Upconversion nanoparticles for sensing pH.

Upconversion nanoparticles (UCNPs) can provide a vehicle for chemical imaging by coupling chemically sensitive dyes and quenchers. The mechanism for coupling of two anthraquinone dyes, Calcium Red and Alizarin Red S, was investigated as a function of pH. The green emission band of the UCNPs was quenched by a pH-dependent inner filter effect (IFE) while the red emission band remained unchanged and acted as the reference signal for ratiometric pH measurements. Contrary to previous expectation, there was little evidence for a resonance energy transfer (RET) mechanism even when the anthraquinones were attached onto the UCNPs through electrostatic attraction. Since the UCNPs are point emitters, only emitters close to the surface of the UCNP are within the expected Förster distance and UC-RET is <10%. The theoretical and experimental analysis of the interaction between UCNPs and pH-sensitive quenchers will allow the design of UCNP pH sensors for determination of pH via IFE.This work was supported by the EPSRC Cambridge NanoDTC, EP/L015978/

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

Upconversion nanoparticles as intracellular pH messengers

Abstract: Upconversion nanoparticles (UCNPs) should be particularly well suited for measurement inside cells because they can be imaged down to submicrometer dimensions in near real time using fluorescence microscopy, and they overcome problems, such as photobleaching, autofluorescence, and deep tissue penetration, that are commonly encountered in cellular imaging applications. In this study, the performance of an UCNP modified with a pH-sensitive dye (pHAb) is studied. The dye (emission wavelength 580 nm) was attached in a polyethylene imine (PEI) coating on the UCNP and excited via the 540-nm UCNP emission under 980-nm excitation. The UC resonance energy transfer efficiencies at different pHs ranged from 25 to 30% and a Förster distance of 2.56 nm was predicted from these results. Human neuroblastoma SH-SY5Y cells, equilibrated with nigericin H+/K+ ionophore to equalize the intra- and extracellular pH‚ showed uptake of the UCNP-pHAb conjugate particles and, taking the ratio of the intensity collected from the pHAb emission channel (565–630 nm) to that from the UCNP red emission channel (640–680 nm), produced a sigmoidal pH response curve with an apparent pKa for the UCNP-pHAb of ~ 5.1. The UCNP-pHAb were shown to colocalize with LysoBrite dye, a lysosome marker. Drug inhibitors such as chlorpromazine (CPZ) and nystatin (NYS) that interfere with clathrin-mediated endocytosis and caveolae-mediated endocytosis, respectively, were investigated to elucidate the mechanism of nanoparticle uptake into the cell. This preliminary study suggests that pH indicator–modified UCNPs such as UCNP-pHAb can report pH in SH-SY5Y cells and that the incorporation of the nanoparticles into the cell occurs via clathrin-mediated endocytosis. Graphical abstrac

Strategies for the design of bright upconversion nanoparticles for bioanalytical applications

In recent years upconversion nanoparticles (UCNPs) received great attention because of their outstanding optical properties. Especially in bioanalytical applications this class of materials can overcome limitations of common probes like high background fluorescence or blinking. Nevertheless, the requirements for UCNPs to be applicable in biological samples, e.g. small size, water-dispersibility, excitation at low power density are in contradiction with the demand of high brightness. Therefore, a lot of attention is payed to the enhancement of the upconversion luminescence. This review discuss the recent trends and strategies to boost the brightness of UCNPs, classified in three main directions: a) improving the efficiency of energy absorption by the sensitizer via coupling to plasmonic or photonic structures or via attachment of ligands for light harvesting; b) minimizing non-radiative deactivation by variations in the architecture of UCNPs; and c) changing the excitation wavelength to get bright particles at low excitation power density for applications in aqueous systems. These strategies are critically reviewed including current limitations as well as future perspectives for the design of efficient UCNPs especially for sensing application in biological samples or cells

Upconversion nanoparticles as intracellular pH messengers

Abstract: Upconversion nanoparticles (UCNPs) should be particularly well suited for measurement inside cells because they can be imaged down to submicrometer dimensions in near real time using fluorescence microscopy, and they overcome problems, such as photobleaching, autofluorescence, and deep tissue penetration, that are commonly encountered in cellular imaging applications. In this study, the performance of an UCNP modified with a pH-sensitive dye (pHAb) is studied. The dye (emission wavelength 580 nm) was attached in a polyethylene imine (PEI) coating on the UCNP and excited via the 540-nm UCNP emission under 980-nm excitation. The UC resonance energy transfer efficiencies at different pHs ranged from 25 to 30% and a Förster distance of 2.56 nm was predicted from these results. Human neuroblastoma SH-SY5Y cells, equilibrated with nigericin H+/K+ ionophore to equalize the intra- and extracellular pH‚ showed uptake of the UCNP-pHAb conjugate particles and, taking the ratio of the intensity collected from the pHAb emission channel (565–630 nm) to that from the UCNP red emission channel (640–680 nm), produced a sigmoidal pH response curve with an apparent pKa for the UCNP-pHAb of ~ 5.1. The UCNP-pHAb were shown to colocalize with LysoBrite dye, a lysosome marker. Drug inhibitors such as chlorpromazine (CPZ) and nystatin (NYS) that interfere with clathrin-mediated endocytosis and caveolae-mediated endocytosis, respectively, were investigated to elucidate the mechanism of nanoparticle uptake into the cell. This preliminary study suggests that pH indicator–modified UCNPs such as UCNP-pHAb can report pH in SH-SY5Y cells and that the incorporation of the nanoparticles into the cell occurs via clathrin-mediated endocytosis. Graphical abstrac

Yb,Nd,Er-doped upconversion nanoparticles: 980 nm versus 808 nm excitation

Yb,Nd,Er-doped upconversion nanoparticles (UCNPs) have attracted considerable interest as luminescent reporters for bioimaging, sensing, energy conversion/shaping, and anticounterfeiting due to their capability to convert multiple near-infrared (NIR) photons into shorter wavelength ultraviolet, visible or NIR luminescence by successive absorption of two or more NIR photons. This enables optical measurements in complex media with very little background and high penetration depths for bioimaging. The use of Nd3+ as substitute for the commonly employed sensitizer Yb3+ or in combination with Yb3+ shifts the excitation wavelength from about 980 nm, where the absorption of water can weaken upconversion luminescence, to about 800 nm, and laser-induced local overheating effects in cells, tissue, and live animal studies can be minimized. To systematically investigate the potential of Nd3+ doping, we assessed the performance of a set of similarly sized Yb3+,Nd3+,Er3+-doped core- and core-shell UCNPs of different particle architecture in water at broadly varied excitation power densities (P) with steady state and time-resolved fluorometry for excitation at 980 nm and 808 nm. As a measure for UCNPs performance, the P-dependent upconversion quantum yield (phi(UC)) and its saturation behavior were used as well as particle brightness (B-UC). Based upon spectroscopic measurements at both excitation wavelengths in water and in a lipid phantom and B-UC-based calculations of signal size at different penetration depths, conditions under which excitation at 808 nm is advantageous are derived and parameters for the further optimization of triple-doped UCNPs are given

Upconversion nanoparticles for sensing pH

Upconversion nanoparticles (UCNPs) can provide a vehicle for chemical imaging by coupling chemically sensitive dyes and quenchers. The mechanism for coupling of two anthraquinone dyes, Calcium Red and Alizarin Red S, was investigated as a function of pH. The green emission band of the UCNPs was quenched by a pH-dependent inner filter effect (IFE) while the red emission band remained unchanged and acted as the reference signal for ratiometric pH measurements. Contrary to previous expectation, there was little evidence for a resonance energy transfer (RET) mechanism even when the anthraquinones were attached onto the UCNPs through electrostatic attraction. Since the UCNPs are point emitters, only emitters close to the surface of the UCNP are within the expected Forster distance and UC-RET is via IFE

Remote Control of Neural Stem Cell Fate Using NIR-Responsive Photoswitching Upconversion Nanoparticle Constructs

Light-mediated remote control of stem cell fate, such as proliferation, differentiation, and migration, can bring a significant impact on stem cell biology and regenerative medicine. Current UV/vis-mediated control approaches are limited in terms of nonspecific absorption, poor tissue penetration, and phototoxicity. Upconversion nanoparticle (UCNP)-based near-infrared (NIR)-mediated control systems have gained increasing attention for vast applications with minimal nonspecific absorption, good penetration depth, and minimal phototoxicity from NIR excitations. Specifically, 808 nm NIR-responsive upconversion nanomaterials have shown clear advantages for biomedical applications owing to diminished heating effects and better tissue penetration. Herein, a novel 808 nm NIR-mediated control method for stem cell differentiation has been developed using multishell UCNPs, which are optimized for upconverting 808 nm NIR light to UV emission. The locally generated UV emissions further toggle photoswitching polymer capping ligands to achieve spatiotemporally controlled small-molecule release. More specifically, with 808 nm NIR excitation, stem cell differentiation factors can be released to guide neural stem cell (NSC) differentiation in a highly controlled manner. Given the challenges in stem cell behavior control, the developed 808 nm NIR-responsive UCNP-based approach to control stem cell differentiation can represent a new tool for studying single-molecule roles in stem cell and developmental biology

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 beta-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/cm(2). 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/cm(2), 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