High-quality water-soluble and surface-functionalized upconversion nanocrystals as luminescent probes for bioimaging

Abstract

Rare-earth upconversion nanophosphors (UCNPs) have great potential to become excellent biological luminescent labels for fluorescence bioimaging. However, it is still difficult to directly synthesize high-quality water-soluble UCNPs bearing appropriate functional groups using a one-step synthetic strategy. Herein, we report a one-step synthetic strategy for high-quality water-soluble and surface-functionalized UCNPs using a hydrothermal reaction assisted by binary cooperative ligands (HR-BCL). In this system, 6-aminohexanoic acid and oleate were introduced to control nuclear generation and crystal growth of small nanoparticles. The UCNPs synthesized here showed high crystalline and intense upconversion luminescence emission. Fourier-transform infrared and nuclear magnetic resonance spectroscopy indicated that 6-aminohexanoic acid and oleate cooperate as surface ligands to co-control the surface of UCNPs. Thus, the water-solubility of the as-prepared UCNPs can be tuned by changing the molar ratio of 6-aminohexanoic acid to oleate. The AA-modified UCNPs provided a free amine content of (6.0 ± 0.2) × 10⁻⁵ mol/g, which renders them dispersible in aqueous solution and allows further conjugation with folic acid (FA) for targeted bioimaging. Furthermore, the amine-functionalized UCNPs show intense near-infrared upconversion luminescence and were successfully applied in the lymphatic capillary bioimaging of small animals with a high signal-to-noise ratio, suggesting that these surface-functionalized UCNPs are promising candidates for luminescent biolabels.

Introduction

Due to the unique 4f or 5f electron structure, some nanocrystals doped with the rare-earth activator ions Er³⁺, Tm³⁺ and Ho³⁺ exhibit upconversion luminescence (UCL) [1], [2], [3], [4], [5]. Such a unique UCL mechanism allows rare-earth nanophosphors to display some special advantages as photoluminescent probes of bioimaging such as a large anti-Stokes shift of several hundred nanometers, absence of autofluorescence from biological samples [6], remarkable light penetration depth in tissue [7] and no photobleaching [7], [8]. Consequently, rare-earth upconversion nanophosphors (UCNPs) have recently become one of the most promising classes of luminescent labels for bioimaging in vitro [9], [10], [11], [12], [13] and in vivo [7], [14], [15], [16], [17], [18], [19], [20], [21].

The practical applications of UCNPs as bioimaging agents make demands on synthetic strategies toward more precise control over several requirements: (I) uniform size and morphology, and high crystallinity (II) good dispersibility in aqueous solution, and (III) the presence of suitable functional groups on the surface that allow further conjugation by active biomolecules. Recently, using a water-soluble ligand or polymer as a surfactant, some water-soluble UCNPs have been reported [15], [22], [23], [24]. For example, Wang et al. prepared water-soluble UNCPs with polyethylenimine (PEI) as the surface ligand [23], [24]. Unfortunately, these methods are still limited by the uncontrollable size and morphology of the as-prepared UCNPs. If using oleic acid (OA) with long alkyl chains as a surface ligand, monodisperse UCNPs with uniform size and morphology have successfully been synthesized through the hydrothermal reaction [25], [26], [27], thermolysis method [28], [29], [30], [31] and solvothermal method [32], [33]. However, such uniform UCNPs are usually hydrophobic due to the OA-coating on the surface, and further post-treatment (including polymer capping, surface silanization, surface ligand oxidation, and ligand exchange) [34], [35], [36], [37], [38], [39] are required. Such two-step conversion synthetic strategies are associated with relatively complicated synthetic processes and post-treatment procedures. Thus, it remains a challenge to develop one-step procedure for preparing high-quality water-soluble UCNPs with uniform size and functional surfaces.

As described above, the method using OA with long alkyl chains as the surface ligand provided high-quality hydrophobic UCNPs with controllable sizes and morphology, and the one-step approach using a hydrophilic ligand with short alkyl chains allowed preparation of water-soluble UCNPs with irregular morphology and size. Our aim was to combine the advantages of these two methods to develop a new one-step synthetic strategy of hydrothermal reaction assisted by binary cooperative ligands (HR-BCL) of OA with long alkyl chains and a hydrophilic ligand with short alkyl chains to prepare high-quality and water-soluble UCNPs with functional surface groups. To cap the surface of UCNPs, the hydrophilic ligand with short alkyl chains should contain a coordinated group (such as carboxylic acid), which bonds with rare-earth ions easily. In light of the fact that 6-aminohexanoic acid (AA) provides a carboxylic acid group to bond the lanthanide ions and an amino group to achieve surface-amino-functionalization of UCNPs, we selected AA and OA as the binary cooperative ligands. These ligands were introduced during a hydrothermal reaction for one-step synthesis of high-quality and water-soluble UCNPs with tunable surface properties (Scheme 1). As expected, regular and uniform cylinder-like upconversion nanocrystals with relatively smooth surfaces were obtained. The effect of AA and the OA ligand on the surface properties, size and morphology of UCNPs was investigated by Fourier-transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy, as well as by transmission electron microscopy (TEM). Moreover, an amino group on the as-prepared UNCPs was confirmed by a standard Fmoc quantification protocol. This amino group could further conjugate with folic acid (FA) for targeted cell imaging. More importantly, we successfully applied water-soluble UCNPs to the in vivo lymphatic imaging of small animals.