Lanthanide-doped up-converting nanoparticles: Merits and challenges

Nano Today (2012) 7, 532—563

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/nanotoday

REVIEW

Lanthanide-doped up-converting nanoparticles: Merits and challenges Anna Gnach a,b, Artur Bednarkiewicz a,c,∗ a

Wrocław Research Center EIT+, ul. Stabłowicka 147, 54-066 Wrocław, Poland Institute of Immunology and Experimental Therapy, PAS, ul. Rudolfa Weigla 12, 53-114 Wrocław, Poland c Institute of Low Temperature and Structure Research, PAS, ul. Okólna 2, 50-422 Wrocław, Poland b

Received 4 June 2012; received in revised form 5 October 2012; accepted 20 October 2012 Available online 16 November 2012

KEYWORDS Lanthanides; Nanoparticles; Biofunctionalization; Up-conversion; Up-conversion enhancement

Summary Due to exceptional photo-physical properties, up-converting nanoparticles (UCNPs) are promising and advantageous alternative to conventional fluorescent labels used in many bio-medical applications. The first part of this review aims at presenting these properties as well as the current state-of-the-art in the up-conversion enhancement, NPs surface functionalization and bioconjugation. In the second part of the paper, the applications of UCNPs and currently available detection instrumentation are discussed in the view of the distinctive properties of these markers. Because the growing widespread use of the biofunctionalized NPs, scarce instrumentation for up-conversion detection is reviewed. Finally, the challenges and future perspectives of the UCNPs are discussed. © 2012 Elsevier Ltd. All rights reserved.

Abbreviations: AHA, 6-aminohexanoic acid; AF, autoflorescence; APTE, (fr.) Addition de Photons par Transferts d’Energie; APTES, 3aminopropyltriethoxysilane; APTMS, 3-aminopropyltrimethoxysilane; CL, cooperative luminescence; CS, cooperative sensitization; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide or 3-(ethyliminomethylene amino)-N,N-dimethyl-propan-1-amine; ESA, excited state absorption; ETU, energy transfer up-conversion; FA, folic acid; FAD, flavin adenine dinucleotide; fNC, functionalized nanocrystal; FRET, Förster resonance energy transfer or fluorescence resonance energy transfer; GFP, green fluorescent protein; GPTMS, (3-glycidyloxypropyl) trimethoxysilane; hCG, human chorionic gonadotropic; HDA, hexanedioic acid; IgG, immunoglobulin G; IBU, ibuprofen; Kd , dissociation constant; LbL, layer by layer; LRET, luminescence resonance energy transfer; MP, multiphoton; MRI, magnetic resonance imaging; NADH, nicotinamide adenine dinucleotide; NHS, N-hydroxysuccinimide or 1-hydroxy-2,5-pyrrolidinedione; NIR, near-infrared; NC, NP, nanocrystal, nanoparticle; OA, oleic acid; PAA, poly(acrylic acid); PEG, poly(ethylene glycol); PEI, poly(ethylene imine); PET, positron emission tomography; PL, poly(L-lysine); PD, penetration depth; PDT, photodynamic therapy; PMAO, poly(maleic anhydride-alt-1-octadecene); PVP, polyvinylpyrrolidone; RGD, arginine-glycine-aspartate tripeptide; SMCC, succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate; SNR, signal-to-noise ratio; TEOS, tetraethoxysilane; TEM, transmission electron microscopy; TGA, thioglycolic acid; TPLSM, two-photon laser-scanning microscopy; TPULSM, two-photon up-conversion laser scanning microscopy; TPUWFM, two-photon up-conversion wide-field microscopy; UC, up-conversion; UCI, up-conversion imaging; UCL, up-converting luminescence; UCNP, up-converting nanoparticle. ∗ Corresponding author at: Wrocław Research Center EIT+, ul. Stabłowicka 147, 54-066 Wrocław, Poland and Institute of Low Temperature and Structure Research, PAS, ul. Okólna 2, 50-422 Wrocław, Poland. Tel.: +48 71 3954 166. E-mail address: [email protected] (A. Bednarkiewicz). 1748-0132/$ — see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.nantod.2012.10.006

Lanthanide-doped up-converting nanoparticles: Merits and challenges

Introduction The rapid development of biological sciences requires continuous improvement of the analytical techniques. There is therefore a growing interest to develop fast, multiplexed, inexpensive and sensitive tests that enable analyzing a few bio-components in one step (‘‘one pot’’ or ‘‘one sample’’) [1,2]. One of the most versatile and sensitive methods relies on static (labels) or active (bioprobes) compounds called markers. Many complementary experimental fluorescent techniques exist which are able to exploit the potential of these markers and offer sensitivity down to a single-molecule [3] and superb, down to nanometers, optical resolution [4,5]. There are many emerging fields that may gain or become possible with the introduction of these innovative methods and materials. Rapid pathogens or drugs detection [6,7], prolonged imaging of biomolecules migration [8], studying cell-signaling or biological interactions in vivo, multiplexing capabilities, that is parallel detection of many different targets or multi-modal labeling and treating of cancerous cells [9,10] are fascinating new possibilities. However, in most cases mentioned above, the progress would be unlikely without the intersection of different disciplines with the rapid progress in nanotechnology and materials science [11]. Although traditional organic dyes, such as fluorescent dyes or proteins, quantum dots or fluorescent beads demonstrate many useful features, their luminescence relies on energy down-conversion, thus have to be excited either by UV, potentially harmful and carcinogenic, or blue—green visible radiation [12]. It is widely known that strong scattering and absorption in tissues and in many biomolecules is inherently limiting the penetration depth of this short-wavelength excitation light [13]. Possible damage to biomolecules or even cell death may be also initiated due to long-term irradiation with photons of relatively high energy. Moreover, under UV-Vis photoexcitation, most of endogenous bio-components, such as flavins, porphyrins, NADH, FAD+ etc., exhibit autofluorescence (AF) [14] which decrease the signal-to-noise detection ratio (SNR) [7]. Due to their nature, different organic fluorescent molecules have broad absorption and emission spectra which overlap between each other and the AF as well. This feature makes them not very suitable for multiplexed biolabeling [15] thus sophisticated and cumbersome spectral deconvolution methods need to be applied. Moreover, organic dyes exhibit short detection times due to their high photo-bleaching rate and resulting chemical degradation [15]. Their fluorescence yield and spectral properties may also be strongly affected by local chemical environment [16] which often complicates the analysis. Converning QDS, which possess size-tunable relatively narrow emission, bright photoluminescence, good photostability, broad ultraviolet (UV) excitation and relatively narrow emission, their use in biofield is restrained because they are toxic [17], chemically instable [18], hydrophobic and expensive [2,17]. A promising alternative to traditional fluorophores and QDs may be lanthanide-doped up-converting nanocrystals. This is because of favorable luminescencent properties

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of these NP, such as photostability, lack of toxicity and mostly because lanthanide ions have the ability to efficiently up-convert NIR radiation into shorter wavelengths [19]. Recently, rare-earth-doped NPs demonstrated their great potential in many fields of biological science including cells and tissue labeling for bioimaging, biodetection, therapy or multiplexed analysis [7,8,20,21]. UCNPs, being excited by near-infrared light, enable detection of signal in AF-free conditions [22] and provide deeper, yet noninvasive penetration of the radiation into biosamples [23]. Despite favorable physico-chemical properties, there are number of issues to be solved before UCNPs can be applied to biological subjects. One of them is the optimization of composition and structure as well as up-conversion enhancement to make these NPs brighter than they currently are. The number of strategies developed for that purpose has been discussed in Section ‘‘Up-conversion enhancement’’. The other prerequisite for a broader use of UCNPs is the availability of commercial instruments that currently are not ready for direct measurements of the optical signals from these materials, neither in the UPC bioassays format nor by routine imaging of the biological samples in the up-conversion mode (Section ‘‘Instrumentation for UC sensing’’). There are also some basic research studies that need further optimization. Most of rare-earth-doped upconverting nanoparticles are synthesized in non-aqueous solutions thus it is essential to make them dispersible in water first, add functional group to further attach biomolecules before use in various bioassays and bioimaging. Different approaches for biofunctionalization and bioconjugation are presented in Section ‘‘Surface functionalization and bioconjugation’’. Once the UCNPs are bio-conjugated with biotargeting molecules, they may become a convenient tool for nanomedicine (Section ‘‘Biological applications of lanthanide-doped UCNPs’’). The motivation to write this review was the need to provide readers with some more insight into physics and spectroscopy of lanthanides in comparison to other conventional and widely used fluorescent probes. The technology of Ln:UCNPs is getting matured and numerous in vitro and in vivo applications suggest huge impact these materials may have in the nearest future. We have therefore uniquely classified these applications and reviewed current, barely available, instrumentation to detect these luminescent probes. We can also see other challenges for UCNPs researches, such as up-conversion enhancement, relation of properties with the architecture of NPs as well as proper biofunctionalization and bio-conjugation, which need further extensive studies.

The features of ideal fluorescent bio-probe There are number of features one would expect from an ideal fluorescent probe, dedicated to either biosensing or bioimaging [24—26]. The probe should demonstrate high and spectrally narrow absorption and emission cross-sections, together with sufficiently large Stokes-shift in order to selectively photo-excite and detect its fluorescence. The ability to create many probes with unique spectral codes and distinguish between them in a single sample is of critical importance for multiplexing (simultaneous detecting

534 many analytes/targets in a sample) capabilities in imaging or the throughput increase of bioassays. The probe should be resistant to photobleaching or bio-chemical degradation to allow for prolonged (i.e. hours, days) fluorescence detection in vivo. Additionally, it should be non-toxic, biocompatible and biospecific to reach selected biological targets (biomolecules, biostructures or specific cells). In the case of exogenous fluorophorescent markers, this targeting capability should be obtained through a simple and flexible bio-functionalization and bio-conjugation of the probe’s surface. The ideal probe should also provide means for ultrasensitive detection [2], which is basically related to the possibility of background or noise diminishing. The simplest and most efficient solution consists in finding the probes that are photoexcitable and luminescent in near infrared (NIR) spectral region — far away from biomolecular AF. The other solution relies on pulsed excitation and time-domain fluorescence measurements, which allows to get rid of AF due to short-living fluorescence originating from cellular components in contrast to expected longer luminescence lifetimes of the probes. In comparison to traditional fluorophores, lanthanideions-doped up-converting nanoparticles meet most of these requirements (Table 1). Most importantly, they are offering a unique property of emitting visible/NIR light following photoexcitation with near-infrared laser light. The up-conversion emission was proven to be much more efficient than the two-photon processes [2] widely used in biological microscopy imaging. NIR photoexcitation allows to get rid of AF of biological matter leading to significant S/N ratio improvement. Additionally, narrow emission bands simplify spectral separation of emission peaks from stray light and different markers, which make them well suited for very sensitive and multiplexed bioassays [26]. Lanthanide ions exhibit sharp luminescence via f—f transitions. Their resistance to photobleaching and absence of blinking effects are another convenient features which are very useful in bioapplications [2,27], since prolonged exposures to excitation light or long-term studies may be carried out. The other features include large Stokes shift, very high chemical and physical stability, low chemical toxicity [28,29], long (micro/milliseconds) luminescence lifetimes. The longevity prevents interferences associated with the spontaneous short-living (nano-seconds) emission of the background or fluorescencent organic dyes. Long luminescence lifetimes are also an excellent measure of biointeractions, where UCNPs act as energy donors in FRET based biosensors [30—32]. Additionally, the luminescence lifetimes in the micro—milli second range simplifies the excitation and detection setups, which allows building portable detection devices for medicine and environment protection. One of the most limiting factors that prevent broader use of the up-converting nanomaterials in bioscience is lack of commercial instruments, which are able to fully exploit the advantages of these highly promising materials. Almost all studies demonstrating the feasibility and suitability of the UCNPs in biodetection and bioimaging have been performed with either home-modified or home-made detectors. A review of the current state-of-the art in UPC detectors can be found in Section ‘‘Future directions and perspectives’’ of this review.

A. Gnach, A. Bednarkiewicz

Lanthanide-doped up-converting nanoparticles as biomarkers Optical properties of lanthanides (as activators of transparent hosts), gained tremendous interest since 1964, when stimulated four-level laser emission from Nd3+ -doped Y3 Al5 O12 garnet was discovered. According to Kaminski, tens of laser channels in UV-Vis-NIR-IR spectral range and hundreds of laser actions have been studied in different hosts (crystals, glasses, fibers, microcrystals, ceramics), with different activators (Ln3+ ions) in different regimes (CW, pulsed, 3- or 4-level laser emission, up-conversion lasers etc.) and different excitation (lamp, laser, laser diode pumping). The luminescent properties of lanthanide-doped matrixes have been also intensively studied in the field of NIR visualization, up-conversion UV-tunable lasers, lighting, X- and gamma-ray detection which fructified in many commercial devices such as energy-saving light bulbs, TV screen phosphors or digital radiography [32—34]. Since the lanthanide physics and spectroscopy are not the major purpose of this review article, the readers are referred to numerous comprehensive papers and books focused on this issues [19,33,35,36]. Some biological applications are discussed in many up-to-date review articles [7,20,21,26,37] as well as in Section ‘‘Biological applications of lanthanide-doped UCNPs’’. It is important to mention the fundamental difference in luminescent properties between Ln-doped materials and the other dyes or QDs, which is manifested in much more efficient anti-Stokes emission, i.e. NIR to Vis-NIR energy conversion (Figure 1). In opposite to two-photon emission (TPE) or second harmonic generation (SHG), the two-photon excitation in lanthanide-doped UCNP engages real mid-electron levels with ␮s-ms lifetimes [33,47]. The up-conversion in lanthanides (either excited state absorption-ESA, energy transfer up-conversion — ETU, cooperative sensitization — CS or cooperative luminescence — CL) relies on sequential absorption of two (or more) low-energy photons. The colloidal Yb3+ /Er3+ and Yb3+ /Tm3+ co-doped NaYF4 nanoparticles demonstrated highly efficient up-conversion luminescence, which seems to be at least one order of magnitude more efficient, then the two-photon absorption of QDs. Maestro et al. [48] compared the relative emission quantum yield of UCNPs (20 nm ␣-NaYF4 :Yb3+ /Er3+ , exc = 980 nm), spherical 4 nm diameter CdSe QDs and gold nanorods. Nevertheless, such behavior may be expected due to different excitation mechanism of up-conversion and two-photon absorption, that are responsible for anti-Stokes emission in the Ln:UCNP and QDs, respectively. The high anti-Stokes emission efficiency and reduced photoexcitation quantum yield threshold allows using a non-laser excitation or two-photon wide-field microscopy [49], which is a unique and great advantage of UCNPs over the other fluorescent labels. Nevertheless, the absolute fluorescence quantum yield (QY) of up-conversion is not very high as compared to conventional fluorescence of organic dyes. The QY in the range of 0.005% to 0.3% were measured for several hexagonal NaYF4: 2% Er3+ , 20% Yb3+ nanoparticles with particle sizes ranging from 10 to 100 nm while a QY of 3% was measured

A comparison of basic chemical, photophysical and functional properties of different fluorescent probes.a

Type of fluorescent probe/Parameter

Organic dyes, fluorescent proteins

Dye-doped silica nanoparticles DDNP

Gold nanorods AuNR

Quantum dots QDs

Quantum beads QBs

Lanthanide chelates

Lanthanide-doped up-converting nanoparticles Ln:UCNP

Size [nm]

Small <5 nm to medium 10 nm

25—5000

10 × 30 nm

2—6 bare, up to ∼30 nm with functional groups Size impacts properties

1200 nm (up to 30 QDs inside a bead)

∼5

>6 nm. typically 20—30 nm

Cost Applications FRETb Multiplexed bioassays Cell imaging Tissue/animal imaging High-resolution imagingc Long term studies therapiesd

Size does not impact emission much

*/**

*/**

***

***

***

**

*/**

*** (D/A) *

too large **

** (A)

*** (D/A) ***

Too large ***

*** (D) **

** (D) **

*** *

* *

** *

Too large Too large

** *

*** ***

** (LSM)

too large

** (LSM)

Too large

* —

ThT

** —

Too large —

**

PDT

*** PDT, ThT, DD

Excitation

UV/Vis

UV/Vis

Vis

UV/Vis

UV/blue—green

UV/Vis

UV/Vis NIR (within tissue optical window)

Emission

Vis (blue—red)

Vis (blue—red)

Vis (yellow-red)

Vis

Vis-NIR

Emission spectra widthe (FWHM in nm)

Single bands

∼multiple bands

Single band

Multiple bands

Vis (green from Tb3+ , red from Eu3+ ) Multiple bands

Multiple bands

∼50—100

50—100 nm

∼30—50

∼30—50 nm

10—20 nm

∼10—20 nm

Medium

High (up to 100 unique assays within single sample with Luminex® technique)

A few spectral codes possible, selective absorption

High, up to 103 —106 spectral codes theoretically possible with 6 colors and 10 intensity levels

A few codes are possible

Medium/high

Multiplexing capabilities

Very broad

No

** (LSM/wide field)

Lanthanide-doped up-converting nanoparticles: Merits and challenges

Table 1

535

536

Table 1 (Continued) Type of fluorescent probe/Parameter

Organic dyes, fluorescent proteins

Dye-doped silica nanoparticles DDNP

Selective absorption possible

Broad emission spectra overlap, internal FRET SiNP provide multi-color coding

Gold nanorods AuNR

Quantum dots QDs

Quantum beads QBs

Lanthanide chelates

Lanthanide-doped up-converting nanoparticles Ln:UCNP

Eu3+ red∼620 nm,

Multiplexing possible without increasing the size of NP

Tb3+ green ∼540 nm,

Broad emission overlap

Dy3+ yellow ∼570 nm, Sm3+ orange 600 and ∼650 nm, Nd3+ NIR ∼880 and 1060 nm, Yb3 + NIR ∼1000 nm, Er3 + ∼1540 nm) Stokes-shift Signal/AF ratiof

* *

* *

*

*** *

*** *

*** CW:*/** TD: ***

*** UCNP: *** DCNP/CW: */** UC/DC TD:***

Luminescence lifetimes

10−10 —10−9 s

10−10 —10−9 s

N/A

100 —102 ns

100 —102 ns

10−6 —10−3 s

10−6 —10−3 s

1-photon luminescence intensity

**/***

***

*

***

***

**/***

**

***

***

**

*/**

**

**

F

F

Environment sensitive ***

***

*

*

F

F

**

***

F

F/MRI/PET[38]

A. Gnach, A. Bednarkiewicz

1-photon absorption Cross-section 2-photon absorption Cross-section Multi-modal detectiong

Type of fluorescent probe/Parameter

Organic dyes, fluorescent proteins

Dye-doped silica nanoparticles DDNP

Gold nanorods AuNR

Quantum dots QDs

Quantum beads QBs

Lanthanide chelates

Lanthanide-doped up-converting nanoparticles Ln:UCNP

Photo stability

Very low

*/**

***

**/*** (photoblinking)

**/***

**/***

***

Chemical stability Water solubility

*/** good

*** After functionalization N/A

*/**

N/A

*** After functionalization N/A

*** good

Clearance

*/** Depends on fluorophore Fast

N/A

N/A

*** After functionalization A few days

Biocompatibility

***

***

***

* - bare

***

Inert or biocompatible (e.g. hydroxyapatites)

*

*** - After shell formation Short-term cytotoxicity

*

*/**

**/*** (depending on composition)

*/**

Key refs.

[39,40]

[41,42]

[43,44]

[45]

a b c d e f g

[46]

Lanthanide-doped up-converting nanoparticles: Merits and challenges

Table 1 (Continued)

[2,19,20]

Low/medium/high - */**/*** - used throughout the table. D- used as FRET Donors, A — used as FRET acceptors. LSM — achievable with Laser Scanning Microscopy only; wide field — achievable with wide-field imaging techniques. PDT- photodynamic therapy, ThT — thermotherapy, DD — drug delivery. FWHM — full width at half maximum. CW- continuous wave, TD — time domain, UCNP — up-converting NP (excitation in NIR), DCNP — down-converting NP (excitation in UV/blue/green). F — fluorescence, MRI — magnetic Resonance Imaging, PET — positron Emission Tomography within single NP.

537

538

A. Gnach, A. Bednarkiewicz

Fig. 1 (a) Schematic representation of different energy transfer mechanisms found in lanthanide-doped materials (GSA — Ground State Absorption, ESA — Excited States Absorption, CR — cross-relaxation or concentration quenching, ETU — Energy Transfer Upconversions, and phonon-assisted ETU with E energy mismatch), (b) A comparison of efficiency of different anti-Stokes emission mechanisms in different materials (APTE — fr. Addition de Photons par Transferts d’Energie, SHG — Second Harmonic Generation, 2Ph ABS — 2 photons absorption). Solid lines represent photons, dashed lines — non-radiative processes. Source: [33].

for a bulk sample [50]. These results may be attributed to the increase of surface-to-volume ratio for smaller nanoparticles leading to increased relative amount of doping ions close to the surface of the nanocrystallite. Additionally, the increase of surface defects close to the outer shell of the NP, may affect the superficial ions as another cause for reduced energy transfer QY. These facts underline the need for developing core-shell UCNPs in order to reduce the de-excitation mechanism for RE-doped NPs as luminescent (bio)labels. The bio-applications of Ln:UCNP have gained tremendous interest relatively recently. While the physics and luminescent behavior of Ln-doped materials is relatively well known, the awareness of some differences in luminescent properties and different NPs preparation of the very same materials in nanoscale is the achievement of recent 10 years. The main drawback of current UCNPs technology comes from their hydrophobicity [51], and lack of functional groups for direct attachment of biomolecules. Despite many

advanced studies have been performed to eliminate the undesirable features, there is still a need to properly modify and tailor the surface of UCNPs. This is urging, because nanotechnology aims at designing nano-platforms, that combine many features, such as highly selective bio-targeting, drug delivery, localized therapy (e.g. PDT, thermotherapy), multi-modal (e.g. MRI, fluorescence, PET) ultrasensitive and prolonged detection [10,52—54]. Despite the huge potential of up-converting nanomaterials for applications to bioscience, improvements are still needed to optimize and enhance their properties for potential commercial applications. Perhaps the most urging improvement in luminescencent nanomaterials that is looked for is the quantum yield of the up-conversion luminescence. While the lanthanide based UCNPs exhibit highest TPE efficiency (NIR-to-VIS/NIR) from all the available other biolabels, the UPC is still much less efficient compared to the competing Stokes emission from single-photon excited

Lanthanide-doped up-converting nanoparticles: Merits and challenges nanomaterials such as organic dyes or QDs. The methods to enhance the up-conversion quantum yield will be discussed in the following section.

Up-conversion enhancement The possibility to enhance the anti-Stokes luminescence from lanthanide-doped nanoparticles has always been an interesting research topic, which was fuelled by the potential of these materials as lasers, phosphors and recently as biolabels as well. Due to excellent photo-stability, efficient NIR-to-Vis conversion and long luminescence lifetimes suitable for time delayed detection, these nanoluminophores appear to be competitive biolabels when compared to organic dyes or quantum dots. Unfortunately, the UCNPs suffer from low-to-medium brightness when compared to conventional biolabels. Thus the up-conversion emission must be further optimized and enhanced, to meet the extremely high sensitivity requirements indispensable for bioassays and bioimaging [55]. There are currently a few ways to improve the upconversion quantum yield, but practically passive and active methods may be distinguished. The passive methods relay on optimizing the dopant concentration, modifying local chemical and structural environment, controlling the distribution of active ions in host material, varying the composition, structure and/or morphology of the up-converting NPs [56]. These parameters should diminish the quenching effects, enhance the absorption of NIR radiation or improve the sensitizer-to-activator energy transfer. The active UPC enhancement relies on the interaction between the upconverting lanthanides pair (e.g. Yb3+ /Er3+ ) with surface plasmons of noble metals.

Increasing the concentration of optically active ions The potentially easiest way to improve the up-conversion quantum yield is to increase the concentration of optically active ions within a single nanoparticle. Although the upconversion processes in singly doped NPS were found to be very weak, it can be efficiently increased by co-doping the activator ions with the so called sensitizer ions [33]. Enhanced energy up-conversion in doubly doped material was first explained by F. Auzel in 1966 [33]. By introduction of the sensitizing ions, it is possible to improve the absorption of excitation radiation and in consequence enhance the ETU as well. The most frequently used sensitizing lanthanide ion is ytterbium. Due to its simple energy scheme, large absorption cross-section at 940—990 nm NIR spectral region, large energy gap (∼10,000 cm−1 ) and long luminescence lifetimes (∼1 ms) of the Yb3+ excited energy states, ytterbium ions play its sensitizing role very well. Yb3+ ions effectively absorb NIR radiation and are further capable of transferring its energy to the activator ions (Tm3+ , Er3+ etc.) in the course of Energy Transfer Up-conversion process (fr. addition de photon par transferts d’energie [57]), which usually requires single activator ions surrounded by at least two or three Yb3+ ions. Despite narrow, atomic-line energy levels are typically found in lanthanides, a number of parasitic non-radiative

539

processes exist, such as concentration quenching or quenching through the impurities, defects or surface ligands [58]. The increased concentration quenching is typical for lanthanides with rich energy level structure, such as Tm3+ , Er3+ , Nd3+ , Pr3+ etc. [36]. This quenching is resulting from a concentration dependent cross-relaxation (CR) process, which involves, in the simplest case, two ions, one in an excited and the other one in the ground states (Figure 1a). For rising activator ions concentration, the effective distance between the ions become smaller, and when respective energy levels are quasi-resonant, the excited ions become non-radiatively depopulated. The concentration quenching manifests itself in shortening the luminescence lifetimes as well as in decreasing the effective luminescence quantum yield enhancement, even though the amount of luminescent centers increases. For this reason, the activator concentration has to be carefully optimized, unless it is done for purpose in order to photo-induce excessive heating [59] like for hyperthermia therapy. Due to simple energy level scheme of Yb3+ ions, these ions are barely affected by CR unlike the other lanthanides are. Despite evident advantages, only a few examples may be found in literature that demonstrates the enhancement of UC process in colloidal nanoparticles through the dopant concentration increase. The group of P. Prasad demonstrated the NIR PL emission from ultrasmall NaYbF4 :2% Tm3+ nanocrystals, to be 3.6 times more intense than the one from conventional 25—30 nm sized NaYF4 :20%Yb3+ /2%Tm3+ nanocrystals, previously synthesized and used for in vitro and in vivo bioimaging. These Yb3+ /Tm3+ -doped UCNPs are of particular interest due to highly efficient NIR-to-NIR conversion, i.e. 980 nm excitation to 800 nm UC emission, since both excitation and emission lines are falling into the optical window of the skin and allow for low scattering and AF free biodetection [22]. Beside the dopant concentration increase, the UC enhancement is usually achieved by the host matrix optimization or surface passivation. Either crystal structure (e.g. cubic vs. hexagonal in NaYF4 ) [60,61], crystal composition (e.g. fluorides vs. oxides) [56] and passive admixtures (e.g. Li co-doping) [62,63] have been applied to increase the UC quantum yield. On the other hand, core/shell structures have been extensively studied [64], since they are known to suppress excited state quenching to the surface defects, impurities, solvents and ligands.

Selection of host material The up-conversion efficiency may be enhanced by a proper selection of host matrix for lanthanide dopants. It is of vital importance for UC efficiency that the host matrix owns lowest energy phonons with highest excitation spectrum. Low-energy phonons assure low non-radiative and multiphonon (MP) losses and increase the luminescence lifetimes [56]. Different host have been compared for their suitability for UPC. Chlorides, bromides and iodides, with phonons energies of ∼144, 172 and 260 cm−1 and low MP relaxation rates are better suited for UPC, then higher phonon matrices such as fluorides or oxides. While fluorides offer ∼355 cm−1 phonons and reasonably low MP relaxation rates, oxides are considered to be least

540 efficient up-converters, with their over ∼600 cm−1 phonons and MP rates comparable to radiative emission rates [65]. On the other hand, the up-conversion may sometimes require phonon assistance (Figure 1a). Concluding, this is a balance between phonon properties and excitation spectra intensity, which make the host appropriate for energy up-conversion in lanthanide-doped materials. Recently different up-converting nanoparticle hosts have been comprehensively reviewed by Haase and Schafer [66]. The up-to-date most studied up-converting material for bio-applications is the Yb3+ and Er3+ co-doped NaYF4 nanocrystalline colloids. The impact of the host matrix type, crystallographic structure, doping [67,68], [Yb3+ ] and [Er3+ ] absolute and relative concentrations ratio [69], nanoparticle size [70,71] and morphology [72] onto the luminescent properties of Yb3+ /Er3+ system has been studied intensively in order to understand the physical background behind the ETU and to optimize its color or quantum efficiency. Since non-radiative de-excitation mechanisms in NaYF4 are suppressed in comparison to other hosts well enough, additionally the excitation spectra are sufficiently broad and strong, as well as MP-assisted energy up-conversion is reasonably high, these hosts have been shown to be the most efficient nanocrystalline host material for UC [73]. Moreover, the NaYF4 materials found an extraordinary interest since low-temperature synthesis protocols have been developed [20,37,74,75], which allow easy tuning of material size and morphology [67,76], facile dispersion and stable colloidal solutions formation [74]. Furthermore, ligand exchange or other functionalization protocols have been mastered for these materials [19] (Section ‘‘Surface functionalization and bioconjugation’’). Due to crystal symmetry, the green upconversion quantum yield of Er3+ ions in Yb3+ /Er3+ co-doped low-temperature cubic ␣-NaYF4 phase is around 10-fold less intense than in high temperature phase, i.e. ␤-NaYF4 NCs. On the other hand, it is easier to obtain small ∼5—7 nm cubic NCs. The hexagonal phase nano-fluorides are more efficient up-converters, but are crystallizing in larger structures with multiple possible morphologies. The higher up-conversion efficiency in ␤ form of NaYF4 material originates from an existence of different environments for Er3+ ions [77]. This multisite formation may be however introduced also by purpose, through the induced host crystal distortions. This can be achieved by intentional modification of the host composition in order to affect the distribution of luminescent lanthanides. As discussed in the next chapter, the possibility to tune and greatly enhance the luminescent properties of lanthanide-doped ABF4 (A+ cation e.g. Na,Li,K, B3+ cation, e.g. Y,Yb,Lu,Gd) fluorides has refreshed the interest of chemists and physicist in these materials. Among others, recent reports on augmented properties of up-converting NaLuF4 derivative of NaYF4 indicated the need for further detailed studies and optimization of these luminescent nanomaterials [78—80]. The sub-10 nm ␤-NaLuF4 nanocrystals doped with 24 mol % Gd3+ , 20 mol % Yb3+ , 1 mol % Tm3+ demonstrated quantum yield of 0.47 ± 0.06% at 800 nm under CW 980 nm excitation, which is only 6 times lower than typically obtained for bulk NaYF4 :Yb/Er crystals Lanthanide doped up-converting nanoparticles as bio-markers.

A. Gnach, A. Bednarkiewicz

Intentional impurities in host matrix Co-doping the up-converting materials with passive or active impurities may be an efficient way to obtain an enhancement of the up-conversion. In passive case, in opposite to active impurities, the doping ions do not participate in energy transfer within the matrix. They however induce distortions to the local symmetry around activators [63,77] or dissociate the lanthanides clusters in the nanocrystals [81], which in consequence leads to the UPC enhancement. The active impurities from the other hand modify the energy transfer rates within the system, and careful optimization of impurity concentration, may enhance the up-conversion efficiency. Li et al. demonstrated up to 10-fold enhancement of UPC in singly Er3+ -doped YAlO3 phosphor by exchanging around 40% of Y3+ ions to larger Gd3+ (0.1159 nm vs. 0.1193 nm), which resulted in expanding the host lattice and distorting the local symmetry [82]. Much higher UPC enhancement indexes were demonstrated by Cheng et al. [63], who co-doped the ␤-NaGdF4 :Yb3+ /Er3+ nanoparticles with different amounts of Li+ ions. In contrast to lithium-free ␤-NaGdF4 :Yb3+ /Er3+ , the green and red UC emission yield intensities of the NPs co-doped with 7Ymol% Li+ ions were enhanced by about 47 and 23 times, respectively. More comprehensive studies on the impact, the alkali ions have on the structure and the spectral properties of up-converting fluoride nanocrystals, have been studied by Dou et al. [83]. Green to blue ratio in Yb/Er co-doped NaYF4 varied in the ranges ∼2—6.5 and ∼0.7—1.7 when, respectively, Li+ and K+ content rose from 20 to 80 mol%, which affected also phase, size, shape and stability of the obtained nanomaterials. While passive impurities affect activator distribution within the UCNP, the active impurities play a critical role in energy transfer. Usually, such an approach leads to important modification of energy distribution as well as to the interaction between respective excited energy levels of the co-doping lanthanides. The most spectacular example of the active Ln3+ impurities improving the UPC quantum yield is co-doping of e.g. Tm3+ and Er3+ activators with the Yb3+ sensitizers, as discussed throughout the manuscript. In consequence of distance variation between respective lanthanides, being modulated by crystal phase, relative concentrations of dopants or through e.g. gadolinium sublattice-mediated energy migration, the relative intensities (so called branching ratios) of emission yield bands change [84] and modify the overall emission color [54].

The concept of core-shell hybrids for UPC enhancement The luminescent properties of down- and up-converting nanoparticles are known to be highly affected by the size of the nanocrystallites. This phenomenon is however somewhat different from quantum confinement effect found in QDs. In lanthanide-doped NPs, the surface to volume ratio increases as 1/D, where D is NPs diameter, thus for nanoparticles, significant amount of optically active ions are found close to or on the NPs surface. Despite the f electrons are shielded by

Lanthanide-doped up-converting nanoparticles: Merits and challenges 5s and 5p orbitals and the f—f transitions in lanthanides are relatively weakly dependent on local chemical/structural environment, it is widely known, the superficial Ln3+ ions may be effectively quenched by surface defects, surface ligands as well as water molecules. For this reason, it is a common practice to passivate the surface of the NPs by shell formation over the solid Ln-doped NPs core. Wide range of shell composition may be found in literature. These are either SiO2 [85], PEG [86] or the un-doped host crystal material [87]. Concerning the NaYF4 UCNPs, most frequently, the un-doped NaYF4 fluorides, stemming from the same structure and composition as that of the (co)doped core, are selected as the shell material. The mesoporous shell allows additionally impregnating it with different molecules, such as drugs or secondary fluorophores to form drug delivery platforms or make biosensors [54,88,89]. The shell formation allows also creating multi-color biolabels or fine tune the color of the obtained emission for multi-color imaging [64,90,91] (Section ‘‘Small animal deep-tissue fluorescence, multi-color and multi-modal imaging’’). During the growth of the shell on the core NP, surface defects of the nanocrystals can be gradually passivated, which results in the enhancement in the overall UC emission quantum yield, decrease of the associated nanoradiative decays as well as renders the NC to be more resistant to quenching by water or ligand molecules [92]. Additionally, quantum yield, crystal phase, lattice strain, down- and upconversion emission properties, energy transfers (e.g. green to red ratio) and UPC saturation threshold within lanthanidedoped luminescent core-shell hybrid nanomaterials may be affected in this way [90,93]. The UPC enhancement index varied from 2 to 5 [94,95] in TiO2 coated Y2 O3 :Yb3+ /Tm3+ or LaPO4 :Er3+ /Yb3+ doped nanoparticles/nanorods core/shell nanostructures, up to 30 in NaYF4 /NaYF4 :Yb3+ /Er3+ like nanostructures [64,90] as compared to the pristine NaYF4 :Yb3+ /Er3+ NPs. Recently, Wang et al. [96] demonstrated much higher ∼300-fold enhancement of up-conversion emission yield in the 10—13 nm large ␣-NaYF4 :Yb3+ ,Er3+ /CaF2 core/shell nanoparticles in comparison to parent ␣-NaYF4 :Yb3+ ,Er3+ NPs. The enhancement was higher than typically found in NaYF4 /NaYF4 core/shell approaches, which was explained by the heterogeneous nature of CaF2 shell. This shell was claimed to prevent RE leakage to local environment as well. The heteroshell of CaF2 was chosen as well by Prasad et al. [97] to coat ␣-NaYbF4 :Tm3+ and reach a 35-fold enhancement of NIR—NIR up-conversion intensity. Linear relation of UPC overall emission quantum yield and the increase of luminescence lifetimes in response to rising shell thickness was convincingly demonstrated by Zhang et al. [92] (Figure 2a—c). The ␣-NaGdF4 core was covered by ␤-NaGdF4 shell through epitaxial layer-by-layer deposition, and while increasing number of shell monolayers, the surface defects were gradually passivated, resulting in the overall UC enhancement (Figure 2d). This resulted from a substantially reduced susceptibility of the up-conversion in UCNPs to the high energy vibrations of water molecules (Figure 2e) The shell thickness not only improves the up-conversion rate but also impacts the distance between the UCNP and the surrounding, which has critical meaning for FRET based sensors (Section ‘‘Bioassays and biosensors’’). From their

541

studies on photophysics and singlet oxygen generation, Wang et al. [98] concluded that the core/shell structured nanoparticles are better than bare cores for FRET applications. More importantly, they deduced a critical shell thickness exists for the best FRET performance, which arises from a trade-off between the opposing optimal conditions for up-conversion and FRET. Interestingly, the core/shell strategies not only induce the UPC quantum yield and luminescence lifetimes enhancement but also may change the energy distribution in the system. Vetrone et al. [64] synthesized NaGdF4 :2%Er3+ ,20%Yb3+ active-core/NaGdF4 :20%Yb activeshell nanoparticles. The up-conversion emission yield of the active-core/active-shell was more intense by a factor of approximately 3 in the green and 10 in the red region compared to active-core/inert-shell, and by a factor of approximately 13 and 20 for the green and red emissions, respectively, in the comparison to core-only nanoparticles. The increased Yb3+ concentration in relation to Er3+ concentration is known to increase the relative red/green emission yield ratio, which is due to the enhanced population of the red emitting (4 F9/2 ) state [99]. The possible concentration quenching was diminished due to spatial separation of ions, by placing them in either core or the shell. These observations were confirmed and expanded by Wang et al. [84], who fine tuned the up-conversion color by careful selection of activators in the core and shell hosts and who modulated the up-conversion by energy migration-mediated up-conversion with Gd3+ ions.

Active up-conversion enhancement It is widely known that plasmons can promote fluorescence from organic dyes and inorganic quantum dots. This effect, called metal enhanced fluorescence (MEF), originates from the electric field enhancement (‘‘concentrating incident light’’) that arises from collective oscillations of electrons at the interface between metal nanoislands and the surrounding — surface plasmon resonance — in the proximity of fluorophore [100]. The MEF is resulting in increased quantum yields and decreased luminescence lifetimes, increased photostability, increased Förster distance between donor and acceptor, as well as may sometimes occur in directional emission yield [101]. While much effort has been given to study MEF in organic dyes, only recently the impact of metallic particles, colloids or surfaces on the upconversion were studied (metal-enhanced up-conversion — MEUC). In many different studies 2- up to 12-fold UPC enhancements were found for up-converting nanoparticles (e.g. NaYF4 :Yb3+ /Er3+ and Yb3+ /Tm3+ NCs) interacting with metals islands, layers, shells or colloidal NPs [102—105]. The general conclusion is that the up-conversion luminescence is more sensitive to and benefits more from the MEF enhancement than the down-conversion photoluminescence. This was rationalized by the numerous MP absorption processes involved in the population of the higher excited states with low energy NIR photons. However, the use of noble metals to enhance the up-conversion quantum yield may be controversial in the in vitro/in vivo bio-tests, since noble metal may, besides enhancing the up-conversion, simultaneously

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A. Gnach, A. Bednarkiewicz

Fig. 2 Up-conversion enhancement with passivating shell strategy. UPC spectra, photographs, luminescence lifetimes and overall UPC quantum yield are presented on panels a, b, c and d, respectively, versus increasing number of shell monolayers. Panel e presents quantitatively the luminescence intensity loss for for NaYF4 :20%Yb3+ ,2%Er3+ core nanocrystals and the corresponding core/shell nanocrystals with 1—6 monolayers of NaGdF4 shell in polar solvents with different amounts of water. Source: Reprinted with permission from Ref. [92].

contribute to the AF and Raman enhancement. In such a case, even though the total fluorescence intensity could be raised, the signal-to-noise ratio may have ultimately and undesirably become lower.

Surface functionalization and bioconjugation Due to hydrophobic ligand (e.g. oleic acid) coordinating NPs after synthesis, their surface is hydrophobic and further functionalization is necessary to achieve biocompatibility and transfer NPs to aqueous solutions. The biocompatibility of NPs means that they are non-toxic, have no effect on the immune system and can perform its desired function without eliciting any local or systemic side effects in cells, tissues or the whole organisms, both in short- and long-term usage. General strategies to make hydrophilic nanoparticles biologically functional require inducing or exploiting an existing NPs surface charge. This is achieved either by affixing surface-bound functional groups or the design of appropriate biocompatible coatings. Such approaches shall guarantee the compatibility with the biological objects, but not yet the specific targeting capabilities. Aiming this feature, bioconjugation has to be applied, which however should not decrease the signal of the luminescent

nanoparticles, should not increase the size of NP [106] too much or reduce the activity of the conjugated biorecognition molecules. It is therefore crucial to develop and optimize strategies towards biofunctionalization and bioconjugation of rare-earth-doped UCNPs that enable to apply them in biosensing and biomedical imaging. The biggest challenges lay in precise controlling of the NPs circulation or clearance time, agglomeration of NPs in cells/organs and predicable/controllable drug release and dosimetry. In general, one may subdivide the strategies of surface functionalization of UCNPs (Figure 3, internal circle) into the following groups: ligand exchange, ligand attraction, ligand oxidation, layer-by-layer assembly, encapsulation in silica shell and host—guest self-assembly [19,20,106]. Ligand oxidation involves oxidation of C C bond by Lemieux-von Rudloff reagent and allows obtaining additional carboxyl group at the surface. Ligand exchange involves replacement of original ligand by a new one, which can provide hydrophilic surface and functional groups for further bioconjugation. Ligand attraction involves adsorption of an additional amphiphilic polymer. Hydrophilic group enables further bioconjugation and provides watersolubility. Silanization is a process of hydrolysis and condensation of siloxane monomers (TEOS, APTES) and can

Lanthanide-doped up-converting nanoparticles: Merits and challenges

543

Fig. 3 General strategies of UCNPs surface modification. The first layer relate to surface biofunctionalization: Ligand oxidation, Ligand exchange, Ligand attraction, Silanization, Layer-by-Layer and Host—guest self-assembly. The next step in UCNPs surface modification is bioconjugation with various biomolecules through direct physisorption, assisted physisorption using prebound molecules, chemical linkage of biomolecules to crosslinkers, direct chemical coupling and targeted binding of biotinylated biomolecules to streptavidin coated NPs via biotin—streptavidin coupling.

provide functional amino groups. Layer-by-Layer assembly is electrostatic adsorption of oppositely charged ions and is limited only to hydrophilic UCNPs. Host—guest selfassembly uses interaction between one ‘‘guest’’ molecule (capping ligand) and cyclic molecule (e.g. cyclodextrin) as ‘‘host’’ reagent to transfer hydrophibic NPs into a water-soluble form. Such prepared NPs may be further loaded with hydrophobic materials [107,108]. Below, all the mentioned surface modification and bioconjugation techniques will be reviewed in brief. One may find detailed information in many reviews on nanoparticles biofunctionalization [19,20,26,37,109]. The next step in UCNPs surface modification is bioconjugation with various biomolecules (Figure 3, external circle) through direct physisorption of biomolecules via non-covalent forces, assisted physisorption using pre-bound molecules, chemical linkage of biomolecules to crosslinkers, direct chemical coupling and targeted binding of biotinylated biomolecules to streptavidin coated NPs via biotin—streptavidin coupling.

Surface functionalization Surface modifications of the NPs are crucial for making them suitable for further use in bioassays and bio-targeted detection. It often provides functional groups (carboxyl, amine, thiol etc.) enabling dispersion and stability of NPs in aqueous solutions, which is indispensable to make them hydrophilic and conjugatable with various biomolecules. Ligand-exchange In ligand exchange approach, the original hydrophobic ligands are replaced by bifunctional molecules such as PEG [110], PAA [28], HDA [111,112], TGA [113], citrate [114] (Table 2). The method is simple and the process does not change the shape or a size of NPs, if only the exchange agents are small [115]. The modified UCNPs usually do not show any significant changes in morphology or luminescence emission yield, they do not aggregate and are stable in aqueous solution for long time [28,110,113,116]

544 Table 2

A. Gnach, A. Bednarkiewicz Molecules used for biofunctionalization of UCNPs.

Molecule

Functional groups

PEG

OH

Poly(ethylene glycol)

PEI

NH

Poly(ethyleneimine)

PAA

Structure

UCNPs

Refs.

NaYF4

[110,120—122]

Y2 O3 Gd2 O3

[123] [124]

NaYF4

[12,125—127]

NaGdF4

COOH

Poly(acrylic acid)

NaYF4

[28,114,122]

NaGdF4 Y2 O3 YF3 GdF3

[116] [123] [128]

PVP Polyvinylpyrrolidone

CO

NaYF4

[117,118,126]

CITRATE

COOH

NaYF4

[114]

Gd2 O3 GdF3 , YF3

[129]

LaF3

[130]

CaF2

[131]

SH, COOH

NaYF4

[113]

COOH

NaYF4

[111,112]

COOH, NH2

NaYF4

[132]

LaPO4

[133]

NH2

Gd2 O3

[134]

NH2

NaYF4

[52,85]

CeF3 LaF3 Gd2 O3 YVO4

[135] [136] [137] [138]

CHITOSAN

TGA

OH, NH2

Thioglycolic acid HDA Hexanedioic acid AHA 6-Aminohexanoic acid PL Poly(L-lysine) APTES 3-AminopropyltriethoxySilane

Lanthanide-doped up-converting nanoparticles: Merits and challenges which makes them ideal candidates for various biological applications. Poly(ethylene glycol) (PEG) and poly(ethylene glycol)-phosphate (PEG-phosphate) are low toxic and low antigenic polymeric ligands which have been used for biofunctionalization of various nanoparticles frequently [51]. Boyer et al. [110] replaced oleate ligand by (PEGphosphate) making NaYF4 UCNPs dispersible in aqueous solvents, chemically stable and therefore also less toxic for cells. Another biocompatible, non-toxic and watersoluble polymer [117] used for UCNPs functionalization is polyvinylpyrrolidone (PVP) [118], which has longer blood circulation time than the widely known PEG [119]. Also hydrophilic poly(acrylic acid) PAA [116] capped NCs showed prolonged colloidal stability in water. Ligand attraction Ligand attraction or capping with secondary organic ligand is the next, often used method of UCNPs surface modification [19,20]. Ligand attraction involves adsorption of an additional amphiphilic polymer onto the nanocrystal surface through the hydrophobic attraction between the original ligand and hydrocarbon chain of the polymer. The amphiphilic ligand intercalates with the hydrophobic segment of original protecting ligand. The hydrophilic part of the polymer sticks out into the water and permits aqueous dispersion and further bioconjugation [19,118]. Moreover, polymer provides stability in various media and avoids unspecific binding of biomolecules [122,126,132,139]. Some of most commonly used ligands in this approach are poly(maleic anhydride-alt-1-octadecene) (PMAO) [139], 6aminohexanoic acid (AHA) [132], PAA [87] or poly(L-lysine) (PL) [134]. A different strategy for introduction of water-solubility onto UCNPs is one-pot synthesis with polymer precursors such as PEI or chitosan which provide additional functional groups [12] for attachment of biomolecules. Unfortunately, these methods are still lacking precision, the size and morphology of the as-prepared UCNPs is controlled. Facile one-pot solvothermal synthesis was used to obtain YF3 :Yb3+ ,Er3+ coated with PAA [128]. The luminescence properties changed as compared to the UCNPs without PAA coating, what important — the NIR luminescence at 831 nm (in ‘‘optical window’’) was stronger then for unmodified ones. A natural biopolymer chitosan has been employed to provide biocompatibility of LaF3 :Eu3+ NPs in a simple, fast one-pot co-precipitation method [130]. Ligand oxidation Another approach to produce water-soluble UCNPs relies on ligand oxidation. This method involves oxidation of the double C C bond of the ligand (e.g. oleic acid) by Lemieux—von Rudloff reagent or potassium permanganate. For this reason, the ligand oxidation is limited to a specific class of ligands [19]. The oxidation has no obvious negative effects on the morphologies, compositions or luminescent capabilities of UCNPs [140]. However, the disadvantage of this strategy is long reaction time and low yield [141]. It is also possible to convert oleic-acid-capped NPs into carboxyl-modified ones through ozonolysis. This process is a simple, clean and flexible method for the functionalization of hydrophobic UCNPs and had no major side-effects on

545

the luminescent properties and morphology of the UCNPs [142]. Layer-by-layer assembly There is one another promising method of NC functionalization. Layer by layer assembly consists of sequential adsorption of oppositely charged polyelectrolytes on NPs surface. LbL assembly seems to be very atractive, being both versatile and simple approach enabling to control the coating thickness of the polyions by choosing the number of layers [143] to obtain biocompatibile water-soluble citrate-coated NPs. Similarly, sequential deposition of anionic poly(acrylic acid) (PAA) and cationic poly(allylaminehydrochloride) (PAH) was demonstrated [144] and proved to have good mechanical stability and efficent NIR-to-visible up-conversion luminescence. But there are some drawbacks of this approach, i.e. only hydrophilic nanocrystals can be functionalized [19] and additional time-consuming washing step is required [26]. Silanization One of the most frequently used method of UCNPs surface modification is surface silanization. The growth of an amorphous silica shell on the UCNPs involves hydrolysis and condensation of siloxane monomers (e.g. tetraethoxysilane TEOS, 3-aminopropyltriethoxysilane APTES). Silanes provide functional amino (e.g. APTES, APTMS) or epoxy (e.g. 3glycidoxipropyl trimetoxysilane GPTMS) groups responsible for interfacial properties such as wetting or adhesion [19]. Silica itself is regarded as biocompatible, its surface has been extensively studied and their porosity can be easily controlled [145]. Unfortunately, some of the reagents used in the course of silica coating are toxic and have to be carefully removed before using the fNPs with silica layer in bioassays. Moreover, additional silica layer on the surface of NPs changes the shape and increases the hydrodynamic size of nanoparticles which can be inappropriate for some of biological appplications like FRET (fluorescence resonace energy transfer) biosensors [111,135]. The precise control over the thickness and shape of the encapsulating SiO2 layer is still an unsolved issue [87]. Another drawback is that the luminescence quantum yield of UCNPs modified with APTES tends to decrease because of free NH2 groups responsible for quenching the emission yield [135]. The most commonly used strategy of silica coating relies on Stöber method [146] and its modifications [135] or on the reverse microemulsion route [52,85]. In the Stöber process, silica is formed in the presence of ethanol and ammonia. Although it is quite simple and effective approach, it is timeconsuming and difficult to control due to the complicated processes involved [147]. Reversed microemulsion system, on the other hand, is based on a homogeneous mixture of water, oil, surfactant (e.g. Igepal CO-520) and TEOS [52,85]. This method enables to control the silica shell thickness with nanometer precision and allows to modify NPs at larger concentration then in Stöber process [147]. Host—guest self-assembly Using host—guest interaction is simple, rapid and efficient method of surface modification of UCNPs [107,108]. Over

546 95% of NPs can be tranfered to water using fast procedure and environmentally friendly reagents [107]. Self-assembly of host molecule (e.g. cyclodextrin) and guest molecule (e.g. oleic acid or adamantaneacetic acid) has several advantages over other methods because it not only enables to obtain hydrophilic UCNPs but provides a hydrophobic layer for loading a hydrophobic molecules (dyes or drug) as well.

Bioconjugation As soon as the UCNPs gain some functional groups (thiol, carboxyl, amine etc.) at their surface, it is possible to decorate them with various biomolecules. These are the antigens or DNA fragments that are responsible for desired functionality of luminescent nanomarkers. These conjugated biomolecules can therefore provide specific bio-recognition of other target molecules or cells. When choosing the proper NPs conjugation strategy, many factors must be taken into account, such as the UCNP matrix, its physico-chemical characteristics, the nature of the NPs’ surface, the properties of functionalizing ligands and groups as well as the type of biomolecule and the desired biotargeting properties. General strategies of bioconjugation There are a few known strategies of NPs bioconjugation with biomolecules (Figure 3). The first method relies on direct physisorption of biomolecules via non-covalent forces [106]. This is a very simple approach which does not allow controlling the number of binding molecules. Moreover, the biological activity of biomolecule being bound in such a way may be changed. This strategy will not suppress non-specific binding because it may target more than one selected group of proteins. This simple, one-step approach was adapted by Nichkova et al. for coating Eu-doped Gd2 O3 NPs with antibodies [148] or by Capobianco et al. to coat UCNPs with heparin [149]. The greatest advantage of this method comes from the fact that the proteins do not lose their activity and the NPs luminescence quantum yield does not change after bioconjugation. In the next method called assisted physisorption, prebound molecules are used [106]. It is non-covalent coupling of two molecules, one of them acting as a mediator between desired biomolecule and the surface of UCNPs. This strategy may help to bind the molecule in a proper orientation, which is of critical importance for bio-specificity and sensitivity. Correct orientation of biomolecules (e.g. antibodies) bound to the UCNPs is crucial for their appropriate activity and for the sensitivity and accuracy of the bioassay (Figure 4). Another popular approach involves chemical linkage of biomolecules to crosslinkers which facilitate highly biospecific coupling of the UCNPs with the biomolecules of interest [106]. The crosslinker may be bound to the surface of UCNPs either by physisorption or chemisorption. Binding biomolecules to the crosslinker is based on the chemical coupling between reactive groups of biomolecules and crosslinker molecules. A lot of potential binding sites on the UCNPs surface limit the possibility to precisely control the whole process of bioconjugation. The chemical linkage through crosslinkers was used so far to bind the UCNPs

A. Gnach, A. Bednarkiewicz with toxic protein [138], streptavidin [137] or antibodies [150]. The drawback of this approach comes from significant increase of biofunctionalized NPs size, which is not desired in some applications (e.g. FRET based biosensors). The next strategy of bioconjugation is a direct chemical coupling of biomolecules to the UCNPs. It is often used for binding oligonucleotides or folic acid (FA) to NPs directly through, mercapto-[106] or amino-[52] groups, respectively, that are available on the NPs surface. Unfortunately, the amount of bound biomolecules cannot be easily controlled, therefore a separation step, like gel electrophoresis, must be included. The use of physisorption method of bioconjugation (both direct and assisted) may lead to some defects and errors. The same problems may arise using chemical coupling as well. Some of the most common mistakes arising during antibodies binding are shown in Figure 4. Another very useful bioconjugation strategy, which is similar to crosslinker technique, involves the targeted binding of biotinylated biomolecules to streptavidin (or avidin) coated NPs via biotin—streptavidin coupling [106]. The biotin-binding proteins, i.e. avidin or streptavidin, act as linker molecules. The affinity of biotin to streptavidin (Kd = 10−15 mol L−1 ) [152] is one of the strongest and most stable non-covalent interactions known in biology, therefore using this specific biotin-protein bond may be helpful in UCNPs’ surface modifications for specific bioapplications. This technique was successfully adapted by several research groups to study applicability of the magnetic UCNPs in cell separation bioassays [137,153], to study quenching of luminescence in functionalized UCNPs [135], to demonstrate use of UCNPs in immunofluorescence assays [133], to develop a novel homogeneous assay technology based on up-conversion luminescence resonance energy-transfer [127,153].

Functional groups used for bioconjugation The strategies of bioconjugation can be also discussed from the perspective of reactive groups involved in the covalent coupling [106]. One of the most common procedures is reaction between primary amine and carboxylic acid groups. It is advantageous over the other approaches because of simplicity of ligands preparation protocol. The use of activators like EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide) is additionally required. Carboxylate particle are activated by EDC, while NHS improves the efficiency of EDC coupling reaction. The next approach is reaction between thiol and maleimide groups in which stable thioether bond is formed at physiological pH. It is useful for bioconjugation of proteins with SH groups (containing cysteine moieties). Coupling of two thiols to form disulfide linkage is as well attractive if one uses crosslinker with the thiol group at its end. The main drawback of this approach comes from the instability of disulfide bond in biological fluids which results in nonspecific binding of proteins which possess cysteine residues in their structure. Another reaction which may be engaged for bioconjugation, between aldehyde and hydrazide groups forms hydrazide bond. The aldehyde group can derive from

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Fig. 4 Typical problems occurring during immobilization of antibodies and binding with UCNPs. (a) Antibodies evenly bound to the surface, in proper orientation (Fc on plate), one antibody may bind one UCNPs, (b) Antibody molecules packed evenly, Fc and Fab on plate, one antibody may bind one UCNPs, (c) Antibodies bound in all orientations, one antibody may bind one UCNPs, (d) Antibodies bound via Fab, the conjugation with UCNPs is impossible, (e) Antibodies bound in greater distance in proper orientation, the interaction between Ab molecules are possible, (f) Antibodies are spaced too widely, (g) as in (e) and orientation is via Fc or Fab, (h) as in (c) and additionally there is less molecules in active orientation, (i) The excess of antibodies leads to multilayered binding, the washing allows to elute the excess. Source: Based on Ref. [151].

crosslinker (e.g. glutaraldehyde) or from oxidation of carbohydrate group. Alternative method is reaction between two primary amines. Unspecific binding may occur for biomolecules that are abundant in functional groups. The novel groups like azido or alkyl have been looked for that reason [26]. A few examples of the most common reactive groups used in bioconjugation are presented in Figure 5. Bioconjugation of nanoparticles The introduction of functional groups to the surface of the particle is essential for covalent attachment of biomolecules that usually enables biorecognition and targeting, examples being proteins, antibodies or polynucleotides [26]. Covalent binding of biomolecules to the surface of UCNPs based on EDC and sulfo-NHS coupling chemistry [154] is often used for that purpose. Generally, this method is used to conjugate UCNPs with IgG [155], avidin, streptavidin [137] or BSA [156] and biotin [127,157]. The use of EDC-NHS chemistry has been demonstrated to facilitate the construction

of various biosensors. Tu et al. [157] prepared UCNPs able to detect avidin using FRET mechanism. The use of activated (by oxidation with sodium m-periodate and ethylene glycol) protein is another approach to binding avidin to UCNPs [158]. UCNPs functionalized by silane with amino groups (APTES, APTMS) can be alternatively conjugated with various proteins via crosslinker as well. For example bis(sulfosuccinimidyl) suberate (BS3) crosslinker was used to conjugate YVO4 :Eu functionalized with APTES in order to study interaction of ␧-toxin of Clostridium perfringens with the cellular membrane [138]. Similar crosslinker-based strategies were used to bind biotinylated LaF3 :Eu to avidin, which was immobilized on cross-linked agarose beads [159]. Using the crosslinker helped to eliminate the risk on nonspecific binding to avidin or the other biomolecules. The use of immunoglobulins, like immunoglobulin G (IgG), has been reported as a way to improve bio-specificity and bio-recognition of Gd2 O3 :Eu3+ UCNPs [148]. This method allows to control the number of binding sites on the surface of the nanoparticles by varying the specific antibody

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Fig. 5 Strategies of bioconjugation of UCNPs in view of reactive groups. The coupling reactions between (a) two thiols form a disulfide bond, (b) carboxylic acid and primary amine form amide bond, (c) thiol and maleimide group form thioether bond, (d) aldehyde group and hydrazide group form hydrazide bond.

concentration. FA and chitosan were also successfully used to bioconjugate NaYF4 :Yb3+ /Er3+ UCNPs to specifically target cancer cells [160].

Biological applications of lanthanide-doped UCNPs Due to unique physical-chemical properties (as discussed in Section ‘‘The features of ideal fluorescent bio-probe’’), the up-converting nanoparticles doped with lanthanide ions can be applied in many fields of biological sciences [2,8,25,26]. Some general observations can be made as presented in Figure 6. In modulation type of applications (Figure 6), the luminescence of lanthanide UCNPs is modified by the other molecules or environment in response to some biological parameters, for example, in hybrid analyte sensors, (Figure 6a) a pH sensitive phenol red dye modulates emission yield of lanthanide ions proportionally to pH content. Most typically, red to green up-converted emission of Er3+ serves as an indicator of analyte concentration. Similarly, lanthanides may also act as energy donors in FRET or LRET based indirect analyte biosensors, where shortening the distance between UCNPs and targeted biomolecule (F-fluorophore, Q-quencher) is responsible for changes in lanthanide luminescence or efficiency of internal FRET (i-FRET). Either competitive analyte (e.g. glucose) sensing (Figure 6b); DNA hybridization between capture, recognition and target (C-, R-, T-, respectively) DNA strands (Figure 6c) or enzyme (Enz) activity (Figure 6d) may be achieved. In such a case, UCNPs offer efficient NIR-to-Vis photoconversion, improving the sensitivity and simplifying the strategies of biodetection.

In direct analyte sensors, the local environment (e.g. content of H2 O2 , drugs or temperature) has proven to directly affect the spectral properties of Ln:NPs as well, leading to changes in emission color (e.g. drug release — Figure 6e), changes in type of emission between narrow or broadband emission (e.g. impact of H2 O2 on Eu2+ and Eu3+ emission — Figure 6f) or changes in spectral shapes, ratios or lifetimes (e.g. temperature — Figure 6g). In most cases, lanthanide-ions-doped NPs are passive luminescent reporters that are basically not susceptible to local chemical environment. In the course of luminescence imaging, for example, the presence of luminescence occurring from bioconjugated UCNPs informs about morphology or structure of targeted organs, cells or sub-cellular structures. The advantages of Ln:UCNPs come from the spatial distribution of these markers accompanied with their high photostability and deeply penetrating NIR photoexcitation-photoemission. By using a nanoplatform integrating fluorine−18 -labeled, up-conversion nanophosphors with T1-enhanced magnetic resonance properties, PET, UCL and MR imaging techniques become simultaneously available. Finally, lanthanide-doped UCNPs may directly and actively affect the cells and tissues, either through localized heat generation or initialization of photodynamic response. In the first case, the NIR radiation is intentionally converted to heat through non-radiative relaxation. In PDT platforms, the lanthanide up-converted emission excites the photosensitizer decorating the surface of NP or its mesoporous shell, which then generate radicals responsible for photodynamic reaction. This group of applications exploits the ability to photo-stimulate lanthanides in NIR region, assuring deep light penetration into the tissues.

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Fig. 6 Three major groups of biological application of lanthanide-doped NPs: modulation, passive and active applications. Source: Parts of figure reprinted with permission from Ref. [161]. © 2011 American Chemical Society.

Simultaneously deep tissue imaging and light/temperature dosimetry may be provided.

Small animal deep-tissue fluorescence, multi-color and multi-modal imaging Bioimaging is a very important technique in biology and medical research, and many new possibilities may open up with the introduction of these photostable markers [162—165]. For instance a prolonged deep tissue in vivo whole body imaging or studying biocomponents (e.g. proteins) migration through tissues or cells could be facilitated. The UCNPs can be excited using NIR light at around 980 nm which has several important advantages over traditional fluorophores such as the absence of photo-damage to living organisms and luminophores themselves, no autofluorescence of biocomponents, highest penetration depth in biological tis-

sues. These features result in drastic improvement in both sensitivity and signal-to-noise ratio of the obtained images or detection signals [68,125,166] and make deep-tissue or whole small animals in vivo imaging feasible. The effectiveness and potential of UCNPs in small animal imaging have been presented recently by Liu et al., who reported a detection limit of around 50 nanocrystals-labeled cells in a whole mouse body photoluminescent imaging. The group demonstrated also high-contrast imaging and around 2 cm penetration depth in a wholebody black mouse experiment [80], even though the absolute emission quantum yield and penetration depth differed strongly depending on the tissue. In fact, some controversy exists about the exact meaning of penetration depth (PD) parameter for up-conversion imaging, since different authors use different definitions of this parameter [80,166,167]. The measurements of PD∼2 mm

550 (for Yb3+ /Tm3+ at 790 nm) and 0.3—0.5 mm (Yb3+ /Er3+ at 650 nm) by Dong et al. [166] made of tissue phantoms seem to be most reliable. Nevertheless the up-conversion signal has been observed from deeper layers in live animals [80]. The struggle to get highest penetration depth is open, but this is not the single or two-photon excitation that matters, but the overlap of excitation and emission lines with optical transmission window of the skin. Recently we have proposed [59,168], which was soon after evidenced by the group of Prasad [167], to use Nd3+ -doped nanomaterials for deep tissue imaging/thermotherapy. This material offers single photon Stokes emission (more efficient than upconversion) at emi ∼ 940 nm under exc ∼ 800 nm excitation, and promise even higher effectiveness than that upconverting nanophosphors. The UCNPs functionalized with FA were effective in the in vitro imaging of cancer cells (e.g. human HT29 adenocarcinoma cells and human OVCAR3 ovarian carcinoma cells), since they are known to over-express folate receptors on the surface [125]. Visualization of tumor in Balb-c mice was performed as well with UCNPs conjugated to neurotoxin CTX from Leiurus quengestriatus. The toxin can specifically bind to complex MMP-2 on the surface of many types of neuroectodermal cancers [169]. The UCNPs was used for real-time UCL bioimaging of lymphatic system in mice as well [170]. New UCNP, based on NaLuF4 which showed about 10-fold stronger luminescence emission yield than NaYF4 , was used to image lymph node in mouse [79]. Also pathogens have been imaged with UCNP technique. For example Wu et al. [171] reported on use of lanthanide-doped UCNPs for imaging of Giardia lamblia, in concentrated environmental water sample and Chen et al. [172], used PEI-NaYF4 :Yb3+ ,Er3+ for in vivo imaging of Caenorhabditis elegans. Another important application of UCNPs in molecular imaging comes from very stable and easily designed multi-color luminescence, to simultaneously map different molecular targets [173,174]. By varying the concentration ratio between the dye attached to the surface of PEGylated or SiO2 coated Yb3+ /Tm3+ or Yb3+ /Er3+ UCNPs, LRET energy transfer mechanism efficiency and up-conversion emission color was intentionally modified for in vivo multicolor imaging of mice [164]. Up to five different colors and thus up to five different biotargets could be simultaneously imaged through the spectral un-mixing. Alternative approach was proposed by Cheng et al., who generated different up-conversion emission colors by modifying concentrations of dopant ions and imaged lymph nodes in mouse (Figure 7), and labeled cancer cells [86]. Niu et al. [175] were able to control the shape and crystallinity of the obtained NPs, which in turn allowed tailoring the color of UC emission from the UCNPs. Another approach to develop multi-color and brighter UCNPs is doping NaYF4 :Yb3+ /Er3+ with Mn2+ ions [54]. Such approaches to multi-color labels offered perfect photo-stability and background free detection in comparison to the other labeling techniques and is best suited for long-term in vivo imaging and kinetic studies of biomolecules migration. The multi-spectral signatures for multi-color long-term imaging have to be obtained within a single nanoparticle, and this can be almost

A. Gnach, A. Bednarkiewicz exclusively achieved with lanthanide co-doped nanoparticles. Thanks to recent advancements in synthesis of UCNPs, not only multi-color but also multi-modal imaging becomes possible (Figure 6, passive). The Positron Emission Tomography (PET) [108,161,176], Computed X-ray Tomography (CT) [120,177—179] or Magnetic Resonance Imaging (MRI) was used in conjunction to deep tissue Up-Conversion Imaging (UCI) [163,180—182]. Each of these techniques has its own advantages and drawbacks. By linking them together, new multi-component tools for high-quality bioimaging become available. These multi-functional UCNPs are likely to be used in early diagnosis and treatment of various diseases by giving more complementary information [177,183]. Recently, much attention is given to T2 -enhanced magnetic resonance. There are two strategies to combine fluorescent and magnetic properties. Either paramagnetic Gd3+ ions are used in host matrix [124,127,163,177], or e.g. Fe3 O4 NPs cores are covered with luminescent shells [178,180]. The inner Fe3 O4 cores and the outer upconverting nanophosphors provide a combination of robust magnetic responsive properties and strong up-conversion fluorescent properties. These materials have great potential applications in drug targeting, bioseparation and diagnostic analysis. The PET imaging has been demonstrated by labeling the (magnetic) up-conversion nanophosphors with F18 isotope [38,161]. In NaYF4 :Yb3+ , Er3+ ,Tm3+ /SiO2 -Au/PEG5000 , the Gd3+ ions are responsible for magnetic properties for MRI and gold NPs act as imaging contrast for CT [177], while in core/shell Fe3 O4 /NaLuF4 :Yb3+ ,Er3+ /Tm3+ magnetic properties come from Fe3 O4 and NaLuF4 enhance positive contrast in CT [178]. UCNPs as contrast agent in CT possess more advantages over predominately used iodinated compounds, which show renal toxicity and are unable to specific targeting [120]. Tri-modal PET-MRI-UC or CT-MRI-UC imaging is of great interest, since MRI alone, while feasible in providing precise anatomical habitus, suffers from low signal sensitivity. Combination of different imaging methods enhances their advantages and improves the quality of in vivo bioimaging and the efficiency of diagnosis. A very important issue in diagnosis and prevention of various diseases is luminescent imaging of lymphatic system. Local lymphatic drainage is one of the routes for the metastasis of cancer cells. The access to the lymphatic vessels is difficult but the use of NIR fluorescent label may overcome most of the limitations of traditionally used optical bioimaging techniques. PET or combined PET/CT with UCL are sensitive enough to study the velocity of transport of UCNPs to lymph node or to recognize the accumulation sites [176,184]. The ability to analyze the distribution of multi-functional UCNPs may be useful as well in toxicity and biocompatibility studies. Whole-body UCL and CT [178] or UCL and PET images [108] of mouse show the potential of a new generation of multi-functional labels. However, it is an on-going challenge to extend the research on multifunctional UCNPs and create NPs capable to target one type of cells in order to deliver and release drugs in a predictable way. It could be a revolutionary tool that would open new perspectives, not only for diagnosis and therapy but for studying fundamental biological processes in vivo as well.

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Fig. 7 Multi-color UCL imaging: (a) UCL emission yield spectra of three NaYF4 solutions (UCNP1: Y:Yb:Er = 78%:20%:2%; UCNP2: Y:Yb:Tm = 78%:20%:2%; UCNP3: Y:Yb:Er = 69%:30%:1%) under NIR laser excitation at 980 nm; (b) a multi-color fluorescence image of three UCNP solutions; (c) a white light image of a mouse subcutaneously injected with UCNPs; (d)—(f) multi-color in vivo images of a nude mouse after injection of three different UCNPs solutions; (g) three colors of UCNPs after spectral unmixing. Source: Reprinted with permission from Ref. [86]. © 2010 Springer.

High resolution in vivo microscopy imaging Besides advantages in whole organism imaging, UCNPs are perfect solutions for high-resolution microscopy. Traditional one-photon confocal and wide-field fluorescence microscopy techniques give excellent lateral resolution, which is however restricted to around 100 ␮m in axial direction due to the usage of UV/blue excitation necessary to photoexcite organic dyes and quantum dots labels. Introducing twophoton laser-scanning microscopy (TPLSM) technique, while expensive due to the need of sophisticated fs-laser sources, allowed achieving ∼500 ␮m penetration depth with an axial resolution of around 2 ␮m [185]. Therefore, the TPLSM, through the photoexcitation of tiny focal volume of the incident laser light and non-linear absorption of two NIR photons, permits to perform high-resolution imaging of living cells in thick tissue samples. Typically, TPLSM exploits organic dyes (exogenous or fluorescent proteins like GFP) or quantum dots due to their high two-photon absorption cross-section and high quantum yields. Despite significant improvement in spatial optical sectioning of TPLSM with the mentioned labels,

there are number of complications that sill remain unsolved. Under photoexcitation, most of the dyes undergo photobleaching which is resulting from radicals being generated in close vicinity of the fluorescent molecules. The other consequence of photobleaching may become a local phototoxicity occurring to the living cells, which is no lesser problem as it comes to long-term cellular imaging in vivo. Moreover, even though the TPLSM uses the NIR radiation and the excitation light falls into the optical transparency window of most tissues, the obtained two-photon fluorescence occurs in blue-Vis spectral region, where both absorption and scattering remain significant. The use of NIR-excitation and NIR emission yield is unfortunately hindered by the lack of appropriate fluorescent molecular labels. Because lanthanide-doped UCNPs demonstrate high photostability (Figure 8), relatively high quantum yield and low toxicity, their use as luminescent labels for microscopy imaging is undoubtedly worth investigating. Yu et al. [186] used laser scanning up-conversion luminescence microscopy (LSUCLM) to obtain three-dimensional NIR-to-Vis visualization of biological samples. However, most vital, from

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Fig. 8 Illustration of improved photostability of UCNP in comparison to conventional confocal microscopy imaging with organic staining dyes. The blue, red and green colors on the images indicate luminescence of DAPI, Dil and UCNPs respectively excited at 405, 543 and 980 nm. Very low power, (a) ∼15, 0.8 ␮W and 19YmW and (b) higher excitation power of 1.6, 0.13 and 19 mW was used, at 405, 543 and 980 nm, respectively, (c) quantitative analysis of the changes in fluorescence intensities of DAPI, Dil and UCNPs in panels (a) and (b). Source: Reprinted with permission from Ref. [186]. © 2009 American Chemical Society.

microscope deep imaging perspective, is the fact that the Ln:NCs offer the possibility of NIR-excitation and NIR emission, as demonstrated by Pichaandi et al. [49] with Yb3+ /Tm3+ co-doped NaYF4 up-converting NC. The photoexcitation at ∼980 nm (through Yb3+ ) and UPC emission at ∼800 nm (by Tm3+ ) allowed increasing the sectioning depth down to ∼600 ␮m and ∼400 ␮m inside agar-milk tissue phantom using two-photon up-conversion laser scanning microscopy (TPULSM) and two-photon up-conversion wide-field microscopy (TPUWFM), respectively, with lateral resolution suitable to distinguish micrometer-sized biological structures. Similar experiments carried out with NIR-to-Vis up-conversion showed complete loss of lateral resolution already at a depth of ∼300 ␮m. It was possible to image capillaries in the mouse’s lung [49] or mouse’s blood vessels (Figure 9) without any negative effects from laser exposure [187,188]. Imaging of cancer cells is yet another promising application of UCNPs conjugated to biomolecules (such as FA, Pyropheophorbide A and c(RGDyK)) which specifically guide UCNPs to tumors such as HeLa [170,182], HT29 and OVCAR3 [125], KB [52,86,108], Panc1 [163], LO2 [108], RAW264.7 [179], U87MG [189], MCF7 [190—192], SK-BR-3 [191]. Tracking of transplanted cells

with UCNPs has been also demonstrated [193]. Such upconverting labels make the method very sensitive — as few as 10 stem cells could be detected in vivo [194] for at least one week after delivery [193].

Drug carriers and therapies After proper design, the UCNPs may become therapeutic agent in cancer therapy or as prolonged drug release mediators. There are some reports on using the lanthanide UCNPs in photothermal [53,183,195] (chemo-, geno-) therapeutic drugs delivery [196] and photodynamic therapy (PDT) of cancer [9,121,189,192,197,198] (Figure 6). In gold decorated NaYF4 :Yb,Er/NaYF4 /silica (core/shell/shell) up-conversion nanoparticles, the upconverted green light was coupled with the surface plasmon of Au leading to rapid heat conversion and efficient destruction of BE(2)-C cancer cells[195]. While most of heating NPs are based on gold or magnetic NPs [53,183], some preliminary studies on the use of Nd3+ -doped NaYF4 nanoparticles in photothermal of NPs therapy were also presented [59]. The absorption spectrum of Nd3+ ions fits the optical transparency window of human tissue offering

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Fig. 9 Subcutaneous up-conversion deep imaging. (a) Surface modification of up-converting Y2 O3 nanoparticles with polyacrylic acid (PAA) and polyethylene glycol (PEG). (b) Excitation under a laser at 980 nm results in luminescence emission observed at 660 nm. (c) Up-conversion image of blood vessels in the mouse ear following tail vein injection of the NPs (10 mg) using an excitation at 980 nm and a laser power density of 550 mW cm−2 . (d) Merged white light and up-conversion images. Source: Reprinted with permission from Ref. [187]. Copyright 2009 Elsevier Ltd.

highest penetration depth, and potentially the optical remote thermometry as well. An example of geno-therapy was demonstrated by conjugating silica-coated NaYF4 UCNPs with siRNA and anti-Her2 antibodies [199]. These NPs were able to transfect cancer cells and silence genes with siRNA. An example of targeted chemotherapeutic drug delivery was demonstrated with PEGylated NaYF4 :Yb3+ ,Er3+ NPs which were loaded by physical adsorption with DOX, a chemotherapy molecule, which was further released and activated by varying pH [54]. Magnetic targeted drug (DOX) delivery was as well achieved by using tri-modal UCNPs encapsulated together with iron oxide NPs (UC-IO/polymer-DOX) [184]. Loading NaYF4 UCNPs with photosensitizers (such as porphirin derivatives Ce6 or TCPP used in PDT) was also demonstrated [121,189,197,198]. The use of UCNP to photoexcite the photosensitized impregnated into the silica shell solves three problems. First of all, the deeply penetrating NIR radiation can be used instead of less penetrative UV or red one. Second, prolonged PS localization

monitoring and light dosimetry becomes feasible in opposite to PS alone. Third, the PSs molecules are less susceptible to photo degradation when embedded into the silica matrix. Prolonged and controlled release of drugs may be of high importance. The LaF3 :Yb3+ ,Er3+ /nSiO2 /mSiO2 microspheres [89] or NaYF4 :Yb3+ ,Er3+ /nSiO2 /mSiO2 [88] microspheres were demonstrated to sustain or control the release of ibuprofen (IBU) that was loaded into the mesoporous shell of the UCNP (Figure 6e). Moreover, the latest studies demonstrated the up-conversion emission quantum yield being proportional to the amount of IBU release [88], forming a platform for drug delivery and drug release monitoring. The UCNPs showed their promise as vaccine carriers as well, since DNA vaccine against foot-and-mouth disease (FMD) gave positive immune response in guinea pigs [200]. The basic idea in using UCNPs for therapeutic procedures comes from the ability to illuminate and trigger e.g. thermotherapy or drug release, with deeply penetrating NIR

554 light. Another great advantage is the fact that simultaneous up-conversion visible light generation supports imaging, diagnosis [196], (thermo-/PDT-) therapy [9,59,198,201] or drug release [88,184] control. These combined features of therapy and diagnostics are called theranostics [11]. Progress in medicine and nanotechnology, including upconverting nanoparticles, may therefore revolutionize the future health care by enabling to create new generation of multifunctional nanoplatforms to simultaneously detect and treat diseases and verify the effects of therapy.

Bioassays and biosensors One of the most promising applications of UCNPs that need further development are bioassays, i.e. immunoassays or DNA microarrays. Basically, these tests relay of Förster Resonance Energy Transfer (FRET) between fluorescencent donor and acceptor molecules (Figure 6b—d). The phenomena, known as Luminescence RET (LRET) or Förster/fluorescence RET (FRET), may open up many new possibilities when UCNPs are used as donor molecules. Many comprehensive reviews exist on FRET techniques in the field of biosensing and bioimaging and intermolecular interactions, like those of Clegg [202], Soukka [31] or Roda [203]. However, the use of UCNPs as energy donors is relatively recent discovery and may revolutionize rapid diagnostics and bio-screening. Despite the progress, a significant attention shall be paid to enhancement of the FRET based assays with lanthanidedoped NP donors. Core-shell [204], beacon sensors [205], plasmon control [206] and NP’s size-tuning [207] strategies have been recently proposed to improve the effectiveness and sensitivity of these assays; however, further research is ongoing. Good examples of immunoassays are lateral flow (LF) assays for detection of E. coli [208] and human chorionic gonadotropin (hCG) [209]. Wang et al. showed the potential of UCNPs in a sandwich-type LRET-based immunoassay for the detection of goat antihuman IgG [210]. The NIRexcited fluorescence emission band of NaYF4 :Yb3+ ,Er3+ (max = 542 nm) partially overlaps with the visible absorption band of the colloidal Au NPs (max = 530 nm), so the LRET mechanism between the two, immunoglobulin conjugated NPs could take place and let detect Ig reaction. Soukka et al. demonstrated the applicability of biofunctionalized and bioconjugated UCNP to the whole-blood tests [31,32], which has enormous importance for rapid testing of many diseases in situ. Simple, homogenous competitive fluorescence-based bioassays were proposed for detection of 17␤-estradiol without any AF from other blood compounds [211]. Recently, a blood test for very important blood biomarker matrix metalloproteinase-2 (MMP-2) has been reported based on homogenous UCNPs — carbon nanoparticles RET assay [212]. These strategies are very promising and may revolutionize and simplify medical analysis. Recently, rapid and sensitive methods for simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus using UCNPs were developed [213]. Another bioassay for detection and quantification of two viruses causing hand, foot and mouth disease (HFMD) — Enterovirus 71 (EV-71) and Coxsackievirus A16 (CV-A16) — was presented by Wu et al. [214]. The homogenous sandwich hybridization assay based

A. Gnach, A. Bednarkiewicz on two-color UCNPs bound to oligonucleotides and magnetic bioseparation was successfully applied for quantitative analysis of viruses in clinical samples. Another example of UCNPs application in diagnostics is array-in-well platform for detection of human adenoviruses [215]. The important field of lanthanide UCNPs application is DNA microarrays [215,216] as well as LF assays [25] characterized as low-cost, rapid, sensitive techniques for detection pathogens, viruses, drugs, antibodies [1,208,215,217] and oligonucleotides [75,140,218]. UCNPs covalently bound to short oligonucleotide (capture DNA) may be used to detect longer oligonucleotide (target DNA) in the presence of reporter DNA labeled with fluorophore whose excitation spectrum overlaps the emission spectrum of the UCNPs [218]. In the presence of the target oligonucleotide, donor to acceptor energy transfer becomes effective because the fluorophore is brought close to the UCNPs. As a result, the light emission from the accepting fluorophore is observed. Based on this principle, Chen et al. [140] demonstrated the suitability of UCNPs conjugated with streptavidin to detect minute amounts of target DNA. Kumar and Zhang demonstrated a modification of this DNA biosensor based on UCNPs as energy donor and an appropriate intercalating dye as energy acceptor [30]. UCNPs can also be useful as biosensors for proteins, enzyme activity, ligand—receptor interactions etc. [27,210,219]. Studies of enzyme activity are very important, for example, to screen new drugs that are enzyme inhibitors or activators. Interesting method based on lanthanide UCNPs and FRET was proposed by Rantanen et al. [220]. In the absence of the enzyme, the fluorophore F and the quencher Q located at the different ends of the substrate molecule (Figure 6d) are close enough to make the internal FRET efficiently occur and emission is initially quenched. If the enzyme is present in the mixture, it hydrolyses oligonucleotide and separates the two fluorophores so that the emission of the fluorophore is recovered. The growing interest in luminescent nanothermometer (LNT) biosensors has been observed recently. Due to their sensitivity in physiological range as well as their nano-size, and thus ability to diffuse throughout living cells and tissues, LNT allow to control photo-thermal therapy, examine mitochondrial or nucleus activity, as well as study intracellular thermodynamics to support future discoveries of the mechanisms of life. Many interesting review articles can be found on this topic [221—224], and lanthanide-doped UCNPs or NPs are one of the most promising nanothermometers due to sufficient sensitivity, small size, high photostability, ratiometric response as well as technical simplicity of the measurements.

Hybrid biosensors Up-converting nanoparticles were also used as biosensors for pH [225], carbon dioxide [226], ammonia [227], mercury [228], glucose [229], cyanide anions [230] and oxygen [25]. Most of these sensors rely on the UC emission yield being modulated (Figure 6a) by analyte sensible organic dyes, such as bromothymol blue for CO2 , ruthenium Ru(II) complex for O2 or phenol red as pH probe. For example, the Ru(II) complex was indirectly excited by the 1 G4 → 3 H6

Lanthanide-doped up-converting nanoparticles: Merits and challenges Tm3+ up-conversion emission yield. Since the excited Ru(II)* complex is effectively quenched by O2 , the observed Ru(II) emission yield was found to vary by ∼75% between pure nitrogen and pure oxygen conditions. The absorption band of ammonia sensitive phenol red rises at 475—600 nm for growing pH value. The phenol red is thus suitable to absorb green (2 H11/2 + 4 S3/2 → 4 I15/2 ) up-conversion emission yield from Er3+ ions in relation to the red one (4 F9/2 → 4 I15/2 ). These types of sensors expand the well-known behavior of some organic dies with the ability to excite them in NIR. Despite simplicity and NIR-to-Vis sensing capabilities, that is appreciated in biology applications, most of these sensors do not provide specificity and up to now, only Hg(II) ions and CN− biosensor was demonstrated in biological samples in vitro. Another type of biosensors relies on the spectral changes occurring to the lanthanide ions themselves, as the effect of changes in local bio-environment (Figure 6e—g). The example of such a sensor is temperature monitoring with nanothermometers [166,224] or drug release [88]. Due to their nature, i.e. shielding of 4f optically active electrons by electrons on 5s2 5p6 orbitals [35], lanthanide ions are weakly susceptible to changes in their surroundings. Nevertheless, the changes occurring to Yb3+ /Er3+ -doped UCNP were sufficient enough to map the distribution of temperature (Figure 6g) in cell culture with accuracy higher than 1 ◦ C and spatial resolution high enough to resolve subcellular components [166,224].

Instrumentation for UC sensing History of QDots dates back to 1980s, but biological potential has been discovered, as soon as Marcel Bruchez et al. in 1998, developed techniques suitable to make these semiconductor nanocrystals biologically compatible [231]. A fascination in biological applications of QDs began as soon as they became commercially available from Evident Technologies LTD in 2003. Since then, hundreds of papers have been published on the use of QDs in bioimaging and biosensing. Their success originates from a few reasons. QDs are very efficient and more photostable than traditional organic fluorophores. Moreover, no modification to analytical instrumentation was necessary, since QDs have UV/blue absorption bands and may be easily excited with a typical Xe lamp built-in into most of well-plate readers or fluorescence microscopes. They may be also excited with UV/blue lasers found in all confocal laser scanning microscopes (LSM). QDs can be detected with the same fluorescence microscopes, well plate readers and other fluorescence instruments and methods, as the organic molecular labels are. However, the QDs emission bandwidths are smaller than those of most organic dyes, which make them more suitable for the multicolor imaging. At that time, the toxic properties of the available bio-functionalized CdSe QDs were neglected in the view of opening-up the novel possibilities. The story of UCNPs is somewhat different. While down-converting lanthanide-doped phosphors were commercially available since 2004 (PhosphorDotsTM www.nanomaterialstore.com), biocompatible up-converting colloids were synthesized by Heer et al. also in 2004 [232], the up-converting bioprobes became available in 2010 only

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(Sunstone® , www.sigmaaldrich.com). Moreover, while offering unique properties such as narrow emission yield lines and an excellent photostability, the UCNPs require some modifications to the reading instrumentation. Although relatively low excitation intensity threshold is necessary for the wide-field luminescence imaging with UCNPs, typically, lamp excitation is not sufficiently strong to induce the UC process, and the use of laser diodes with emission wavelength of ∼980 nm is required. Fortunately, due to the progress in telecommunication and military applications, the convenient, powerful and cheap InGaAs/AlGaAs/GaAs laser diodes are available in this spectral region already. The UCNPs could have gained the same impact on biosciences as QDs had, if only the UCNPs could be accessible to broader bio-scientific community early enough, and, what is equally important, the commercial imaging instrumentation is available. The available instrumentation for UPC detection can be grouped into two categories, namely plate/strips readers and imagers. The readers include 2D scanners for LF strips and well plate readers. The imagers consist of the microscopes (either wide-filed or LSMs) and small animal wide-field imagers. While the scheme of UCNPs detection is simple, there are no commercial instruments dedicated for up-conversion detection, neither in bio-assays format nor as UC imagers [31]. Thus, all available commercial fluorescence instruments require a customization. This is mostly due to lack of appropriate excitation light source at ∼980 nm dedicated for these instruments. The UPC technique requires the inverse filters, namely long-pass dichroic filters have to exchange the short-pass conventional filter (Figure 10a).

Microscope imagers Two-photon imaging is an excellent and powerful tool to image both morphology and functioning of cellular structures, tissue slices and living animal, with spatial resolution better than traditional confocal microscopy [233]. Conventionally, the two-photon technique requires expensive femtosecond lasers to excite organic dyes, fluorescent proteins, metal complexes and semiconductor quantum dots. Current advancements in laser diode light sources in NIR region allow the up-converting nanoparticles to be routinely used for two-photon type NIR-to-Vis/NIR imaging. Basically, the two-photon LSM fulfill the technical requirements of the UCNP used as biological probes. However, there are a few technical issues that need to be solved, when the LSM microscopes are to be adopted for UC imaging. These modifications include the exchange of femtosecond lasers to continuous wave 980 nm excitation sources. Additionally, the biological samples do not absorb 980 nm radiation, nor two-photon absorption in molecular dyes or QDs with this wavelength is effective, thus background free detection, ultimate sensitivity, photo-bleaching free and long-term high-resolution imaging may be easily achieved. It has to be mentioned however, the CW laser diodes routinely used to photoexcite Ln:NPs are not as powerful as femtosecond lasers. Secondly, due to low UC emission quantum yield, the sensitivity of the system has to be optimized. While

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A. Gnach, A. Bednarkiewicz

Fig. 10 Schemes of methods and instrumentation suitable for UCNPs detection. (a) Explanation of reverse dichroic filter and time gated detection. A scheme of Laser Scanning Microscope (b), wide-filed imaging microscope (c) and in vivo imaging system (d) modified to meet the requirements of up-converting nanoparticles.

the UC emission quantum yield is higher than TPE from organic dyes for example, the highest penetration depth is achieved with NIR-to-NIR lanthanide-doped NP labels, such as e.g. Yb3+ /Tm3+ UCNPs exc = 980 nm/emi = 800 nm or Nd:NPs exc = 800 nm/emi = 860 nm. However, most of photodetectors in LSMs are optimized for visible region, with a significant sensitivity drop at around 800 nm. Furthermore, the efficient up-conversion is usually accompanied with relatively long risetimes of luminescence, which comes from the mechanism of UC and photo-physics of lanthanide-doped materials, and may sometimes lead to apparently decreased UC emission quantum yield with fast laser scanning technique. The next technical issue to adopt LSM for UC imaging originates from long luminescence lifetimes of lanthanide ions. The principle of LSM imaging relies on fast scanning the excitation beam with two galvano-mirrors and collecting the spot induced fluorescence with a photodetector (usually a single or an array of photomultiplier tubes). Due to long luminescence lifetimes, low excitation threshold of UCP, relatively low absorption cross-section of Ln ions at NIR and deep penetration of NIR radiation into tissues, the spatial confinement of the excitation beam is not as tight as in the two-photon microscopy. Thus, some cross-talk between neighbor locations may be observed and

needs optimization. Yu et al. [186] studied the feasibility of using LSM (FV1000 Olympus) for small photo bleaching, background free up-conversion and NIR fluorescence imaging. This was achieved by introducing a reverse excitation dichroic mirror (short pass, edge 850 nm, Figure 10a) and a confocal pinhole. The other confocal LSM were similarly modified to perform UC imaging [163,190,234]. Moreover, Xiong et al. [190], demonstrated that Confocal Z-scan UC imaging of tissue slices showed no AF signal even at high penetration depth (∼600 ␮m). The UC LSM imaging of UCNPs may be therefore conducted with reasonably moderate customization of the conventional confocal imaging system (Figure 10b), in order to provide more details about complex biological samples [186]. Thanks to relatively simple modifications to LSM, as well as due to the convenience and low cost of laser diodes in NIR region at 800 or 980 nm, the UC dedicated microscopes shall be available in nearest future to trigger the broader use of this sophisticated technique. Due to the efficiency of the UC mechanism, wide-field imaging of up-conversion is also possible (Figure 10c). However, first tests of custom modified wide-field upconversion microscope equipped with a sensitive CCD detector for UCNPs high-resolution imaging were not as promising as for UC LSM [125]. In addition, this kind of

Lanthanide-doped up-converting nanoparticles: Merits and challenges modified microscope fails to provide depth discrimination and three-dimensional visualization capability [186]. However, the wide-field imaging with UCNPs seems to be better suited for small animal in vivo imaging (Section ‘‘In vivo small animal imaging systems’’). Since lanthanide-doped materials offer long luminescence lifetimes, the time gated detection with Ln:UCNPs. (Figure 10a) is of great interest for deep in vivo imaging as well as for bio-assays. The time gated detection relies on a difference between luminescence lifetimes of lanthanides (∼␮s-ms) and that of the AF (ps-ns) found in organic molecules. The possibility to delay the detection after short excitation pulse allows to completely get rid of tissue AF background signal and thus improve detection sensitivity. Many time-gated ultra-sensitive cameras exist on the market, but they are suited for short-living (∼ns) chromophores, while are not dedicated for long-living lanthanides. We are aware of just one time gated camera system, dedicated for lanthanide materials (Photonic Research Systems, http://www.prsbio.com), which offers 0.2 ms time-gating resolution. The system has proven to work for lanthanide Eu3+ cryptates on DNA arrays, but no wider use of this system is known to the authors. Further development would be necessary if the up-converting NPs are to be excited. The technical problem lays in lack of a powerful pulsed excitation source at 980 nm. Currently available CW laser diodes, when triggered to work in pulsed mode, lose their average power and may not be sufficiently intense for wide-field time gated imaging.

In vivo small animal imaging systems There are at least a few in vivo small animal imaging systems available in the market (Table 3). Most of these systems are equipped with: 1. A very sensitive low noise (deep)cooled back-thinned cameras sensitive up to 300—1050 nm or typically in the Vis-NIR region, with the ability to acquire a single frame by accumulation of photons from tens of milliseconds to minutes and sometimes to hours. 2. Spectral filtering through either a set of a few bandpass filters within a filter wheel (Pearl® Impuls, iBox® , Carestream In vivo FX Pro), or a tunable filter (MaestroTM ) for higher flexibility. The filters, combined with the sensitive B&W camera, provide VIS-NIR spectral range of detection with a tunable band wavelength and up to ±20/40 nm bandwidth, available for most advanced tunable-filter systems (MaestroTM ). The spectral unmixing allows for identification and separation of multiple fluorophores and removal of AF background. 3. Homogenous illumination system based on a halogen lamp (NightOWL LB 983), xenon lamp (CareStream) or laser light source (Pearl® Impulse FieldBriteTM Xi), ring LED based illuminator or fiber like input port (gooseneck or fiber — most of the systems). The latter options may be potentially suitable to attach a custom light source, like Nd:YAG lasers, multi LED light sources, or the 980 nm laser diode being indispensible for up-conversion imaging.

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The in vivo imaging systems are sometimes equipped with height corrected signal intensity, automated animal heater unit, radiographic and radioisotopic imaging screens, rotation stages, computer controlled zoom, aperture and focus of the lens. The systems offer great flexibility to capture whole animal multi-fluorescence images and false-color the white light images to localize different fluorescence probes (Figure 10d). These systems can be easily used in combination with a number of molecular dyes, fluorescent proteins, NIR dyes and modern QD labels. Unfortunately, they are not ready to explore the advantages of up-conversion labels by default. This is due to up-conversion phenomenon, which requires relatively high excitation at 980 nm and efficient blocking of the excitation photons combined with a reverse excitation dichroic mirror (short pass). The conservative limit for human skin exposure at 980 nm equals 726 mW cm2 [235]. In most studies, 100—200 mW cm−2 at 980 nm was applied. Therefore, imaging of a small animal on a stage 20 cm × 20 cm large requires at least 400 cm2 × 100—250 mW cm−2 ∼40 to 100 W of optical power at the output of the illuminator. Higher excitation power density (1.25 W cm−2 ) was used at 915 nm excitation instead of 980 nm for NaYbF4 : Yb3+ /Tm3+ to reduce thermal problems, which may accompany 980 nm excitation [236] and high absorption coefficient of water molecules at this wavelength. Additionally, even though the absorption crosssection of Yb3+ sensitizer is lower at 915 nm in comparison to 980 nm, the 915 nm excitation may be suitable for even deeper penetration of excitation light. Moreover, high homogeneity of laser excitation and elimination of laser speckles are the additional prerequisites for repeatable and highquality UC imaging. Single report on custom build in vivo imaging system, similar to the commercial ones, was presented by Liqin Xiong [190]. More numerous studies exist, that demonstrate the feasibility of customization of commercial imagers to perform up-conversion in vivo imaging [86]. Most frequently not only MaestroTM In Vivo Imaging System was adopted for UCNP imaging [86,162,164,165,183,236] but also Carestream Multispectral FX In Vivo Imaging System was used for that purpose [108,120]. The customization was achieved by applying 980 nm CW laser diode(s) into the illumination ports of these systems. Further, a short pass (edge of transmission at ∼850 nm) emission filter was placed in front of the (EM)CCD camera. Up to 5 W optical power 980 nm LD provided around 100—200 mW cm−2 power density. Such power was sufficient to image mice and the dissected organs in order to study the accumulation of UCNPs at different time points post intravenous injection. Depending on the sensitivity of the camera and excitation power, the acquisition time of 1 up to 10 s was sufficient. A schematic design of such in vivo imaging systems combined with X-ray imaging possibility is presented in Figure 10c and d.

Well plate and lateral flow strip readers Since the up-conversion quantum yield strongly depends on excitation power density and collection setup, no reliable quantitative comparison is possible between bio-assay results obtained by different groups on different, custom built or custom modified instruments. Lack of commercial

558 Table 3

A. Gnach, A. Bednarkiewicz Small animal imaging systems available commercially.

Company

Instrument models

Web pages

Li-COR®

Pearl® Impuls

http://www.licor.com

Carestream Molecular Imaging

In Vivo MS FX PRO In Vivo Xtreme

http://www.carestream.se

Caliper

MaestroTM Imaging Systems

http://www.caliperls.com

PerkinElmer Berthold Technologies

NightOWL LB 983 in vivo Imaging System

https://www.berthold.com

UVP

®

TM

iBox Explorer , iBox® ScientiaTM , iBox® SpectraTM

well plate and LF readers is a serious drawback and limitation for UC technology development in point of care (POC) bio-analytical test [237]. In 2007, a prototype of rapid virus infection antigen test based on UPC technology was presented by Mokkapati et al. [238]. The UplinkTM system comprises a portable reader with a built-in IR (infrared) laser and immunoassay devices (disposable cassettes containing test-specific LF strips). The results showed excellent agreement (>90%) in reproducibility test, but up to now, the device has not been yet commercialized. The advantages of using UPC technology for immunoassays were recognized back in 2000 by Niedbala et al. [239]. His group modified a 96-well, fluorescence microtiter plate reader (Packard Instruments, Meridian, CT) with a fiber coupled 1.2 W NIR laser attached to the original detection unit through the long pass filter. The 980 nm light was directed to the sample through a bifurcated fiber bundle and collected back to a PMT tube [208]. In 2005, Soukka et al. modified Plate ChameleonTM instrument (Hidex Ltd., Turku, Finland) [153,240] by replacing original excitation xenon flash lamp module with IR CW laser diode that was suitable to deliver 200 mW at 980 nm. The filter and mirrors were replaced accordingly, to direct visible radiation to the photodetector. A simple optical reader was developed in house by L. Huang et al. [241] for rapid and sensitive quantification of LF strip with up-converting phosphor (UCP) particles as reporters. Some other reports on customized microplate readers were recently presented by Ylihärsilä et al. [215] and P. Corstjens et al. [242] to study genotype human adenoviruses and detect nucleic acid sequences of human Papillomavirus type 16 infections, respectively. The use of up-converting nanoparticles promises simpler, faster and more sensitive readout. Not only these features could be beneficial for the patients, whose compliance with doctors’ instructions would result in more effective therapy, but also the chance to pre-screen multiple target analytes (DNA, viruses, cytokine proteins etc.) within the sample could result in earlier diagnosis and thus better targeting and more successful treatment, lower curing costs and lower side-effects. The ability to bring the analytical instrumentation from specialized central laboratories to point-of-care (POC) would bring cost reduction of the whole treatment, which could be beneficial for national health systems capabilities as well [237].

http://www.uvp.com/

Future directions and perspectives A wide range of applications exploiting the advantages of lanthanide up-converting nanoparticles in vitro remain undoubtedly highly promising and competitive to traditional approaches. Numerous studies report negligible or low toxicity of lanthanide-doped UCNPs which is encouraging future explorations of UCNPs for biomedical applications (Section ‘‘Biological applications of lanthanide-doped UCNPs’’). To fully exploit the great potential of lanthanide-doped UCNPs in the future, a continuous, interdisciplinary, both basic and applied research is required. NaYF4 fluoride matrices seem to be a material of choice due to highest up-conversion rate among known UCNP hosts. However, fluorides are missing charge transfer or f-d absorption bands, which may sometimes be useful for direct Stokes excitation in UV/blue spectral range. Lanthanide-doped vanadates [243], oxides [134], calcium phosphates [244] were alternatively proposed for bioapplications. Not too many demonstrations of bio-applications of these materials exist and the research on suitable (other than NaYF4 or CaF2 fluorides) bio-oriented hosts for lanthanide doping is still in its infancy [66]. There is a great scope for development of nanoparticles that are easy to bio-functionalize and bio-conjugate, which possess high colloidal stability, high up-conversion efficiency and tailorable optical properties. Engineered multi-functional nanoparticles that bridge different imaging modalities (fluorescence, MRI, PET) as active probes or contrast agents form another class of highly promising composite materials [38,163,181]. In addition to luminescent label function, some other nanoparticles give premise to study pH [225] or temperature [224] through some fluorescence ratiometric approaches within the same NP. The ability to probe many biotargets within the same sample volume (e.g. in rapid whole blood assays) is also very attractive research direction, but currently remains unresolved unless unique features of lanthanide ions (i.e. long luminescence lifetimes or NIR-to-Vis emission) are utilized [31,32]. Even though spectral unmixing of known fluorescent taggants is well known in microscopy, this approach usually requires ‘sterile’ experimental conditions and will not be suitable for homogenous assays as lanthanide-doped UCNPs do [32]. Multi-target imaging is also of vital importance

Lanthanide-doped up-converting nanoparticles: Merits and challenges for long-term studies of biodistribution and migration of biocomponents in living cells and tissues or whole animals [164]. One may expect, these multi-modal and multi-target applications will stimulate further development in both materials and detection techniques dedicated for widest exploitation of the enhanced properties. A lot of research was devoted to bio-functionalization and bio-conjugation of other types of nanoparticles (e.g. quantum dots, magnetic NPs and other). Because of different nature of the dielectric NPs, these procedures have to be adjusted to the specific materials. As was indicated in recent nanotoxicology studies, the physical and chemical properties of the NPs, such as size, shape, NP stability and purity, NP surface charge and chemistry, are of critical importance for the ability to manage the diffusion, circulation and clearance time. Nevertheless, further studies need to be done to generate sufficiently large and comparable data to quantify and conclude about safety of the NPs. Lanthanides offer numerous unique spectral properties like long luminescence lifetimes of emission, large Stokes shift and narrow absorption and emission bands. These properties put new technical requirements to the bioimaging and biosensing instruments. Therefore, miniaturization of biosensors and other high throughput screening devices used in biological application or development of lab-on-a-chip assays equipped with lanthanide-doped nanoparticle based indicators is a challenge for the next years. This is especially true, since no commercial, ready-to-use bio-assay or bioimaging instruments exists in the market. Another important issue, concerning lanthanide-doped nanomaterials is standardization of synthesis and measurement protocols to allow for quantitative comparison of spectral properties and QY for different hosts, phases, morphologies, core-shell architectures, co-dopant absolute and relative concentrations. Similar problems concern measurement protocols (e.g. optical excitation density, exact excitation wavelength, penetration depth etc.), since currently it is almost impossible to compare results from different labs. One cannot forget the potential harmfulness and toxicity of nanoparticles. It is obvious that nanotechnology develops much faster than methods able to quantify or predict its potential adverse effects on humans and environment. Unfortunately, many nanotechnology discoveries have been commercialized, much before any concern about their toxicity was raised. While many nanomaterials have been toxicity tested since then, new products emerge like the lanthanide-doped up-converting nanoparticles dedicated for bio-applications. These luminescent nano-labels offer significant advantages over traditional biomarkers. Current knowledge about the toxicity of these NPs is modest but promising. Nevertheless, a versatile physicochemical characterization and long-term toxicity studies still need to be performed before introducing the Ln:UCNPs for wide spread use in drug screening, bioassays or bioimaging.

Conclusions The current revolution in life sciences is strongly dependent on the availability and development of new advanced tools that enable studying biological processes. We are strongly convinced, the lanthanide up-conversion nanoparticles

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become a serious option in this respect in comparison to traditional organic fluorophores and quantum dots. Bio-functionalization of UCNPs enables exploiting their potential and overcomes limitations of current markers technology such as toxicity, hydrophobicity, lack of biocompatibility etc. The possibility of specific targeting (e.g. to reach cancer cells or specific tissue) is also enabled after surface modification of UCNPs. In this review, we recalled the unique properties of UCNPs, most commonly used techniques of UCNPs surface functionalization and bio-conjugation. The choice of the method depends, first of all, on further application of nanoparticles which follows fitting for purpose rule. We have reviewed and made some classifications of currently existing applications into passive, active and modulation techniques. We have also examined current state-of-the-art in up-conversion enhancements indicating the further need for optimize composition and architecture of novel luminescent nanomaterials. We have also underlined the need for dedicated instrumentation, since bio-functionalized lanthanide-doped UCNPs are continuously inspiring scientists and applications at the edge of biology, chemistry, physics and biomedical engineering.

Acknowledgments The work was supported by Wrocław Research Centre EIT+ within the project ‘‘The Application of Nanotechnology in Advanced Materials’’ — NanoMat (POIG.01.01.02-02-002/08) financed by the European Regional Development Fund (Operational Programme Innovative Economy, 1.1.2). A.B. acknowledges support from Polish National Science Centre Grant No. N N507 584938.

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