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Surface modifications for photon upconversion based energy transfer nanoprobes Elina Andresen, Ute Resch-Genger, and Michael Schäferling Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00238 • Publication Date (Web): 14 Mar 2019 Downloaded from http://pubs.acs.org on March 18, 2019

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Langmuir

Surface modifications for photon upconversion based energy transfer nanoprobes Elina Andresen ‡, §, Ute Resch-Genger ‡, *, Michael Schäferling ‡, †, * ‡BAM Federal Institute of Materials Research and Testing, Division Biophotonics, Richard-Willstätter-Str. 11, D-12489

Berlin, Germany

§Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, D-12489 Berlin, Germany

New address: Münster University of Applied Sciences, Department of Chemical Engineering, Stegerwaldstr. 39, D48565 Steinfurt

†

*Corresponding Authors, Email: [email protected]; [email protected]

ABSTRACT: An emerging class of inorganic optical reporters are near infrared (NIR) excitable lanthanide-based upconversion nanoparticles (UCNPs) with multicolor emission and long luminescence lifetimes in the range of several hundred of microseconds. For the design of chemical sensors and optical probes that reveal analyte-specific changes in their spectroscopic properties, these nanomaterials must be combined with sensitive indicator dyes that change their absorption and/or fluorescence properties selectively upon the interaction with their target analyte, utilizing either resonance energy transfer (RET) processes or reabsorption-related inner filter effects. The rational development of UCNPs-based nanoprobes for chemical sensing and imaging in biological environment requires reliable methods for the surface functionalization of the UCNPs, the analysis and quantification of surface groups, a high colloidal stability of UCNPs in aqueous media as well as chemically stable attachment of the indicator molecules, and suitable instrumentation for the spectroscopic characterization of the energy transfer systems and the derived nanosensors. These topics are highlighted in the following feature article and examples of functionalized core-shell nanoprobes for the sensing of different biologically relevant analytes in aqueous environments will be presented. Special emphasis is dedicated to intracellular sensing of pH.

INTRODUCTION Photon upconversion (UC) materials are capable to convert near infrared (NIR) light (typically 800−1000 nm) into shorterwavelength (visible (vis) or ultraviolet (UV)) luminescence. This anti-Stokes or up-converted emission is an interesting feature particularly for luminescence applications in complex biological matrices such as immunoassays in whole blood, or for cellular and in vivo imaging because the NIR-excited UC luminescence (UCL) is more or less free of an autofluorescence background.1-3 In addition, the NIR excitation enables a high penetration depth in biological matter due to the reduced absorption of biological components in this wavelength region and minimizes scattering and thus image blurring and the photodamage of cells and tissue. Moreover, the long lifetimes of the emissive lanthanide ions are ideal for time gated detection and lifetime multiplexing which has been recently demonstrated in in vivo imaging studies.4-6 Photon UC occurs e.g. in inorganic crystalline materials doped with lanthanide ions. François Auzel recognized in the 1960s that this process involves the sequential absorption of multiple photons (typically two to three) and several energy transfer steps between the involved lanthanide ions. 7-9 This excitation power density-dependent process accounts for multicolor emission bands with long excited state lifetimes in the range of a few hundred of microseconds. In addition, lanthanide-based upconverters also reveal down-converted or down-shifted emission bands at wavelengths > 1000 nm suitable for shortwave infrared (SWIR) imaging.10, 11 In the past years, several extensive review articles have highlighted the synthesis, properties, and possible applications of lanthanide-doped UC nanocrystals and the underlying UCL mechanisms.11-15 Typically, highly efficient upconverting systems are composed of a host matrix with a suitable crystal lattice of low lattice phonon energy, and two chemically different dopants, one acting as sensitizer that absorbs the NIR excitation light and one acting as activator that emits light of shorter wavelength. The host lattice does not only accommodate the sensitizing and emitting lanthanide ions but also plays a crucial role for the UC process as e.g., its crystal phase controls the position of and the distances between the different dopant ions in addition to dopant ion concentration and the symmetry of the crystal field faced by these ions and hence the Stark splitting of their energy levels.16 The sensitivity of UCL to crystal phase and size can be even elegantly exploited for monitoring the formation and phase transformation of UCNP during particle preparation.17

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The host matrix should possess the following properties: i) the size of the lattice lanthanide replaced by the dopant lanthanide ions should be relatively similar to avoid lattice distortions, ii) it should show a high chemical and thermal stability and transparency in the optical window of NIR excitation and lanthanide emission, and iii) it should show low phonon energies to prevent UCL quenching. Moreover, when the host lattice consists of paramagnetic lanthanide ions like Gd3+ (e.g. NaGdF4)18 it can also affect e.g., UCL sensitization by organic antennae dyes by favoring intersystem crossing.19 Excellent and broadly used host materials are rare-earth oxysulfides (e.g. Y2O2S, La2O2S) and fluorides (e.g. YF3, NaYF4), which keep UCL-quenching multiphonon relaxation rates low. There are three main UC mechanisms relevant for generating UCL: (a) excited state absorption (ESA) or multistep (or sequential) absorption, (b) energy transfer UC (ETU), and (c) photon avalanche (PA).16, 20, 21 The ESA process occurs within a single ion, hence it is independent of dopant concentration.21 In ETU, energy is transferred from an excited donor ion to an acceptor ion that is already in an excited state, thereby producing a doubly excited acceptor ion.22 ETU is up to two orders of magnitude more efficient compared to the stepwise absorption of multiple photons by the same lanthanide ion, (e.g., Er3+ versus Yb3+/Er3+ in SrF2).9 ETU efficiency depends on the distance between these two ions and thus relies on their concentration.21 ETU enables to separate the function between dopants, with one dopant acting as absorbing sensitizer and the other as light emitting activator and to tune optical properties through dopant engineering by varying parameters such as the combination of dopants, and thereby emission color, and by tuning the population of donors and acceptors. PA is a process involving ESA of the incident light and cross-relaxation (CR) energy transfer (ET) between two lanthanide ions. For generating UCL in UCNP through PA, a threshold excitation power is required20 and the concentration of the dopant lanthanide doping ions should be high enough for the ion-ion interactions to cause efficient CR ET which populates the metastable state and induces ESA. Particularly, hexagonal phase nanocrystals of NaYF4 doped with Yb3+ as sensitizer, that has a relatively large molar absorption coefficient or absorption cross section for a lanthanide ion, and Er3+ or Tm3+ as activator, show high UC efficiencies upon 980 nm excitation and several sharp emission bands in the blue (~ 475 nm), green (~ 550 nm), red (~ 660 nm) and NIR (~ 800 nm) spectral range. These materials became a kind of “workhorses” for the development of UC nanomaterials for bioimaging and sensing. In Yb3+-based co-doped NaYF4 crystals, UCL originates commonly from an ETU mechanism involving the long-lived (up to 1 ms) excited energy level of Yb3+ to the higher energy levels of the Er3+ ions that exhibit properly matching energies. The hexagonal NaYF4:Yb3+,Er3+ or NaYF4:Yb3+,Tm3+ nanoparticles are typically prepared by a co-precipitation23, 24 or a solvothermal25, 26 synthesis. Systems with efficient UCL under typically used low excitation power densities are obtained with dopant concentrations of around 20% for the sensitizer (Yb3+) and 1-3 % for the activator (Er3+ or Tm3+). Since the late 1990s, UC nanoparticles (UCNPs) have become one of the most attractive research fields within nanoscience with a variety of applications emerging such as in vitro and in vivo bioimaging, photodynamic and photothermal therapy, or security encoding.13, 27-32 Nanocrystalline materials with a particle size < 100 nm that are dispersible in water and colloidally stable in the respective matrix are of special interest for biomedical and bioimaging applications.33, 34 Nanometer-sized UC materials show, however, a much weaker UCL than the corresponding bulk materials due to surface quenching effects, which are favored by solvent and ligand molecules containing moieties with high energy vibrational modes such as OH vibrations.35, 36 This can render UCL intensity dependent on UCNP size or surface-to-volume ratio particularly for bare UCNP.36 Therefore, many efforts have been dedicated to minimize UCL quenching and enhance the brightness of UCNPs by a core-shell design, shielding the emitting lanthanide ions from quenchers on the particle surface, particularly water molecules, by a surface passivation shell.18 With a sufficiently thick and tight surface passivation shell and by minimizing/excluding sources of moisture and other sources of hydroxyl ions during UCNPs synthesis, nanocrystals can be obtained that have UCL quantum yields closely matching that of the respective bulk material of 5-10 %.18, 37 The growing interest in nanometer-sized photon-UC materials in the past years has evoked enormous progress in the design and synthesis of ultra-small, monodisperse, and bright UCNPs with controllable optical properties. These advances have paved the way for UCNPs as promising alternatives to conventional labels such as organic dyes and semiconductor nanoparticles (quantum dots). This is also triggered by the unbeatably broad wavelength region of the luminescence of UCNPs covering the UV/vis/SWIR as the up- and down-shifted emission can be simultaneously utilized.10, 18 Concurrently, this boosted the development of methods for the quantitative characterization of the optical properties of different core/shell particle architectures in application-relevant environments at excitation power densities used for bioimaging assays. Other studies focused on sensing applications, spectroscopic and microscopic studies38 and the comparison of material performance.39, 40 A high colloidal stability of UCNPs in aqueous matrices can be achieved by using hydrophilic ligands or by coating of UCNPs stabilized with hydrophobic ligands either with biocompatible polymer or silica shells. These surface modifications can also be utilized to introduce reactive groups such as amino groups for the further covalent attachment of chemical sensitive dyes or biomolecules.41 By combining UCNPs with indicator dyes that selectively change their absorption and/or fluorescence properties upon interacting with their target analytes. UCL can be made responsive to various chemical species. The sensitized excitation of the indicator via UCL can be achieved via distance-dependent non-radiative processes such as resonance energy transfer (RET). Frequently, this process is also referred to as “Förster Resonance Energy Transfer” or “Fluorescence Energy Transfer” (FRET) or “Luminescence Resonance Energy Transfer” (LRET) in connection with luminescent lanthanide species. Alternatively, excitation of the indicator dye can occur by reabsorption (termed also inner filter effect-based sensing).39, 42-45

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It is an essential prerequisite for the design of UC-based nanoprobes and nanosensors to understand the different modes of interactions of UCNPs with their environment, affecting the UCL intensities/relative distributions of the different emission bands and the UCL lifetimes, and to control quenching processes46 and possible interferences by external factors such as temperature.47 Moreover, for RET-based probes, where the indicator dye typically acts as RET acceptor for one of the several UCL bands,45 the distribution of the emissive lanthanide ions within the nanoparticle must be considered, that can each act as individual RET donors.48 This implies that RET involves mostly lanthanide ions at or close to the UCNPs surface whereas lanthanide ions in the UCNPs center do not contribute to RET, because they are too far away from the UCNPs surface.49 Thus, the RET process depends on particle size, dopant concentration, and architecture including the spatial arrangement of the emitting lanthanide ions and the thickness of a passivation and/or ligand shell.48, 50 As already indicated, the luminescence of Yb3+, Er3+-doped UCNPs strongly depends on temperature. Particularly, the ratio between the intensities of the green emission lines at 525 and 545 nm is very sensitive to temperature. These two emission lines originate from energetically closely spaced states of Er3+ ions, which are thermally coupled. Therefore, UCNPs can be employed for the measurement of the local temperature with high spatial, temporal and thermal resolution and sensitivity. Different groups have extensively studied and discussed the basic mechanisms causing the thermal sensitivity of Yb3+ ,Er3+doped UCNPs 51-56 and the impact of environmental factors.57 Water-dispersible UCNP nanothermometers for imaging of temperature gradients in living HeLa cells were obtained by polymer coating of NaYF4:Er,Yb.53 Seidlmeier et al. compared temperature sensing with UCNPs of varying size, dopant concentration, and core/shell structures and utilized these for temperature sensing in a range from 25 °C to 45 °C with a precision less than 0.5 °C in HeLa cells. 58 UC-RET systems are also promising sensitizers for photodynamic therapy (PDT) of cancer, which produce reactive oxygen species such as singlet oxygen by irradiation with NIR light. Wang et al.59 have attached the photosensitizing dye Rose Bengal to core/shell UCNPs for singlet oxygen gen-eration upon 980 nm excitation. This application field was recently reviewed by M. Hamblin31 and Qui et al.32 However, thermometry and PDT are not within the scope of this article. A rational development of UCL nanoprobes for chemical sensing and imaging in biological environment requires reproducible methods for the functionalization of UCNP surfaces, reliable tools for the analysis and quantification of surface groups and the attached indicator molecules, as well as methods for spectroscopic characterization of the UCNP-RET system and the derived nanoprobes. In the following, we will highlight these topics in this feature article and show examples of functionalized core-shell nanoprobes for the sensing of pH and other biologically relevant analytes in different environments, particularly in live cells. RET-based sensor systems using biomolecular recognition elements, e.g. DNA- or immunoassays, will not be covered in this feature article. GENERAL STRATEGIES FOR SURFACE FUNCTIONALIZATION — HOW TO CHOSE YOUR METHOD UCNPs which are applied in biological environment must show a high dispersibility and stability in aqueous media. Additionally, suitable functional surface groups for subsequent conjugation of biomolecules or probe molecules are compulsory. Because the majority of high-quality and monodisperse UCNPs are synthesized in organic solvents by the thermal decomposition method or the modified solvothermal method, the surfaces of these UCNPs are capped by hydrophobic organic ligands such as oleic acid.60-62 Therefore, different approaches for surface functionalization and engineering of as-synthesized hydrophobic UCNPs have been developed and established in the past years.63-65 Strategies to generate conjugable UCNPs can be generally divided into two main groups: ligand exchange and encapsulation techniques (Figure 1). Both strategies do not affect the spectral position of the UCL bands, yet can influence the intensities or the relative spectral distribution of the different UCL bands and hence emission color and the respective decay kinetics.63 In addition, ligands like oleic acid with unsaturated C=C-bonds can be directly oxidized with the aid of a Lemieux-von Rudloff reagent to yield carboxylic acid-functionalized UCNPs with good water stability and bioconjugatability.25 However, this oxidation process produces MnO2 that can quench the UCL.66 In any case, it must be kept in mind that except for surface modification strategies involving silica chemistry, the surface ligands are commonly only coordinatively bound to the UCNPs surface atoms. Hence, they can desorb from the UCNPs surface e.g., upon dilution or protonation and can be partly or completely replaced by stronger binding molecules present in the nanoparticle environment. Moreover, also functional groups present in the ligand shell at the UCNPs surface like OH, NH, and COO– groups can quench UCL by favoring nonradiative multiphonon relaxation in a distant-dependent manner. Ligand exchange strategies The most convenient and straightforward technique is to exchange hydrophobic native capping agents such as oleic acid (OA) with hydrophilic molecules or polymers. The ligand-exchange processes reported in literature occur either in a one-step procedure accomplished by direct substitution of the primary ligands with the new ligands or via ligand-free UCNPs as an intermediate. Atoms or molecular structures like O, COO–, N, PO33–, and SO33– can coordinate lanthanide ions, with more than one coordinating motif per ligand enhancing the binding strength due to multivalency effects. A direct substitution can be carried out adding secondary ligands with an affinity to rare earth ions higher than those of the primary capping agent, so that a stronger interaction between the nanoparticles and the new ligands is established. This approach is often challenging, time-consuming and requires often a considerable excess of the new ligand and high temperatures.64 Moreover, frequently,


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the ligand exchange is not complete, leading to a not well-defined surface chemistry, which can affect the reproducibility and in some cases also subsequent (bio)functionalization steps. Brahmaiah et al. reported a general ligand exchange strategy to functionalize UCNP with both hydrophobic and hydrophilic ligands starting from methyl oleate-capped UCNPs.67 The presence of methyl groups in the close neighborhood of the carboxyl groups favors weak binding of the ligand to the nanocrystal surface, which can be exploited for ligand exchange with a variety of organic molecules, e.g. dicarboxylic acids. The approach via a ligand free intermediate provides the opportunity to introduce a variety of new capping ligands in a controlled manner and thus to tune the surface properties specifically. Such systems were investigated for oleic acid capped UCNPs and can be obtained either by removal of the oleic acid in acidic environment (pH < 4) to obtain water-dispersible ligand-free (or rather H3O+ capped) UCNPs68 or by introducing weakly coordinating ligands like BF4-. This provides UCNPs with a good long-term and storage stability in polar, hydrophilic solvents as DMF or acetonitrile.69 BF4- can be then easily replaced by a hydrophilic ligand. This presents the method of our choice for comparative spectroscopic studies.35, 36, 48, 63 The use of bifunctional molecules in a ligand exchange approach is currently one of the most popular strategies for modifying hydrophobic surface for bioapplications. Such bifunctional ligands possess one functional group capable for tight binding to the particle surface and a second hydrophilic functional group, providing good water dispersibility and an anchor site for subsequent dye conjugation. A large number of organic molecules and polymers which meet these requirements have been investigated for the surface modification of UCNPs in direct or ligand-free intermediate approaches (Table 1). Polyethylene glycol (PEG) diacid62, PEG-phosphate70, 3-mercapropropionic acid2, hexanedionic acid71, 6-aminohexanoic acid, alendronic acid72, 2-aminoethyl dihydrogen phosphate have been utilized as secondary surface modifications. Schäfer et al.73 reported

Figure 1. Overview of different strategies for surface functionalization and engineering of biocompatible UCNP.


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Langmuir Table 1. Examples of surface functionalization via ligand exchange. Native ligand

Surface

Exchange

functionality

conditions

—PO33–

—OH

180°C / 320°C

73

PEG diacid

—COO–

—COOH

rt, 48 h

62

(surface coordinating group)

Intermediate ligand

New ligand

Coordinating functionality

HEEDA (-NH2)

—

HEDP

Oleyl amine (-NH2)

—

Ref.

Oleyl amine (-NH2)

—

HDA

—COO–

—COOH

240°C, 1.5h

71

Oleic acid (-COOH)

—

Citrate

—COO–

—OH

220°C, 3h

84

Oleic acid (-COOH)

—

—COO–

—SH

rt, over night

2

Oleic acid (-COOH)

—

PVP

—C=O

—

100°C, 6h

32, 70

Oleic acid (-COOH)

—

PEG-phosphate

—PO33–

—PEG

70°C, 5h

85

PAA

—COO–

—COOH

240°C, 1.5h

76

Oleic acid (-COOH)

—

3-mercapto- propionic acid

rt,

Oleic acid (-COOH)

Citrate

PAH-PSS-PAHPSS

—NH2

—SO32–

Oleic acid (-COOH)

BF4–

O–phospho-Lthreonine (OPLT)

—PO33–

—COOH

rt, 2h

72

Oleic acid (-COOH)

BF4–

Alendronate

—PO33–

—NH2

rt, 2h

72

Oleic acid (-COOH)

BF4–

PEG-phosphate

—PO33–

—OH

rt, 2h

72

Oleic acid (-COOH)

BF4–

PAA

—COO–

—COOH

rt

7779

PVP (-C=O)

—

PEI

—NH2

—NH2

80°C, 2h

75

PVP (-C=O)

—

PAA

—COO–

—COOH

80°C, 2h

75

Oleic acid

—COO–

—

Oleylamine

—COO–

—

Hexanoic acid

—COO–

—

Oxalic acid

—COO–

—COOH

Malonic acid

—COO–

—COOH

10 min, rt

67

Succinic acid

—COO–

—COOH

Adipic acid

—COO–

—COOH

Sebacic acid

—COO–

—COOH

Methyl oleic acid (-COOH)

—

20 min/layer

63

the first synthesis of NaYF4:Yb3+,Er3+/Tm3+ UCNPs in the coordinating solvent N-(2-hydroxyethyl)ethylenediamine (HEEDA), and further modified the UCNPs surface with 1-hydroxyethane-1,1-diphosphonic acid (HEDP) to obtain water-dispersible UCNPs. The removal of the original capping ligand, however, required high temperatures (180 or 320 °C) and a vacuum (p < 0.1 mbar). Another strategy to generate water dispersible UCNPs is the replacement of the capping ligands with water-soluble organic polymers bearing functional groups which strongly coordinate to the particle surface. This includes polyvinylpyrrolidone (PVP)70, 74, poly(acrylic acid) (PAA)66, 75-79, branched polyethylene imine (PEI)75, poly(amidoamine) (PAMAM)80, and multidentate thiolate-grafting polymers81. The attachment of polymers with reactive amino- or carboxylic acid groups such as PEI and PAA enables the conjugation of functional molecules to the UCNPs. In 2006, Wang et al.82 demonstrated a direct ligand exchange with PEI yielding water-dispersible and biocompatible UCNPs. PEI is a highly branched hydrophilic polymer with primary, secondary and tertiary amino groups which stabilize the UCNPs in solution due to high positive net charge. The primary amino groups on the surface allow covalent bonding of biomolecules via conventional active ester/carbodiimide chemistry. The cytotoxic effect of free PEI is greatly reduced in the surface-bound state.83 Therefore, it was shown that cell viability is insensitive to PEI-coated UCNPs even after two weeks of exposure.82 Polymer coatings can also be achieved via layer-by-layer (LBL) deposition, where alternatively charged polyions are sequentially adsorbed onto the oppositely charged nanoparticle surface utilizing electrostatic attraction. This method provides precise control over the thickness and charge of the layers and generates a stable shell. In one of the first reports on the use


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of this coating technology, Wang et. al.86 functionalized UCNPs by sequential deposition of positively charged poly(allylalamine hydrochloride) (PAH) and negatively charged poly(styrene sufonate) (PSS) resulting in hydrophilic NPs with stable amino-rich surfaces. However, this modification requires UCNPs which are already hydrophilic. Encapsulation strategies The encapsulation strategy relies on the wrapping of the as-synthesized nanoparticles by an additional coating layer (e.g., polymeric or silica shells), thereby conserving the initially used capping ligands.63, 64 Table 2 summarizes examples for such surface modifications. These additional shells, that increase the UCNPs hydrodynamic diameter, which can be important for all biological applications where size matters like cellular uptake and imaging studies, can provide reactive functional moieties for the conjugation of sensing molecules and colloidal stability. For this purpose, amphiphilic molecules87 like phospholipids, amphiphilic polymers63, 88 like poly(isobutylene-alt-maleic anhydride) modified with dodecylamine (PMA), pyridine (Py-PMA) or polyethylenglycol (PEG-PMA) or silica coatings are commonly used. The coating with amphiphilic polymer shells involves the use of polymers with both hydrophobic and hydrophilic segments in their backbone. The hydrophobic surface ligand intercalates with the hydrophobic segment of the copolymer by mixing the UCNPs with the amphiphilic polymer. Accordingly, the hydrophilic part of the copolymer forms a hydrophilic shell, rendering the nanoparticles dispersible in aqueous media. Yi et al. 88 presented a coating based on ligand attraction between the native oleyl amine capping agent and an amphiphilic layer of 25% octylamine and 40% isopropylamine modified polyacrylic acid (PAA). The polymer coating is formed by hydrophobic interactions of the octyl and isopropyl groups of PAA with the octadecyl group of oleylamine on the nanoparticles surface. Surface silanization is a frequently used technique for a stable surface modification of a broad variety of materials including UCNPs.64 Silica is chemically inert, optically transparent and has a low cytotoxicity. Furthermore, it exhibits the potential to functionalize the inner pore systems and/or the external surface. In 2015, Liu et al. have reviewed the developments in the area of UCNP@silica structures including the coating with dense (dSiO2) or mesoporous silica (mSiO2) layers.92 The synthesis of UCNPs@dSiO2 can be performed in either of the following ways: a water-in-oil reversed microemulsion or a Stöber-like process utilizing the controlled hydrolysis and polycondensation of precursors such as tetraethoxysilane (TEOS) to yield a close amorphous silica layer. The thickness of the silica layer can be adjusted by varying the amount of silica precursor. Subsequent treatment with organosilanes yields surfaces with the desired functionality for the covalent attachment of sensor molecules or targeting ligands on the silica surface, e.g. amino groups via 3-aminopropyl triethoxysilane (APTES) or N-(3-trimethoxysilyl) propyl)ethylene diamine (TMED). Additionally, carboxy93, alkyne94 and azide94 modified silica coating have been reported. Dense silica shells have been grown in a Stöber-like procedure on hydrophilic PVP-stabilized βNaYF4:Yb3+/Er3+ nanoparticles, thereby taking advantage of the affinity of the PVP chains to silica.70, 90 PVP on the surface of the particles improves their stability in ethanol and its affinity to silica enables a uniform silica shell growth on the particle surface. The most commonly used technique for the coating of hydrophobic nanoparticles with a thin shell is the inverse microemulsion route.89, 95 This method exploits chemical reactions in hydrophilic cavities formed by a homogeneous mixture of ammonia, cyclohexane, surfactant, and TEOS. Thin silica shells grown in a reverse microemulsion often have the limitation Table 2. Examples of surface functionalization using encapsulation strategy Native ligand

Surface

Encapsulation

functionality

conditions

New ligand

Coordinating modality

Oleyl amine (-NH2)

Octylamine + isopropylamine modified PAA

Hydrophobic interactions

COOH

Oleic acid (-COOH)

1,2-distearoyl-sn-glycero-3phospho-ethanolamine-N[methoxy(PEG)] (ammonium salt) (DSPE)


PEG

Oleic acid (-COOH)

Py-PMA

(surface coordinating group)

PMA PEG-PMA


COOH COOH COOH + PEG

Intercalation at room temp Intercalation at room temp Intercalation at rom temp

Ref. 88 63, 87

63

Oleyl amine (-NH2)

dSiO2

Encapsulation

—

Inverse microemulsion

89

PVP

dSiO2

Encapsulation

—

Stöber-like

70, 90

Oleic acid (-COOH)

dSiO2

Encapsulation

—

Inverse microemulsion

63

Oleic acid (-COOH)

mSiO2

Encapsulation

—

Stöber-like

91


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of a lower colloidal stability and hence an enhanced aggregation tendency due to the dynamic nature of the reverse micelles and the presence of excess surfactants, as was shown in earlier reports.96 For example Liu et al.95 have reported that the silicacoated NaYF4 particles from reverse micelles were stable in water for only one day, indicating a tendency to aggregate once removed from the stabilizing micelles which kept them apart during growth. A mesoporous silica shell can be grown around UCNPs using cetyltrimethylammonium bromide (CTAB) as the stabilizing surfactant and organic template for the formation of mesoporous silica in the sol–gel reaction, offers many sites for the accommodation of functional biomolecules and drugs onto the surface of UCNPs.91 Problematic for silica coated UCNPs is the the presence of quenching OH groups in the silica shell close to the UCNPs that can affect the intensity and the spectral distribution of UCL. This effect is more severe in the case of mesoporous silica shells allowing the water molecules to reach the UCNP surface leading to non-radiative relaxation of the excited states of the lanthanides. COMMON METHODS FOR SURFACE GROUP ANALYSIS AND QUANTIFICATION The surface chemistry of nanoparticles largely determines their colloidal stability, biocompatibility (e.g., formation of a protein corona97), and potential toxicity98 as well as further processing steps like the attachment of analyte-responsive dyes or target-specific biomolecules.99-104 Generally, the efficiency of surface functionalization is influenced by the respective conjugation strategy, e.g., direct labeling of surface groups or covalent attachment via linkers such as heterobifunctional PEG molecules, or non-covalent strategies including coordinative or electrostatic interactions.103, 105 The properties of the molecules to be attached (e.g., chemical nature of the reactive group(s), bulkiness, hydrophilicity, and charge), and the characteristics of the particle surface (e.g., charge and morphology), which can affect the accessibility of reactive surface groups are other important factors.103, 106-109 In the last years, the importance of the analysis and quantification of surface functionalities including capping ligands has been increasingly recognized for nanomaterials, 110 particularly for semiconductor quantum dots with chemical constituents like Cd2+ or Pb4+ which are hazardous to health and the environment. Meanwhile a large toolbox of label-free and label-based more or less quantitative methods of varying information depth is available for the analysis of dispersed or dried samples.111 Typical examples of comparably simple methods requiring only inexpensive instrumentation are electrochemical methods such as conductometry and potentiometry for (de)protonable functionalities such as COOH and NH2103 and optical assays104, 106, 112, 113 as well as fluorophore-labeling approaches.103, 107, 114 Methods requiring more sophisticated instrumentation are thermogravimetric analysis (TGA) with mass loss, mass spectrometry (MS) or Fourier Transform Infrared Spectroscopy (FTIR) detection, X-Ray photoelectron spectroscopy (XPS),115 different mass spectrometry techniques such as time-of-flight secondary ion mass spectrometry (ToF-SIMS),115 and quantitative nuclear magnetic resonance spectrometry (qNMR; performed in solution either with dispersed or dissolved particle or with solid samples).102, 116-118 The latter principally absolute method presents our golden standard for surface group quantification as it can provide quantitative information on the total number of a certain chemical functionality. This requires, however, a specific NMR signal of the functional group(s) to be quantified that occurs at a magnetic field where no interferences from the particle matrix occur. Also, qNMR can only be used in the absence of paramagnetic species like Gd3+ in the case of UCNPs. For surface group analysis and quantification, the choice of suitable analytical methods depends on the aim, i.e., whether the total number/concentration of certain functional groups is desired or the number/concentration of accessible functional groups.103 The former, that is relevant e.g. for the colloidal stability and charge of the nanoparticles, can be best obtained with label-free methods or methods using very small reporters like H+ or OH-. In the case of the latter, which is important for the attachment of functional molecules like indicator dyes and/or bioligands, the size, shape, and charge of the reporter must be considered relative to that of the functional molecule to be eventually bound to the nanoparticles.103 The quantification of the density or amount of organic capping ligands such as oleic acid on inorganic particles such as UCNPs can be easiest performed with TGA, measuring the relative mass loss during heating under nitrogen atmosphere as shown by us and the group of T. Hirsch for a series of UCNPs.63 In the case of UCNPs capped with a mixture of organic ligands, however, this provides no information on the relative amount of the different types of ligands present. TGA can also be tricky for porous materials like mesoporous silica119 or nanomaterials with porous surfaces like some UCNPs systems with organically modified silica coatings. In order to obtain a more detailed chemical information on the thermally released species, TGA needs to be combined with FTIR or MS. The determination of the ligand density requires also knowledge of the particle concentration, which can be determined e.g., by inductively coupled optical emission spectrometry (ICP-OES) in the case of UCNPs. The methods developed by us for quantifying different types of functional groups on inorganic and organic nanomaterials like semiconductor quantum dots, laponites, and polymer particles can be also adapted to UCNPs.102, 103, 107, 109, 113, 120 This is currently assessed by us for carboxyl and amino groups using electrochemical titration methods, fluorophore labeling approaches like the Fluram test (for primary amines) and other reactive dyes, here quantifying the amount of unbound dyes in the supernatant after removal of the stringently washed UCNPs, and our newly developed multimodal cleavable probes.120 These versatile and modularly built molecules consist of a reactive group, undergoing a biorthogonal reaction with the surface group to be quantified, a quantitatively cleavable linker like the reductively cleavable disulfide motif, and a multimodal re-


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porter that generates a photometric or fluorometric signal.120 Moreover, these probes contain heteroatoms like sulfur of fluorine for quantification with other analytical techniques like ICP-OES or F19-NMR for straightforward method validation. These probes can be readout when bound to the particle surface and after being cleaved off from the nanomaterial in solution. In the case of the disulfide linkers, the respective thiol groups formed on the particle surface can be quantified with an optical assay or by ICP-OES for a straightforward mass balance. In this context, currently the Ellman´s test, which has been evaluated by us for quantifying thiol ligands and thiol groups on different types of nanoparticles,113, 120 is assessed regarding its suitability for UCNP-based materials. SENSOR SYSTEMS: BASICS CHALLENGES As the focus of our research is the development of UCNPs-based nanomaterials for (bio)chemical sensing, as a first step, we investigated the influence of aqueous media on the photophysical properties and the stability of UCNPs. Here, in addition to the already mentioned challenges regarding the colloidally stability of UCNPs in biologically relevant environments, further problems such as surface quenching by water molecules must be considered.35-37, 121 In addition, there are also reports on the dissolution of the NaYF4 host lattice particularly for UCNPs with coordinatively bound ligands under high dilution conditions.122-125 The latter, that can lead to the release of potentially hazardous fluoride and lanthanide ions, only recently raised concerns regarding a potential toxicity of UCNPs in addition to general nanotoxicity issues.126, 127 Further aspect that should be considered in term of biocompatibility of UCNPs is also the usage of toxic solvents (e.g. octadecene and oleic acid) during synthesis. Therefore, the biocompatibility and potential toxicity of UCNPs need to be fully addressed before their extensive bioapplications in vitro and in vivo. Surface coatings and quenching by water It is well known that water can quench the luminescence of lanthanide ions and lanthanide-doped nanocrystals due to the high energy vibrational modes of its OH-groups favoring multiphonon relaxation.11 We addressed the mechanisms of waterbased quenching of UCL of UCNPs in excitation power density-dependent UCL studies and by luminescence decay measurements. This revealed the importance of shielding the Yb3+ ions from water molecules.35, 121 UCL measurements of both bare and silanized UCNPs in H2O and D2O show that a silica shell provides only little protection against quenching water molecules. However, silica shells can also contain OH groups. Systematic UCL studies with bare UCNPs and different ligand exchange and encapsulation strategies (e.g., silica, DSPE, PMA, Py-PMA, PEG-PMA) in H2O and D2O from our group and the group of T. Hirsch revealed that all surface modifications consisting of an additional layer on top of the original oleate ligand lead to a brighter green luminescence compared to the intensity of the red emission, regardless of the type of ligand used, and thus reduced UCL quenching by water molecules.35, 63 Increasing the thickness of the surface protecting shell might provide better shielding against OH-vibrations, but it simulataneously increases the spatial separation between the UCNPs surface and the tethered molecules and impairs RET efficiency. Therefore, ligand exchange involving the replacement of the primary capping ligands seem to be the more promising strategies to facilitate efficient UC-based RET sensing systems. An alternative are coatings with polymers which can be achieved e.g. by multidentate thiolate-grafting of a polymer on the UCNPs surface81 to yield a thin polymer shell. Really quantitative information on which extent such coatings can provide an efficient surface passivation required for the use of UCNPs in bioanalytical applications is still missing. Degradation The biocompatibility of materials intended for use in medicine is equally important to their functional properties. Bio-compatible NPs must be nontoxic and chemically and colloidally stable in vivo, with long blood-circulation times, and they should be excreted from the body after treatment. Although UCNPs composed of fluoride materials have been formerly considered to be chemically stable, a low degree of particle constituting ion dissolution from cubic-phase nanoparticles has been observed in water.124, 128 The hexagonal-phase NaYF4 nanocrystals have been considered to be thermodynamically more stable than the cubic-phased crystals, but still fluoride ion dissolution along with the other constituents of the hexagonal NaYF4:Yb3+,Tm3+ particles has been observed in water.124 The group of T. Soukka studied the disintegration of hexagonal NaYF4:Yb3+,Er3+ UCNPs coated with the polymer PAA and with thin and thick silica shells in aqueous media at different UCNPs concentrations.123 This revealed that only the thick silica shell slowed down the dissolution of UCNPs. Our group presented a stability screening of differently sized hexagonal NaYF4:Yb3+,Tm3+ UC nanoparticles in the bioanalytically relevant buffer PBS (pH 7.4) at different temperatures.122 The release of fluoride- ions that was determined potentiometrically, upon storage and aging in PBS buffer was more pronounced for smaller particles due to the greater surface/volume ratio and for higher temperatures. UCNPs aging also resulted in changes of the UCL and downconversion (DC) luminescence intensities and lifetimes. Correlation of fluoride ion release with lifetime measurements and the determined integrated decay curves revealed that the luminescence decay of the 800 nm UC emission of Tm3+ can present a suitable parameter for the monitoring and screening of the stability of Tm3+-based UCNPs.122 For other UCNPs, also the lifetime of the Yb3+ emission seems to be well suited for this purpose.


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General studies of RET systems The origin of RET in UCNPs-based core-shell systems is still discussed controversially, yet typically referred to non-radiative Förster resonance energy. The most frequently used RET acceptors for UCNPs are organic dyes because they offer substitution pattern control of their absorption and fluorescence properties and particularly the spectral position of their absorption and emission bands. Moreover, there exists meanwhile a large toolbox of conventional and stimuli-responsive organic dyes with reactive groups for their covalent attachment to ligands and surface groups. In addition, many dyes contain functional groups like carboxy and sulfonate groups that can directly coordinate to lanthanide ions.48 A prerequisite for a successful RET is the spectral match between the absorption band of the energy acceptor and one of the emission bands of the UCNPs. In addition, an efficient RET requires a short distance between energy donor and acceptor. Hence, as previously mentioned, also UCNPs size, chemical composition, and particle architecture, particularly the thickness of an often used inactive surface passivation shell, present key paFigure 2. A) Schematic representation of the two-step ligand48, 49, 129, 130 Also, the energy acceptor exchange process assisted by NOBF4. ; B) FRET efficiencies rameters for RET efficiency. of organic dye-capped NaYF4 (20% Yb,2% Er) UCNPs density matters. Systematic studies from our group and the group of T. Hirsch dispersed in DMF with and without an additional inactive shell consisting of NaYF4. This was done for the model dyes revealed a size dependence of the RET efficiency for bare rose bengal (RB) and sulforhodamine B (SRB). (Reprinted NaYF4:Yb3+,Er3+ UCNPs with sizes between 10 nm and 43 nm with permission from Reference 48 Copyright 2017 American decorated with surface-adsorbed dyes in an aprotic organic solChemical Society). vent.48 The surface functionalization procedure employed is illustrated in Figure 2A. It relies on the exchange of the initial oleic acid capping ligand first for weakly coordinating BF4-. NOBF4 is then replaced by an organic dye bearing carboxy and/or sulfonate functionalities. This procedure was developed by the group of T. Hirsch to bring the UCNP donor and the RET accepting dye molecules in close neighborhood. Subsequently, we studied the influence of UCNP size and hence surface-to-volume ratio on RET efficiency studied for differently sized core and core-shell UCNP (NaYF4: 20% Yb3+, 2% Er3+) and different labeling densities of the model dyes rose bengal (RB) and sulforhodamine B (SRB).48 The absorption bands of both dyes overlap with the green UCL. Time resolved fluorescence measurements of these dye-functionalized UCNP revealed a considerable shortening of the lifetime of the green emission of the UCNP donor as a clear hint for RET, with the size of this reduction depending on UCNP size and particle architecture. As the UCNPs size decreases, i.e., at larger surface-to-volume ratios, two competing processes occur. On the one hand, an increasing percentage of the UCNPs Er3+ ions become involved in RET, as the distance between in increasing number of Er3+ and the surface-bound dyes are now within the Förster radius, thereby increasing RET efficiency. However, on the other hand, the surface-to-volume ratio increase leads to more luminescence quenching at the UCNP surface, which reduces the donor quantum yield and lifetime and hence, also RET efficiency. Utilizing time-resolved UCL measurements for RET studies of a size series of bare (unshelled) UCNP with sizes of 21 nm, 26 nm, and 31 nm, our group and the Hirsch group observed the strongest shortening of the donor lifetime for 21 nm-sized UCNPs, yielding a RET efficiency of 60 %.48 Moreover, as the RET efficiency correlates with the quantum yield of the donor, the group of T. Hirsch grew thin inactive passivation shells of about 1 nm thickness around differently sized UCNP cores to enhance their quantum yield. Subsequently, they together with our group performed similar RET studies with a set of core and core-shell UCNPs. The results, shown in Figure 2B highlight the influence of such an inactive shell on the resulting RET efficiencies. For particles < 17 nm, a thin shell improved the RET efficiency by up to 40 %. This underlines the strong influence of surface deactivation processes on UCL of small UCNPs. For larger UCNPs, e.g., the previously as most efficient ones identified 21 nm-sized UCNPs, such a thin shell did not lead to a higher RET efficiency. Mély´s group129 presented a similar study with assembled UCNP–organic dye RET systems in aqueous dispersions and investigated luminescence decays and spectra with varying UCNPs sizes and quantities of acceptor dyes grafted onto the surface with the aid of a polymer shell. They proposed a Monte Carlo simulation for the quantitative prediction of RET-behavior of UCNP-organic dye systems. Only recently, the group of M. Kumke presented a study of four different types of core and core-shell NaYF4-based UCNPs co-doped with Yb3+ and Tm using the dye methyl-5(8-decanoylbenzo[1,2-d:4,5-d´[bis([1,3]dioxole)-4-yl)-5-oxopentanoate (DBD-6) as acceptor. Fluorescence intensities and decay kinetics were measured for assessing RET.131 This study revealed the influence of the UCNP architecture on the UCL features and the energy transfer to the dye in a similar way as reported by


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us and the group of T. Hirsch for UCNPs co-doped with Yb3+ and Er3+.48 UCNPs with an inert shell showed the strongest luminescence, but their RET efficiency was the lowest (17 %). UCNPs with Tm3+ dispersed only in the shell revealed the highest RET efficiencies of about 51 % despite the reduced luminescence efficiency due to surface quenching. Wang et al.59 reported that the RET efficiencies in core-shell particles of the type NaYF4:Yb3+,Er3+@NaYF4 can be improved by tuning the thickness of the shell layers and identified a critical shell thickness of 6 nm for optimal RET performance for this particle size. Ding et al. 132 investigated the dependence of the relative contribution of RET and reabsorption in dyedecorated UCNPs on the thickness of an inert NaYF4 shell between the active UCNP core and dyes. By optimizing the dopant concentrations and the core–shell structure for higher excitation power densities, Drees et al. 133 observed enhanced UC- emission as well as a strongly increased sensitized acceptor fluorescence. In addition, Hildebrandt´s group49 analyzed the efficiency of RET between Er3+ ions distributed over either the core or the shell of UCNPs and Cy3.5 dye acceptors. The RET phenomenon was also studied for UCNP-quantum dot (QD) system.134-137 Mattsson et al. demonstrated for the first time that UCNP-to-QD RET is possible with protein conjugated UCNPs.138 By combining the UCNPs with QD RET acceptors with very strong spectral overlap with the UCNP donors, they can overcome the main drawbacks of UCNPs for RET, namely the very low UCL quantum yields of the UCNP RET donor. These nanobiosensors were used for the quantification of biological recognition, here biotin-streptavidin interactions, at low nanomolar concentrations. Rubio-Retama et al.139 used time-resolved fluorescence spectroscopy to analyze the UCNP-to-QD RET process as a function of UCNP-QD distance by varying the

Figure 3. Main resonance energy transfer (RET)-based detection strategies utilizing upconversion nanoparticles and analyteresponsive chromophores illustrated on Yb3+, Er3+-doped UCNP in combination with an indicator: A) Fluorogenic sensor with analyte-responsive fluorescence of the indicator induced by LRET from the UCNP; B) Indicator impacts UCNP emission acting as acceptor for LRET. Addition of analytes cuts the linkage between UCNPs and acceptors, leading to the recovery of the emission.; C) and D) colorimetric indicator, showing a significant change (either intensity or wavelength) after reacting with analyte, impacts UCNP emission by reabsorption (inner filter effect). In this case an analyte-responsive fluorescence of the indicator can occur.


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thickness of the silica shell separating the UCNPs and QDs. They present a model to estimate the contribution of every Er3+ ion to the final RET response which enables to develop different strategies for improving RET efficiency. Recently, Capobianco´s group reported the correlation of distance estimations and time-resolved RET analysis in UCNP-dye nanostructures and underlined the importance of distinguishing between the photoluminescence properties of the emitting lanthanide ions located at or near the UCNP surface and in the more shielded core.140 For this study, they used tetragonal LiYF4:Tm3+/Yb3+ UCNPs surface-modified with a lipid bilayer incorporating photochemically switchable azobenzene-modified lipid molecules. which can absorb the blue Tm3+ emission. This can then trigger the release of payload incorporated into the lipid bilayer. As a proof-of-concept for the functioning of this nanoconstruct as a potential light addressable drug delivery vehicle, the dye Nile red was encapsulated in the lipid bilayer as a model for hydrophobic drugs. Excitation of this nanocarrier at 980 nm lead to the release of Nile Red as determined by monitoring fluorescence spectra of the dye. Time-resolved spectroscopy on the system revealed that the energy transfer mechanism is predominantly radiative (reabsorption) with a minor contribution of nonradiative RET. Generally, an effective RET is expected to decrease the intensity of the UCL band involved in RET. The excited state population of the different energy levels of the emissive lanthanide ions, however, reveal a complex interplay, which can complicate this situation. Moreover, the reliable identification of RET processes in UCNP-dye systems is often hampered by the relatively large UCL contribution of lanthanide ions in the UCNPs center, that do not participate in RET, because they are too far away from the RET acceptors at the UCNPs surface. This can lead to a very weak quenching of the UCL intensities and a strong UCL background signal, reflecting the contribution of unquenched relative to RET-quenched lanthanide emitters.48, 49 Thus, often time-resolved measurements of UCL of the UCNPs donor are utilized as a clear hint for the occurrence of RET as shown e.g. by us and the group of T. Hirsch.48 Over the last years, RET has been successfully used as detection tool for a variety of (bio)chemical analytes. One strategy for achieving chemoresponsive UC nanomaterials is the combination of UCNPs with fluorogenic indicators with analyte-responsive fluorescence induced by RET from the UCNPs to the dye, as shown in Figure 3A. As the RET efficiency is highly dependent on the distance between the donor and acceptor, UCNPs-based nanosensors can also be designed in the opposite manner, with a target-induced increase in donor-acceptor distance, leading to the recovery of UCL as presented in Figure 3B. Another option is to combine UCNPs with colorimetric indicators or colorimetric and fluorogenic molecules. In this case, the sensing mechanism is based on the change (either intensity or wavelength) of the absorption of the indicator after reaction with the analyte (Figure 3C and 3D). Thereby, the resulting inner filter effect modulates the intensity of this emission in dependency on the analyte concentration. In the case of a fluorogenic indicator, also an extra fluorescence band can appear. In this case, the distance between UCNPs surface and indicator is not relevant. Both strategies, triggering or blocking the RET process, thus attenuating or recovering the UC emission can be used for sensing of specific analytes. SENSOR SYSTEMS: APPLICATIONS In the past years several UCNP-based nanoprobes for the determination of protons, metal ions, anions and gases have been investigated. As UCL are per se is only responsive to changes in temperature52, 53 and not to chemical stimuli, the design of UCL nanosensors requires the combination of UCNPs with suitable sensitive indicator dyes that can recognize the respective target and signal its presence by specific changes in its optical properties. This can then result in a modulation of the fluorescence intensity of certain UCL bands of the UCNPs, thereby making UCL dependent on the targeted ion concentration.

Table 3. UCNP-based probes used for intracellular sensing of pH. RET donor

RET acceptor

Surface functionalization

Detection strategy

Cell type

Ref.

NaYF4: Yb3+,Er3+

pHrodo Red

Silica shell

ratio of the emission intensity I590/550

HeLa

141

NaYF4: Yb3+,Tm3+

Fluorescein

Silica shell

Recovery of UC-blue emission upon protonation; ratio of the relative emission intensity I519/465

HeLa

142

NaYF4: Yb3+,Tm3+

Xylenol orange

Silica shell

Quenching of UC-blue emission upon protonation; ratio of the emission intensity I450/646

HeLa

143

NaYF4: Yb3+,Er3+

pHrodo Red

Organic polymer (PEI)

ratio of the emission intensity I590/550

MDA-MB231

144

NaYF4: Yb3+,Tm3+

Fluorescein

Organic polymer (PEI)

Recovery of UC-blue emission upon protonation; ratio of the relative emission intensity I475/645

QBC939

145


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Sensing of pH The combination of UCNPs with pH-sensitive dyes (indicators) leads to optical pH nanosensors which can be used for intracellular pH sensing and maybe also for in vivo imaging or for the fabrication of sensor films by incorporation in polymer matrices.146 The pH value is one of the basic parameters characterizing biological systems because it impacts structure and function of all present biologically active macromolecules and ionic species. The cytoplasmic pH regulates the capability of ion transport, cell proliferation, and migration. An abnormal acidic pH is characteristic of cellular dysfunctions such as apoptosis or cancer. A reduced pH between 6 and 4 occurs also in certain cellular compartments such as endosomes or lysosomes.147 Thus, using pH sensitive nanoprobes in biological samples requires, both colloidal and chemical stability at acidic conditions. Surface protonation has a great impact on the surface charge and the zeta potential of the particles and can induce their aggregation and precipitation. Moreover, as outlined above, the decomposition of UCNPs represents another major problem if they are exposed to acidified environments for a longer period of time. The functionalization with pH indicator dyes can be achieved optionally via simple ligand exchange procedures or by the attachment to small organic linker molecules on the particle surface. For example, Boyer et al. have shown that NaYF4 UCNPs can be modified with PEGphosphonates by a ligand exchange procedure.85 The coated particles were applied for cellular imaging. Bisphosphonate alendronic acid was used as reactive linker molecule to attach fluorescein isothiocyanate (FITC) on the particle surface via amide coupling.72 Esipova et al. coated NaYF4:Yb3+,Er3+ nanoparticles with polyglutamic porphyrin-dendrimers.78 The porphyrin unit exhibits a pH dependent absorption at 660 nm. This results in a pHdependent change of the red/green emission ratio in a pH range from 8.5 to 5.0. In this case, an emission-reabsorption inner filter mechanism is assumed. The polyanionic dendrimers enhance the dispersibility of the nanoprobes. Nevertheless, it is questionable whether phosphate- or carboxylic acid-based monomolecular surface layers are really stable in acidic media of high ionic strength, that contain also a high concentration of concurring phosphate anions, which are typical conditions in biological samples. Hence, for intracellular sensing and imaging amino-functionalized polymer (e.g. PEI) or silica coatings have been used to enable both chemical protection of the crystals and covalent attachment of dye molecules. Table 3 shows examples from the literature using UCNP-based probes for intracellular sensing of pH. In the first approach for referenced intracellular pH sensing utilizing UCNPs, we conjugated the commercial pH indicator pHrodo Red (as NHS ester) to hexagonal NaYF4:Yb3+,Er3+ crystals with axes of 28 × 36 nm, coated with an aminosilane shell of ~ 3 nm thickness. 141 The pH-dependent ratiometric signal is most likely generated by RET from the UCNPs to the fluorogenic indicator dye. A referenced read out can be achieved by measuring the ratio of the green UCL emission (550 nm) and the sensitized red emission of the indicator emission (590 nm), which rises with decreasing pHs. As comparatively low and constant excitation powers have been applied, the excitation-

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Figure 4. A) Scheme of ratiometric core-shell UC nanoprobes for intracellular pH sensing; B) Comparison of the ratiometric pH response of aminosilane coated (black squares) and PEI coated (red squares) UCNPs conjugated to pHrodo Red in cell culture medium. Excitation at 980 nm.; C) Ratiometric imaging of pH probes reveals their localization in three types of microenvironment. Panel A shows localization of UCNPs by means of its green emission (550 nm) using 980 nm excitation, panel B sensitized UC-RET emission from pHrodo Red, panel C shows outlines of the cell in transmitted light, and panel D shows an overlaid ratiometric image of pH-nanoprobes with different ratio depending on the localization. Different intensity ratios indicate localization of the nanoprobes in extracellular medium (ctrl), small endosomes, large endosomes, and lysosomes. Scale bar 10 μm. (Reprinted with permission from Reference 144. Copyright 2017 American Chemical Society).


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power density dependence of the UCL, that could affect the ratiometric sensor response, can be neglected The UCNPs were efficiently internalized by HeLa cells by endosomal uptake and the green reference signal as well as the red pH-dependent signal could be detected. Both individual particles and particle clusters were visible in the cells. An increased red to green ratio was observed in case of the particle clusters located in the acidic endosomes. The synthesis of the nanoprobes and the fluorescence microscope experiments were carried out in cooperation with the Department of Biochemistry/Biotechnology and the Laboratory of Biophysics at the University of Turku. Later, the silica coated sensor system was varied by replacing Er with Tm as emitter and attaching fluorescein142 (using FITC as reactive dye) as indicator or xylenol orange143 (via amide coupling) as indicator. In the second step we used branched PEI coatings to provide a higher number of reactive primary amino groups for the conjugation of the pHrodo Red dye (Figure 4).144 The PEI coating was also expected to improve the colloidal stability of the particles. The highly positive zeta potential of the PEI shell reduces particle aggregation, whereas the zeta potentials of the aminosilane coated UCNPs was only slightly negative at neutral pH and reached zero around pH 5. In both core–shell systems a decrease of the luminescence lifetime of the green UC emission was observed after the conjugation of the dye. The shorter lifetimes indicate that RET is at least partly involved in the sensitization of the indicator dye (e.g. a RET efficiency of 38 % was calculated in the case of the silica coated particles), but other mechanisms such as UCL reabsorption cannot be excluded. The limited RET efficiencies lead to rather low ratios between the sensitized sensor signals and the reference signals. Much stronger reduction of the luminescence lifetime of the 475 nm UC emission and therefore a higher RET efficiency (around 78 %) has been reported for NaYF4:Yb3+,Tm3+@PEI in combination with fluorescein. 145 However, we could achieve two improvements by using PEI compared to the silica coated particles: the enhancement of the cellular uptake of the nanoprobes and higher signal to reference ratios, due to the increased labeling density of the pH-sensitive dyes. The nanoprobes were internalized after 16 h by all cells under observation. Again, an intracellular compartmentalization in endosomes or lysosomes was observed, but PEI promotes also endosomal escape. Therefore 5% of the nanoprobes resided in a neutral microenvironment. Sensing of metal ions Heavy metal ions, such as Hg+ are known as extremely toxic chemicals which can accumulate in human bodies leading to a severe nervous system damage. Selective detection and bioimaging of these ions are of great importance for the biological systems. On the other hand, metal ions such as Fe3+, Cr3+, Cu2+, Zn2+, and Ca2+ are essential trace elements and play a vital role in the physiology and biochemistry of organisms and the cell. The imbalance of these metal ions is implicated in various disorders. 148-150 Monitoring the level of these ions is thus important for health and environmental concerns. UCNPs-based nanosensors can present useful tools for the highly sensitive and selective detection of these ions in vitro and in vivo. Many examples are summarized in Table 4. Calcium ions Liu and co-workers151 constructed an efficient UC nanoprobe to detect and image Ca2+. This probe is composed of an inert core with an emissive inner shell and inert outer shell and the commercial calcium indicator Fluo-4 attached directly on the surface via the carboxylic groups of the aminopolycarboxylic acid (BAPTA) moiety (Figure 5A). In the absence of Ca2+, the fluorophore Fluo-4 is coordinated to lanthanide surface atoms of the UCNPs and acts as an energy acceptor leading to 80% quenching of the green UCL band. Upon addition of Ca2+, Fluo-4 coordinates the Ca2+ ions and is removed from the UCNP surface as indicated by the recovery of UCL. Copper ions Huang et al.152 constructed a RET-based UCNPs probe for Cu2+ by assembling sulfonated porphyrins onto SiO2-coated UC nanorods. The addition of Cu2+ results in a significant red shift of the maximum absorption of the porphyrin from 515 to 545 nm, due to the formation of a complex with Cu2+. As the maximum absorption at 545 nm overlaps with the UC emission at 545 nm of the β-NaYF4:Yb3+,Er3+,Gd3+@SiO2 nanorods, RET occurs between the nanorods and the TSPP-Cu2+ complex, resulting in a decrease of UCL at 545 nm. The red UCL emission was used as reference. Shi and co-workers153 employed a similar strategy to detect Cu2+ by grafting a rhodamine B derivative onto UCNPs coated with mesoporous silica and applied this nanosensor for the monitoring subcellular distribution of Cu2+ in living cells (Figure 5B). Addition of Cu2+ causes a ring-opening of the rhodamine-B derivative, causing color change from colorless to pink and an enhancement of the fluorescence emission. Upon 980 nm excitation, the intensity of green UC emission decreased gradually while a new weak emission appeared at 580 nm which increased with the increasing concentration of Cu2+, corresponding to the RET from UCNPs to the RBH–Cu2+ complex. Chromium ions Liu et al. 154 reported a ratiometric fluorescence sensor for Cr3+ utilizing RET from LiYF4:Yb3+, Ho3+,Ce3+ UCNPs with a LiYF4 shell to rhodamine B hydrazine embedded into an amphiphilic surface layer, involving the green emission of the UCNPs. The reaction of Cr3+ ions with the dye results in a color change from green to yellow to orange and to red with increasing analyte concentration, that can be detected by naked eye.


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Iron ions Wei et al. 155 reported a nanoprobe for the detection of Fe3+ in live cells based on the attachment of a Nile red derivative onto the surface of PEG-modified Gd3+-doped core-shell UCNPs. Upon addition of Fe3+ ion, the emission intensities of green UCL, red UCL and NIR UCL all gradually decrease, which is attributed to the increased spectral overlap between UCL emission and the broad absoption band of Nile red, which is located in the 450-650 nm range. Both green and red UCL emission intensity at 540 nm and at 654 nm decreased linearly with the amount of Fe3+ from 0 to 30 μM and the detection limit was calculated to be 89.6 nM. Zinc ions Peng et al.156 constructed a sensor for the detection of Zn2+ by assembling a positively charged Zn2+ -indicator dye on the surface of PAA-coated UCNPs electrostatically. In the absence of Zn2+ the absorption band of the dye matches with the Tm3+- emission band at 475nm, resulting in strong quenching of the blue UC-emission by reabsorption. Upon binding of Zn2+, the dye undergoes a blue shift in absorption which results in the recovery of the blue UCL emission band. Hu et al. developed multifunctional UCNPs loaded with an organic fluorescent probe for the in situ detection of endogenous Zn2+ and the control of Zn2+-induced singlet oxygen generation for guided photodynamic therapy. The reported theranostic nanoprobe is based on the combination of two successive radiative energy-transfer processes under NIR laser irradiation: (i) from UCNPs to Zn2+sensing molecule L and(ii) from the complex [Zn2+/L]. to activate photosensitizer RB by absorbing green fluorescence of complex [Zn2+/L]. The activated RB can successively produce 1O2 to trigger photodynamic therapy. Mercury ions Different strategies for the development of UCNP-based Hg2+ nanosensors have been reported. This includes nanosensors, combining UCNPs with Hg2+-sensitive rhodamine dyes which do not absorb at wavelengths where UCL occurs and are non-

Figure 5. Schematic illustration of the synthetic procedure and the proposed sensing mechanism of A) Ca2+ (Reprinted with permission from Reference 151. Copyright 2015 American Chemical Society); B) Cu2+ + (Reprinted with permission from ref 153. Copyright 2016 Royal Society of Chemistry); C) Hg2+ (Reprinted with permission from ref 161. Copyright 2015 Royal Society of Chemistry); D) Cysteine and CN- (Reprinted with permission from Reference 166. Copyright 2014 American Chemical Society).


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Table 4. Overview of UC-based nanoprobes for detection of different metal ions. Analyte

Energy donor

Ca2+

NaYF4:Yb3+, Er3+ @NaYF4

Energy acceptor

Surface functionalization

Detection strategy

Ref.

Fluo-4

Ligand exchange of oleic acid with Fluo-4; direct dye attachment

Recovery of UC-green emission upon analyte binding

151

NaYF4@

Cu2+

β-NaYF4: Yb3+,Er3+,Gd3+

Meso-tetra(4-sulfonatophenyl)porphine dihydrochlorid (TSPP)

Silica shell

Quenching of UC-green emission upon analyte binding

152

Cu2+

NaYF4: Yb3+,Er3+@Na YF4

Rhodamine B hydrazine

Mesoporous silica shell


153

Cr3+

Ce3+-doped LiYF4:Yb3+, Ho3+@LiYF4

Rhodamine B hydrazine

Encapsulation with amphiphilic polymer

NaYF4: Yb3+,Er3+,Tm3+ @NaGdF4

Nile red

PEGylated

derivative

amphiphilic polymer

Zn2+

NaYF4:Yb3+, Tm3+@NaYF4

Zn2+ responsive dye

Organic polymer (PAA)

Zn2+

NaGdF4: Yb3+, Tm3+@NaYF

Zn2+ responsive dye

Amphiphilic molecule

Hg2+

β-NaYF4: Lu3+,Gd3+, Yb3+, Er3+

Fe3+

Hg2+

Hg2+

NaYF4: Yb3+,Er3+ NaYF4: Yb3+,Er3+,Tm3+

Hg2+

NaYF4: Yb3+,Er3+,Tm3+

Hg2+

NaYF4:Yb3+, Er3+,Tm3+

Yb3+,Er3+,Tm3+ @NaGdF4

visual color change as function of analyte

154


155

Recovery of UC-blue emission upon analyte binding

156

Decrease of blue UCL intensity upon analyte binding

162

Quenching of UC-green emission upon analyte binding; Rhodamine B-derivative

Silica shell

Rhodamine B thiolactone

Polyethylene-blockpoly(ethylene glycol) + 2dioleoyl-sn-glycero-3phosphoethanol-amine-N(succinyl) polymer film


157

[RuII(bpy)2(thpy)]PF6

Amphiphilic polymer-coating


158

Ruthenium complex N719

Ligand exchange of oleylamine with the ruthenium complex N719; direct dye attachment


159

Thiazole derivative dye

Modification of oleic acid with γ-cyclodextrin

Ruthenium complex

Mesoporous silica with additional PEI layer

NaYF4: Hg2+

Quenching of UC-green emission upon analyte binding;

new emission band from chromophore upon analyte binding

Recovery of UC-green emission upon analyte binding; ratio of the UCL (I540/I803) as the detection signal Recovery of UC-green emission upon analyte binding

142

160

161

emissive. Upon addition of Hg2+- ions, the absorption of the dye is shifted to 550 nm, thereby matching with the green UCL band, and its emission is turned on. Examples of such system were presented by Li et al.157, who incorporated Rhodamine B thiolactone into a thin amphiphilic polymer film on the UCNPs surface and by Wu et al. 142, who incorporated a rhodamine B derivative into a silica shell coated onto UCNPs. The second group of UC-based Hg2+ sensors is based on the combination of UCNPs with metal-complexes or dyes which undergo a blue-shift in the absorption upon binding with Hg2+, leading to a decrease in the spectral overlap between the green UCL of the UCNPs and the absorption band of the acceptor; this results in a recovery of the green UCL.158,159,160,161 An example for this detection strategy is illustrated in Figure 5C. 161


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Oxygen sensing The development of improved NIR probes for the measurement of intracellular oxygen and imaging of tissue oxygen supply in vivo is of considerable interest for tumor diagnostics and the development of cancer therapies. Several nanosensors with oxygen-sensitive dyes as RET acceptors have been meanwhile presented in the literature. These are either based on Tmdoped UCNPs in combination with ruthenium complexes, particularly ruthenium(II)-tris(4,7-diphenyl-1,10-phenanthroline) [(Ru(dpp)3]2+ or on Er-doped UCNPs combined with platinum porphyrins. Liu et al. synthesized a triethoxy-silane derived ruthenium complex [Ru(phen)2phen-Si]Cl2, which was converted with tetraethoxysilane to form an organically-modified silica shell on the UCNPs surface.163 These particles were then coated with aminopropyltriethoxysilane to introduce amino groups on the surface to improve the colloidal stability and provide anchor groups for bioconjugation. However, in this work the ruthenium complex was directly excited at 460 nm and not via RET. Another O2-sensitive ratiometric RET sensor was obtained by utilizing a mesoporous silica shell to entrap a positively charged ruthenium complex in the negatively charged silica pores by electrostatic forces.164 A high RET efficiency was achieved using these hollow structures for dye loading, as indicated by the significant quenching of UCL. The absorption of the ruthenium complex around 460 nm overlaps with the two UCL bands at 450 and 475 nm of the UCNPs and the RET-excited red luminescence of [Ru(dpp)3]2+ at 613 nm is strongly quenched by oxygen. Although the linearity and sensitivity of the oxygen response of this nanosensor is restricted compared to directly excited free [Ru(dpp)3]2+, this nanosensor could signal various oxygen levels in cells. Moreover, also hypoxic conditions in the brain of zebrafish embryos could be detected. An alternative approach relied on NaYF4:Yb3+,Er3+ UCNPs and a platinum(II) octaethyl-porphyrin as oxygen-sensitive probe.165 In this work, the hexagonal NaYF4 crystals were modified with an amphiphilic silane with a 18C alkyl chain. This forms a thin self-assembled monolayer on the spherical UCNPs after silane condensation. Then the platinum porphyrin was loaded on the particles via hydrophobic interactions together with paclitaxel, an anticancer drug used in chemotherapy. Also in this case, oxygen sensing was only done by direct excitation of the platinum porphyrin at 532 nm and not via excitation of the UCNPs and a RET process or reabsorption. Therefore, the proof is still missing that these UCL mediated RET approaches can be utilized for oxygen sensing. Other examples for analyte sensing Cystein/ Homocystein The group of Li presented a UCNPs-based nanoprobe for the amino acids cystein/ homocystein.166 They utilize yolk-shell nanostructures with UCNP as movable core and a mesoporous silica shell with a colorimetric reporter system occupying the hallow cavities (Figure 5D). Quantitative detection of cysteine or homocysteine is based on RET from the UCNPs to the reporters, where the UC luminescence is decreased upon analyte binding. Thereby, the UCL intensity ratio at 540 to 800 nm decreased with the addition of cysteine. Cyanide Cyanide (CN–), a highly toxic anion with a strong affinity for transition metals (e. g. Fe3+) and deleterious effects on many biological functions, is applied in numerous chemical processes, such as electroplating, plastic manufacturing, gold extraction, tanning and metallurgy.167 There are several reports on highly selective and sensitive CN− sensors based on UCNPs in combination with chromophoric chemodosimeter-type molecules. A chemodosimeter is a system that changes its optical properties irreversibly upon interacting with an analyte.29 The sensing mechanism in these systems utilizing Ho3+ or Er3+ -doped UCNPs relies on the RET-promoted strong quenching of the green UC emission by the CN–-sensitive molecules with significant absorbance at around 540 nm, which overlaps with the green emission of the Ho3+ or Er3+ activator ions. When adding CN−, the absorption of the indicator decreases gradually, resulting in a less efficient RET. Hence, the green UC emission of NPs is stepwise recovered. Using this UCL-turn-on strategy, the group of Li reported a cyanide nanosensor consisting of a CN–-responsive chromophoric iridium(III) complex embedded into an amphiphilic polymer shell covering the surface of UCNPs168 or loaded into a porous silica shell of yolk−shell structured nanoparticle with NaLuF4:Yb,Er,Tm as the inner yolk.166 Zhao et al. 169 constructed a UC-based nanosensor using the CN-sensitive organic chemodosimeter phenothiazine–cyanine deposed onto the γ-cyclodextrin cavity of surface-modified UCNPs. CONCLUSION AND OUTLOOK The majority of recent research and development activities in the area of UC nanomaterials is mainly focused on bio-imaging applications. This is mainly triggered by the potential of these NIR-excitable materials to minimize the autofluorescence background and scattering and their potential as spectral converters, providing several luminescence bands in the UV/vis/NIR/SWIR. In this respect, an increasing number of concepts for the rational tuning of UCNP properties have emerged that focus on the identification of particle architectures including chemical composition and dopant ion concentrations with a optimum for


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applications at different excitation power densities as utilized in assays, for sensing, or high resolution optical microscopy. This includes the development of multi-functional nanoprobes by combining core-shell UCNP systems and stimuli-responsive dye molecules. This can considerably expand the areas of applications of lanthanide-doped UCNPs and paves the way for chemical UC nanosensors. Alternatively, this strategy can also be exploited for dye-based sensitization of UCL to boost UCNP brightness, here utilizing strongly absorbing dye molecules as donors and UCNPs as acceptors, respectively.11 The growing interest in the development of UC nanoprobes for chemical sensing is demonstrated by the increasing number of publications on this topic in the past years. As summarized in this article, several approaches have been meanwhile reported for ratiometric dual wavelength sensors which can be intrinsically referenced, thereby exploiting the multi-color nature of UCL. Examples are the sensing of temperature, pH, oxygen and ionic species summarized in this article. Except for temperature sensing, the sensor principle is based on a RET process between the UCNP core and a responsive (indicator) dye attached on the UCNP surface. The fundamental challenges of such core/shell systems are several-fold. First it must be considered that the photophysics of UCNPs are governed by the complex interplay of several energy transfer processes involving a multitude of energy levels of the different lanthanide ions constituting the respective UCNPs. The efficiency of these processes depends on inter-ion distances and hence crystal lattice, dopant concentration, surface quenching effects (and hence UCNP size), and excitation power density. The latter can affect the desired ratiometric sensing, as the ratio of the green and red emission bands depends on this parameter as has been shown by us and other groups in several publications. 35-37, 63 Hence, reliable ratiometric sensing can be achieved only at more or less constant excitation power densities or in a low excitation power density range. This is often disregarded in the design of UCNP-based nanosensors. Moreover, this implies restrictions on the excitation power range exploitable for sensing as e.g. certain UCNP sizes and excitation power densities favor the red over the green emission particularly in UCL-quenching by water as demonstrated by us. Moreover, for distance-dependent RET, generally a small distance between the NP surface and the dye is required for a high RET efficiency. This is a prerequisite to obtain usable signal responses and sufficient luminescence intensities from the indicator dyes in the UCNPbased sensors. On the other hand, compact and tight surface shells are needed to protect the UCNP core from surface quenching processes and for some applications, also from chemical decomposition. Furthermore, as only lanthanide ions located at distances within or close to the Förster radius can participate in efficient RET, the particle size and particle architecture must be optimized to prevent a too large UCL background of unquenched emitting lanthanide ions in the inner core. Polymer and silica shells can be prepared with thicknesses of a few nanometer and enable both, a close proximity of the dyes to the surface and some protection of the core. However, silica or organic polymer shells cannot completely exclude UCL quenching by water. Particularly, small organic ligands with reactive groups enable the direct attachment of the indicator on the UCNP surface with very close proximity. In this case, an efficient RET can be achieved, but problems can arise regarding the stability of such shells in complex biological sample matrices. For example, changing pH or high ionic strength can lead to a detachment of the ligand together with the coupled indicator dye from surface. In this respect, there is still a considerable need in systematic studies in optimum particle architectures and surface chemistries. For example, more multi-shell particle architectures with differently doped active and inactive shells and different sizes need to be systematically assessed in conjunction with surface chemistries that enable a close distance between the lanthanide donors and the dye acceptors and provide sufficient protection against dissolution in application-relevant media. Such systems need to be studied with quantitative methods such as integrating sphere spectroscopy38, 39 and with time-resolved luminescence measurements at application-relevant excitation power densities to identify optimum systems for typical sensing and imaging applications. Moreover, as recently shown by the group of M. Haase together with us,37 the systematic optimization of UCNP synthesis and shell growth can provide an additional handle to improve the efficiency of RET-based UCNP sensors by improving the donor quantum yields.

ACKNOWLEDGMENT URG gratefully acknowledges fruitful collaborations with the research groups of T. Hirsch (University of Regensburg), M. Haase (University of Osnabrück), and T. Soukka (University of Turku) as well as financial support by the German Research Council (DFG; grants RE 1203/18-1 and RE 1203/20-1 (M-Eranet project NANOHYPE)). Moreover, COST Action CM1403, the European upconversion network from the design of photon-upconverting nanomaterials to (biomedical) applications, is gratefully acknowledged. MS thanks T. Soukka and T. Näreoja (University of Turku) as well as DFG for a Heisenberg fellowship. EA and MS gratefully acknowledge also financial support by DFG (grant SCHA 1009/17-1). REFERENCES 1. Kuningas, K.; Päkkilä, H.; Ukonaho, T.; Rantanen, T.; Lövgren, T.; Soukka, T., Upconversion Fluorescence Enables Homogeneous Immunoassay in Whole Blood. Clin. Chem. 2007, 53 (1), 145-146. 2. Nyk, M.; Kumar, R.; Ohulchanskyy, T. Y.; Bergey, E. J.; Prasad, P. N., High Contrast in Vitro and in Vivo Photoluminescence Bioimaging Using Near Infrared to Near Infrared Up-Conversion in Tm3+ and Yb3+ Doped Fluoride Nanophosphors. Nano Lett. 2008, 8 (11), 3834-3838.


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AUTOR INFORMATION Elina Andresen studied chemistry and graduated with a MSc from the Technical University Berlin in 2017. She is currently pursuing her PhD at the Federal Institute for Materials Research and Testing (BAM) in Berlin in division Biophotonics. Her research topic is the surface modification of upconverting nanoparticles for bioanalytical applications and the development of upconversion-based nanoprobes for biosensing. Ute Resch-Genger obtained her PhD in Physical Chemistry in 1989 at Hahn-Meitner-Institute (HMI) Berlin under the supervision of Prof. A. Henglein and H. Weller on the photophysics of colloidal semiconductor quantum dots. After a two-year postdoc with Prof. M.-A. Fox at the University of Texas at Austin from 1989-1991 on the spectroscopic characterization of self-made porphyrines, porphyrine-bipyridine-RuO2 clusters, and cadmium and zinc benzenethiolate clusters and a brief stay at HMI, she became a researcher at the Federal Institute for Materials Research and Testing (BAM). After leading the working group Fluorescence Spectroscopy for a couple of years, since 2012, she is head of the division Biophotonics. In addition, she is co-chair of the steering committee of the Methods & Applications in Fluorescence (MAF) conference series and member of the editorial advisory board of Bioconjugate Chemistry, editorial board of the journal MAF, and the Conference Committee of the Workshop Colloidal Nanoparticles for Biomedical Application at BIOS SPIE. She is also involved in several international standardization activities of luminescence measurements. Her research interests include functional nanomaterials, photophysics of molecular and nanoscale emitters, signal enhancement and multiplexing strategies, optical biomarker analysis, spectroscopic methods for surface analysis, and traceable methods for the characterization of the signal relevant optical properties of luminescent materials. This includes also the standardization of fluorescence measurements including the development of reference materials for optical methods. Michael Schäferling obtained his PhD in Organic Materials Chemistry in 2001 at the University of Ulm under the supervision of Prof. P. Bäuerle. From 2000-2002 he was engaged as project scientist for microarray technology and surface chemistry development at the Thermo Hybaid GmbH in Ulm. In 2002 he continued his academic career as Assistant Professor at the Institute of Analytical Chemistry at the University of Regensburg (Director: Prof. O.S. Wolfbeis). He finished his habilitation in 2008 and was 2013-2014 visiting scientist (FiDiPro fellow) at the Division of Biochemistry/Biotechnology at the University of Turku. From 2014 to 2018 he was as Heisenberg fellow of the DFG at the Federal Institute for Materials Research and Testing (BAM) in Division Biophotonics. Since 2019 he is Professor for Photonic Materials at the Department of Chemical Engineering at the Münster University of Applied Sciences. His research activities include luminescence imaging methods, chemical sensor materials, nanoprobes, and luminescent molecular probes for enzymatic assays.


Surface Modifications for Photon-Upconversion ... - ACS Publications

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