Lanthanide-doped nanomaterials for luminescence detection and imaging

Trends in Analytical Chemistry 62 (2014) 123–134

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Trends in Analytical Chemistry j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a c

Lanthanide-doped nanomaterials for luminescence detection and imaging Shiguo Wang, Leyu Wang * State Key Laboratory of Chemical Resource Engineering, Beijing Key Laboratory of Environmentally Harmful Chemical Analysis, Beijing University of Chemical Technology, Beijing 100029, China

A R T I C L E

I N F O

Keywords: Downconversion Dual-mode luminescence Functionalization Lanthanide-doped nanomaterial Luminescence Multicolor emission Nanocomposite Optical imaging Theranostics Upconversion

A B S T R A C T

Lanthanide-doped inorganic nanomaterials are a prominent class of nanocrystals with multicolor emissions that are an essential tool for optical imaging in analytical chemistry. Based on the excellent luminescence of lanthanide-doped nanomaterials, novel nanocomposites with diverse sizes (sub10 nm) and properties, such as luminescence-plasmon, magnetism-luminescence, and upconversion (UC) and downconversion (DC) dual-mode luminescence, have been designed and prepared. These functional nanocomposites have been achieved by functionalization of lanthanide-doped nanomaterials with various types of polymers, responsive moieties, and targeting molecules. Here, we summarize the recent advancements in application of lanthanide-doped luminescence nanocomposites in analytical chemistry and highlight the challenges for future research. Precisely regulated and multicolor-emission lanthanidedoped nanocomposites will be a next thrust for applications in theranostics. Future applications of lanthanide-doped nanocomposites will benefit from such research. © 2014 Elsevier B.V. All rights reserved.

Contents 1. 2.

3.

4.

Introduction ........................................................................................................................................................................................................................................................ General requirements for lanthanide-doped nanocomposites ......................................................................................................................................................... 2.1. Host materials, activators and sensitizers .................................................................................................................................................................................. 2.2. General requirements ........................................................................................................................................................................................................................ 2.3. Surface functionalization .................................................................................................................................................................................................................. 2.4. Mechanism for modulating luminescence ................................................................................................................................................................................. Lanthanide-doped nanocomposites for detection ................................................................................................................................................................................. 3.1. Disease markers ................................................................................................................................................................................................................................... 3.1.1. Avidin-based detection ..................................................................................................................................................................................................... 3.1.2. Nucleic acids ......................................................................................................................................................................................................................... 3.1.3. Enzyme activity ................................................................................................................................................................................................................... 3.1.4. Glutathione (GSH) .............................................................................................................................................................................................................. 3.2. Glucose ................................................................................................................................................................................................................................................... 3.3. Metals ions ............................................................................................................................................................................................................................................ 3.4. Reactive oxygen species (ROS) ........................................................................................................................................................................................................ 3.5. Hazardous chemicals ......................................................................................................................................................................................................................... 3.6. Fingerprint and document security .............................................................................................................................................................................................. 3.7. Temperature .......................................................................................................................................................................................................................................... Lanthanide-doped nanocomposites for bio-imaging ........................................................................................................................................................................... 4.1. Surface functionalization .................................................................................................................................................................................................................. 4.2. Dual-mode UCL and DCL imaging ................................................................................................................................................................................................. 4.3. In vivo multicolor and multifunctional imaging ....................................................................................................................................................................... 4.4. Ultra-small lanthanide-doped nanoparticles for bio-imaging ............................................................................................................................................

* Corresponding author. Tel.: +86 10 64427869; fax: +86 10 64427869. E-mail address: [email protected] (L. Wang). http://dx.doi.org/10.1016/j.trac.2014.07.011 0165-9936/© 2014 Elsevier B.V. All rights reserved.

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Summary and prospects ................................................................................................................................................................................................................................. 133 Acknowledgments ............................................................................................................................................................................................................................................ 133 References ............................................................................................................................................................................................................................................................ 133

1. Introduction One goal of analytical chemistry is to probe the chemical constituents, interactions and distributions in complex systems (such as environmental, biological, medical and therapeutic systems) by means of sensitive, selective and specific sensors. Luminescence imaging represents an essential tool to visualize analytes and subcellular structures in real time. To achieve the goal, many luminescent detectors have been explored and developed using conventional organic fluorescent scaffolds and emerging nanomaterials [1]. Among them, lanthanide-doped inorganic nanomaterials are a prominent class of nanocrystals with multicolor emission. Due to the unique 4f n inner shell configurations of Ln 3+ ions, lanthanide-doped nanomaterials, rich in magnetic and optical properties, can perform ultrasensitive in vitro and in vivo bioassays with excellent photostability, narrow emission bands, and high chemical stability. Generally, lanthanide-doped nanomaterials are categorized into lanthanide-doped nanorods (NRs), nanospheres (NSs), nanoparticles (NPs) and quantum dots (QDs) according to different sizes and morphologies. Especially, lanthanide-doped NPs (the majority of lanthanide-doped nanomaterials) are categorized into upconversion NPs (UCNPs) and downconversion NPs (DCNPs) based on the mechanism of luminescence. DCNPs in DC luminescence (DCL) are involved in the process that obeys Stokes’ law: absorbing a shortwavelength light and releasing long-wavelength emission through lanthanide doping. UC is the process that absorbs two or more lowenergy photons, such as near-infrared (NIR) photons, and releases a high-energy luminescence photon [2]. Different from conventional organic dyes (single- or twophoton dyes), dye-contained fluorescent NPs (e.g., dye-doped silica NPs) and luminescent lanthanide complexes used as probes for optical imaging, both DCNPs and UCNPs possess unique tunable optical properties through variation of lanthanide dopants and host matrices, such as multicolor and multifunctional imaging [3]. Furthermore, UCNP-based UC luminescence (UCL) imaging shows excellent optical features, such as narrow anti-Stokes shifted light and low autofluorescence background. Especially, after the surface modification with various types of polymers, antennas, and targeting molecules, such as folic acid, antibodies, DNA and RNA, the functionalized lanthanide-doped nanocomposites with low toxicity and excellent tissue-penetration depth because of longwavelength irradiation have been excellent candidates for further application in analytical chemistry, and biochemical, medical and materials fields [3]. Lanthanide-doped nanocomposites have found wide applications from solid laser and display devices to quantitative and qualitative analysis and in-vivo detection, cell imaging, animal imaging, and other advanced applications [3–7]. Considering the rapid development of lanthanide-doped nanocomposites in most areas of analytical chemistry, we review the advancements of functionalized lanthanide-doped nanocomposites, discuss their representative applications in analytical chemistry, and highlight the challenges for future research and development. Precisely regulated and multicolor-emission lanthanide-doped nanocomposites will be a next thrust for future applications. 2. General requirements for lanthanide-doped nanocomposites 2.1. Host materials, activators and sensitizers Conventionally, the host matrices, sensitizer, and activator are the major components influencing the optical properties of DCNPs

and UCNPs. Up to now, in the UC luminescent system, hexagonalphase (β-phase) NaYF4 is one of the most efficient host materials emitting strong UCL via co-doping with Yb3+-Er3+ or Yb3+-Tm3+ ion pairs. Meanwhile, LaVO4 and LaF3 DCNPs with special photochemical properties remain efficient hosts for DC luminescence emitting strong green or red DC luminescence via doping with Ce3+-Tb3+ ion pairs or Eu3+ ions, which have found wide application in analytical chemistry. Much attention has been paid to the following research: (i) doping UCNPs and DCNPs with new rare-earth (RE) ion couples; and, (ii) exploration of new host materials; To date, fluorides and metal oxides, such as NaYbF4, NaGdF4, NaLuF4, LiLuF4, Y2O3, Gd2O3, Y2O2S, CaF2 and YF3, have been employed as novel host materials for the doping of Ln3+ to achieve the desirable DC or UCL due to their low phonon energies. With recent research on lanthanide co-doping of Yb3+-Er3+, Yb3+-Tm3+, Gd3+, and Nd3+, the typical DC and UC luminescent systems have found more and more applications in analytical chemistry. 2.2. General requirements With NPs being introduced into analytical chemistry, the range and the limit of detection (LOD) of analytical chemistry have been greatly broadened due to the enhanced permeability and retention (EPR) effect of nanomaterials. The ideal nanocomposites for analytical application should have several basic features, such as relatively small average particle size (sub-50 nm) with low toxicity, good dispersibility and biocompatibility and high chemical stability as well as excellent optical features. Furthermore, to uncover the secrets of the biology via analytical chemistry, it is essential to understand many functions of analytes in living systems by means of luminescent imaging. However, due to the complexity of the microenvironment of living cells and animals, this task remains challenging. UCNP nanosensors absorbing in the NIR region and nanosensors emitting in the NIR region are expected to be superior to conventional fluorescent probes due to the following advantages: (i) low phototoxicity; (ii) high chemical stability and excellent photostability; (iii) enhanced tissue-penetration depth: NIR photons travel through tissue much more efficiently than those in the visible range; and, finally, (iv) low autofluorescence, narrow absorption bands, and tunable colors with a broad choice and high extinction coefficients. These prominent properties make UCNP nanosensors ideal imaging candidates that are particularly suitable for detection at deeper sites to afford deep tissue and live animal imaging with high spatial resolution and high extinction coefficients against conventional methods. 2.3. Surface functionalization Lanthanide-doped nanocomposites for detection and bioimaging should be water-soluble but their applications are often greatly limited due to the hydrophobic surface of nanomaterials, such as surfaces with oleic acid (OA) or oleylamine (OAm). Thus, surface functionalization of lanthanide-doped nanomaterials from

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Scheme 1. Representative ways of surface functionalization (a) and core-shell structure (b) of lanthanide-doped nanoparticles (NPs). Green spheres are lanthanide-doped NPs. The lanthanide-doped NPs (inside blue sphere in Scheme 1a) are coated by different ligands (blue strips), such as oleic acid (OA) or oleylamine (OAm) or polymers. APTES, 3-aminopropyltriethoxysilane; GSH, Glutathione; PAA, Poly(acrylic acid); PAH, Poly(allylamine hydrochloride); PEG, Poly(ethylene glycol); PEI, Poly(etherimide); PMMA, Poly(methylmethacrylate); PSI, Polysuccinimide. Dyes and other NPs are detailed in Table 1.

hydrophobic to hydrophilic ones and engineering lanthanidedoped NPs with additional functional groups (such as -COOH, -NH2, -SH, and -OH) for further conjugation with antennae is prerequisite before bio-application. To date, ligand exchange, ligand oxidation, ligand attraction and layer-by-layer (LBL) assembly encapsulation (with amphiphilic polymers and lipids) are the general ways for the functionalization of hydrophobic lanthanide-doped NPs. However, these methods are not so simple and general to every NP system. There are therefore several novel, simple ways for surface engineering (Scheme 1a), such as surface silanization [8,9], or coating with carboxylated phospholipid [10], chitosan (CS) [11], poly-(aspartic acid) (PASP), polysuccinimide (PSI) [12,13], peptide [14], poly-(acrylic acid) (PAA) [9,15], modified PEG [16], and polyetherimide (PEI) [9]. Furthermore, for luminescence enhancement, the compact coreshell architecture (Scheme 1b) is usually adopted: the lanthanidedoped active core is covered with one or several shells (un-doped inert shell or doped active shell) with similar lattice constants. To give a highly selective, sensitive response to different analytes, there is great need to conjugate the responsive moiety (antenna [17] is adopted in this review) to the surface of hydrophilic lanthanidedoped NPs (Scheme 2a). The following ways are usually used: (i) (ii) (iii) (iv)

covalent conjugation; ligand exchange or ligand modification; non-covalent adsorbing via hydrophobic interactions; and, LBL modification.

With such surface functionalization and compact core-shell architecture, the lanthanide-doped NPs tend to accumulate in abnormal or diseased tissues and gain a longer blood-residence time through the enhanced permeability and retention (EPR) effect, which allows the lanthanide-doped NPs to provide sensitive detection (such as low LOD and broad linear range) and accurate imaging. 2.4. Mechanism for modulating luminescence As the bands of green luminescence (with emission maxima at 542 nm) or red luminescence (with maxima at 660 nm) of UCNPs

strongly overlap the absorption band of certain fluorescent dyes or NPs, such fluorescent dyes and NPs could be utilized as acceptors of lanthanide-doped NPs. For antenna-conjugated lanthanidedoped nanocomposites, the mechanism of detection is mainly through modulating the energy-transfer process from lanthanidedoped NPs to antenna by the analytes. Upon the addition of varied concentrations of analytes, the elaborated designed antennae on the surface of the NPs will be triggered and transfer the energy emitted from lanthanide-doped NPs, which will lead to changes in the emission intensity or wavelength of luminescence. Thus, the variation in concentration could be precisely and directly read out by luminescence changes. There are several ways to modulate the energy transfer, such as fluorescence resonance-energy transfer (FRET), quenching and surface-plasmon resonance (SPR) [17]. However, FRET or quenching is most commonly used. The commonly used acceptors are gold NPs (AuNPs), Au nanoclusters (AuNCs), graphene or graphene oxide (GO) [19], carbon NPs (CNPs), rhodamine (Rh) [15] and cyanine (Cy) [1]. Based on this mechanism, many nanosensors were designed and applied to detection and delivery. 3. Lanthanide-doped nanocomposites for detection Rapid, qualitative analysis of bioactive substances, disease markers, hazardous chemicals, pathogens, and bacteria is one of the main topics of analytical chemistry (Fig. 1a). Detecting such analytes in different environments, cells or animals is very important for the advancement of analytical chemistry. Existing luminescent nanosensors with lower LODs than those using other methods have increased the applications of lanthanide-doped nanocomposites. Up to now, many lanthanide-doped nanosensors have been successfully developed for the detection of analytes (Table 1). 3.1. Disease markers 3.1.1. Avidin-based detection Many early works focused on the detection of avidin by lanthanide-doped nanocomposites due to the high affinity between avidin and biotin (Ka = 1015 M−1) [20,21]. Li’s group and Chen’s group explored this detection system early. To date, the avidin-biotin

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Scheme 2. Fabrication of antenna-modified lanthanide-doped nanocomposites: cyanine dye (IR-806) and the NaYF4-Yb3+-Er3+ nanoparticles (NPs) (a); cyanine dye (hCy7) and the NaYF4-Yb3+-Er3+-Tm3+ NPs (b). {Reprinted with permission from [17] (©2012 Nature Publishing Group) and [18] (©2013 American Chemical Society)}.

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Fig. 1. Lanthanide-doped nanoparticles for detection and imaging (a), and in vivo multicolor imaging (b). (b) Reprinted with permission from [16] (©2011 American Chemical Society).

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Table 1 Representative analytes that have been detected by lanthanide-doped nanocomposites Analytes

Nanoparticles (size, nm)

Hg2+

NaYF4: Yb3+, Er3+ (50) NaYF4: Yb3+, Tm3+ (50) ZrO2-Eu3+, ZrO2-Tb3+ (<5) LiLuF4:Yb,Er@LiLuF4 (50.7 + 2.8) LaF3: Ce3+,Tb3+ (15–20) NaYF4: Yb3+, Er3+ (50) NaYF4: Yb3+, Er3+ (10–100) NaYF4: Yb3+, Ho3+ (7) LaF3:Ce3+,Tb3+ (15–20) NaYF4: Yb3+, Er3+ (1 μm (L.) × 200 nm (d.m.) NaYF4: Yb3+, Er3+ (800 nm (L.) × 200 nm (d.m.) NaYF4: Yb3+, Er3+ (20)

Hg2+

LaVO4: Eu3+(50)

Avidin Avidin β-hCG Glucose Glucose Cu2+ Fe3+ Fe3+ K+ Pb2+

Surface layer

Antennae

Linear range and LOD 0.5–370 nM, −0.5 nM

Aqueous solution

[20]

AEP Avidin

FITC-labeled avidin Biotinylated β-hCG antibody RBITC-labeled APBA GO-CS RBH RBD PASP Chromoionophore ETH 5294 Chromoionophore ETH 5418 RBT

3 nM 0–13.5 nM, 0.17 nM

H1299 Cancer Cells Cell imaging

[21] [22]

0.5–25 mM, −0.5 nM 0.56 to 2.0 μM, 0.025 μM 1–10 μM, 1 μM 5–400 μM, 1.2 μM 0.5–100 μM, 0.306 μM 0.1–10 mM

Aqueous solution Human serum Aqueous solution Aqueous solution Drinking water The whole blood

[23] [19] [24] [25] [26] [27]

0.1 mM–1 μM

Sheep plasma and artificial industry wastewater Tap water

[28]

Tap water samples and cell culture medium In a mouse model HepG2 and HeLa Cells Human serum Aqueous solution Human serum Aqueous solution Mixed water samples

[30]

Real water samples Aqueous solution Human plasma Human plasma and whole blood samples NIH3T3 cells Laboratory tap water, kitchen tap water and swimming pool water Fingerprint

[36] [37] [38] [39]

Glucose PAA-ConA – γ-CD PASP – –

Dithizone

40 nM–4 μM, 32 nM

hCy7 MnO2 nanosheets Dopamine Capture DNA PMPD TAMRA Au NPs

83.5 nM 0–10 mM, 0.9 μM 1–75 μM, 0.29 μM 7.8–78 nM, 7.8 nM 0.1–6 nM, 0.036 nM 0–100 nM, 0.18 nM 0–35.2 μM

TNT Diazinon Thrombin MMP-2

NaYF4: (25) NaYF4: Yb3+, Tm3+@NaYF4 (−30) NaYF4: Yb3+, Er3+ (60) NaYF4: Yb3+, Er3+ (<50) NaYF4: Yb3+, Er3+ (20–40) NaYF4: Yb3+, Er3+ (28) NaYF4: Yb3+, Er3+ (106–142) nanoflowers NaYF4: Yb3+, Er3+ (100) LaVO4: Eu3+ (400–800) NaYF4: Yb3+, Er3+ (50) NaYF4: Yb3+, Er3+ (30–50)

PAA-EGDMA – PAA PEI

APTS – Thrombin aptamer Polypeptide

44 μM–3.96 mM, 0.43 nM 0.33 nM–0.3 μM, 0.23 μM 0.5–20 nM, 0.18 nM 0.72–36 nM, 0.72 nM

HOCl NH3

NaYF4: Yb3+, Er3+, Tm3+ (25) NaYF4: Yb3+, Er3+ (30–60)

PAA –

RBD NaHCO3, H2O2

0–120 μM, 0.32 μM 0.5–50 μM, 11 nM

Cocaine

NaYF4: Yb3+, Er3+ (260)

PAA

LBA, CBA

33 nM

GSH GSH DNA DNA DNA TNT

Yb3+,

Er3+,

Tm3+

Ref.

Biotinylated-Au NPs

Carboxylated phospholipid Poly (MMA-co-MAA) P-PEG Azelaic acid PAA Probe DNA Probe DNA Citrate PSI

MeHg+

Applications

Biotin

5 nM–10 μM, 3.7 nM

[29]

[18] [31] [32] [33] [34] [34] [35]

[15] [40] [41]

β-hCG, β sub-unit of human chorionic gonadotropin; AEP, 2-aminoethyl dihydrogenphosphate; APBA, 3-amino phenyl boronic acid; APTS, 3-aminopropyltriethoxysilane; AuNP, Gold nanoparticle; AuNR, Gold nanorod; CBA, Cocaine-binding aptamer; CD, Cyclodextrin; ConA, Concanavalin A; CS, Chitosan; EGDMA, Ethylene glycol dimethacrylate; FITC, Fluorescein isothiocyanate; GSH, Glutathione; GO, Graphene oxide; HOCl, Hypochlorous acid; LBA, Lysozyme-binding aptamer; LOD, Limit of detection; MAA, Methacrylic acid; MeHg+, Methylmercury; MMA, Methyl methacrylate; PAA, Pol (acrylic acid); PEI, Polyethylenimine; PMPD, Poly-m-phenylenediamine; PSI, Polysuccinimide; RBD, Rhodamine B derivative; RBH, Rhodamine B hydrazide; RBITC, Rhodamine B isothiocyanate; RBT, Rhodamine B thiolactone; TAMRA, Carboxytetramethylrhodamine.

interaction has been employed as a model system for many detection systems. Herein, we select several typical lanthanide-doped nanocomposites detection systems based on avidin to illustrate the detection mechanism. In 2005, Li’s group initiated the UCNP-based FRET biosensor for avidin with NaYF4:Yb3+-Er3+-biotin NPs as energy donors and Aubiotin NPs as energy acceptors [20]. AuNPs were adopted in FRETbased detection due to their broad absorption spectrum in visible light overlapping with the emission wavelength of UCNPs. The intensity of UCL varied linearly with the concentration of avidin in the solution in the range 0.5–370 nM. Chen’s group synthesized a sensitive time-resolved FRET nanosensor to detect avidin with a record-low LOD of 3.0 nM based on sub-5 nm amine-functionalized tetragonal ZrO2-Ln3+ NPs [21]. The intense long-lived luminescence of this nanosensor (1.82 ms), achieved by using a low phononenergy ZrO2 host, was crucial for in situ detection and targeted imaging to get accurate quantification, as the autofluorescence of biological tissues is short-lived (in the ns range). Besides the streptavidin-biotin interaction, there are various reports on immunoassay for antigens, antibodies and other disease markers [22]. Very recently, based on the streptavidin-biotin interaction, Chen’s group [22] conjugated avidin to the surface LiLuF4. Yb 3+ -Er 3+ @LiLuF 4 core/shell UCNPs detected the biotinylated disease-marker β sub-unit of human chorionic gonadotropin (βhCG) with an LOD of 3.8 ng mL−1, comparable to the β-hCG level in the serum of normal humans. By successive LBL injection of shell precursors through a thermal decomposition route, the novel

LiLuF4:Ln3+ core/shell UCNPs showed typically high absolute UC quantum yields (5.0% and 7.6% for Er3+ and Tm3+, respectively). As it is challenging to enhance the quantum yields of UCNPs, this method gave a way to achieve UCNPs with high UC quantum yields, which is crucial for enhancing the LOD, the sensitivity of detection, accurate quantification and extensive applications in biomedical fields. 3.1.2. Nucleic acids Since the elucidation of the structure of the DNA double helix by Watson and Crick in 1953, nucleic acids (NAs) have played a crucial role in decoding, storing, regulating and transmitting genetic information. Rapid, sensitive, and quantitative identification of sequences and structures of short oligonucleotides (DNA/RNA) has become an urgent task for the investigation of expression levels of genes in cells, which provides a solid basis for the diagnosis of genetic and infectious diseases. As the solid base for identifying sequences and structures of DNA/RNA, the well-established DNA hybridization-based techniques hold great promise for a variety of DNA-detection sensors. Li’s group [33] developed a new method for DNA assay combining UC fluorescence with magnetic separation. Magnetic NPs were modified with capture DNA and phosphor NPs were modified with probe DNA with an LOD of 0.5 nM in the linear range 0.5–370 nM, which promoted the applications of magnetic NPs and lanthanidedoped nanocomposites. Liu’s group [34] developed an interesting DNA sensor by choosing PEI-modified NaYF4:Yb3+-Er3+ UCNPs as the

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energy donor and poly-m-phenylenediamine (PMPD) NSs as the energy acceptor. PMPD NSs were conjugated to probe DNA on the surface UCNPs through the π-π stacking interaction. In the absence of target DNA, UCNPs were almost non-luminescent due to the FRET process. In the presence of target DNA, the π-π stacking interaction between PMPD and UCNPs was dramatically weakened due to the formation of double-stranded DNA (dsDNA) between the target DNA and the probe DNA. As a result, the energy acceptors PMPD were separated from UCNPs. The luminescence of UCNPs was restored. The UCNP-dsDNA-PMPD nanosensor showed a linear range of 0.1–6.0 nM for target DNA with an LOD of 0.036 nM in an aqueous buffer. This nanosensor was also applied to human serum with high precision and pronounced specificity. Most current methods of detecting DNA with low LOD are performed under laboratory conditions, which are not so rapid, timeconsuming and highly sensitive. Developing novel, rapid detection methods (e.g., a UC microplate-reader system, a high-throughput detection system and paper-based devices) with low cost and ease of use is highly desirable and challenging.

3.1.3. Enzyme activity Quantification of enzyme activity and protein is important for disease diagnosis and therapy. Diverse methods with lanthanidedoped nanosensors have been to image activities of enzyme in vivo, such as tumor-associated lysosomal protease, thrombin, matrix metalloproteinases (MMPs), and other proteins. The limit of quantification (LOQ) of these lanthanide-doped nanosensors was much lower than that of other reported methods of assays, such as enzymelinked immunosorbent assay (ELISA), the CdSe/ZnS-fluorescent method and the electrogenerated chemiluminescence method, and is therefore of great significance for accurate quantification, clinical diagnosis and other biomedical applications. In 2011, a novel aptamer biosensor [38] for thrombin based on FRET from thrombin aptamer-functionalized UCNPs to CNPs was developed. CNPs were conjugated to the surface of PAA-functionalized UCNPs through the π-π stacking interaction. In the absence of thrombin, the luminescent of UCNPs was almost silenced due to the quenching ability of CNPs [38]. Upon addition of thrombin, the thrombin aptamer on the surface of UCNPs was induced to form a quadruplex structure, which dramatically weakened the π-π stacking interaction between CNPs and UCNPs. As a result, the energyacceptor CNPs were separated from the UCNPs. The luminescence of the UCNPs was restored. The UCNP-aptamer-CNP nanosensor showed a linear range of 0.5–20 nM for thrombin with an LOD of 0.18 nM in an aqueous buffer. This nanosensor was also applied to spiked human serum samples with a slightly higher LOD (0.25 nM) for thrombin with good selectivity over other proteins. It is worth noting that CNPs have a similar sp 2 electronic structure to graphene, so allowing CNPs to be an energy acceptor. Furthermore, unlike two-dimensional graphene with a relatively large plane, the smaller size of zero-dimensional CNPs is more biocompatible. The same strategy was applied to detect MMP-2 enzyme activity by the same group [39]. CNPs were conjugated to the surface of UCNPs by a polypeptide chain (GHHYYGPLGVRGC), which contains both the MMP-2 specific substrate domain (PLGVR) and a π-rich motif (HHYY). In the presence of MMP-2, the polypeptide chain on the surface of UCNPs could be specifically recognized and cleaved by MMP-2, resulting in restoration of the luminescence of UCNPs. The UCNP-polypeptide-CNP nanosensor showed an LOD of 0.72 nM in the range 0.72–36 nM in human plasma and whole-blood samples. This LOD is at least one order magnitude lower than that of other reported nanosensors for MMP-2, so the UCNP-polypeptide-CNP nanosensor could be used to determine the level of MMP-2 in vivo and in real samples.

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3.1.4. Glutathione (GSH) GSH is a very important thiol-containing biomolecule, biomarker and endogenous antioxidant with concentration as high as 10 mM in vivo that has been implicated in many defensive systems and diseases, such as cancer, heart problems, leukocyte loss, HIV, psoriasis, aging and liver damage. Fluorescence-imaging analysis based on lanthanide-doped nanocomposites has attracted increasing attention, as it offers an appealing approach to the visualization of GSH at the cellular level. To date, detection of GSH by UCNP probes relies on FRET or quenching [31,32]. FRET efficiency from the functionalized UCNPs to the antenna is modulated through variation in the concentration of GSH. In 2011, Liu’s group [31] reported a novel manganese-dioxide (MnO2) nanosheet-coated NaYF4:Yb3+-Tm3+@NaYF4 UCNP nanosensor for rapid, selective detection of GSH in aqueous solutions and living cells based on the FRET process from UCNPs as donor to MnO2 nanosheets as quencher. In the absence of GSH, the luminescence of UCNPs is quenched by MnO2 nanosheets. Upon the addition of GSH, GSH was oxidized by MnO2 nanosheets to generate glutathione disulfide (GSSG) through thiol-disulfide exchange, which rendered the recovery of luminescence of UCNPs with an LOD of 0.9 μM. Lv’s group [32] developed another novel probe for rapid, economic, sensitive, selective detection of GSH in aqueous solution and human-blood serum based on linkage of UCNPs and dopaminequinone through hydrogen bonding and electrostatic interaction, which proved to be a competitive method due to the lower LOD of 0.29 μM in the linear range 1–75 μM than the MnO2-modified UCNPfluorescent method, the CdSe/ZnS-fluorescent method and the CdTeGO amplified electrogenerated chemiluminescence method [32]. This method of Lv’s group, with such a low LOD, gives highly sensitive detection of GSH and accurate results, which are very important for bio-detection and imaging. 3.2. Glucose As important bioanalytes, glucose and ATP play various roles in bioreactor monitoring, energy storage and clinical diagnosis. There are several reports on determination of the levels of glucose by lanthanide-doped nanocomposites [19,23]. Li’s group [23] synthesized a water-soluble LaF3:Ce3+, Tb3+ NP to monitor the levels of glucose based on FRET from glucose-modified LaF3:Ce3+-Tb3+ NP to rhodamine B isothiocyanate (RhBITC) that had been conjugated with 3-aminophenyl boronic acid (APBA). In the absence of glucose-modified LaF3:Ce3+, Tb3+ NP, RhBITC and LaF3:Ce3+, Tb3+ NP were fluorescent. Upon the addition of glucose-modified LaF3:Ce3+, Tb3+ NPs into RhBITC, binding of glucose with APBA would bring LaF3 and RhBITC into appropriate proximity and hence induce energy transfer with no fluorescence. The proposed LaF3:Ce3+, Tb3+ NP-RhBITC FRET model promoted the development of lanthanidedoped nanosensors in analytical chemistry. In addition to AuNPs, graphene has been adopted as another energy-acceptor candidate in FRET-based detection due to its strong electron-capturing and superquenching ability to fluorescence or luminescence (sp2 electronic hybrid and a large conjugate plane allow for graphene non-radiative transfer of electronic excitation energy from donors to the π system of graphene). Liu’s group [19] constructed a nanosensor NaYF4:Yb3+-Er3+ UCNPs-GO by covalently attaching concanavalin A (ConA) and chitosan (CS) to UCNPs and GO. In the absence of glucose, energy from UCNPs was transferred to GO. However, in the presence of glucose, glucose is more competitive than CS for ConA, which results in the inhibition of the FRET process. As a result, the luminescence of UCNPs recovered with an LOD of 0.025 μM in the range 0.56–2.0 μM. It is notable that, despite the advantages of GO over other kinds of energy acceptors, research into sensors based on GO and

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lanthanide-doped nanomaterials is still in its infancy due to the challenges of functionalization, process control, and intracellular uptake of GO-based materials.

3.3. Metal ions Participation of metal ions is essential in diverse fields, such as food analysis, environmental monitoring, metabolism and clinical diagnosis. Cu2+, Fe3+ and K+ are essential nutrients for humans and other mammals due to their importance in the intracellular metabolism. Pb2+, Hg2+ and methylmercury (MeHg+) are highly toxic heavy metals that pose serious consequences for living organisms, and environmental and human health. Selective, sensitive detection of these metal ions via nanosensors is of vital importance. For selective detection and imaging of Cu2+, UCNP sensors have been designed by taking advantage of the Cu2+-responsive antenna RB-hydrazide molecules [24]. Zhang et al. [24] designed a nanosensor for detection of Cu2+ by choosing NaYF4:Yb3+-Er3+ UCNPs as donor and RB-hydrazide as acceptor. Through hydrophobic interactions with the oleic-acid ligand on the surface of UCNPs, the RB-hydrazide molecules were non-covalently adsorbed to the surface of NPs. In the absence of Cu2+, there is no FRET as the fluorescence of RB is silenced. In the presence of increasing concentrations of Cu2+, the fluorescence intensity of RB-hydrazide increases dramatically due to a delocalized xanthene moiety of the RB group generated by the Cu2+ trigger, while the luminescence intensity of UCNPs nanosensor centered at 521 nm and 539 nm decreases gradually due to FRET from UCNPs to fluorescent RB. The red emission of UCNPs with little change acts as a reference to the luminescence changes, which allows for ratiometric detection of Cu2+. Based on self-calibration of green emission and red emission intensity in the different concentrations of Cu2+, ratiometric imaging based on the UCNP nanosensor is a more precise and practical method than conventional fluorescent methods or atomic spectroscopic techniques for detection, and it allows quantitative measurement of metal-ion levels and provides accurate data for bio-applications. Our group found that PASP-coated LaF3:Ce3+-Tb3+ NPs could act as a luminescent nanosensor for Fe3+ over other cations due to the formation of a PASP-Fe3+ complex on the NP surface [26]. Upon the addition of Fe3+, the luminescence of the nanosensor was gradually quenched in a linear range 0.5–100 μM with an LOD of 0.306 μM and it was applied to detect Fe3+ in drinking water. Later, in a different approach, the group headed by Zhu and Burda developed a NaYF4:Yb3+-Ho3+ UCNP nanosensor for Fe3+ based on the FRET process from γ-cyclodextrin (CD)-modified NaYF4:Yb3+-Ho3+ (CDUCNPs) as donor to Fe3+-responsive antenna rhodamine B derivative (RBD) molecules as acceptor [25]. Without Fe3+ in aqueous solution, the CD-UCNPs showed a characteristic luminescence spectra centered at 542 nm and 646 nm, while RBD was non-fluorescent. Upon being chelated with Fe3+, RBD was strongly fluorescent due to the formation of the open-ring form triggered by the oxygen atoms of the amide groups in RBD. In the presence of varying concentrations of Fe3+, the green emission of UCNPs was partially quenched. However, the red emission of UCNPs at 646 nm stayed nearly unchanged, which made this nanosensor a ratiometric detector of Fe3+. This method acts as a typical model to construct UCNP ratiometric nanosensors based on the following mechanism: red emission acts as inert reference signal while the green light varies with the concentration of the analytes. Based on self-calibration of green emission and red emission intensity in presence or absence of analytes, ratiometric imaging based on lanthanide-doped nanosensors is a precise, practical method for detection, when compared with conventional detection methods. It is interesting to note that the CD pocket could act as the vital nexus between UCNPs and RBD, which

protects the Fe3+-responsive antenna from the harsh environments and thus prevents photobleaching and photodegradation. It is worth noting that various levels of H+ and K+ in blood or cells play a significant role in maintaining the normal functions of cell organelles. Through incorporating NaYF4:Er3+-Yb3+ UCNRs, H+sensitive chromoionophore ETH 5294, and specific ionophores together in hydrophobic polymer matrices, Qin’s group [27] designed an optode NaYF4:Yb3+-Er3+ NRs-ETH 5294 that could selectively detect K+ in a whole-blood sample [27]. Upon the addition of K+, the K + exchanged with hydrogen ions in order to conserve electroneutrality within the optode, which triggered the chromoionophore changing from its protonated form to its deprotonated form. Concomitantly, the luminescence intensity ratio of peak 656 nm to peak 542 nm increased due to the quenching of red emission by the protonated form of ETH 5294 and to the quenching of green emission by the deprotonated form of ETH 5294. This novel ratiometric detection based on UCNRs eliminates the background absorption and autofluorescence of the biological sample. In a similar way, the same group designed a Pb2+ nanosensor with the linear range 0.001– 10 mM at pH 5 in industrial wastewater by incorporating Pb2+ ionophore with NR-ETH 5418 [28]. There are also several nanosensors that have been reported to monitor Hg2+ [29,30,42] and MeHg+ [18]. Our group [30] developed a UC FRET sensor for the detection of Hg2+ in water based on the rhodamine B thiolactone (RBT)-functionalized NaYF4:Yb3+-Er3+ (UCNP@RBT) nanocomposites. Upon excitation at 980 nm, this UCNP nanosensor gave an ultrasensitive, selective, rapid response to Hg2+ in water free of interfering background signals, when compared with conventional methods, such as fluorescent probes, and atomic spectroscopic techniques. Zhou’s group developed a glutathionecapped Eu3+-doped CdS nanosensor for detecting Hg2+ in river water, lake water and tap water [42]. Compared with transition-metal ions, lanthanide ions are not easy to dope in QDs due to the different ionic radii and charge. However, lanthanide-doped QDs can retain sharp emission signals, tunable colors, and the long lifetime of lanthanide ions with μs-scale or ms-scale lifetimes, which have a significant effect on imaging resolution and LOD. These merits of lanthanidedoped QDs are also favorable for time-resolved detection and imaging. It is interesting to note that the MeHg+ nanosensor (Scheme 2b) contained heptamethine cyanine dye (hCy7) as responsive antenna and was applied to mice [18]. In the absence of MeHg+, there is FRET from UCNPs to hCy7, during which the red emission centered at 660 nm was silenced while the luminescence intensity of the UCNP nanosensor centered at 540 nm and 800 nm was unchanged. In the presence of increasing concentrations of MeHg+, the luminescence intensity of the nanosensor centered at 800 nm decreased dramatically due to another FRET process from UCNPs to hCy7 triggered by the specific interaction between MeHg+ and hCy7, while the luminescence intensity of the UCNP nanosensor centered at 540 nm and 660 nm was unchanged. Thus, ratiometric emission at 800 nm to 660 nm acted as a detection signal, allowing for sensitive detection of MeHg+. The hCy7-UCNP nanosensor could monitor MeHg+ within the liver and the spleen of mice both ex vivo and in vivo and provided an LOD as low as 0.18 ppb in aqueous solution. As more and more lanthanide-doped nanosensors are developed for monitoring intracellular metal ions, their limitations in wavelength and optical properties should be noted and rectified by alternative wavelengths. For DCNPs, excitation wavelengths are mainly limited at UV that will damage cells or animal tissues, while, for UCNPs, the excitation sources are generally limited to 980 nm and cause strong absorption of water and sample overheating; excitation at alternative wavelengths (such as an 800-nm excitation source for Cy-sensitized NaYF4:Yb3+-Er3+ UCNPs [17] or Nd3+-Yb3+doped UCNPs [43]) and spectral conversion under broadband excitation are challenging [17].

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3.4. Reactive oxygen species (ROS) ROS are a short-lived, high-reactive family, including hydrogen peroxide (H2O2), the superoxide anion radical (O2•−), ozone (O3), hydroxyl radical (•OH), singlet oxygen (1O2), peroxy radical (ROO•), and hypochlorous acid (HOCl, generated by the myeloperoxidase-H2O2Cl− system) [1]. They are mainly produced by leaking of electrons in cells. Since the “free radical theory” of aging and associated degenerative diseases was proposed by Harman in 1956, the deleterious or beneficial roles of free radicals on cell constituents and on the connective tissues are continually argued. They are closely connected with healthy physiological events (such as metabolism) and neurodegenerative disorders (such as Parkinson’s disease, aging and cancer). In 2014, Joachim et al. developed a nanosensor for HOCl detection choosing PAA-modified NaYF4:Yb3+, Er3+, Tm3+ UCNPs as energy donor and rhodamine B derivative as energy acceptor [15]. This novel ratiometric nanosensor was applied to visualization of HOCl in the MPO-H2O2-Cl− system in NIH3T3 cells with an LOD of 0.32 μM in the linear range 0–120 μM, which laid a solid foundation for highlysensitive detection of ROS due to the high chemical and photo stability of lanthanide-doped nanosensors. Detecting ROS by lanthanide-doped nanosensors provides an effective approach to real-time, non-invasive, sensitive and selective image dynamics changes of radicals, which will further advance the understanding of the biological roles of radicals. However, to date, the UCNP nanosensors for ROS are still rare due to the short lifetimes of ROS. Thus, development of highly-sensitive, rapid detection systems for ROS is very challenging.

3.5. Hazardous chemicals As serious pollution sources of water and potential homeland security threats, the aqueous solution of ammonia (ammonium hydroxide), Hg2+, nitroaromatic explosives and organophosphate pesticides are hazardous chemicals that are highly toxic to human and animal health. Thus, rapid, sensitive, selective detection of these hazardous chemicals is in great demand for homeland security and public safety. Without a light source for excitation and no autofluorescence of samples, chemiluminescence (CL) detection has proved to be a powerful method. In 2012, Lin’s group [40] introduced NaYF4: Yb3+Er3+ UCNPs into CL detection. A CL resonance-energy transfer (CRET) system NaHCO3-NH4OH-NaYF4:Yb3+-Er3+ UCNPs–H2O2 (NNNH) was constructed for highly sensitive, selective measurement of ammonia in water samples by choosing a novel energy donor-acceptor pair (HCO4- and NaYF4:Yb3+-Er3+ UCNPs). The enhancement of CL is produced from the emission of UCNPs excited by the decomposition energy of HCO4− catalyzed by ammonia. This UCNP-based CL system showed a wider linear range of 0.5–50 μM for ammonia with a lower LOD of 1.1 × 10−8 mol L−1 (S/N = 3) than the single CL-detection system, which shows robust potential for detecting ammonia in laboratory and kitchen tap water, Wanquan River water, swimming pool water and lotus pond water. Nitroaromatic explosives, such as 2,4,6-trinitrotoluene (TNT), 2,4,6-trinitrophenol (TNP), 2,4-dinitrotoluene (DNT) and nitrobenzene (NB), are common, powerful explosives with low safety coefficients. In 2013, our group designed a novel SPR-enhanced UCL TNT sensor by using PSI-functionalized UCNF and cysteaminemodified AuNPs [35]. In the absence of TNT, the UCL at 661 nm enhanced gradually upon increasing the concentration of Au-NPs from 0 mg mL−1 to 38 mg mL−1 due to SPR effects. In the presence of TNT, the red UCL was further enhanced dramatically due to further aggregation of the AuNPs and UCNFs. This novel sensor has excellent selectivity for TNT with a wider linear range of 0–8.0 mg mL−1

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(R2 = 0.9984) for TNT than the conventional detection methods and that is crucial for improving the LOD and accurate quantification. In 2014, our group designed another NaYF4:Yb3+-Er3+ UCNPs nanosensor for TNT [36]. In this nanosensor, ethylene-glycoldimethacrylate (EGDMA)-modified UCNPs were conjugated with 3-aminopropyltriethoxysilane (APTS). Upon the addition of TNT, APTS bound with TNT through amino groups on the UCNPs, the nakedeye visible green UCL of the UCNPs was dramatically quenched over other nitroaromatics including TNP, DNT, and NB, with an LOD of 9.7 ng/mL and linear range of 0.01–9.0 mg/mL. The present studies served as a novel, easy strategy to fabricate the UCL sensors with highly selective recognition ability in aqueous media and are desirable for label-free analysis of TNT in mixed solution independent of immunoassay and molecularly-imprinted technology and complicated instruments. Our group designed a bifunctional nanosensor Fe3O4-LaVO4:Eu3+ for organophosphate pesticides based on molecularly-imprinted polymer (MIP) NSs. The sensor giving a low LOD of 0.23 μM was fabricated by assembling recognition sites of template organophosphate pesticides to the surface of NSs containing luminescent LaVO4:Eu3+ nanocrystals and magnetic Fe3O4 NPs [37]. Despite these advancements and strategies for detecting hazardous chemicals, developing fast, sensitive and reliable detection systems and more facile detection techniques (e.g., high-throughput diagnosis techniques) is still challenging and highly desirable for public safety and homeland security. 3.6. Fingerprint and document security During criminal investigations, visible, impression, and latent fingermarks are often used. Latent fingermarks, especially, have attracted increasing attention. There is a mixture of water, salts, fatty acids, free amino acids, lysozyme, peptides, and other proteins. In 2014, Yuan’s group developed a novel UCNP nanosensor to detect fingerprints with cocaine powder through recognizing lysozyme in the fingerprint ridges by conjugating lysozyme-binding aptamer (LBA) to the PAA-modified UCNPs [41]. This novel method can avoid background fluorescence and thus allows for imaging fingerprints on a wide range of complex surfaces, such as marble, Petri dishes and patterned coins, and it is superior to conventional fluorescent methods, AuNP methods and magnetic Fe3O4 NPs. 3.7. Temperature Temperature is closely linked with industrial processes and cellular events, including cell division, apoptosis and death, so accurate measurement of temperature distributions in the environment and within living cells is necessary for industrial applications and quantitative descriptions of cellular events. It is also reported that pathological cells maintain a higher temperature than normal cells because of their enhanced metabolic activity. Changes in them at the cellular level have a great effect on diseases and pathologies. UCNPs mainly display two narrow emission bands, each of which has its own temperature dependence (the green fluorescence band of the Er3+ dopant ion especially changes with temperature). The ratio of the intensity of two of these bands provides a reference signal for optical sensing of temperature in living cells, which conventional fluorescence probes and nanosensors could not satisfy. The intensity of the green emission (510–530 nm) of NaYF4:Yb3+-Er3+ NPs remained virtually constant while the yellow-green emission (530– 560 nm) and the red emission changed dramatically, which made ratiometric sensing a particularly reliable method for temperature determination in human embryo kidney cells. Capobianco’s group found that the internal temperature of the living HeLa cancer cells was from 25°C to its thermally induced death at 45°C by using NaYF4:Er3+-Yb3+ NPs [44].

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4. Lanthanide-doped nanocomposites for bio-imaging

4.2. Dual-mode UCL and DCL imaging

Another attractive application of lanthanide-doped nanocomposites in analytical chemistry is bio-imaging. Surface functionalization and bio-imaging of UCNPs have been summarized in several reports [3]. In this review, we focus on the recent progress of bio-imaging of lanthanide-doped nanocomposites. Bioimaging based on lanthanide-doped nanocomposites includes tracking the intracellular distribution, tumor targeting, lymphatic imaging, vascular imaging, tumor angiogenesis analysis, cell tracking, bacteria imaging and Giardia-cyst imaging [9]. However, intracranial glioblastoma targeting across the blood brain barrier (BBB, one of the most exclusive biological barriers) [45] and multicolor and multifunctional imaging [45] are challenging.

Recently, dual-mode UCL and DCL imaging received increasing attention [43,47]. The group headed by Zhang and Zhao [43] prepared core/shell1/shell2/shell3-structured NaGdF4:Nd3+/NaYF4/ NaGdF4:Nd3+, Yb3+-Er3+/NaYF4 nanocrystals for dual-mode UC-DC luminescence upon excited at 800 nm. The in-vivo, high-contrast DC imaging of a whole body nude mouse was achieved by this multifunctional nanocomposite. By coating mixed DC-UC hydrophobic NPs with a polymer through in-situ cross-linking polymerization, our group [47] fabricated the ZnS:Mn 2+ -NaYF 4 : Yb3+-Er3+ multifunctional nanocomposites with simultaneous DC and UC emission. These multifunctional nanocomposites were applied to label HeLa cells for dual-mode cell imaging. 4.3. In vivo multicolor and multifunctional imaging

4.1. Surface functionalization For bio-imaging, a wide range of lanthanide-doped nanocomposites have been prepared. Silica coating has turned out to be one of the most effective ways to functionalize NPs. So far, there are two main easy strategies for coating a silica layer on the surface of NPs: the Stöber method, and reverse microemulsion. Silica coating by reverse microemulsion was superior to the Stöber method, due to the good dispersity and the biocompatibility of silica-coated NPs. In the typical mechanism, TEOS hydrolysis occurs in the aqueous microdroplets of the water-in-oil emulsion. The silica-coated UCNPs were successfully used as a bio-tag for cancer-cell imaging [8]. For their outstanding amphiphilicity, biocompatibility, and bioconjugatability, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamineN-(succinyl) (carboxylated phospholipid) [10] and polyethyleneblock-poly-(ethylene glycol) (PE-b-PEG) were selected as the coating material. Through ultrasound-assisted encapsulation technology, hydrophobic lanthanide-doped NPs (DC LaVO4:Eu3+, LaF3: Ce3+-Tb3+ and UC NaYF4:Yb3+-Er3+), QDs (ZnS:Mn2+), YPO4 nanoplates and hydroxyapatite NRs were successfully encapsulated in the carboxylated phospholipids and polymers micelles with one particle per micelle. Carboxylated phospholipid-functionalized NaYF4:Yb3+-Er3+ NPs were bioconjugated with folic-acid antibody (FA, an overexpressed receptor on the surface of tumor cells) to provide the luminescent NPs specifically with immune labeling of HepG2 liver cancer cells. As chitosan [11] is one of the most occurring natural biopolymers with good biocompatibility, biodegradability, low toxicity, good availability, and antibacterial activities toward mammalian cells, O-carboxymethyl chitosan (OCMC)-functionalized NaYF4:Yb3+-Tm3+Er3+ NPs were bioconjugated with FA antibody so the luminescent NPs could specifically recognize the HeLa cells. PASP is essentially a highly biocompatible layer of poly-amino acid, but chemically more robust than other polymers [12,46]. Our group developed a general strategy to functionalize hydrophobic lanthanide-doped NPs (DC LaVO 4 :Eu 3+ , LaF 3 : Ce 3+ -Tb 3+ and UC NaYF4:Yb3+-Er3+), noble metal (Ag), QDs (ZnS:Mn2+), and magnetic oxide (Fe3O4) NPs through coating the lanthanide-doped NPs with this new type of water-soluble, biodegradable functional material, PASP. After functionalization with PASP on the surface of lanthanidedoped NPs, these nanocrystals are highly stable in water, biocompatible, and bioconjugatable with antenna molecules. Individual PASP-wrapped UC NaYF4:Yb3+-Er3+ NPs were bioconjugated with FA so the luminescent NPs could specifically recognize the HeLa cells. This surface modification of hydrophobic NPs will attract great interest in bio-imaging from the fields of chemistry, materials, nanobiotechnology, and nanomedicine. In a parallel work, Wen’s group [14] functionalized a series of lanthanide-doped NPs by coating a short synthetic peptide (RE-1) to form a stable coating layer on the surface of the NPs, which effectively cancels out their autophagy-inducing activity.

An attractive development of bio-imaging would be the possibility to design multifunctional lanthanide-doped nanocomposites and report two or more different signals simultaneously for multiparameter imaging in living cells or animals. Multifunctional lanthanide-doped nanocomposites are highly desirable for many important technological applications, ranging from magnetic recyclable catalysis to multimodal imaging, and simultaneous diagnosis and therapy. Several lanthanide-doped nanocomposites exhibiting different properties, such as magnetism-luminescence (Fe3O4/ lanthanide-doped NPs) [48,49], SPR-UCL (NaYF 4 /Au) [35] and multicolor lanthanide-doped nanocomposites have been reported. Another attractive development of bio-imaging would be the possibility to design multicolor lanthanide-doped nanocomposites. By varying lanthanide ions in the host matrices, a broad choice of emission wavelengths, such as NIR-to-UV, UV-to-visible, NIR-to-visible, NIR-to-NIR, and NIR-to-NIR-II emission, is available, and enables multicolor imaging and high detection sensitivity. It is well established that both of UCNPs and DCNPs could emit multicolor or white emission (balance with yellow-blue light for DCNPs and red-greenblue light for UCNPs) that is very important for white LEDs due to the color stability and high color purity of white light. The group headed by Shao and Liu [16] developed a flexible method to tune the UC spectra of UCNPs for in-vivo five-color imaging (Fig. 1b) in nude mice. UCNPs were first conjugated with mPEG-PMHC18 polymers by hydrophobic interactions between hydrocarbon chains of the PMHC18 and the oleic acid layer. Then mPEG-PMHC modified UCNPs were loaded with RhB, R6G and Tide Quencher 1 (TQ1) by non-covalent adsorption. Multicolor imaging was achieved by subcutaneously injecting five different UCNP solutions (NaYF4:Yb3+Er3+, NaYF4:Yb3+-Er3+/RhB, NaYF4:Yb3+-Er3+/R6G, NaYF4:Yb3+-Er3+/ TQ1, and NaYF4:Tm3+-Er3+) into the backs of nude mice. 4.4. Ultra-small lanthanide-doped nanoparticles for bio-imaging For biological imaging and avoiding the interference with cellular systems, lanthanide-doped NPs of small or ultra-small size (comparable to the biomolecule NPs are labeling) are always desirable. However, precise size control over the ultra-small or ultrathin (sub-10-nm) lanthanide-doped NPs will give distinctive properties (such as properties of polymers) [50]. However, most of the reported lanthanide-doped NPs have a single size in the 15– 40 nm range; furthermore, there is a paradox between the size and the brightness of UCNPs: size reduction significantly reduces the UC emission brightness due to quenching from the surface quencher and the low extinction coefficient and the low quantum yield of NPs. Our group [46] presented an easy, in-situ, one-pot Gd3+-exchange strategy to enhance the UCL of NaYF 4 :Yb 3+ -Er 3+ NPs about 29 times with unchanged particle size (11.8 nm). By varying the

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concentration of OAm, basic surfactants (Yb3+:F− ratio) and reaction temperature, Cohen’s group [50] developed sub-10-nm NaYF4:Yb3+-Er3+ UCNPs (4.8 nm) with significant brightness compared with existing UCNPs for single-molecule imaging, so that these ultra-small protein-sized UCNPs could probe subcellular structures with greater tissue-penetration, increased accessibility and reduced interference with biomolecule function, binding events and trafficking. Thus, tremendous efforts will be made to prepare UCNPs with small particle size and strong luminescence.

Project of Fundamental Research of China (2011CB932403), the Fundamental Research Funds for the Central Universities (ZZ1321), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1205). We also thank the “Public Hatching Platform for Recruited Talents of Beijing University of Chemical Technology” for support.

5. Summary and prospects

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Due to the unique optical properties of lanthanide-doped NPs (especially the low autofluorescence of UCNPs upon excitation at 980 nm), luminescence imaging has turned out to be an essential tool for detecting analytes. We summarized the application of lanthanide-doped nanocomposites in analytical chemistry mainly in two aspects: detection and bio-imaging. We also demonstrated the principles of fabricating functionalized lanthanide-doped nanocomposites and the exploitation of them for further developments as well as fundamental studies. However, such a summary was incomplete and awaits more innovative, comprehensive research. In comparison to the conventional detection systems, the lanthanide-doped nanocomposite systems are more functional with new opportunities and challenges for future development: (1) the ultra-thin (sub-10-nm) lanthanide-doped NPs will show distinctive properties [50], tremendous efforts will be made to improve the quantum yield of UCNPs and to prepare UCNPs with small particle size and strong luminescence; (2) transferring lanthanide-doped NPs from hydrophobic to hydrophilic by using polymers or biocompatible molecules always decreases the brightness of NPs, so functionalization of their surface while retaining their emission intensity is a challenge for fundamental research; (3) lanthanide-doped QDs retaining long lifetime, sharp emission signals and a broad choice of emission wavelengths need to be further exploited; (4) construction of multifunctional, precisely photo-regulated, and multicolor emission lanthanide-doped NPs systems with NIRto-UV, NIR-to-visible, NIR-to-NIR, and NIR-to-NIR-II emission by doping lanthanide ions or conjugation with different organic fluorescent dyes will receive more attention; (5) the LOD of the existing luminescent nanosensors needs to be lowered for complex systems; (6) imaging exclusive biological barriers (e.g., BBB) is still challenging; and, (7) though lanthanide-doped NPs possess the potential for invivo detection, imaging and therapies due to the low toxic nature (e.g., Gd3+ chelates are frequently used contrast agents for magnetic resonance imaging ), little systematic assessment of their toxicity is available. Before widespread use in medical assays, drug screening and clinical applications, more efforts should be devoted to toxicity experiments of lanthanide-doped NPs, such as quantitative correlations between physicochemical properties and toxicity. In conclusion, luminescence detection and imaging based on lanthanide-doped NPs will be great stimuli for the new generation of diagnostics. Systematic studies of functionalized lanthanidedoped nanocomposites are essential for future applications. Acknowledgments This research was supported in part by the National Natural Science Foundation of China (21475007, 21275015), the State Key

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