Nanomaterial-based fluorescent probes for live-cell imaging

Trends in Analytical Chemistry xxx (2014) xxx–xxx

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Trends in Analytical Chemistry journal homepage: www.elsevier.com/locate/trac

Review

Nanomaterial-based fluorescent probes for live-cell imaging Qing Li, Lin Liu, Jin-Wen Liu, Jian-Hui Jiang ⇑, Ru-Qin Yu, Xia Chu ⇑ State Key Laboratory of Chemo/BioSensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China

a r t i c l e

i n f o

Keywords: Fluorescent imaging Fluorescent probe Intracellular detection Intracellular nanosensor Live-cell imaging Nanobiosensor Nanomaterial Nanosensor Sensor design Transduction

a b s t r a c t Nanomaterial-based fluorescent probes represent a significant approach to intracellular detection with high spatiotemporal resolution. We review the properties of various nanomaterials that can be used for intracellular nanosensors in terms of the sensor design and the approaches to delivery of nanosensors based on engineering their surfaces. We also review general strategies for these nanosensors based on the transduction mechanisms of the fluorescence signal. Ó 2014 Elsevier Ltd. All rights reserved.

Contents 1. 2.

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5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nanomaterials used in intracellular fluorescent biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Type 1 nanomaterials with intrinsic fluorescent properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Type 2 nanomaterials with intrinsic quenching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Type 3 nanomaterials that are non-fluorescent and non-quenching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intracellular delivery of nanobiosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Non-targeted delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Targeted delivery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analytical strategies for intracellular fluorescent nanobiosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Intensity-based sensing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Dual-wavelength ratiometric and single-wavelength detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Fluorescence wavelength-shift and lifetime detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary and prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Visualization and monitoring molecular and physical events in live cells represent a key approach to understanding cell biology and have a profound influence on progress in biomedical sciences. There is a constant need for bioanalytical and biomedical sensors that can achieve quantitative, selective signals for biological mole-

⇑ Corresponding authors. Tel./Fax: +86 731 88664085 (J.-H. Jiang); Tel./Fax: +86 731 88821916 (X. Chu). E-mail addresses: [email protected] (J.-H. Jiang), [email protected] (X. Chu).

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cules or physical variables in live cells with high spatiotemporal resolution. To this end, fluorescent spectroscopic techniques offer sensing for intracellular signaling and intracellular analysis due to their non-invasiveness and high sensitivity. A number of organic dyes and fluorescent proteins have been developed as powerful molecular probes. However, these molecular probes may have limitations in making reliable intracellular measurements. Their poor photobleaching resistance may limit their use in long-term tracking in many applications. Their broad emission spectra and largely-shifted excitation bands may hinder practical application for multiplexing of different fluorophores with the requirement for multiple excitation wavelengths [1]. Also, their possible chemical

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interactions or steric hindrance with biomolecules may cause biotoxicity or perturbation to the systems being investigated [2]. The development of nanotechnology has fueled the possibility of designing fluorescent nanomaterials for intracellular sensors. Nanomaterial-based fluorescent probes have certain superior performance that makes them a promising alternative to organic dyes or fluorescent proteins for intracellular sensors. These nanomaterials have dimensions typically 1–200 nm. Larger particles generally have much lower efficiency of uptake by the live cells and may be quickly cleared from circulation by macrophages and eventually destroyed in animal experiments [3]. Some nanomaterials show tunable, narrow-absorption and luminescence peaks, with sizecontrolled synthesis generating diverse optical properties [4], and some display electron-accepting or energy-accepting properties that can act as signal-transduction components [5]. On the other hand, organic dyes can also be encapsulated with a large amount of nanomaterials to overcome optical instability and bio- toxicity. Besides the superior optical properties, including strong emission, high photostability and improved multiplexing capability, the nanomaterials can also be modified by flexible surface chemistry to give excellent biocompatibility. Furthermore, surface functionalization is able to increase selective delivery of the nanosensors to specific cells or even to subcellular organelles to realize effective intracellular imaging. Typically, the construction of nanomaterialbased fluorescent biosensors starts with the synthesis of the nanomaterials in one of three ways – top down, bottom up and a combination of top down and bottom up [6] – followed by designed surface-chemistry modifications. The precisely-controlled preparation of size, shape, chemical composition, crystal structure, and surface chemistry of the nanomaterial-based biosensors is a key step to obtaining unique properties and high performance in intracellular imaging applications. In this article, we first discuss the properties of various nanomaterials and classify them based on their applications in fluorescent live-cell imaging. Second, we outline the existing intracellular delivery strategies and the mechanisms of transport into cells or organelles. Finally, we focus discussion on the design of nanomaterialbased biosensors for fluorescent live-cell imaging and overview how they are used for measurement of different intracellular analytes. In this context, this review may provide new directions for future development and some perspective to the intracellular sensing field. 2. Nanomaterials used in intracellular fluorescent biosensors A wide variety of nanomaterials have been used for the construction of fluorescent biosensor platforms for live-cell imaging, which can be classified into three types:  Type 1 includes nanomaterials that possess intrinsic fluorescent properties and can serve as fluorescence reporters for live-cell imaging;  Type 2 comprises intrinsically fluorescence-quenching nanomaterials that can be designed as quenchers in imaging applications; and,  Type 3 are non-fluorescent, non-quenching nanomaterials that serve as a matrix for immobilizing fluorescent probes on the surface or encapsulating fluorescent dye in their interior. It is important to note that this classification is applied to the elementary nanomaterials only. In biosensor development, two or more nanomaterials of these three types can be combined into nanocomposites to enhance the performance for live-cell imaging. 2.1. Type 1 nanomaterials with intrinsic fluorescent properties Type 1 nanomaterials possess intrinsic fluorescent properties and can deliver fluorescence signals in intracellular detection.

Typically, such fluorescence-reporting nanomaterials include transition-metal semiconductor quantum dots (QDs) [7], silicon dots [8], carbon materials {e.g., carbon dots (CDs) [9], nanodiamonds [10], graphene QD [11] and near-infrared (NIR) single-walled carbon nanotubes (SWCNTs) [12]}, metal nanoclusters (NCs) (e.g., Au, Ag, Cu, and Pt) [13,14], lanthanide-doped nanocrystals [e.g., Eu(III), Sm(III), Tb(III), and Gd(III)] [15,16], and dye-encapsulated silica [17] or polymer nanoparticles (NPs) [18]. Because these fluorescent nanomaterials are predominant in imaging applications, Table 1 summarizes their properties. QDs exhibit the quantum-confinement effect due to their physical dimensions being smaller than the exciton Bohr radius. The most significant properties of transition-metal semiconductor QDs include high quantum yield (QY), broad absorption allowing single-wavelength excitation, narrow, symmetric size-dependent emission bands, and high photostability [7,20]. QDs have been widely used for labeling cells or tissues, and real-time cell tracking [23–25]. For example, QDs can be used for simultaneous labeling of multiple cells for long-term multicolor imaging, and the cells can remain stably labeled for over a week as they grow and develop [23]. Moreover, specific peptides or antibodies can be modified on the surface of QDs, which can be specifically recognized and internalized into the cells. It was reported that QDs functionalized with EGF cell-surface receptors could be used to investigate receptor-mediated signal transduction in different cancer cell lines, and they were demonstrated to be brighter and more photostable than organic dyes [25]. However, the excellent performance of QDs in bioimaging applications is hindered because they suffer from some significant shortcomings, such as photoblinking and toxicity [21]. The phenomenon of photoblinking can decrease QY during measurement. The biological toxicity of QDs originates from the diffusion of heavy metals, such as Cd, from the core of the QDs [7]. To address the toxicity issue, carbon QDs [carbon dots (CDs)], silicon QDs (silicon dots) and graphene QDs have been developed and they promise to exhibit undisputed cyto-compatibility [8,11,21,22]. CDs have been demonstrated in successful uses in fluorescence imaging of cells, taking advantage of their fluorescence brightness at the individual dot level, and their high photostability. They can also be used at different excitation wavelengths [26]. For example, polyethylenimine-functionalized CDs were shown to be water soluble and taken into cells, displaying tunable fluorescent emission at various excitation wavelengths, suggesting their potential in gene delivery and bioimaging [27]. More importantly, CDs are very brightly multi-photon fluorescent, with twophoton cross-sections in the NIR (800–900 nm) orders of magnitude larger than those in the benchmark organic dyes. It was reported that aminopolymer-functionalized CDs could be taken up by cells, and the internalized CDs exhibited bright fluorescence emissions under two-photon excitation with a femtosecond pulsed laser at 800 nm [28]. CDs also exhibit non-blinking, size- and excitation-wavelength-dependent photoluminescence, presumably because of the quantum effect and the different emissive traps on the surface of CDs. Their QY is still not comparable to QDs and highly dependent on the surface passivation [9]. Some of particular fluorescent carbon materials such as nanodiamonds can be luminescent in a region spanning visible and IR wavelengths due to their nitrogen vacancy center, and SWCNTs can have very large Stokes shift and bright NIR emission fluorescence, which may be very attractive for high-resolution images [20]. To apply silicon dots to bioimaging, extensive efforts have been undertaken to realize their aqueous dispersibility, because most such silicon dots are covered with hydrophobic moieties (e.g., styrene, alkyl and octene) on their surfaces and not well dispersible in water. These silicon dots can be modified to become well dispersible in water by grafting water-soluble hydrophilic molecules or coatings, such as micelles or polymers [29–31]. This modification makes silicon dots

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Q. Li et al. / Trends in Analytical Chemistry xxx (2014) xxx–xxx Table 1 Typical properties of fluorescent nanomaterials Nanomaterials

Size

Absorption spectra

Emission spectra

Tunability of emission

Excitedstate lifetimel

Quantum yield

Cell toxicity

Ref.

Almost the entire visible spectrum to NIR Broad emission wavelengths from 400 nm to IR band In the visible, extending into NIR

Size-dependent

ns–ls

Variable 90%

High

[7,20]

Size-dependent

ns–ls

Influenced by surface Low oxidation, 10%60%

[8,21]

Size and kex dependent

0.1–10 ns

Low

[9,21]

UV-vis absorption

From NIR to UV regions

Size-dependent

ps–ls

Low

[7,13]

<100 nm Upconversion fluorescence nanoparticles Fluorescent 1–100 nm nanodiamonds

NIR

In the visible range

By varying the dopanthost combination

100 ns to milliseconds

Dependent on surface passivation, 0.43%60.1% Increases with the cluster size decreasing, 2%70% 0.005%0.3%

In the visible spectral region

Size-dependent

10–20 ns

99%

Low

[10,19,20]

Graphene 1–10 nm quantum dots

Typically in UV region

Broad emission spanning visible and IR wavelengths From the visible to NIR region

Dependent on size, pH,excitation, solvent, concentration

0.24–20 ns

2%46%

Low

[11,22]

Quantum dots

2–10 nm

From the first longwavelength band continuously to the UV UV-vis absorption

Silicon dots

1–8 nm

Carbon dots

1–10 nm

Upon the near-UV or visible spectral region

Metal nanoclusters

<2 nm

Medium [7,15]

UV-vis, Ultraviolet-visible; NIR, Near-infrared; IR, Infrared.

a new, low-toxicity nanomaterial suitable for long-term, real-time cellular imaging [32–34]. It was reported that (3-aminopropyl)trimethoxylsiliane could be used as a precursor for preparing silicon dots with strong fluorescence (20–25%) and a small size (2.2 nm). These silicon dots could preserve stable, bright fluorescence during irradiation for some time (e.g., 180 min). However, the fluorescence of fluorescein isothiocyanate (FITC), CdTe and CdSe/ZnS was completely quenched within 15 min, 60 min, and 160 min under the same irradiation. Moreover, silicon dots were found to show negligible cellular toxicity in cell-viability assays and excellent pH stability [34]. Nevertheless, silicon dots still suffer the limitations that they usually possess inferior QY, their QY is significantly influenced by surface oxidation, and it is difficult to tune their fluorescence. Fluorescent metal NCs comprise up to 100 atoms and their size is smaller than the Fermi wavelength of electrons, so they exhibit molecular-like properties, including discrete electronic states and size-dependent fluorescence. Fluorescent metal NCs can also have good biocompatibility, low toxicity, more efficient renal clearance, tunable emissions, and excellent photostability, so they are promising fluorescent labels for biological applications [11]. Moreover, the long Stokes shift of metal NCs can avoid spectral overlapping and thus enhance imaging contrast. Among the various metal NCs, fluorescent AgNCs and AuNCs are widely used for cell imaging due to their high stability and well-developed methods of synthesis. For example, it was reported that aptamer AS1411 could be integrated with the poly (cytosine) template as the scaffold for AgNC synthesis. The synthesized AgNCs exhibited a red-color emission with QY of 40.1%, and could be internalized into the cells and stain the nucleus because of the binding of AS1411 to nucleolin that could trigger receptor-mediated endocytosis [35]. Besides the DNA-templated NCs, AuNCs templated by protein conjugated to folic acid were also reported for targeted imaging of oral carcinoma cells. The synthesized AuNCs emitted bright fluorescence with a peak at 674 nm, enabling their applications for imaging in the NIR region [36]. Despite the successes, fluorescent metal NCs are still hampered by their relatively low fluorescence, polydispersity, and the difficulties in modifying and functionalizing them. Upconversion NPs (UCNPs), mainly based on lanthanide-doped nanocrystals, can emit high-energy photons upon irradiation with NIR light. Compared with traditional down-conversion fluorescence nanomaterials, UCNPs provide deeper tissue penetration

and avoid cellular autofluorescence background. By varying the dopant-host combination, UCNPs can also be tunable for emission wavelengths [15]. The efficient two-photon excited NIR-to-visible upconversion fluorescence of UCNPs raised high expectations of their utility as a contrast agent for intracellular fluorescence imaging and studies of single-cell dynamics. UCNPs can be non-bleaching and non-toxic, so they are ideal candidates for live-cell imaging [37]. For example, UCNP-loaded live cells were transplanted into a live-mouse model with a cryoinjured hind limb to study their distribution and activity. After intramuscular injection, the UCNP-loaded cells were still unambiguously identified at least 1300 lm deep with a high signal-to-background ratio in the fully vascularized live tissue [38]. Dye-doped nanomaterials commonly use biocompatible polymer or silica NPs as the matrix. These nano-containers can load a large number of dye molecules to achieve their high fluorescence intensity. Embedded fluorophores can also show improved resistance against photobleaching and quenching due to their encapsulation in the protective matrix [39]. Furthermore, with each NP incorporating an individual dye, dye-doped nanomaterials offer the advantage of a multiplexing assay, which can facilitate simultaneous detection of a greater range of analytes. In addition, it is possible to fabricate multicolor NPs that can be excited by a single excitation wavelength using multiple dyes having large overlapping absorption spectra [17,18]. Cancer cell-specific aptamer-conjugated silica NPs could be used for multiplexed monitoring of cancer cells. Tuning the doping ratio of the three fluorescence resonance-energy transfer (FRET) dyes was able to provide multiple colors upon excitation with a single wavelength. These silica NPs enabled simultaneous, sensitive detection of multiple cancer-cell targets [40]. These fluorescent NPs can be directly used for intracellular tracking of a biological events. Alternatively, by coupling with a fluorescence-quenching or a fluorescent donor and acceptor pair, the fluorescent NPs can be used to develop biosensor-based fluorescence activation or energy transfer. 2.2. Type 2 nanomaterials with intrinsic quenching Type 2 comprises fluorescence-quenching nanomaterials that are themselves non-fluorescent but can quench the fluorescence

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from fluorophores. These nanomaterials can be used as quenchers for developing fluorescence-activated nanobiosensors. Typically, most of these nanomaterials, despite the position of their absorption peak, are capable of quenching the fluorescence from different dyes with varying emission wavelengths, indicating the role of non-resonant energy transfer or electron transfer in the quenching mechanisms. Because of their advantage as universal quenchers, fluorescence-quenching nanomaterials eliminate the difficulty in selecting wavelength-matching fluorophore and quencher with conventional fluorescence-activated biosensors. So far, fluorescence-quenching nanomaterials have included gold NPs (AuNPs) [41,42] or nanorods [43], carbon nanomaterials, such as carbon nanotubes [44], carbon NPs [45] and graphene [46–51], MoS2 nanosheets [52,53] and WS2 nanosheets [54,55], copper-oxide nanobelts [56], and polyaniline nanofibers [57]. Because of the consideration of low cytotoxicity, fluorescence-quenching nanomaterials widely used for intracellular sensors are AuNPs or gold nanorods, and carbon nanotubes or graphene. The use of these nanomaterials for intracellular biosensors provides not only a universal fluorescence quencher but also a nano-carrier for the targetresponsive probes to improve the delivery. A typical example of an intracellular biosensor based on the fluorescence-quenching nanomaterials is the Nanoflare. Nanoflares take advantage of the ultrahigh efficiency of fluorescence quenching of the AuNPs versus different dyes, enabling the construction of multi-color fluorescent sensors for intracellular detection. Moreover, because of the vast resource of functional nucleic acids currently available, the combination of AuNP-based fluorescence quenching and the flexible design of DNA probes may create a powerful tool for in-vitro detection and live-cell imaging [58]. Graphene and graphene oxide (GO) represent alternative fluorescence-quenching platforms for intracellular imaging studies [59,60]. For example, nano-sized polyethylene glycol (PEG)-grafted GO was shown to exhibit intrinsic fluorescence quenching and was used for non-covalently attaching a hydrophobic aromatic cancer drug via p-p stacking. This nanocomposite could provide a useful vehicle for cancer-drug delivery and cellular imaging [59]. 2.3. Type 3 nanomaterials that are non-fluorescent and non-quenching Type 3 is typically a carrier nanomaterial that does not have fluorescent nor fluorescence-quenching properties, and serves as only an inert inorganic or organic matrix. The fluorescent probes can either immobilize on the surface or be encapsulated in the pores or the interior of a polymer matrix or a self-assembled nanostructure. Common Type 3 nanomaterials include polymer NPs [61] or nanofibers [62], magnetic NPs [63], silica NPs [5] and porous nanomaterials [64]. The motivations to use an inert carrier nanostructure for surface immobilization of target-responsive fluorescent probes are the possibility of enhanced cellular uptake and increased biocompatibility. Moreover, the co-decoration of fluorescent probes and target-directed signal molecules on the carrier nanostructure can improve intracellular cytosolic delivery and even allow subcellular localization. Such improved efficiency in cellular uptake and targeted delivery may also increase the concentration of the fluorescent probes in the localization area of the target, thus enhancing detection sensitivity and imaging contrast. A most important feature of Type 3 nanomaterials is that the fluorescent probes immobilized or encapsulated are themselves indicators of the intracellular target. They are able to show varied fluorescence signals in response to the target, due to the formation of new compounds (or complexes) or the change of spatial localization relative to the nanomaterials. In contrast, dyes doped in Type 1 nanomaterials are non-reactive to the target, and their

molecular structure or spatial localization relative to the nanomaterials will display no variation in response to the intracellular target. According to the response mechanisms, Type 3 nanomaterials can be further divided into two categories:  the first typically uses the fluorescent probes immobilized on the nanomaterial surfaces so that the probes are directly exposed to the surroundings and can react with the target to generate enhanced or quenched fluorescence signal; and,  the second, in contrast, typically involves the use of either porous nanomaterials with pores capped by target-responsive agents or target-responsive nanomaterials. The dyes can be entrapped or be co-entrapped with a fluorescence quencher in the nanomaterials at a high concentration, so that the dyes exhibit weak fluorescence due to homo-quenching or hetero-quenching effects. When the nanomaterials react with the target, the dyes entrapped can be released due to degradation of the nanomaterials or uncapping of the pores. The second category of Type 3 nanomaterials is very similar to the design of smart drugs, in which the drugs are released via the target-responsive degradation of the matrix or the removal of the capping agents [65,66]. 3. Intracellular delivery of nanobiosensors A key issue in using nanomaterials as intracellular sensors is to transport them into cells or organelles by passing through the plasma membrane and escaping from endosomal entrapment. Existing intracellular delivery strategies can be divided into physical delivery strategies (e.g., microinjection, electroporation, and gene gun) and biochemical delivery strategies [67]. Because physical delivery technologies usually have a high rate of cellular mortality, and require specific instruments to operate, their applicability is very limited [5]. In this setting, biochemicaldelivery strategies show more versatility and higher throughput due to the flexible tailorability of nanomaterial-based biosensors, and can be classified into two main strategies: non-targeted delivery and targeted delivery. 3.1. Non-targeted delivery There is increasing evidence that NPs could spontaneously penetrate cell membranes and be transported into cells of various types through endocytosis, even though there is no specific interaction presented by surface-modified ligands. Such a non-specific endocytosis of NPs is sometimes called non-targeted delivery. The known endocytotic processes could be subdivided into four categories: phagocytosis, macropinocytosis, and caveolae-dependent and clathrin-mediated endocytosis [68]. Different endocytosis mechanisms may preferentially take up NPs of varying sizes. Phagocytosis is able to engulf solid particles with diameters >750 nm by forming internal phagosomes, while smaller NPs ranging from a few to several hundred nanometers (nm) are internalized by pinocytosis or macropinocytosis. Clathrin-mediated endocytosis is probably the primary mechanism for intracellular uptake of NPs with diameter <100 nm, in which the NPs are firstly internalized into small endocytic vesicles and then fused with early endosomes [69]. This is why NPs of 20–50 nm are taken up more rapidly than smaller or larger particles. Besides the size-dependent effect, surface chemistry, shape and surface charge may also affect the intracellular internalization efficiency of NPs [70]. For example, NPs with a positive charge can bind to the negatively-charged cell surface through electrostatic interactions, so we expect positively-charged NPs to be endocytosed more

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efficiently than negatively-charged particles [71]. Furthermore, the presence of hydrophobic pockets on the surface may also facilitate the uptake of NPs due to the hydrophobic interactions with the cell membrane. A very exceptional example of cellular uptake of NPs is AuNPs modified with high-density oligonucleotides. Despite the extremely negatively-charged surface, these NPs are internalized into different cells very efficiently [72]. It was revealed that the uptake of polyvalent oligonucleotide-functionalized NPs was assisted by scavenger receptors that mediated the cellular entry and appeared to be conserved across cell species [73]. To improve the delivery efficiency of NPs into cells, non-targeted delivery strategies were developed through decorating with cellpenetrating peptides (CPPs). The majority of CPP sequences are derived from natural sequences and comprise polycationic or hydrophobic amino acids [74]. These amino-acid residues can facilitate cellular uptake of NPs through electrostatic and hydrophobic interactions with plasma membrane. Commonly-used CPPs are Tat-peptide (GRKKRRQRRRPPQ), penetratin and Octa-arginine (R8) [75]. Cell-penetrating peptides can occasionally deliver NPs directly to the cytoplasm via an energy-independent mechanism, while, in most situations, endocytosis plays a dominant role in the cellular internalization of CPP-modified NPs [76–78]. Hydrophilic arginine-rich CPP-modified QDs were easily delivered into human lung-cancer cells [79], and TAT-modified AuNPs could negotiate the intracellular membrane barriers [80]. In all cases, short incubation times were enough for the transmembrane delivery. However, the NPs were typically sequestered within endocytic vesicles and more incubation time was required for escape from these vesicles. Furthermore, CPPs can be modified with fatty acids, cholesterol or cysteamine in order to enhance the endosomal escape [68].

3.2. Targeted delivery In contrast to CPP-mediated internalization that delivers NPs into cells of different types, targeted delivery of NPs into a specific cell type can be achieved by ligand-mediated delivery. This typically involves decorating the NP surface with functional ligands that bind to specific receptors on the cell surface. The ligands include small molecules, such as folic acid [81], aptamers [82– 84], peptides [85–87], and proteins or antibodies [88,89]. Confocal cell images showed that transferring (TF)-conjugated AuNPs were internalized into the cellular plasma of human nasopharyngeal carcinoma (NPC) cells over control cells. The control studies were performed with fluorescence-labeled albumin and competition experiments with natural full-length transferrin (holo-TF) to verify that the internalization of the TF-coupled NPs was mediated via the specific TF-receptor interaction pathway [90]. A 41-nt aptamer sgc8c had remarkable specificity in terms of binding to the CCRFCEM cells and is over-expressed as a membrane tyrosine kinase receptor. Sgc8c aptamer-modified AgNCs showed specific targeting and endocytosing capabilities to CCRF-CEM cancer cell over control cells [91]. With surface-modified ligands that may coordinate together to exert multivalent interactions with the receptors expressed on cell surface, the functionalized nanomaterials can specifically bind to the corresponding receptor-expressing cells and then are efficiently internalized by the target cell through receptor-mediated endocytosis. Besides intracellular delivery, targeting of specific subcellular organelles is sometimes desired for nanomaterial-based biosensors. Subcellular organelle targeting presents a more significant challenge than intracellular delivery. Specific signal peptides, such as nuclear localization signal (NLS), mitochondrial localization signal, and endoplasmic reticulum (ER) signal [90], have delivered NPs into the corresponding organelles. The ER signal peptide-modified NPs enhanced the endocytosis and accumulated in both the

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ER and endosomal compartments within 2 h, enabling intracellular trafficking of the ER [92]. Mitochondrial targeting can also be achieved using NPs modified with lipophilic cations, such as triphenyl phosphonium (TPP) derivatives [80], and MITO-Porter liposome that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. The use of poly-L-lysine coating for NPs facilitated their escape from endolysosomal compartments. Furthermore, with the assistance of TPP, TPP-conjugated poly-L-lysine-coated NPs could selectively target the mitochondria for imaging and even anti-cancer therapy [93]. Golgi apparatus and endoplasmic reticulum may express certain abnormalities or characteristic traits associated with diseases. Conjugating with toxins such as Shiga or cholera toxins can deliver NPs to Golgi apparatus and endoplasmic reticulum [94]. 4. Analytical strategies for intracellular fluorescent nanobiosensors Basically, a fluorescent nanobiosensor comprises two components: an analyte recognizer and a signal transducer. Due to the tailorability of nanomaterials, the recognizer and the signal transducer can be separate or a single sensing entity in a nanobiosensor. According to the signal-output mode, intracellular fluorescent nanosensors can work in three different sensing mechanisms: intensity based; wavelength shift; and, lifetime based. 4.1. Intensity-based sensing Interaction of the analyte with the nanosensor within a sensing event can result in enhancement (activation) or quenching (deactivation) of the intensity of the fluorescence signal. In principle, fluorescence-signal intensity can be modulated by the analyte using different strategies, and can be grouped into five approaches: (1) analyte-induced change of distance between a fluorescent donor and a fluorescent acceptor or quencher; (2) analyte-controlled release of fluorescent reporters; (3) analyte-induced activation of fluorescence reporter; (4) analyte-specific quenching of fluorescence reporter; and, (5) analyte-mediated deactivation of fluorescence quenchers. The most widely used strategy for fluorescence-intensity modulation is the analyte-induced change of distance between a fluorescent donor and a fluorescent acceptor or quencher. It is known that the efficiency of energy transfer or electron transfer depends on distance, so alteration of distance between a fluorescent donor and a fluorescent acceptor or quencher can generate detectable fluorescence-signal enhancement or decrease. There are three common approaches for analyte-induced changes of distance between a fluorescent donor and a fluorescent acceptor or quencher, as illustrated in Fig. 1. The first approach is the competitor-displacement assay (Fig. 1A), in which fluorescent donors and fluorescent acceptors or quenchers are coupled in a nanosensor via the interaction between a capture probe and a ligand that can compete with the analyte for the capture probe. The analyte, if present, can bind to the capture probe, usually at higher affinity than that for the ligand, so the analyte will displace the ligand and release the label of fluorescent acceptors or quenchers away from the nanosensor, resulting in a distance between the fluorescent donors and the fluorescent acceptors or quenchers with a concomitant recovery of fluorescence signal. For example, Mirkin and co-workers have developed AuNP-based sensors for detecting mRNA in cells. DNA-capture probes modified on AuNPs are pre-hybridized with

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dye-labeled DNA-reporter probes with the fluorescence of dyes quenched by AuNPs. Target mRNA can displace the reporter probes because of the formation of more stable duplexes between the target mRNA and the capture probes on AuNPs, releasing the dye labels from AuNPs and activating the fluorescence signal. Using different fluorophores, multiplexed detection of multiple mRNA targets can be achieved in cells [58]. Tang and co-workers (Fig. 2) reported a FRET system-based competitor-displacement assay for quantitative analysis of glucose and imaging of glucose consumption in live cells. A FRET system based on apo-GOx (inactive form of glucose oxidase)-modified AuNPs and dextran-FITC (dextran labeled with FITC) was constructed to realize the highly sensitive detection and imaging of glucose in live cells. Due to the higher affinity of apo-GOx for glucose over dextran, once glucose was added in the system, the quenched fluorescence of FITC recovered. This sensor monitored glucose at a low level in live cells with higher selectivity over other monosaccharides [95]. Chen and co-workers reported a displacement assay for detection of mercury ions. Rhodamine B isothiocyanate (RBITC) was modified on the surface of AuNPs and the fluorescence was quenched by AuNPs. Due to higher affinity of Hg2+ toward isothiocyanate (ITC), binding with ITC induced detachment of RBITC from the AuNP surface, so triggering a remarkable enhanced fluorescence of RBITC. This displacement assay was highly robust and recyclable (since it could be regenerated by resupplying free RBITC into the AuNP solutions and reuse for detecting Hg2+). This sensor

has been applied in monitoring Hg2+ in complex samples, such as river water and live cells, with high sensitivity and specificity [96]. The second approach is the cleavage assay (Fig. 1B), in which fluorescent donors and fluorescent acceptors or quenchers are conjugated in a nanosensor via covalent linkers. The analyte, if present, can cleave the linker and separate the fluorescence acceptors or quenchers away from the fluorescent donors, thereby activating the fluorescence signal. This design has been widely implemented for intracellular determination of analytes that have cleaving activity, such as enzyme [97], organic thiols [98], and reactive oxygen species (ROS) [99]. Our group (Fig. 3) recently reported a graphene-peptide conjugate as a nanosensor platform for intracellular imaging of apoptotic signaling. GO is used as a high-efficiency quencher and a nanocarrier, and the nanosensor is constructed by covalent conjugation of fluorophore-labeled substrate peptides of caspase-3 and TAT CPPs on GO. The nanosensor allows efficient delivery into cells because of the hydrophobic interactions between GO, the cell surface and the CPP-mediated cellular uptake. On cleaving the substrate peptides by caspase-3, remarkable, activated fluorescence signals can be obtained [97]. The recovery of the fluorescence of fluorescent-quenched dyes from the AuNPs can also be realized through the analyte cleaving the linking group between the quenched dye and the AuNPs. Fluorescein-labeled hyaluronic acid (HA) end-capped with a dopamine group was grafted onto the surface of AuNPs via Au-catechol bonds and the fluorescence of the dye was quenched by NP surface-energy transfer (NSET). Release of the dye from the surface of the AuNPs and

Fig. 1. (A) Competitor displacement assays; (B) cleavage assay; and, (c) conformational change assay.

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Fig. 2. Apo-GOx-modified gold nanoprobe for glucose sensing and cellular imaging.

fluorescence recovery can be achieved by cleaving HA in the presence of ROS. Compared to other fluorescent probes, this sensor was more sensitive in response to superoxide anion (O2) and hydroxyl radical (OH), and applied in real-time intracellular LPS-induced ROS detection within macrophage cells [99]. Lu and co-workers (Fig. 4) realized the DNAzyme for intracellular applications. A 13-nm AuNP was functionalized by uranylspecific 39E DNAzyme, while the 30 end of the enzyme strand was functionalized with a thiol group (SH) for immobilization onto the AuNP. The fluorescence of the Cy3 at the 50 end modified in the substrate strand is quenched by both AuNP and the molecular quencher modified at the 50 end substrate strand. In the presence of UO2+ 2 , the DNAzyme cleaves the substrate strand, thus releasing the Cy3-labeled shorter product strand and increasing the fluorescence. This sensor is the first DNAzyme-based probe of metal ions in live cells [100]. The third approach is the conformational change assay (Fig. 1C), in which fluorescent donors and fluorescent acceptors or quenchers are conjugated in a nanosensor via linkers that are selectively responsive to the analyte. Upon binding to the analyte, the linker undergoes a conformational change that induces a change in the distance between the fluorescent donors and the fluorescent acceptors or quenchers, thus leading to a substantial change in fluorescence signal. The conformation changes in biomolecular

recognition are widely known as the adaptive fitting mechanism, and there are many examples including the interactions between protein receptors and ligands, hairpin-structured DNA probes and target DNA or RNA, as well as aptamer probes and protein or small-molecular targets [101]. Two groups have reported the use of dye-labeled hairpin DNA probed for functionalization of AuNPs for mRNA imaging in live cells. The hairpin DNA probes are immobilized on AuNPs via S-Au bond self-assembly. Hybridization between the loop region with target DNA results in the opening of the stem region, drawing the fluorophore label far away from AuNPs and generating a restored fluorescence signal [102,103]. Seo and co-workers reported a novel molecular beacon (MB) to detect mRNA in cancer cells. The MB employed an organic dye as the fluorescent reporter and a graphite NP (GN) as the quencher, the pyrene-modified MB was attached on the GN through p-p stacking and the fluorescence of the dye was quenched by the GN. Hybridization between MBs with target survivin mRNA resulted in the stem region opening and generating a restored fluorescence signal. The introduction of the GN enhanced the biological stability and the transfection of the MB into the cells. The MB could successfully image the target survivin mRNA inside the cancer cells [104]. Besides the distance-modulation strategy for the design of intensity-based nanosensors, there are four other strategies that

Fig. 3. Graphene-oxide peptide conjugate as intracellular protease sensor for caspase-3 activation imaging in live cells.

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Fig. 4. Intracellular uranyl probe based on fluorescent DNAzyme-modified gold nanoparticles.

are also routinely used for live-cell fluorescent imaging, as shown in Fig. 5. The first strategy is analyte-controlled release of fluorescent reporters, such as fluorescent donors and fluorescent acceptors or quenchers that are entrapped together in a matrix nanomaterial, such as degradable polymer NPs, liposomes and mesopore-silica NPs (MSNs), displaying quenched fluorescence (Fig. 5A). The analyte, if present, can trigger degradation of the matrix, formation of pores in the matrix, or removal of the capping agents, thereby releasing the fluorescent donors and the fluorescent acceptors or quenchers and activating the fluorescence signal inside or outside the matrix nanomaterials. For example, Ju and co-workers (Fig. 6) reported telomeraseresponsive MSNs for imaging intracellular telomerase activity. Fluorescein and black-hole-quencher molecules are co-entrapped in positively-charged MSNs, the pores of which are blocked by a DNA primer for telomerase. The primer probes can be extended in the presence of telomerase, and undergo a conformation change to form a rigid hairpin-like structure that has much weaker affinity to MSNs and is detached from the MSN surface, releasing fluorescein and recovering its fluorescence in the cells [105]. Kwon and co-workers reported polymeric NPs for imaging protein kinase activity in single live cells. A polyion-induced complex (PIC) composed of positively-charged polymer (Cy5.53-PEI-kemptide25) and negatively-charged polymer (PAA) self-assembled into the polymeric NPs through electrostatic interaction, and NIR fluorescence intensity was quenched due to the short distance between Cy5.5 fluorochromes. When phosphorylated by protein kinase A PKA, the serine moieties of Cy5.53-PEI kemptide25 incorporated the negatively-charged phosphate groups, due to charge unbalance, PIC NPs dissolved, resulting in NIR fluorescence recovery [106]. The second strategy for fluorescence-intensity modulation is analyte-induced activation of the fluorescence reporter. The analyte, if present, can react with the non-fluorescent probes to form a fluorescent product, thus activating the fluorescence signal (Fig. 5B). For example, the fluorescence of FITC can be modulated by pH in the range 5–9, and, with increasing pH values, the fluorescence can be changed from dark to light. Ma and co-workers reported a pH sensor based on CDs for measurement of the intracellular pH. CDs serve as an excellent delivery matrix due to their biocompatibility and small size, while FITC and pH-insensitive rhodamine B isothiocyanate (RBITC) were covalently modified on the surface of CDs. There was dramatic enhancement of the fluorescence of FITC when pH increased from 6.0 to 8.0 in HeLa cells, while RBITC served as a reference reporter and showed little change. This sensor has monitored intracellular pH of intact HeLa cells and the pH fluctuations associated with oxidative stress [107].

The photophysical properties and the photoactivation of QDs are influenced by many factors, including surface ligand, solvent, temperature, pH and concentration [108]. For example, the surface ligand of QDs can effectively passivate the surface defects, and ensure the bright fluorescence of NPs. The low pH will dissociate the ligand from the ZnS outer shell, then increase contact between the nanocrystal surface and the solution, induce a higher non-radiative relaxation rate and quench the PL intensity of QDs. Gundersen and co-workers reported mercaptoacetic acid (MAA)-capped CdSe/ZnSe/ZnS semiconductor-nanocrystal QDs for sensing pH based on this mechanism, with increases in intracellular pH leading to enhanced QD fluorescence intensity. The NPs were incubated with the cells and were taken up by endocytosis, remaining located within the acidic lysosomes. The PL intensity of the QDs within the lysosomes was rather low, but, upon adding chloroquine, the pH of the lysosome became more alkaline, and an enhancement of the PL intensity of QDs could be detected [109]. The third strategy for fluorescence-intensity modulation is analyte-specific quenching of fluorescence reporter. The analyte, if present, can quench the fluorescence of the NPs, resulting in a decreased fluorescence signal (Fig. 5C). Sun and co-workers reported that photoexcited CDs are excellent electron donors and acceptors, and that the photoluminescence of CDs could be quenched efficiently by electron acceptor or donor molecules [110]. Recently, through surface functionalization, CDs were endowed with molecular recognition ability and applied in sensing, and were reported in detecting intracellular ions [111,112]. For example, Tian and co-workers (Fig. 7) designed a dual-emission CdSe@C-QD nanocompound integrated into an organic molecule N-(2-aminoethyl)-N,N,N0 tris(pyridin-2-ylmethyl)- ethane-1,2diamine (AE-TPEA) specific for Cu2+; under single wavelength excitation, the fluorescence reporter QDs (C-QDs) functionalized with AE-TPEA emitting blue fluorescence quenched when selectively recognizing Cu2+ in live cells while the reference CdSe/Zns QDs emitting red fluorescence stayed constant. The nanohybrid showed good dispersibility, photostability, and low toxicity against dualemission organic fluorophores for monitoring intracellular metal ions [112]. The specific interaction with NCs (via the metal core or ligand shell) can influence the luminescence properties of the fluorescent metal NCs. For example, the high percentage of Au+ or Ag+ present in the surface of NCs can interact with Hg2+ by formed a strong metallophilic bond between them, and quench the luminescence of Au/AgNCs, which can be used for Hg2+ detection. Nienhaus and co-workers developed a simple strategy to synthesize NIR-emitting (QY: 2.9%) dihydrolipoic acid (DHLA)-capped AuNCs, with ultrasmall hydrodynamic diameter (3.3 nm), good

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Fig. 5. (A) Analyte-controlled release of fluorescent reporters; (B) analyte-induced activation of fluorescence reporter; (C) analyte-specific quenching of fluorescence reporter; and, (D) analyte-mediated deactivation of fluorescence quenchers.

colloidal stability and low cytotoxicity toward HeLa cells. These DHLA-AuNCs were capable of sensing Hg2+, the limit of detection (LOD) was 0.5 nM, and they were applied in imaging intracellular Hg2+ in HeLa cells [113]. The ligand shell of the NC surface can pro-

vide good protection and avoid luminescence quenching induced by the aggregation of the NCs. Based on this mechanism, the quenching of the luminescence can be related to the concentration of the analytes that interact with the ligand and destabilize the NCs.

Fig. 6. Switchable fluorescent imaging of intracellular telomerase activity using telomerase-responsive mesoporous silica nanoparticle.

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Fig. 7. CdSe@C-TPEA nanohybrid detection of Cu2+-based dual-emission ratiometric fluorescence.

Huang and co-workers prepared bright, yellow-emitting DNAAgNCs. In the presence of the hydroxyl radical (OH), which was generated by Fenton reagent, the template DNA was cleaved, resulting in spontaneous aggregation due to the lack of DNA stabilization and further the quenching of the AgNC fluorescence. It was demonstrated that the quenching efficiency increased with OH quantity. Furthermore, these DNA-AgNCs could penetrate through cell membranes to monitor intracellular OH in human bone-marrow neuroblastoma cells [114]. The fourth strategy for fluorescence-intensity modulation is analyte-mediated deactivation of fluorescence quenchers, in which the quenchers are tethered on the surface of fluorescent NPs. The analyte, if present, can react with the quenchers to form a product with low-efficiency fluorescence quenching, restoring the fluorescence signal (Fig. 5D). For example, Liu and co-workers (Fig. 8) reported MnO2-nanosheet-modified UCNPs for detecting intracellular glutathione. UCNP/MnO2 nanosheet assemblies were formed by electrostatic interactions. The absorption band of MnO2 nanosheets overlaps with the emissions of the UCNPs in the UV and blue spectral regions, and almost completely quenched fluorescence of UCNPs with non-emission, the recovery of upconversion emission to the glutathione (GSH) mediated reduction of MnO2 to Mn2+, leading to the decomposition of the MnO2 nanosheets, and dequenching of the UCNPs. This system can monitor the GSH levels in live cells [115]. An electron transfer from the coating molecules to the valence band of an excited QD can result in emission quenching. If the electron donor is bound to a substrate, this process may be interrupted and the emission restored. Long and co-workers showed that oxidized Cytochrome c (Cyt c) efficiently quenches the fluorescence of the CdSe/ZnS QDs, electrostatic interactions cause the absorption of the Cyt c on the surface of the QDs and the achieved proximity allows the Cyt c to quench the QD emission through the electrontransfer mechanism. However, the reduced form of Cyt c, incubated with negatively-capped QDs, leads to the recovery of the fluorescence of the QDs. Subsequent addition of O readily 2 reduced the oxidized Cyt c to the reduced form of Cyt c, then altering the quenching of QDs by the oxidized form and leading to enhanced fluorescence, the sensor is capable of fluorescent imaging of a superoxide radical with high sensitivity and specificity in live cells, without interference from other ROS or relevant intracellular components [116]. 4.2. Dual-wavelength ratiometric and single-wavelength detection The above analytical strategies for fluorescence-intensity-based sensing can be implemented in single-wavelength imaging or dualwavelength ratiometric detection. The problem of single-wavelength intensity-based sensing is the absence of calibration, the fluctuation of excitation-light intensity, fluorescence-imagecollecting parameters, probe concentration and intracellular delivery efficiency, all having great impact on the image contrast. The development of fluorescent ratiometric nanobiosensors can

therefore realize self-calibration by incorporating dual-wavelength measurements; one reference reporter provides the information concerning fluctuations of experimental parameters while the signal reporter provides the information concerning analyte concentration. In principle, nanosensors with fluorescence turn-off architecture are widely implemented for live-cell imaging applications using the single-wavelength intensity-based sensing mode, though strict controls of the experimental parameters are required. However, single-wavelength imaging using nanosensors with fluorescence turn-off architecture, such as the nanosensing strategy of analyte-specific quenching of fluorescence reporter, are not recommended, because it may lead to false-positive signals due to low efficiency in intracellular delivery of nanosensors. To design a reliable nanosensor for such applications, one can introduce into the nanosensor an internal reference fluorescence reporter, which is not responsive to the analyte and provides a stable reference signal, in order to achieve ratiometric measurements. For example, our group (Fig. 9) has reported a single-excitation and dual-emission fluorescent nanocomplex (DEFN) of AuNC-decorated silica NPs as a novel nanosensor for live-cell imaging of highly ROS (hROS). The DEFN can be excited at a single wavelength (405 nm) and gives well-resolved, dual-emission signals, one from the silica NPs entrapping the internal reference dye CF 405S, and the other from the AuNCs with their fluorescence sensitively quenched in response to hROS, including OH, ClO and ONOO in live cells [117]. The sensitive indicator probe and the non-sensitive reference dye can be encapsulated into the nanomatrix, and can build in a correction for environmental effects; they have been applied for sensing Zn2+,Ca2+,Cu2+, Cl, and H2PO 4 [118–121]. Biscup and coworkers reported a nanosensor incorporating a chloride-sensitive dye (lucigenin) and a reference dye (sulforhodamine derivative) into polyacrylamide NPs. In the presence of chloride ions, the fluorescence intensity of lucigenin reversibly decreased due to a collisional quenching process, and quenching of lucigenin was found to be 53 M1 lower than free lucigenin (KSV = 250 M1) determined by Stern-Volmer constant KSV. The ratiometric nanosensor was applied for monitoring the response to changing chloride concentrations in the fibroblast cells [118]. Kopelman and co-workers reported (Fig. 10) NP PEBBLE sensors for imaging of intracellular free calcium ions. Ca2+-sensing dye rhodamine probes and the reference dyes Hilye Fluor 647 were loaded into polymer matrix of the PEBBLEs. Rhodamine showed a stable fluorescence enhancenment when selectively binding Ca2+ at near-neutral pH, and the fluorescence of Hilye Fluor 647 remained constant [119]. For other nanosensing strategies, ratiometric detection can be used to improve their stability. For example, in nanosensors based on distance-modulation strategies, ratiometric detection can be easily implemented by using Förster resonance-energy transfer (FRET) between a fluorescent donor and a fluorescent acceptor. By co-entrapping fluorescent donors and fluorescent acceptors

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Fig. 8. Intracellular glutathione detection using MnO2-nanosheet-modified upconversion nanoparticles.

Fig. 9. Using the DEFN of gold-cluster-decorated silica particles for detection of intracellular hROS.

with FRET interactions in the matrix nanomaterials, nanosensorbased analyte-controlled release of fluorescent reporters can also be used in ratiometric imaging. For example, Gang and co-workers (Fig. 11) reported a novel QDs-fluorescent protein (FP) probe for sensing intracellular pH. The pH sensors comprised carboxylfunctionalized QDs conjugated to pH-sensitive mOrange FP, the QDs served as the donor and the FP served as the acceptor. Under QD excitation at 400 nm, the QD-mOrange probe demonstrated strong energy transfer, and mOrange exhibited sensitized emission at 560 nm. With reduction in pH, the emission intensity of mOrange decreased and the emission intensity of QD increased, due to the change in the mOrange absorbance reversibly modulating the emission from the pH-insensitive QD. With pH increase from 6 to 8, the ratio of acceptor emission to donor emission increased more than 12-fold. The QD-FP probe was applied with visualization of the acidification of intracellular endosomes through polyarginine-mediated endocytosis. Furthermore, by tailoring the FP, the probe can be designed for sensing different pH ranges, and the QD-mOrange M163K FRET system has been applied for cytosol pH mapping [122]. Willner and co-workers (Fig. 12) reported another smart FRET probe for monitoring the intracellular 1,4-dihydronicotinamide adenine dinucleotide (phosphate) cofactors (NAD(P)H), the

Fig. 10. Nanoparticle PEBBLE sensors for quantitative nanomolar imaging of intracellular free-calcium ions.

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acceptor Nile Blue (absorbance: 630 nm) was covalently linked to bovine serum albumin-functionalized QDs (emission: 635 nm), and the fluorescence of QDs quenched through energy transfer. In the presence of NADH, the Nile Blue was reduced and lacked absorbance in the visible spectral region, so the fluorescence of the quenched QDs recovered. However, this sensor has poor reproducibility due to uncontrollable modification of QDs [123]. UCNPs are promising donors for FRET applications. The NIR excitation and large anti-Stokes shifts can avoid direct excitation of the down-conversion acceptor, which enlarges detection sensitivity and simplifies subsequent analysis. Furthermore, biological materials do not absorb NIR light, and can also avoid attenuating the excitation of the UCNPs by surrounding biomolecules. Li and co-workers described a highly-selective, sensitive Hg2+ sensor by using chromophoric ruthenium complex-assembled nanophosphor (N719-UCNPs). The FRET system was constructed using the spectral overlap between the absorbance of the complex N719 and the emission spectrum of Er3+-doping UCNPs. When Hg2+ was added, a significant blue shift for the absorption maximum of N719 destroyed the FRET system, resulting in recovery of the upconversion luminescence emission of UCNPs at 541 nm. The nanosensor exhibited capability of monitoring the concentration and the distribution of Hg2+ in live cells [124].

nanosensors in intracellular imaging. Both approaches can overcome the fluorescence uncertainties arising from fluctuations of experimental parameters. For example, CdTe QDs capped with Lcysteine have been reported to show alterations of emission colors in response to pH variations. When the pH values decrease from 7.0 to 6.8, QDs undergo a decrease in the band-gap energy, causing a 16-nm red shift of the emission color accompanied with quenched fluorescence signals [125]. In contrast to intensity-based or wavelength-shift detection, fluorescence-lifetime imaging requires very specific instruments. However, there are many nanomaterials with fluorescence lifetime much longer than organic dyes, which allow differentiation of nanomaterials using autofluorescence of biomolecules (lifetime 2–3 ns), so offering advantages for nanomaterial-based sensors for live-cell imaging applications. For example, QDs, which show long lifetimes in the range 10–100 ns, have been demonstrated to sense analytes in live cells. QDs modified with chloride ionophore lucigenin display a dramatic decrease in fluorescence lifetime due to electron transfer from QDs to lucigenin. On reacting with the chloride ion, QDs exhibit substantially increased fluorescence lifetime, which enables the determination of chloride ions inside cells with high sensitivity and high selectivity [126]. 5. Summary and prospects

4.3. Fluorescence wavelength-shift and lifetime detection In addition to intensity-based sensing, fluorescence wavelength-shift and lifetime detection can also be used for

Nanomaterial-based fluorescent sensors create a powerful platform for detecting molecular and physical events in live cells with high spatiotemporal resolution. The versatility of nanomaterials as

Fig. 11. Quantum dot-fluorescent protein FRET probes for sensing intracellular pH.

Fig. 12. Nile-Blue-functionalized CdSe/ZnS QD sensing of NADH-based FRET.

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color-tunable, long-wavelength emitting and intensity-enhanced fluorescent reporters or as low-background universal fluorescence quenchers can provide high imaging contrast and detection sensitivity for intracellular detection. Moreover, the engineerability of nanomaterials by structure design and surface modification has furnished us with intracellular nanosensors with high biocompatibility, excellent stability and superb targeted delivery performance. We reviewed some of the typical nanomaterials widely adopted for intracellular sensor development according to their functions in the nanosensors. We also discussed the cellular uptake mechanisms and surface modifications that allow desired intracellular delivery. Specifically, we reviewed the analytical strategies for various intracellular nanosensors in detail. These discussions may provide a guide for the design of nanosensors for live-cell imaging applications. The applications of nanosensors in live cells are still faced with several challenges. First, despite of the existence of delivery strategies, uptake of nanosensors by live cells can be very variable. Size, monodispersity and surface properties of the nanosensors, the state of cells and even inter-cell differences will all have significant effects on the delivery efficiency. Further developments of dual-wavelength ratiometric nanosensors may provide a solution to the problems. Second, the cellular surroundings are highly heterogeneous with many components exhibiting spatially-differentiated concentration, so intracellular distribution of nanosensors in target-localized areas and subcellular structures is crucial for unbiased determination of intracellular analytes. Third, unlike in-vitro sensors that can be coupled with various signal or target-amplification technologies, intracellular sensors typically only have limited sensitivity, so that many low-abundance biomolecules are still undetectable. Fourth, cellular autofluorescence presents a barrier to sensitivity in intracellular detection, so there is invariably a need for low toxicity, multicolor and high brightness NIR or two-photon fluorescent nanomaterials. Nanomaterial-based fluorescent sensors have the advantages over in-situ expressed fluorescent protein sensors in their ability to probe native cells without the need for gene transfection. Future studies will focus on development of nanosensors into high-performance intracellular detection platforms that allow spatiotemporal interrogation of cellular signal events in great detail, thereby offering insight into complicated biological mechanisms in cell biology. Acknowledgments This article was supported by National Natural Science Foundation of China (21025521, 21035001), National Key Basic Research Program (2011CB911000), the Hunan Provincial Natural Science Foundation of China (10JJ7002). References [1] H. Yuan, J.K. Register, H. Wang, A.M. Fales, Y. Liu, T. Vo-Dinh, Plasmonic nanoprobes for intracellular sensing and imaging, Anal. Bioanal. Chem. 405 (2013) 6165–6180. [2] L. Baù, P. Tecilla, F. Mancin, Sensing with fluorescent nanoparticles, Nanoscale 3 (2011) 121–133. [3] D.E. Owens III, N.A. Peppas, Opsonization, biodistribution, and pharmacokinetics, Int. J. Pharm. 307 (2006) 93–102. [4] W. Zhong, Nanomaterials in fluorescence-based biosensing, Anal. Bioanal. Chem. 394 (2009) 47–59. [5] M.J. Ruedas-Rama, J.D. Walters, A. Orte, E.A.H. Hall, Fluorescent nanoparticles for intracellular sensing: a review, Anal. Chim. Acta 751 (2012) 1–23. [6] A. Tuantranont, Nanomaterials for sensing applications: introduction and perspective, in: G. Urban (Ed.), Applications of Nanomaterials in Sensors and Diagnostics, Springer-Verlag, Berlin, pp. 1–16. [7] A.P. Demchenko, Nanoparticles and nanocomposites for fluorescence sensing and imaging, Methods Appl. Fluoresc. 1 (2013) 022001–022028.

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Please cite this article in press as: Q. Li et al., Trends in Analytical Chemistry (2014), http://dx.doi.org/10.1016/j.trac.2014.03.007