Surface functionalization of up-converting NaYF₄ nanocrystals with chiral molecules

Abstract

I report surface modification of up-converting, Er³⁺ and Yb³⁺ co-doped β-NaYF₄ nanoparticles with four types of chiral molecules with proven molecular chiral recognition abilities: L-cysteine, L-penicillamine, L-arginine and L-glutathione. The presence of chiral ligands near to the nanoparticle surfaces was confirmed based on Fourier transform infrared spectroscopy and circular dichroism measurements. The performed post synthetic treatment of nanoparticle surfaces led to stable water solutions, and the best results regarding the long term colloidal stability were found for nanoparticles functionalized with L-penicillamine. The selected chiral molecules were also found to strongly influence the spectroscopic properties of NaYF₄:Er³⁺,Yb³⁺ nanoparticles, including the change in power dependence slopes for blue, green and red emission and luminescence lifetimes. Nevertheless, the high up-conversion emission intensity was preserved after functionalization. The obtained results open up new perspectives for possible applications of up-converting β-NaYF₄ NPs as optical markers in chiral recognition and sensing of chiral drugs.

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Introduction

The efficient transfer of nanosized fluorescent markers to the aqueous phase is an essential step for future application of those nanoparticles (NPs) in bionanophotonics,^1,2 and especially in nanomedicine. The most important requirements for the phase transfer reaction are (i) the long term colloidal stability of the NPs in a water environment, (ii) preservation of high luminescence intensity and (iii) the possibility of further conjugation of the functionalized NPs to biologically important molecules. Additionally, in any bionanophotonics related application of NPs the use of UV or high power light excitation should be avoided, due to the increased autofluorescence background, decreased light penetration depth and possible biological specimen destruction.^3,4 The inorganic NPs co-doped with pairs of lanthanide ions, e.g. Er³⁺/Yb³⁺, Tm³⁺/Yb³⁺ or Ho³⁺/Yb³⁺, give the advantage of an up-conversion process in which the NIR light is converted to the higher energy visible emission through sequential absorption and energy transfer processes.^5,6 Several surface functionalization techniques have been already introduced in order to obtain water solutions of lanthanide doped NPs, including ligand exchange, oxidation, surface silanization or layer-by-layer assembly.⁷ Simultaneously, the possibility of bioconjugation of those NPs for sensing applications has been studied and ready to use solutions have been presented.⁸ Zhu et al.⁹ fabricated up-converting NPs based sensing platform for the detection of lysosome and DNA, while Yuan et al.¹⁰ used up-converting Tm³⁺/Yb³⁺ co-doped NPs for the detection of thrombin in the NIR region of light. The biosensing with the use of lanthanide doped NPs has focused on the remote optical detection of proteins,^11,12 nucleic acids^13,14 or disease markers,^15,16 while the possibility of the molecular recognition remained neglected. Optical receptors for chiral recognition processes are preferred due to the high sensitivity and easily accessible equipment of the fluorescence based techniques.¹⁷ The application of inorganic NPs as optically active receptors is usually accomplished by the functionalization of NPs surfaces with chiral organic ligands,¹⁸ such as L-cysteine, L-penicillamine or L-glutathione. The majority of former research has concerned the ligand induced optical activity in plasmonic NPs and semiconductor quantum dots (QDs). Shemer et al.¹⁹ showed that the interaction of chiral double stranded DNA scaffold with silver NPs can induce chirooptical effects related to metal plasmon resonance. In contrast, the distortion of the gold cluster surfaces due to chiral L-glutathione molecules resulted in much stronger circular dichroism (CD) signals.²⁰ In the case of semiconductor QDs the CD signals in the visible region of light were attributed to the electronic states of QDs themselves. Moloney et al.²¹ reported first demonstration of strongly white-emitting D- and L-penicillamine capped CdS QDs, with strong CD signals in the range 200–390 nm. Ligand induced CD and circularly polarized luminescence (CPL) have been studied in different QDs systems up to date, including CdSe,^22,23 CdS²⁴ and CdTe²⁵ QDs, and sensing chiral drugs with the use of N-acetyl-L-cysteine methyl ester capped CdSe@ZnS QDs has been also introduced.²⁶ In the case of lanthanide doped NPs the mechanisms for NPs + chiral ligand interaction are suspected to be rather different, than those of metallic NPs or QDs. The intra-configurational 4f–4f transitions in lanthanide ions are insensitive to the changes in local chemical environment, due to the shielding by filled higher energy orbitals. However, as indicated by Richardson in 1980 (ref. 27) several types of 4f–4f transitions observed in lanthanide ions are predicted to be particularly favorable for optical activity studies. One of the highest emission dissymmetry factor (difference in left- and right-handed circularly polarized emission intensity) values were in fact reported for lanthanide ions complexes.^28,29 The optical activity in those systems resulted from coupling interactions between lanthanide ions and dissymmetric environment provided by the ligand molecules. The CPL response of Eu³⁺ and Tb³⁺ complexes, have proven to be sensitive to the local environment geometry,³⁰ while M. Herren and M. Moriota³¹ showed that the CPL of NIR Er³⁺ ions emission can be used to distinguished between two different rare-earth sites in GMO crystals. On the other hand, Wei et al.³² reported the procedure of up-converting NaYF₄ NPs surface modification with cysteine molecules and showed their good in vitro biocompatibility, however, they did not discuss the optical activity of the obtained systems.

With this in mind I have synthesized hydrophobic, highly luminescent Er³⁺ and Yb³⁺ ions co-doped β-NaYF₄ NPs, and further modified their surfaces with four types of chiral molecules: L-cysteine, L-penicillamine L-arginine and L-glutathione. The selection of the types of active ions and the crystal matrix was dictated by the highest up-conversion efficiency in such systems,³³ while the choice of chiral molecules was made due to their zwitterionic nature, small size and proved biological importance in chiral recognition reactions.^34,35 The epoxidation of oleic acid carbon–carbon double bond allowed me to further attach the selected chiral molecules near to the surface of β-NaYF₄:2%Er,20%Yb NPs, and transfer them to the nontoxic water environment. The functionalized NPs were characterized with respect to their long term colloidal stability, and the presence of chiral molecules near to the NPs surface was confirmed based on FT-IR and CD spectroscopy. Finally, I have investigated in detail the influence of the performed surface functionalization and dispersing NPs in water on the down- and up-converted spectra of Er³⁺ ions after 976 nm laser diode excitation, by measuring the power dependent luminescence spectra and decay kinetics.

Experimental

The hexagonal structure β-NaYF₄ NPs doped with 2% of Er³⁺ and 20% of Yb³⁺ ions were synthesized based on protocol described by Abel et al.³⁶ Briefly, first the lanthanide acetate precursor was prepared starting from lanthanide oxides (2 mmol) and acetic acid, with the use of a microwave reactor (MAGNUM II ERTEC). Next, the obtained dried acetate precursor salt was mixed under inert atmosphere at 140 °C with 12 mL of oleic acid and 30 mL of octadecene to form clear, homogenous solution. After initial degassing, the reaction temperature was lowered to 50 °C and the methanol solution of ammonium fluoride (8 mmol) and sodium hydroxide (5 mmol) was added dropwise. The obtained mixture was maintained at 60 °C for 45 min followed by the methanol evaporation. Next, the reaction temperature was increased to 300 °C for the synthesis of β-NaYF₄:2%Er,20%Yb NPs for 60 min under inert atmosphere. After the NPs formation the mixture was allowed to cool down to room temperature, followed by the precipitation with ethanol and isolation by centrifugation. After washing three times with excess of ethanol, the obtained NPs were dispersed in chloroform at the concentration of 100 mg mL⁻¹. For surface functionalization with chiral molecules we used the carbon–carbon double bond epoxidation method,³² 100 mg of NaYF₄:2%Er,20%Yb NPs was mixed for 3 hours with 20 mL of cyclohexane, 10 mL of dichloromethane and 25 mg of 3-chloroperoxybenzoic acid under inert atmosphere at 40 °C. Next, 0.2 mmol of the selected chiral ligands (L-cysteine, L-penicillamine, L-arginine or L-glutathione) was added, and the stirring was continued for another 5 hours at room temperature. The obtained surface modified NaYF₄:2%Er,20%Yb NPs were collected by centrifugation and washed twice with excess of ethanol. Finally, the NPs were dispersed in distilled water with the concentration of 15 mg mL⁻¹. The NPs functionalized with L-cysteine, L-penicillamine, L-arginine or L-glutathione will be named in the text as NPs-LCys, NPs-LPen, NPs-LArg and NPs-LGlu, respectively. For the measurements of down-converted emission spectra of Er³⁺ ions at 1524 nm the NPs were re-dispersed in heavy water in order to avoid water absorption present in this spectral range. The morphology and crystal structure of the as-synthesized and chiral ligands functionalized NPs were characterized with a STOE diffractometer with Ge-filtered CuK_α1 radiation and a FEI Tecnai G² 20 X-TWIN transmission electron microscope (TEM). The average size of NP and crystal lattice parameters were evaluated based on Rietveld refinement of X-ray powder diffraction (XRD) data and the ICSD 51916 standard pattern. The colloidal stability of NPs in water solutions after surface functionalization was evaluated based on measurements of their hydrodynamic diameters (D_H), polydispersity index (PdI) and zeta-potential (ς-potential) with the Malvern Zetasizer Nano. For the D_H and PdI measurements we used the Dynamic Light Scattering (DLS) method with the detection angle of 173° in optically homogeneous square fused silica cells. All measurements were performed at 25 °C and each value was obtained as an average of three runs with at least 10 measurements. The DTS (Nano) program was applied for data evaluation. The ς-potential values were measured at 25 °C and each value was obtained as an average of three subsequent runs of the instrument with at least 20 measurements. The circular dichroism (CD) spectra of surface modified NPs and free ligands in water solutions were recorded with a Jasco J-815 spectropolarimeter equipped with a Jasco Peltier-type temperature controller (CDF-426S/15). The CD measurements were performed at 20 °C, and each spectrum was obtained as an average of three subsequent measurements. The Fourier transform infrared (FT-IR) spectra were recorded with a Bruker Optic GmbH Vertex 70v vacuum spectrophotometer in the range between 500–4000 cm⁻¹ and with the resolution of 0.4 cm⁻¹ for the dried samples. The emission spectra and power dependence studies of as-synthesized and surface functionalized NaYF₄:2%Er,20%Yb NPs colloidal solutions were obtained with an Ocean Optics USB2000 fiber coupled spectrophotometer in visible range, and with an Ocean Optics NirQuest 512-2.2 fiber coupled spectrophotometer in the infrared range, using excitation with a 976 nm laser diode (5W, CW, Spectra Laser, Poland). For power dependent studies we used un-focused beam excitation in the range of 500 mW up to 4500 mW, what roughly correspond to low/moderate light intensities of 2.5–20 W cm⁻². The luminescence kinetics was measured with an Edinburgh Instruments FLS 980 spectrofluorometer with a 976 nm fractionated laser diode (8W, CW, Spectra Laser, Poland).

Results and discussion

The morphology, size distribution and crystal structure of as-synthesized and surface functionalized NaYF₄:2%Er,20%Yb NPs were characterized with TEM and XRD techniques. Fig. 1 shows the TEM pictures of oleic acid capped NaYF₄:2%Er,20%Yb NPs (Fig. 1a and b), and after surface functionalization with L-penicillaminee molecules (Fig. 1c). Additional higher magnification TEM images of NaYF₄:2%Er,20%Yb NPs before and after surface treatment are presented in the (ESI) in Fig. S1a and b,† respectively. The used epoxidation method did not affect the surface of the NPs, as can be observed for NPs treated with strong acid in ligand removal procedure.^37,38 Additionally, the epoxide ring, as highly reactive species, can easily and rapidly react with important functional groups such as –OH, –SH, –NH₂ or –COOH,³⁹ and since during the functionalization procedure the ligand shell covering is not removed from the NPs surface, the unwanted aggregation of NPs is less possible. The studied NaYF₄:2%Er,20%Yb NPs exhibited very good size and shape uniformity. Based on TEM images I have calculated the mean size of NPs to be 38.8 ± 2.2 nm (see ESI Fig. S2† for size distribution histogram).

Fig. 1 TEM images of as-synthesized NaYF₄:2%Er,20%Yb NPs (a and b) and after surface functionalization with L-penicillamine molecules (c), together with XRD spectra of as-synthesized NaYF₄:2%Er,20%Yb NPs compared to standard ICSD 51916 pattern for hexagonal β-NaYF₄ (d).

The analysis of the crystal structure of the obtained NPs was performed based on the ICSD 51916 standard pattern. The obtained NaYF₄:2%Er,20%Yb NPs had a hexagonal crystal structure of sodium yttrium fluoride (space group P6̄(174)), lattice parameters equal to a = 5.97 Å and c = 3.51 Å, and the mean grain size of 32 nm were calculated based on Rietveld refinement. The mean size of NaYF₄:2%Er,20%Yb NPs calculated based on TEM images was in a good agreement with the one calculated based on XRD pattern. The performed surface functionalization led to stable water solutions of NaYF₄:2%Er,20%Yb NPs, the colloidal stability, and the successful modification of NPs surface was confirmed by the DLS, ς-potential measurements and FT-IR spectroscopy. Table S1† presents the values of hydrodynamic diameters (D_H), polydispersity index (PdI) and ς-potential measured for chloroform solution of as-synthesized NaYF₄:2%Er,20%Yb NPs together with corresponding values for water solutions of NPs just after surface functionalization and after 30 days of storage. It should be emphasized that the particle size measured by DLS is always larger because the hydrodynamic diameter is determined. The as-synthesized NaYF₄:2%Er,20%Yb NPs in chloroform solution showed D_H values at the level of 82 nm, and low PdI equal to 0.157. The increase in hydrodynamic diameter of NPs after surface functionalization is related to the partial aggregation of NPs in water suspensions, and most probably could be diminished by the addition of the reducing agents that prevent disulfide formation and by the change of the solution pH. The highest D_H and PdI values were observed for NPs functionalized with larger L-Arg and L-Glu molecules, those NPs also showed further aggregation after storage (Table S1†). NPs-LCys and NPs-LPen presented increased colloidal stability, with smaller degree of aggregation and low PdI index. However, after 30 days of storage the NPs-LCys aggregated in water solution, most probably due to the disulfide formation. The NPs functionalized with L-Pen molecules showed, however, stable in time D_H and PdI values, with simultaneous high ς-potential equal to +24.3 mV, which proves their water colloidal stability (Table S1†). Penicillamine has been also shown to be less prone to dimerization than cysteine,⁴⁰ which makes this molecule an interesting agent for surface functionalization targeting biological applications. The good dispersibility of NPs-LPen was also visible in the TEM images (Fig. 1c and S1b†), where the particles are homogeneously distributed and no agglomerates are observed. The change in the type and structure of the organic molecules attached to the NPs surface was further analyzed based on FT-IR spectra. As shown in Fig. 2a the as-synthesized NaYF₄:2%Er,20%Yb NPs, with oleic acid molecules attached to the surface, showed two sharp peaks at 2921 cm⁻¹ and 2852 cm⁻¹, which can be assigned to the asymmetric and symmetric vibrations of methylene (CH₂) group in the long alkyl chain of oleic acid, respectively. This feature is apparently lost in the spectrum of NPs after surface functionalization treatment (Fig. 2b–e) suggesting the disappearance of the –HC=CH– group.^41,42 In the FT-IR spectrum of NPs with attached chiral molecules the broad band at approximately 1640 cm⁻¹ is attributed to the amide vibration, while peak at ∼1550 cm⁻¹ to the vibration of carbonyl (C=O) groups. Similar FT-IR spectra of NaYF₄ NPs,³² CdSe@ZnS QDs²⁶ and ZnO NPs⁴³ with attached L-cysteine molecules have been reported previously, which also supports the hypothesis of efficient attachment of the chosen chiral molecules to the epoxidized oleic acid molecules.

Fig. 2 FT-IR spectra of NaYF₄:2%Er,20%Yb NPs before (a) and after functionalization with chiral molecules: L-cysteine (b), L-penicillamine (c), L-arginine (d) and L-glutathione (e).

Fig. 3 CD spectra of NaYF₄:2%Er,20%Yb NPs after functionalization with L-cysteine (a), L-penicillamine (b), L-arginine (c) and L-glutathione (d) compared with CD spectra of free molecules in water. Insets show enlarged region between 190 nm and 300 nm.

The presence of the chiral molecules attached to the NPs was further confirmed with the CD measurements. The CD spectra of NaYF₄:2%Er,20%Yb NPs before (Fig. S3†) and after functionalization with L-cysteine, L-penicillamine, L-arginine and L-glutathione compared with CD spectra of free molecules in water are shown in Fig. 3. The corresponding absorption spectra of studied samples are presented in Fig. S4.† To my knowledge, investigations of ligand induced optical activity of up-converting NaYF₄ NPs have not been reported previously. The as-synthesized NPs did not show any optical activity in the whole spectral range (Fig. S3†), while for all four types of the investigated NPs-chiral molecules systems the CD spectra of functionalized NPs corresponded roughly with those of free molecules in water (Fig. 3). However, in the case of larger L-penicillamine, L-arginine and L-glutathione molecules some slight differences could be noticed. For NPs-LPen I have observed the most pronounced change in the CD spectra, with the maximum shifted to shorter wavelengths (Fig. 3b). The changes in the CD spectrum of those molecules could be due to the shift of the conformational equilibrium²⁶ after attaching them near to the NPs surfaces.

The epoxidation process and the presence of chiral molecules near to the NPs surface influenced also their up-converted emission spectra and kinetics. Fig. 4 shows the concentration corrected up- and down-conversion spectra of the studied NPs systems obtained with 976 nm laser diode excitation. All of the studied NPs systems exhibited four sharp emission lines in the visible part of the spectra, the peaks at 408 nm, 525 nm, 550 nm and 654 nm were assigned to the up-converted transitions of ²H_9/2 → ⁴I_15/2, ²H_11/2 → ⁴I_15/2, ⁴S_3/2 → ⁴I_15/2 and ⁴F_9/2 → ⁴I_15/2 in Er³⁺ ions, respectively, while the emission line at 1524 nm to the down-converted emission of Er³⁺ ions associated with the ⁴I_13/2 → ⁴I_15/2 electronic transition (for energy levels diagram see ESI Fig. S5†). For colloidal, inorganic NPs synthesized in the hydrophobic environment, one of the main problem after surface functionalization is the decrease of emission intensity due to the solvent and surface attached ligands quenching effect. However, as I have used the epoxidation process rather than ligand exchange, the initial NPs surface coating was preserved, and thus the concentration corrected emission intensity did not decrease strongly after the surface functionalization. I have also observed increase of emission intensity at 1524 nm after NPs functionalization and dispersing them in heavy water. The infrared emission associated with Er^{3+ 4}I_13/2 → ⁴I_15/2 electronic transition arises from 976 nm photon absorption by Yb³⁺ ions (²F_7/2 → ²F_5/2) followed by the Yb³⁺ → Er³⁺ (⁴I_15/2 → ⁴I_11/2) energy transfer, and nonraditive depopulation of ⁴I_11/2 level down to the ⁴I_13/2 one (Fig. S5†). The increase in ⁴I_13/2 → ⁴I_15/2 emission intensity at 1524 nm after surface treatment should thus be the result of the enhanced nonradiative processes due to the change of the ligands and solvent environment. However, it must be kept in mind that absolute integral emission intensity measurements are to some extent susceptible to errors due to the determination of the NPs concentration in the solutions (weighting of the dried NPs) and inevitable fluctuations in laser power intensities during experiments. In order to get a better insight into the Er³⁺ ions emission mechanisms change upon NPs surface functionalization, we have performed power dependence studies, calculated intensity ratios of blue (²H_9/2 → ⁴I_15/2) to green (²H_11/2 → ⁴I_15/2, ⁴S_3/2 → ⁴I_15/2) and red (⁴F_9/2 → ⁴I_15/2) to green emission lines, and measured the decay kinetics of ²H_11/2, ⁴S_3/2 and ⁴F_9/2 excited states.

Fig. 4 Up- (a) and down-conversion (b) spectra of as-synthesized and surface functionalized NaYF₄:2%Er,20%Yb NPs after 976 nm laser diode excitation. The intensities have been corrected for different concentration of NPs before and after surface treatment.

For up-converting NPs the dependence between the intensity of visible emission (I_VIS) and excitation power (P) is determined by the competition between the linear decay and up-conversion process for the depletion of the intermediate state, and can be expressed as: I_VIS ∼ Pⁿ,^44,45 where n is the number of infrared photons absorbed per visible photon emitted. For the domination of linear decay process the n values for visible emission are usually equal 2, or are between 2 and 3, which indicates that two or mixed two and three photon processes are required for the populating of the emitting state. However, with increasing excitation power, up-conversion depletion of the intermediate state becomes more prominent, and the n gradually becomes close to unity, followed by the up-conversion saturation. For the power dependence slope values smaller than 1 quenching due to thermal effects occurs. I have performed the power dependence studies of up-converted emission of as-synthesized and surface functionalized NaYF₄:2%Er,20%Yb NPs after 976 nm laser diode excitation, and the double logarithmic plots (I = f(P)) for blue, green and red emission are presented in Fig. 5a, b and c, respectively. Surprisingly, slope values for blue emission at 408 nm were equal to 1.13, 0.85, 1.11, 1.24 and 1.45 for NPs-LCys, NPs-LPen, NPs-LArg, NPs-LGlu and as-synthesized NPs, respectively (Fig. 5a). For up-converted emission of such a high energy, one would expect to observe three or even four photon processes, however the saturation of one of the lower lying emitting levels can effectively diminish the slope values for UV and blue up-converted emission.^46,47 Suyver et al.⁴⁷ presented a model where the slopes of up-conversion double logarithmic plots were equal to 1, 2 and 3 for low power limit, and were gradually reduced from 3 to 1, from 2 to 2/3 and from 1 to 1/3 at high excitation regimes. Additionally, the power density required to observe the change in the slope values from higher than 1, to a slope of 1 was different for the different up-conversion emission bands.^46,47 Stecher et al.⁴⁶ demonstrated slope values ∼1.6 for blue (362 nm and 452 nm) up-converted emission of Tm³⁺ ions. The single-photon excitation of higher lying Tm³⁺ ions energy level (¹G₄) was possible due to the saturation of red emitting ³F₃ and ³H₄ ones.⁴⁶ Similar saturation and “super saturation” up-conversion processes were reported for Er³⁺,Yb³⁺ co-doped nanomaterials.^48,49 Authors reported diminishing power dependency slope values to ∼1 for blue, and to ∼2 for deep UV Er³⁺ emission, where one would expect three- or even five photon processes, while using low pumping powers. The metastable character of red emitting energy level in Er³⁺ ions, characterized by very long luminescence lifetimes, was responsible for the saturation of ⁴F_9/2 energy level for very low pumping powers, what further results in the decrease of slopes of both red and blue emission in comparison to the expected values. In fact, a closer look at the power dependence of red emission at 654 nm of as-synthesized NaYF₄:2%Er,20%Yb NPs (Fig. S6†) revealed the saturation regime of ⁴F_9/2 energy level at high excitation powers, which can be the reason for the observed low slope values for blue Er³⁺ emission, which is the consequence of ⁴F_9/2 → ²H_9/2 transition (Fig. S5†). The observed saturation process at low excitation powers can be in fact considered as an advantage of the studied systems. Based on the work of H. Liu et al.,⁵⁰ who investigated the power dependency of up-conversion quantum yield values, materials with high balancing power density (power at which the slope values of up-conversion emission change from ∼2 to ∼1) are desirable to increase the up-conversion intensity for power densities far away from the saturation. Nevertheless, for NPs dispersed in water, heating due to the water absorption of 976 nm laser light and emission quenching due to thermal effects cannot be neglected. The decrease in slope values for green and red emission was also observed for functionalized NPs when compared to the as-synthesized ones (Fig. 5b and c). The observed slope values close to 2 for both green and red emission of as-synthesized NaYF₄:2%Er,20%Yb NPs indicate the typical two photon mechanism, in which first an Yb³⁺ ion is excited (²F_7/2 → ²F_5/2) and then it transfers the energy to neighbor Er³⁺ (⁴I_15/2 → ⁴I_11/2) by the energy transfer up-conversion process.⁴⁴ A next 976 nm photon can further populate the ⁴F_7/2 energy level in Er³⁺ ion followed by the multiphonon relaxation to ²H_11/2 and ⁴S_3/2 levels, which results in green emission. The pathway for population of the red emitting ⁴F_9/2 level can be, however, twofold. The ⁴F_7/2 energy level can be nonradiatively bridged with emitting ⁴F_9/2 one (NR₁ at Fig. S5†), or the nonradiative transition from ⁴I_11/2 → ⁴I_13/2 can occur (NR₂), and a second photon then excites Er³⁺ ion from ⁴I_13/2 up to ⁴F_7/2 level. The decrease in slope values after surface functionalization can be attributed to the change in the up-conversion luminescence bridging intermediate level from ⁴I_11/2 for NPs with oleic acid molecules at the surface, to ⁴I_13/2 for functionalized NPs³⁸ due to the increase in nonradiative transitions. The most pronounced decrease in n values was observed for NPs functionalized with L-penicillamine, with the change from 1.45 to 0.85, from 1.91 to 1.57 and from 2.15 to 1.53 for blue, green and red emission, respectively, when compared to NPs with oleic acid molecules at the surface. Different behavior was observed for down-converted Er³⁺ emission at 1524 nm (Fig. 5d), for which the slope values were slightly higher for functionalized NPs, with again the highest value for NPs-LPen, which, together with the observed increased NIR emission in those NPs, supports the hypothesis of enhanced nonradiative transitions, which populate the NIR emitting ⁴I_13/2 energy level.

Fig. 5 Double logarithmic plots of the luminescence integral intensity of blue (a), green (b), red (c) and infrared (d) bands for as-synthesized and surface functionalized NaYF₄:2%Er,20%Yb NPs vs. power of the excitation laser beam.

After functionalization of the NaYF₄:2%Er,20%Yb NPs surfaces I have also observed the increase in blue to green (B/G) and red to green (R/G) emission intensity ratio (Fig. 6). The spectroscopic R/G parameter can be used as a characteristic value for Er³⁺ and Yb³⁺ co-doped materials, as it depends on the morphology, crystal structure, active ions relative dopant concentration and, what is the most important in our case, on the surface state of NPs.^51,52 The decrease of green emission, with respect to both blue and red emission lines, upon surface functionalization can be related to the ligand-dependent processes which affect the population of green emitting (²H_11/2 and ⁴S_3/2) energy levels. The energy difference between green (⁴S_3/2) and red (⁴F_9/2) emitting levels is approximately equal to 3000 cm⁻¹ and can be effectively bridged with the O–H vibrations in water molecules, thus increasing the red emission. Additionally, the NR₂ processes (Fig. S4†) can be enhanced when NPs are dispersed in water due to the similar energy difference between ⁴I_11/2 and ⁴I_13/2 energy levels. Similar effect was observed by Meesaragandla et al.⁵³ after functionalization of NaYF₄ NPs surface with dicarboxylic acid molecules and transferring the NPs to water dispersions. The increase in the population of ⁴F_9/2 energy level would also facilitate the ⁴F_9/2 → ²H_9/2 transitions, and thus the enhanced blue emission should be observed, which was actually the case for surface functionalized NPs (Fig. 6a). The influence of the surface related quenching on the B/G and R/G Er³⁺ ions emission ratios was also observed for NPs with different sizes.⁴⁸ As the surface to volume increased both red and blue emission intensities were increased in respect to the green one. Again, the increased nonradiative bridging by ligand and solvent molecules of the ⁴I_11/2 → ⁴I_13/2 transition lead to the increase in both red and blue Er³⁺ emission. Beside increase in the R/G values after surface functionalization, I have also observed the change in general trend for R/G values power dependence. For as-synthesized NPs with oleic acid molecules on the surface the R/G ratio increased from 0.5 up to 0.68 with the excitation power, while for NPs having chiral ligands attached near to the surface this relationship was reversed, and increased excitation power resulted in lowering the R/G values from 0.91 to 0.83 (Fig. 6b). There are two phenomena which can be responsible for the observed changes in R/G ratio with rising excitation power. These are either local temperature rise, or population increase and saturation of intermediate levels in the course of energy transfer up-conversion process. For as-synthesized NPs increase in R/G with the excitation power rise is due to the observed saturation of ⁴F_9/2 energy level (Fig. S6†), while for functionalized NPs the increased population of red emitting level is achieved due to the ligand and solvent induced nonradiative transitions (NR₂ in Fig. S5†), the R/G ratio power dependence tends to saturate at lower excitation powers.

Fig. 6 Power dependence of blue to green (B/G) (a) and red to green (R/G) (b) emission intensity ratio for as-synthesized and surface functionalized NaYF₄:2%Er,20%Yb NPs.

Finally, I have investigated the luminescence decays of green and red emission of Er³⁺ ions after 976 nm pulsed laser diode excitation, and the decay curves for as-synthesized and functionalized NPs are presented in Fig. 7. All of the studied samples exhibited single exponential behavior, and the values of luminescence lifetimes (τ), which were calculated based on fitting the experimental results with y = A exp(−t/τ) + B curve, are included in Fig. 7. The as-synthesized NPs showed long Er³⁺ ions luminescence lifetimes, with values of 372 μs, 369 μs and 529 μs for emission at 525 nm, 550 nm and 654 nm, respectively. The surface functionalization resulted in decrease in luminescence lifetimes values for both green (²H_11/2 → ⁴I_15/2, ⁴S_3/2 → ⁴I_15/2) and red (⁴F_9/2 → ⁴I_15/2) emission, however this effect was more pronounced for higher energy transitions. Additionally, the τ values only slightly varied with the change of chiral molecules attached near to the NPs surface, and were equal to ∼132 μs, ∼131 μs and ∼412 μs for emission at 525 nm, 550 nm and 654 nm, respectively. This is reasonable, since for the preparation of those samples we used epoxidation process and no change in the size or surface structure of NPs was observed. The emission lifetimes of ²H_11/2 and ⁴S_3/2 states were shortened by ∼60% after surface functionalization, while for red emission I have observed only ∼20% lifetime shortening. In general, changing the solvent to water results in increase in nonradiative relaxations, and thus the observed luminescence lifetimes are shortened. The observed changes in luminescence lifetimes values, and more pronounced nonradiative depopulation of green emitting levels after surface functionalization, are consistent with the results of power dependence and luminescence intensity ratio values measurements presented earlier in the text.

Fig. 7 Luminescence decay curves of ²H_11/2 (a), ⁴S_3/2 (b) and ⁴F_9/2 (c) excited states of Er³⁺ ions for as-synthesized and surface functionalized NaYF₄:2%Er,20%Yb NPs after 976 nm pulsed laser diode excitation.

Conclusions

In summary, I have presented an aqueous phase transfer procedure for up-converting Er³⁺,Yb³⁺ co-doped hexagonal NaYF₄ NPs based on the use of commercially available, small and biologically important chiral molecules: L-cysteine, L-penicillamine, L-arginine and L-glutathione. The most promising results, regarding the long term colloidal stability, were obtained for NPs with epoxidized oleic acid ligands at the surface, and further attached L-penicillamine molecules. Those NPs showed sustained during storage low PdI index (∼0.2) and the highest surface potential at the level of +24 mV. The efficient attachment of chiral molecules near to the NPs surfaces was also confirmed based on FT-IR and CD spectroscopy, the latter also revealed the strongest interaction between NPs and L-penicillamine molecules, which was visible as shift in peak maximum in CD spectra. Additionally, the performed surface functionalization did not strongly affect the overall emission intensity, and water solution of NPs exhibited bright up-conversion luminescence after 976 nm laser diode excitation. The change of the surface attached molecules and dispersing solvent resulted also in the increase in NIR emission of Er³⁺ ions at 1524 nm. The changes in the important spectroscopic parameters, which characterize the up-converting NPs systems, such as: luminescence intensities, blue to green, red to green emission ratios and the luminescence decays were related to the increase in nonradiative transitions upon surface functionalization and changing the solvent to water. Again, I have observed the strongest effect for NPs-LPen, which showed the highest decrease in power dependence slopes for blue, green and red emission when compared to as-synthesized NPs, and simultaneously the highest corresponding value for down-converted emission. The results presented within this article constitute an advance in the search for new types of biologically important ligands for surface functionalization of up-converting β-NaYF₄ NPs. Additionally, since it was already proven that the hybrid NPs composed of inorganic, luminescent cores with attached chiral molecules can be applied as optical markers in chiral recognition and sensing chiral drugs, the present results open new perspectives for possible applications of up-converting β-NaYF₄ NPs. The initial CPL signals originating from chiral molecules binding to the NPs surfaces could by changed in shape or intensity upon reacting with e.g. chiral drugs, providing the specific sensing of o certain target molecules. Additionally, as in surface functionalized NPs the NIR Er³⁺ emission was enhanced in comparison to as-synthesized NPs (Fig. 4b), the increase of ⁴I_13/2 Er³⁺ ions energy level population could thus act in favor of possible applications in NIR to NIR molecular recognition sensing with studied NPs systems.

Acknowledgements

DW acknowledges the support from the Ministry of Science and Higher Education under grant “Iuventus Plus” in years 2015–2017, project no. IP2014 050273.

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Footnote

† Electronic supplementary information (ESI) available: High resolution TEM images, size distribution histogram, characterization of nanoparticles colloidal stability, CD spectra of as-synthesized nanoparticles and energy level diagram. See DOI: 10.1039/c5ra19496b

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