Composite particles with magnetic properties, near-infrared excitation, and far-red emission for luminescence-based oxygen sensing

Oxygen sensing, magnetic, and upconversion luminescence properties are combined in multi-functional composite particles prepared herein by a simple mixing, baking, and grinding procedure. Upconverting nanocrystals are used as an excitation source and an oxygen indicator with far-red emission. The composite particles are excited with near infrared (NIR) laser light (980 nm). The visible upconversion emission is converted into an oxygen concentration-dependent far-red emission (<750 nm) using an inert mediator dye and a platinated benzoporphyrin dye. This concept combines the advantages of NIR excitation and far-red emissive indicator dyes, offering minimized auto-fluorescence and enhanced membrane permeability. Additional functionality is obtained by incorporating magnetic nanoparticles into the composite particles, which enables easy manipulation and separation of the particles by the application of an external magnetic field. Researchers from Austria and Germany have synthesized a composite of optical nanometer material particles that enable measurement of the amount of oxygen in a system. Torsten Mayr and colleagues from the Graz University of Technology and the University of Regensburg combine a light-emitting dye with inorganic nanoparticles that contain traces of erbium and ytterbium atoms. The composite particles absorb incoming near-infrared light and re-emit it at a shorter wavelength in the far red. The efficiency of this process, known as upconversion, is dependent on the concentration of oxygen in the environment. Such an optical oxygen sensor has the advantages of being contactless and non-invasive. In addition, the re-emitted far-red light is not reabsorbed or scattered by biological matter. The concept could therefore find applications in medical diagnostics and biotechnology research.


INTRODUCTION
Oxygen is essential for nearly all living systems, thus it is one of the most important chemical species on earth. Knowledge of the actual oxygen concentration is of particular significance in areas such as medical diagnostics, biology, biotechnology, marine research, and environmental analysis 1 . In recent years, optical chemical sensors have proven to be valuable and flexible tools for online monitoring and control of analyte concentrations 2 . Optical sensor systems for oxygen are typically based on gas-permeable polymer matrices containing oxygen-sensitive probes. Such a design allows for the fabrication of sensor films or spots, which can be placed inside any optical transparent vessel, enabling a contactless and non-invasive readout for subsequent data processing and further analysis 3 . Another approach is the application of particles with micro-or nanometre dimensions and oxygensensing capabilities [4][5][6] . Such particles combine the benefits of sensor layers with the flexibility of dissolved indicators. Nanoparticles can easily be applied by adding them to a corresponding measurement setup or reaction chamber [7][8][9] . A promising class of materials for sensor development is upconverting crystals because of their near-infrared (NIR) excitation.
Lanthanide-doped inorganic upconverting luminescent nanoparticles (UCLNPs) are capable of emitting visible light upon NIR excitation 10 . UCLNPs consisting of hexagonal-phase NaYF 4 acting as a crystalline host material and ytterbium(III) and erbium(III) dopants as sensitizer and activator ions, respectively, are the most efficient upconverting phosphors known to date 11 . The generation of upconversion luminescence follows a photophysical process called energy transfer upconversion 12 . The excitation energy (usually at a wavelength of 980 nm) is absorbed by Yb 3+ sensitizers and then sequentially transferred to Er 3+ activators, which results in anti-Stokes emission. These emission bands are fairly narrow with a full-width-at-half-maximum (FWHM) typically less than 20 nm. In contrast to simultaneous two-photon absorption, which requires expensive high-power femtosecondpulsed lasers, UCLNPs are excited by low-cost, low-power continuous wave (CW) laser diodes. NIR excitation causes minimal photo-damage to biological specimens and does not excite endogenously fluorescent moieties in biological matter.
Herein, we present multifunctional particles, which combine oxygen sensing, magnetic, and upconversion luminescence properties in one unit. The sensing functionality enables the quantitative detection of gaseous and dissolved oxygen. In contrast to other sensor systems based on upconversion luminescence mainly using green or red emission, we employ an oxygen sensitive, far-red/NIR emitting platinum(II)tetraphenyltetrabenzoporphyrin (PtTPTBP) dye. This dye facilitates penetration of the emitted light from biological materials attributed to the reduced scattering compared to green and red light. The magnetic functionality enables easy manipulation and separation of these particles from the sample, as well as control over their concentration at particular positions by applying an external magnetic field 28 . Recently, the application of magnetic sensor particles without upconversion capabilities as flexible analytical tools in microfluidic devices was reported by our group 29 . The present sensing scheme based on the utilization of UCLNPs as nanolamps for indirect excitation of an oxygen-quenchable probe minimizes autofluorescence from the sample matrix.
PtTPTBP was synthesized by our group 30 . All other reagents and organic solvents were of the highest grade available.

Synthesis of hydrophobic NaYF 4 nanoparticles doped with Yb/Er
Hydrophobic, lanthanide-doped NaYF 4 nanocrystals were prepared via a modified procedure as reported by Zhang et al. 31 YCl 3 ·6H 2 O (0.78 mmol), YbCl 3 ·6H 2 O (0.20 mmol), and ErCl 3 ·6H 2 O (0.02 mmol) were used for the synthesis, which were dissolved in approximately 5 mL of methanol by sonication. The resulting clear and optically transparent solution of rare earth chlorides in methanol was transferred into a 50 mL flask, mixed with 8 mL of oleic acid and 15 mL of 1-octadecene under an atmosphere of nitrogen and heated to 160°C. A homogeneous, clear solution was formed after 30 min at 160°C under vacuum. The reaction mixture was then cooled to room temperature and 10 mL of methanol containing NaOH (0.25 M) and NH 4 F (0.4 M) was added immediately. The colloidal dispersion was heated to 120°C and stirred for 30 min. The resulting colloid was refluxed at approximately 325°C for 15 min. After cooling to room temperature, the UCLNPs were precipitated by addition of approximately 20 mL of ethanol and isolated via centrifugation at a relative centrifugal force (RCF) of 1000 g for 5 min. The pellet was washed several times by dispersing it in small amounts (approximately 0.5 mL) of chloroform and cyclohexane, followed by precipitation by the addition of a large excess (approximately 15 mL) of ethanol and acetone. A white solid was obtained, which could easily be re-dispersed in cyclohexane to form a clear dispersion.
Synthesis of the NaYF 4 shell YCl 3 ·6H 2 O (1 mmol) was dissolved in approximately 5 mL of methanol by sonication in a 50 mL flask, and mixed with 8 mL of oleic acid and 15 mL of 1-octadecene. The solution was heated to 160°C under an atmosphere of nitrogen. A homogeneous, clear solution was formed after 30 min at 160°C under vacuum. The reaction mixture was then cooled to 80°C. UCLNPs (ytterbium/erbium (Yb/Er) or ytterbium/thulium (Yb/Tm)) dispersed in cyclohexane were then added and the cyclohexane was evaporated. The mixture was cooled to room temperature and then 10 mL of methanol containing NaOH (0.25 M) and NH 4 F (0.4 M) was added immediately. The colloidal dispersion was heated to 120°C and stirred for 30 min under an atmosphere of nitrogen. The resulting colloid was refluxed at approximately 325°C for 15 min. After cooling to room temperature, the UCLNPs were precipitated by the addition of approximately 20 mL of ethanol and isolated via centrifugation at a RCF of 1000 g for 5 min. The pellet was washed several times by dispersing in small amounts (approximately 0.5 mL) of chloroform and cyclohexane, followed by precipitation by the addition of a large excess (approximately 15 mL) of ethanol and acetone. A white solid was obtained, which could be easily redispersed in cyclohexane to form a clear dispersion. The size of the obtained monodisperse, hexagonal crystals was approximately 40 nm, which was verified by transmission electron microscopy (TEM) images.

Preparation of sensor particles
Typically, 100 mg of PPSQ was dissolved in 500 µL of chloroform. Then, 10 mg of UCLNPs and 0.5 mg of organic dyes were added and the mixture was homogenized. In the case of magnetic sensor particles, an additional 5 mg of magnetite nanoparticles (EMG1300) were added and mixed thoroughly. After complete removal of the solvent, the blend was sintered for 4 hours in a drying furnace at 230°C. Finally, the composite material was manually grounded in a mortar until a fine powder was obtained. The microparticles were dispersible in a 1:1 mixture of ethanol and water.
Luminescence measurements-setup and fitting of the fluorescence spectrophotometer Fluorescence measurements were performed with a Hitachi F-7000 fluorimeter from Metrohm Inula GmbH (Vienna, Austria, http://www. metrohm.com) equipped with a red-sensitive photomultiplier tube R9876 (PMT) from Hamamatsu Photonics (Herrsching am Ammersee, Germany, http://www.hamamatsu.com). A self-made fitting (Supplementary Figure S1) was installed, enabling the excitation with a diode laser. The fitting allows for thermostatted and stirred measurements. A 120 mW laser diode with a peak emission at 980 nm, an NIR interference filter (980 nm, FWHM: 10 nm) from Melles Griot GmbH (Bensheim, Germany, www.cvimellesgriot.com), and a collimator lens were used for luminescence measurements.
Oxygen partial pressure was adjusted using a gas mixing device of Vögtlin Instruments (Aesch, Switzerland, www.red-y. com), nitrogen gas, and compressed air. The gas mixing device was triggered by a self-made LabVIEW code.

Transmission electron microscopy
Upconverting nanoparticles have been characterized by TEM studies performed by using a 120 kV Philips CM12 microscope from FEI (Hillsboro, OR, USA, www.fei.com).

RESULTS AND DISCUSSION
The composition and preparation of sensor particles are schematically shown in Figure 1a. We used PPSQ as a sensor matrix. This ladder-like polymer is used in electronics as a semiconductor, insulator, or organic light emitting diode material, as well as a catalyst for chemical reactions 32,33 . Its properties, including an amorphous structure and appropriate oxygen permeability, are similar to those of ormosil glasses, which have been used for optical oxygen sensors. This polymer enables easy preparation of sensor layers and particles by mixing and sintering of the components.
PtTPTBP, used as an indicator, offers similar sensitivity toward oxygen as the platinum complex of the widely used tetrakis-(pentafluorophenyl)porphyrin, but possesses better photophysical properties, such as higher photostability, molar absorbance, and quantum yield. Another advantage is its emission above 720 nm with a peak maximum at 770 nm, which is not absorbed by biological matter. In principle, this approach could be suitable for subcutaneous in vivo measurement of oxygen because the NIR excitation as well as the far-red emission efficiently penetrate into the tissue up to a few millimetres. To excite PtTPTBP by NIR via UCLNPs, a mediator is necessary as shown in Figure 1b. This mediator dye, MFR, does not respond to changes in oxygen concentration and has the potential to pass the energy of the UCLNP emission to the indicator dye. The mediator dye absorbs the green emission of the UCLNPs and further excites the oxygen-sensitive dye, PtTPTBP, via non-radiative energy transfer. The emission intensity of PtTPTBP, which is dependent on the oxygen partial pressure in the sample, can then be monitored. Spectral properties of the employed dyes are shown in Supplementary Figure S3.
UCLNPs are known for their low-quantum yield 34 ; therefore, the most important step in the light cascade is the absorbance of the upconversion emission by the mediator. This step was investigated using PPSQ particles with embedded UCLNPs. Figure 2a shows the normalized upconversion luminescence spectra of particles with and without MFR. In the presence of MFR, the green emission of the UCLNPs is significantly reduced and a broad MFR emission peak approximately 600 nm appears. We assume that MFR is excited via absorption of the green light via radiative energy transfer. This assumption is based on two experimental findings: first, the green emission of the UCLNPs is not quenched completely despite the MFR concentration of 0.5%, which is significantly higher than the Er concentration of 0.2% in the particles. In the case of resonance energy transfer (RET), for such high acceptor concentrations, a stronger reduction of the green UCLNP peak is expected. Note that a higher concentration of MFR did not result in stronger quenching of the green emission. Second, the emission peak of MFR is weak. By excitation via RET, the peak area of the MFR emission corresponds to the reduced area of the green UCLNP emission. We have proven in previous experiments that this is not attributed to self-quenching of MFR. To exclude quenching by water, the measurements were performed in an ethanol (EtOH)/deuterium oxide (D 2 O) mixture. RET between UCLNPs and MFR should change the luminescence lifetime of the green upconversion emission. However, there was no significant change in the lifetime of particles containing only NaYF 4 (Yb, Er) and particles consisting of NaYF 4 (Yb, Er) and MFR (Figure 2b). As a consequence, the mechanism for exciting the mediator by NIR excitation can be ascribed to an inner-filter effect or radiative energy transfer. For further improvement of the upconversion luminescence, the NaYF 4 (Yb, Er) crystals were coated with an inert shell of NaYF 4 of approximately 7 nm (Supplementary Figure  S4) to prevent non-radiative decay caused by surface defects and polymer surroundings. Consequently, an enhancement of the luminescence intensity of the green emission compared to NaYF 4 (Yb, Er) without a shell is found 35,36 . In Figure 2c, the emission spectra of the sensor particles are plotted, revealing a change of luminescence intensity in aerated and deoxygenated solutions. In the absence of oxygen the green UCLNP peak is significantly less pronounced and the fluorescence of MFR vanishes completely, indicating a complete energy transfer from MFR to PtTPTBP. The inset in Figure 2c shows the quenching of the PtTPTBP emission by oxygen.
Significant advantages of magnetic particles include enhanced sensor signal attributed controllable accumulation of the particles, the possibility of generating in situ sensor spots at any desirable location and the possibility of easily retaining the sensor particles in a flow. In general, two opposite effects are observed by the introduction of magnetite into the sensor particles. First, for dispersed particles a decrease of the luminescence intensity by a factor of approximately 6 as a result of the high lightabsorbing properties of the magnetic nanoparticles is observed ( Supplementary Figures S5a and S6). Second, collected magnetic sensor particles show an emission increase by a factor of 10 compared to dispersed sensor particles (Supplementary Figure  S5b). As a result, the sensor signal is enhanced by a factor of 5/3. After removing the permanent magnet, the sensor particles could be re-suspended in the sample by stirring.
The response of collected magnetic sensor particles exposed to air-saturated and oxygen-free solutions is depicted in Figure 3a. The particles are magnetically collected in the focus of the 980 nm laser light source. The oxygen-dependent emission of PtTPTBP was detected at a wavelength of 760 nm to 780 nm. A significantly higher signal to noise ratio of~35 was obtained in comparison tõ 13 for non-magnetic dispersed particles (Supplementary Figure S7). Calibration curves are shown in Figure 3b. The magnetic sensor particles showed a good response, especially at low oxygen concentrations (up to 100 hPa O 2 ). The calibration curve was fitted with a modified two-site model 37 , assuming the existence of two fractions of dye molecules within the sensor particles; one fraction is quenchable by oxygen and the other is not. A K SV value of 0.0445 hPa −1 was obtained. The sensitivity expressed by I 0 =I air −1 is 1.8. In comparison, the reported oxygen-sensing film employing UCNLPs as nanolamps by Achatz et al. showed a significantly lower sensitivity with a K SV value of 0.0101 hPa −1 and a I 0 =I air −1 value of 0.25 21 . The higher sensitivity (K SV = 0.0607 hPa −1 ) of nonmagnetic sensor particles is most likely attributed to the quenching effects by the incorporated magnetite nanoparticles.

CONCLUSION
In summary, we herein reported luminescence-based oxygen sensor composite particles using upconverting nanocrystals as an excitation source and a dye with far-red emission to minimize autofluorescence in biological samples. For signal enhancement, magnetic particles were embedded into the composite, enabling the collection of the sensor particles by a permanent magnet at any desired position to form an in situ sensor spot. The manually ground micrometre-sized particles were easily prepared, and smaller sizes were obtained by automated milling. An increase in the signal was achieved by incorporating a directly bound mediator dye into the particle.
In principle, this concept could be useful for applications in biological materials, such as cell cultures, tissues, or subcutaneous measurements. Both NIR excitation and far-red emission offer the advantage of good penetration efficiency into biological matter. In addition, NIR excitation generates low or practically zero autofluorescence. Furthermore, UCLNP magnetic sensor particles are suggested to be applied in microfluidic systems for monitoring of miniaturized cell cultures.