plasma membrane in live

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organisms, several responsive probes have been developed for the detection of various biological species on cell surface. Despite the significant progresses in this field, the number of the responsive

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fluorescence probes is limited. More importantly, responsive probes for the detection of full spectrum of biological processes on cell plasma membrane are not available. Therefore, it is necessary to develop new cell plasma membrane targetable probes for the detection of other biological species, such as proteins (including enzyme) and extracellular DNA/RNS. In summary, membrane-related fluorescent probes have become very attractive tools for the visualisation of cell plasma membrane biology, and more and more membrane fluorescent probes have been developed in recent years. It is likely that in the foreseeable future, work in this area will prosper for many years. We expect that this review will contribute to inspire the rapid development of excellent fluorescence probes for the visualisation of cell plasma membrane biology and biophysics. Acknowledgments 44

The authors gratefully acknowledge the financial support from National Natural Science Foundation of China (Grant 21775015), Australian Research Council (DE170100092), and National Health and Medical Research Council (APP1175808).

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Figure Captions: Fig. 1. Cell plasma membrane structure (A), strategies using phospholipid bilayer structure (B) and cell membrane proteins (C) as the targeting sites for the design of cell plasma membrane fluorescent probes. Fig. 2. Overview of fluorescence probes for cell plasma membrane. Fig. 3. Chemical structures fluorescent probes for membrane based on lipophilic moieties as targeting unit.

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Absorption (B) and emission spectra (C) of MemBright probes (200 nM) in the absence (dashed

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lines) or presence of DOPC vesicles. (D) Turn-on mechanism of the MemBright probes. Reprinted

Fig. 5. Chemical structures fluorescent probes for membrane based on lipophilic moieties as

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Fig. 6. Chemical structures fluorescent probes for membrane based on lipophilic moieties as

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Fig. 7. (a) Laser-scanning luminescence confocal microscopy images of living HeLa cells incubated with complexes 1-4. (b) Images of the cells costained with complexes 1-4 and Cell Mask (CM). (c) Images of the cells costained with complexes 1-4 and MitoTracker (MT). Reprinted with permission from Ref. [60]. Copyright 2018 Royal Chemistry Society. Fig. 8. Chemical structures of AIE-based fluorescent membrane probes. Fig.9. Illustration of Cell Membrane Staining and Cell Necrosis Induced by Compound TPE-MEM Reprinted with permission from Ref. [69]. Copyright 2019 American Chemical Society. Fig. 10. (A) Schematic of Pent-TMP for plasma membrane. (B) Image of zebrafish with Pent-TMP. Reprinted with permission from Ref. [70]. Copyright 2020 Wiley. Fig. 11. Chemical structures of polymer membrane anchoring reagents based on chemistry lipophilic structure. Fig. 12. Chemical structures of two red emitting membrane anchoring polymers (S2, M2) based on 63

chemistry lipophilic structure. Fig. 13. Schematic of ALP-dependent cell membrane anchorage of DNA-lipid-P. Reprinted with permission from Ref. [83]. Copyright 2019 Springer Nature. Fig. 14. Chemical structures of cell plasma membrane labeling reagents based on cholesterol. Fig. 15. Chemical structures of membrane labelled fluorescent probes (1P, Chol-PEG-Cy5) based on cholesterol. Fig. 16. Chemical structure of toc-fApt, designed by using tocopherol as the targeting unit.

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Fig. 17. Chemical structure of fluorescent probes (P-IID, BPDPA-Zn) for imaging cell surface

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phosphatidylserine.

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Copyright 2005 American Chemical Society.

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Fig. 18. Multicolor imaging of CP fusion proteins. Reprinted with permission from Ref. [106].

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Fig. 19. (A) Structures of fluorescent probes, FCTP, FCATP, and FCANB, for labelling PYP. (B)

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Structure of fluorescent probe, CBG-549-QSY7, for labelling SNAP. Fig. 20. Schematically illustration the design principle and expression of the semisynthetic sensor

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proteins on the surface of HEK 293T cells (A, B). Cell imaging of human carbonic anhydrase II

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(HCA) with the semisynthetic fluorescent sensor proteins (C). Reprinted with permission from Ref. [115]. Copyright 2011 American Chemical Society. Fig. 21. Schematic illustration the procedure for the solvatochromic fluorogenic probe sensing cell surface proteins. Reprinted with permission from Ref. [116]. Copyright 2014 American Chemical Society. Fig. 22. Cellular location of PTL in different cell lines after 48 h of incubation: (A) SK-BR-3 cell line, (B) MCF-7 cell line. The false colors of PTL, Dil (cell membrane stain) and Hoechest 33258 (nucleus stain) are green, red and blue respectively. Reprinted with permission from Ref. [119]. Copyright 2013 Royal Chemistry Society. Fig. 23. Schematic illustration the design strategy of the ligand-directed acyl imidazole (LDAI) approach (a). Chemical structures of LDAI (1, 2) and LDT (3) reagents (b). Reprinted with 64

permission from Ref. [124]. Copyright 2012 American Chemical Society. Fig. 24. Schematic illustration of narrow emissive semiconducting polymer and Pdot bioconjugates for specific targeting cell surface proteins. Reprinted with permission from Ref. [132]. Copyright 2013 American Chemical Society. Fig. 25. Schematic illustration the SYL3C DNA aptamer for recognizing the human cell expressed epithelial cell adhesion molecule (EpCAM) protein on the cancer cell surface. Reprinted with permission from Ref. [146]. Copyright 2013 American Chemical Society.

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Fig. 26. Schematic illustration the trifunctional probe for catching cancer cells via targeting the αvβ3 integrin overexpressed on their surface. Reprinted with permission from Ref. [150]. Copyright

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2012 American Chemical Society.

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Fig. 27. Structure of the lipid-DNA probes (A) and the working principle of cell-anchored

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lipid-DNA probes for ratiometric fluorescent sensing of extracellular pH (B). Reprinted with

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permission from Ref. [152]. Copyright 2014 American Chemical Society.

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Fig. 28. Schematic illustration of cell surface-anchored DNA nanomachine as reversible and tunable pH sensor. Reprinted with permission from Ref. [171]. Copyright 2018 American Chemical Society.

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Fig. 29. Chemical structures of membrane-targeting fluorescent probes for Ca2+.

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Fig. 30. Chemical structure and the design concept of K+ fluorescent probe TLSHalo. Reprinted with permission from Ref. [154]. Copyright 2016 American Chemical Society. Fig. 31. Structures of membrane-targeting fluorescent probes for Fe2+, Cu2+, Zn2+, and Hg2+. Fig. 32. (A) The structure of the photocontrollable fluorogenic probe HGMem-3. B) and C) Schematic illustration of working mechanism of the membrane-anchored probe HGMem-3 for imaging Hg2+ near cell membrane. Reprinted with permission from Ref. [205]. Copyright 2018 Wiley. Fig. 33. Structures of membrane-targeting fluorescent probes for reactive small molecule. Fig. 34. Schematic illustration of working mechanism of the membrane-anchored probe Mem-NO. Reprinted with permission from Ref. [229]. Copyright 2018 Royal Chemistry Society. 65

Fig. 35. Schematic illustration of the membrane-anchored probe (ANRP) for real-time monitoring of the release of CO from living cells. Reprinted with permission from Ref. [237]. Copyright 2019 Royal Chemistry Society. Fig. 36. Structures of membrane-targeting fluorescent probes for biological macromolecules. Fig. 37. The structure of the membrane-anchored and furin-responsive probe (MFP) and the corresponding surface-associated cleavage of the probe by furin on the cell plasma membrane. Reprinted with permission from Ref. [157]. Copyright 2014 Wiley.

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Fig. 38. Structures of membrane-targeting fluorescent probes for membrane microdomains.

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Fig. 39. Structures of membrane-targeting fluorescent probes for membrane microdomains.

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Fig. 40. Structures of membrane-targeting fluorescent probes for membrane viscosity.

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Fig. 41. Structures of membrane-targeting fluorescent probes for membrane tension.

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lP

na

ur

Jo

Fig. 24

90

of

ro

-p

re

lP

na

ur

Jo

Fig. 25

91

of

ro

-p

re

lP

na

ur

Jo

Fig. 26

92

of

ro

-p

re

lP

na

ur

Jo

Fig. 27

93

of

ro

-p

re

lP

na

ur

Jo

Fig. 28

94

of

ro

-p

re

lP

na

ur

Jo

Fig. 29

95

of

ro

-p

re

lP

na

ur

Jo

Fig. 30

96

of

ro

-p

re

lP

na

ur

Jo

Fig. 31

97

of

ro

-p

re

lP

na

ur

Jo

Fig. 32

98

of

ro

-p

re

lP

na

ur

Jo

Fig. 33

99

of

ro

-p

re

lP

na

ur

Jo

Fig. 34

100

of

ro

-p

re

lP

na

ur

Jo

Fig. 35

101

Fig. 36 OH O

OH N

N

O C11H23

O

1 O

O

K O HN

R-V-R-R-S-V-K

N H

HO

N

NH N

S

O

S

HO

O

NH

O

O O

MFP

OH

O

O

H N E-V-N-L-D-A-H-F-W-A-D-R-K N H NH

O

O

of

O

R-D

O

5

3

ro

O

NH

O

S

OH N

N

N

O

H N

NH2

-p

-MAP

O

O O

re

N N

ur

N O O P O O

N

O N N

O2N

Jo

N

N

na

N

lP

N

2 Br-

Q12

Flu7

102

NO2

O N

of

ro

-p

re

lP

na

ur

Jo

Fig. 37

103

of

ro

-p

re

lP

na

ur

Jo

Fig. 38

104

of

ro

-p

re

lP

na

ur

Jo

Fig. 39

105

of

ro

-p

re

lP

na

ur

Jo

Fig. 40

106

of

ro

-p

re

lP

na

ur

Jo

Fig. 41

107

Highlights •

Cell plasma membrane structures and functions were introduced

•

Fluorescence probes for cell plasma membrane labelling were reviewed

•

Responsive probes for biomolecules detection on cell plasma membrane were summarized

•

Challenges and future perspectives for the development of cell plasma membrane fluorescence probes were discussed

Jo

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of

Graphical Abstract

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Jo

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: