Design, Synthesis and Surface Modification of Lanthanide-Doped Nanoparticles for FRET-Based Biosensing Applications

This thesis describes the preparation, surface modification, and application of lanthanide doped upconversion luminescent nanoparticles (UCNPs) in bioanalytical sensing and imaging based on time-resolved Förster resonance energy transfer processes (FRET). Chapter 1 provides an overview of optical properties of lanthanides and highlights outstanding aspects of luminescence phenomena occurring in trivalent lanthanide ions with respect to chemical sensing. Down- and upconversion luminescence are defined and UCNPs are introduced and characterized as a unique class of nanomaterials that show exceptional potential for bioanalytical applications. FRET processes using UCNPs as energy donors are introduced and current issues that limit the more widespread implementation of ratiometric measurements with UCNPs in bioanalytical applications are addressed. In Chapter 2 the aim of the work is presented as the investigation of FRET between UCNP donors and organic dye acceptors based on lifetime changes of the upconversion luminescence, with respect to a detailed characterization of the effect of interplay between particle architecture and surface modification on the FRET efficiency. The comprehensive understanding of energy transfer processes is needed to design an efficient FRET nanoprobe applicable in biosensing and –imaging. Challenges regarding the choice of the type of surface modification to transfer hydrophobic nanoparticles into hydrophilic ones are described in Chapter 3. Amphiphilic coatings, encapsulation with inorganic materials, and ligand replacement are introduced as commonly used techniques to fabricate UCNPs that display colloidal stability in buffers and biological media. Advantages and disadvantages of the different methods are critically discussed and suggestions and examples for the application of each single technique depending on and tailored towards the desired individual applications are given. Chapter 4 presents a study of the effect of nanocrystal size on the time-resolved FRET efficiency from UCNPs acting as energy donors to organic dyes as acceptors. Ligand exchange was selected for the attachment of the two acceptor dyes rose bengal and sulforhodamine B to the UCNP surface, which enables the shortest possible donor-acceptor distance and high, reproducible dye loading. UCNPs with diameters in the range of 20 - 25 nm were identified as the ones that yield the highest FRET efficiencies based on lifetime measurements of the upconversion luminescence. Lower FRET efficiencies at both smaller and larger UCNP sizes were ascribed to an increasing competition of surface quenching and lower amounts of FRET donors within Förster distance to the acceptor dye on the particle surface, respectively. Comparison with conventional ratiometric intensity measurements illustrates the independence of the lifetime based approach on inner-filter-effects, particle concentration and excitation power. The information gained from these FRET studies was the basis for the design of the upconversion FRET nanoprobe for the metabolite adenosine triphosphate (ATP), which is described in Chapter 5. Different surface modification strategies of core-shell UCNPs were investigated for the subsequent attachment of a structure switching ATP-responsive aptamer. Ligand exchange with poly(acrylic acid) represented the best compromise between colloidal stability, reduced surface quenching and increased distance to the FRET acceptor propidium iodide (PI). In presence of ATP the aptamer formed a G-quadruplex, which was recognized by the dye PI. The spectral shift of the absorption spectrum of PI bound to the G-quadruplex led to minimized background absorption and influence of unspecific binding. Successful FRET to the bound PI in close proximity to the UCNPs was shown by the reduction of the lifetime of the UCNP emission for ATP concentrations between 0.2 and 1.0 mM. The nanoprobe was selective for ATP and showed no cytotoxic effects. Eventually, Chapter 6 provides a concise discussion of the main results and insights gained within this thesis with respect to ideal particle design and surface functionalization for upconversion luminescent energy transfer processes in bioanalytical applications. Future perspectives as well as remaining challenges in the field are critically addressed

Photosensitiser functionalised luminescent upconverting nanoparticles for efficient photodynamic therapy of breast cancer cells

Photodynamic therapy (PDT) is a well-established treatment of cancer in which cell toxic reactive oxygen species, including singlet oxygen (1O2), are produced by a photosensitiser drug following irradiation of a specific wavelength. Visible light is commonly used as the excitation source in PDT, although these wavelengths do have limited tissue penetration. In this research, upconverting nanoparticles (UCNPs) functionalised with the photosensitiser Rose Bengal (RB) have been designed and synthesised for PDT of breast cancer cells. The use of UCNPs shifts the required excitation wavelength for the production of 1O2 to near infrared light (NIR) thus allowing deeper tissue penetration. The system was designed to maximise the production of 1O2via efficient Förster resonance energy transfer (FRET) from the UCNPs to the photosensitiser. Highly luminescent NaYF4:Yb,Er,Gd@NaYF4 core–shell UCNPs were synthesised that exhibited two main anti-Stokes emission bands at 541 and 652 nm following 980 nm irradiation. RB was chosen as the photosensitiser since its absorption band overlaps with the green emission of the UCNPs. To achieve efficient energy transfer from the nanoparticles to the photosensitiser, the functionalised UCNPs included a short L-lysine linker to attach the RB to the nanocore yielding RB-lysine functionalised UCNPs. The efficient FRET from the UCNPs to the RB was confirmed by luminescence lifetime measurements. The light emitted by the UCNPs at 541 nm, following excitation at 980 nm, generates the 1O2via the RB. Multi-photon and confocal laser scanning microscopies confirmed the internalisation of the RB-lysine-UCNPs by SK-BR-3 breast cancer cells. Cell viability studies revealed that the RB-lysine-UCNPs induced low dark toxicity in cells prior to PDT treatment. Importantly, following irradiation at 980 nm, high levels of cell death were observed in cells loaded with the RB-lysine-UCNPs. Cell death following PDT treatment was also confirmed using propidium iodide and confocal microscopy. The high drug loading capacity (160 RB/nanoparticle) of the UCNPs, the efficient FRET from the UCNPs to the photosensitiser, the high level of accumulation inside the cells and their PDT cell kill suggest that the RB-lysine-UCNPs are promising for NIR PDT and hence suitable for the treatment of deep-lying cancer tumours

Multiband emission from single β-NaYF4(Yb,Er) nanoparticles at high excitation power densities and comparison to ensemble studies

Ensemble and single particle studies of the excitation power density (P)-dependent upconversion luminescence (UCL) of core and core-shell β-NaYF4:Yb,Er upconversion nanoparticles (UCNPs) doped with 20% Yb3+ and 1% or 3% Er3+ performed over a P regime of 6 orders of magnitude reveal an increasing contribution of the emission from high energy Er3+ levels at P > 1 kW/cm2. This changes the overall emission color from initially green over yellow to white. While initially the green and with increasing P the red emission dominate in ensemble measurements at P < 1 kW/cm2, the increasing population of higher Er3+ energy levels by multiphotonic processes at higher P in single particle studies results in a multitude of emission bands in the ultraviolet/visible/near infrared (UV/vis/NIR) accompanied by a decreased contribution of the red luminescence. Based upon a thorough analysis of the P-dependence of UCL, the emission bands activated at high P were grouped and assigned to 2–3, 3–4, and 4 photonic processes involving energy transfer (ET), excited-state absorption (ESA), cross-relaxation (CR), back energy transfer (BET), and non-radiative relaxation processes (nRP). This underlines the P-tunability of UCNP brightness and color and highlights the potential of P-dependent measurements for mechanistic studies required to manifest the population pathways of the different Er3+ levels.Peer Reviewe

Particle-size-dependent upconversion luminescence of NaYF4: Yb, Er nanoparticles in organic solvents and water at different excitation power densities

A systematic study of the luminescence properties of monodisperse beta-NaYF4: 20% Yb3+, 2% Er3+ upconversion nanoparticles (UCNPs) with sizes ranging from 12-43 nm is presented utilizing steady-state and time-resolved fluorometry. Special emphasis was dedicated to the absolute quantification of size- and environment-induced quenching of upconversion luminescence (UCL) by high-energy O-H and C-H vibrations from solvent and ligand molecules at different excitation power densities (P). In this context, the still-debated population pathways of the F-4(9/2) energy level of Er3+ were examined. Our results highlight the potential of particle size and P value for color tuning based on the pronounced near-infrared emission of 12 nm UCNPs, which outweighs the red Er3+ emission under "strongly quenched" conditions and accounts for over 50% of total UCL in water. Because current rate equation models do not include such emissions, the suitability of these models for accurately simulating all (de)population pathways of small UCNPs must be critically assessed. Furthermore, we postulate population pathways for the F-4(9/2) energy level of Er3+, which correlate with the size-, environment-, and P-dependent quenching states of the higher Er3+ energy levels

Time-dependent luminescence loss for individual upconversion nanoparticles upon dilution in aqueous solution

Single-particle luminescence microscopy is a powerful method to extract information on biological systems that is not accessible by ensemble-level methods. Upconversion nanoparticles (UCNPs) are a particularly promising luminophore for single-particle microscopy as they provide stable, non-blinking luminescence and allow the avoidance of biological autofluorescence by their anti-Stokes emission. Recently, ensemble measurements of diluted aqueous dispersions of UCNPs have shown the instability of luminescence over time due to particle dissolution-related effects. This can be especially detrimental for single-particle experiments. However, this effect has never been estimated at the individual particle level. Here, the luminescence response of individual UCNPs under aqueous conditions is investigated by quantitative wide-field microscopy. The particles exhibit a rapid luminescence loss, accompanied by large changes in spectral response, leading to a considerable heterogeneity in their luminescence and band intensity ratio. Moreover, the dissolution-caused intensity loss is not correlated with the initial particle intensity or band ratio, which makes it virtually unpredictable. These effects and the subsequent development of their heterogeneity can be largely slowed down by adding millimolar concentrations of sodium fluoride in buffer. As a consequence, the presented data indicate that microscopy experiments employing UCNPs in an aqueous environment should be performed under conditions that carefully prevent these effects

Particle-Size-Dependent Förster Resonance Energy Transfer from Upconversion Nanoparticles to Organic Dyes

Upconversion nanoparticles (UCNPs) are attractive candidates for energy transfer-based analytical applications. In contrast to classical donoracceptor pairs, these particles contain many emitting lanthanide ions together with numerous acceptor dye molecules at different distances to each other, strongly depending on the particle diameter. UCNPs with precisely controlled sizes between 10 and 43 nm were prepared and functionalized with rose bengal and sulforhodamine B by a ligand-exchange procedure. Time-resolved studies of the upconversion luminescence of the UCNP donor revealed a considerable shortening of the donor lifetime as a clear hint for Forster resonance energy transfer (FRET). FRET was most pronounced for 21 nm-sized UCNPs, yielding a FRET efficiency of 60%. At larger surface-to-volume ratios, the FRET efficiency decreased by an increasing competition of nonradiative surface deactivation. Such dye-UCNP architectures can also provide an elegant way to shift the UCNP emission color, since the fluorescence intensity of the organic dyes excited by FRET was comparable to that of the upconversion emission of smaller particles

Europium-doped GdVO4 nanocrystals as a luminescent probe for hydrogen peroxide and for enzymatic sensing of glucose

The authors describe the preparation of Eu3+-doped GdVO4 nanocrystals (NCs) by precipitation of the Cd3+(Eu3+)-citrate complex which was then converted to the respective vanadate by dialysis. The fractions of Eu3+ ranged from 5 to 100 mol%. The NCs were characterized by XRD, TEM, ICP-OES and dynamic light scattering which revealed that they possess superior colloidal stability in aqueous solutions in that no precipitation can be observed even after several months. The NCs display red and largely red shifted fluorescence (peaking at 618 nm) on photoexcitation at around 300 nm. Fluorescence is strongly quenched by hydrogen peroxide. It is also shown that the fraction of doping with Eu3+ strongly affects quenchability. Most efficient quenching by H2O2 is observed if the NCs are doped with 50% of Eu3+. The findings were exploited to develop a fluorometric assay for H2O2 that works in the 5 to 250 mu M concentration range, with a limit of detection as low as 1.6 mu M (at a signal-to-noise ratio of 3). The probe was further employed to design a highly sensitive enzymatic assay for glucose via measurement of the quantity of H2O2 formed as a result of the catalytic action of glucose oxidase. (C) 2016 Elsevier B.V. All rights reserved

Quantitative assessment of energy transfer in upconverting nanoparticles grafted with organic dyes

Upconverting nanoparticles (UCNPs) are luminophores that have been investigated for a multitude of biological applications, notably low-background imaging, high-sensitivity assays, and cancer theranostics. In these applications, they are frequently used as a donor in resonance energy transfer (RET) pairs. However, because of the peculiarity and non-linearity of their luminescence mechanism, their behavior as a RET pair component has been difficult to predict quantitatively, preventing their optimization for subsequent applications. In this article, we assembled UCNP-organic dye RET systems and investigated their luminescence decays and spectra, with varying UCNP sizes and quantities of dyes grafted onto their surface. We observed an increase in RET efficiency with lower particle sizes and higher dye decoration. We also observed several unexpected effects, notably a quenching of UCNP luminescence bands that are not resonant with the absorption of organic dyes. We proposed a semi-empirical Monte Carlo model for predicting the behavior of UCNP-organic dye systems, and validated it by comparison with our experimental data. These findings will be useful for the development of more accurate UCNP-based assays, sensors, and imaging agents, as well as for optimization of UCNP-organic dye RET systems employed in cancer treatment and theranostics

Water dispersible upconverting nanoparticles: effects of surface modification on their luminescence and colloidal stability

We present a systematic study on the effect of surface ligands on the luminescence properties and colloidal stability of β-NaYF4:Yb3+,Er3+ upconversion nanoparticles (UCNPs), comparing nine different surface coatings to render these UCNPs water-dispersible and bioconjugatable. A prerequisite for this study was a large-scale synthetic method that yields ∼2 g per batch of monodisperse oleate-capped UCNPs providing identical core particles. These ∼23 nm sized UCNPs display an upconversion quantum yield of ∼0.35% when dispersed in cyclohexane and excited with a power density of 150 W cm−2, underlining their high quality. A comparison of the colloidal stability and luminescence properties of these UCNPs, subsequently surface modified with ligand exchange or encapsulation protocols, revealed that the ratio of the green (545 nm) and red (658 nm) emission bands determined at a constant excitation power density clearly depends on the surface chemistry. Modifications relying on the deposition of additional (amphiphilic) layer coatings, where the initial oleate coating is retained, show reduced non-radiative quenching by water as compared to UCNPs that are rendered water-dispersible via ligand exchange. Moreover, we could demonstrate that the brightness of the upconversion luminescence of the UCNPs is strongly affected by the type of surface modification, i.e., ligand exchange or encapsulation, yet hardly by the chemical nature of the ligand

Particle-Size-Dependent Förster Resonance Energy Transfer from Upconversion Nanoparticles to Organic Dyes

Upconversion nanoparticles (UCNPs) are attractive candidates for energy transfer-based analytical applications. In contrast to classical donor–acceptor pairs, these particles contain many emitting lanthanide ions together with numerous acceptor dye molecules at different distances to each other, strongly depending on the particle diameter. UCNPs with precisely controlled sizes between 10 and 43 nm were prepared and functionalized with rose bengal and sulforhodamine B by a ligand-exchange procedure. Time-resolved studies of the upconversion luminescence of the UCNP donor revealed a considerable shortening of the donor lifetime as a clear hint for Förster resonance energy transfer (FRET). FRET was most pronounced for 21 nm-sized UCNPs, yielding a FRET efficiency of 60%. At larger surface-to-volume ratios, the FRET efficiency decreased by an increasing competition of nonradiative surface deactivation. Such dye-UCNP architectures can also provide an elegant way to shift the UCNP emission color, since the fluorescence intensity of the organic dyes excited by FRET was comparable to that of the upconversion emission of smaller particles