High-quality water-soluble and surface-functionalized upconversion nanocrystals as luminescent probes for bioimaging
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.
Section snippets
Materials
All the chemicals used were of analytical grade and were used without further purification. Deionized water was used throughout. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (abbreviated as EDC) and N-hydroxysulfosuccinimide sodium salt (abbreviated as sulfo-NHS) were purchased from Sinopharm Chemical Reagent Co. (China). 9-Fluorenylmethyl chloroformate (abbreviated as FmocCl) and folic acid (FA) were obtained from Acros. 6-aminohexanoic acid and oleic acid were obtained from
Synthesis and characterization of UCNPs
It is well known that the selection of an appropriate surfactant to stabilize a micelle and/or to control the growth of nanocrystals is very important [25], [26], [40]. OA is commonly used as the chelating agent and surfactant. Thus, the as-prepared nanocrystals will inevitably be hydrophobic, because the hydrophobic alkyl tails of the surfactant capped nanocrystals extend into the nonpolar continuous-phase solvent. In this present study, to obtain water-soluble UCNPs bearing appropriate
Conclusion
In summary, we have demonstrated a new one-step synthetic strategy for high-quality surface-functionalized upconversion nanophosphors (UCNPs) via a hydrothermal reaction assisted by binary cooperative ligands (HR-BCL) of hydrophilic 6-aminohexanoic acid (AA) and hydrophobic oleate ligands, which hold the surface of the nanoparticles simultaneously, and significantly affect the morphology, size, crystallinity and surface properties of the nanocrystals. By carefully controlling suitable
Conflict of interest statement
None.
Acknowledgments
The authors thank National Natural Science Funds for Distinguished Young Scholars (20825101), National Natural Science Foundation of China (91027004), Shanghai Sci. Tech. Comm. (1052nm03400 and 10431903100), IRT0911, Shanghai Leading Academic Discipline Project (B108) and the CAS/SAFEA International Partnership Program for Creative Research Teams for financial support. We thank Kaiyun Ye for help and discussions.
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