| Florescence resonance energy transfer (FRET) is recognized as a nanoscale "spectroscopic molecular ruler". As an important homogeneous analytical technique, which is simple and sensitive, it has been widely used in immunoassay, nucleotide hybridization, interactions of biological macromolecules, and other biomedical analysis. Nevertheless, traditional FRET with one-photon excitation more suffer from some disadvantages. The sensing system is susceptible to interference with autofluorescence from biological specimens and scattered light, owing to excitation typically in UV-Vis range. Besides, in case of spectral overlap of excitation between the energy donor and the acceptor, the excitation light of the donor usually would directly excite the acceptor, resulting in a false positive signal.Upconversion nanoparticles (UCNPs), which is a kind of lanthanide-ion-doped phosphor with anti-Stokes luminescent nature, can sequentially absorb two or more photons of lower energy (typically at 980 nm) and emit UV-vis light. This unique characteristic minimizes background signal from autofluorescence and scattered light, thus improves the optical penetration depth in biological samples, and reduces photo-damage and photo bleaching. Also, under 980 nm excitation light the co-excitation of the donor-acceptor pair is thoroughly eliminated. For these reasons, UCNPs can be promising energy donors in FRET-based assays. Furthermore, FRET can improve signal-to-background ratios via the luminescent off-on assay model, which can remedy the inadequate luminescent efficiency of UCNPs. Combining the strongpoints of these two tools, UC-FRET will become a robust approach in analysis of biological specimens with complex matrices and in vivo analysis. A variety of sensor platforms and probes based on UC-FRET have been employed in many areas in biomedicine.Despite the great success and the ongoing increase of interest with UCNPs-based bioassay since 2005, there is an intractable problem with UCNPs as energy donor due to its inherent shortcoming, i.e., the clash between energy-transfer efficiency and other performances of the nanoprobe such as luminescent efficiency, biocompatibility, stability and flexibility. UC-FRET nanoprobes with small-molecule energy acceptor normally have good stability, biocompatibility and flexibility in application in cell and in vivo. However, only those emission centers near the surface of the nanoparticles are in the effective energy-transfer range and quenchable. As a result, the efficiency of energy transfer is poor and the donor emission is incompletely quenched, impairing the assay sensitivity. In recent years, in order to improve the energy transfer efficiency, sub-10 nm homogeneous UCNPs were used as the energy acceptor of FRET to shorten the donor-acceptor distance. But these materials are susceptible to severe environmental quenching due to small diameter and large specific surface area, result in poor luminescent efficiency. On the other hand, several kinds of inorganic nanomaterials, such as gold nanomaterials, carbon nanomaterials (e.g. graphene and carbon nanoparticles) and planar atomic crystals, have been employed as energy acceptors. They extend the range of energy transfer to 20-30 nm and exhibit improved quenching performance against UCNPs, thanks to extremely intense absorption or high conductivity, as well as the surface energy-transfer mechanism. But the introduction of nanosized quenchers may compromise the thermodynamic stability and biocompatibility. In the meanwhile, their bulky size is also undesirable in applications in cellular and tissue analysis. According to the mentioned above, aiming to improve the energy transfer efficiency of the UC-FRET system while maintaining other features, this dissertation proposes the strategy to use sandwich-structure UCNPs (SWUCNPs) as the energy donor to construct a series of new upconversion nanoprobes through selecting or designing and synthesizing proper energy acceptors, and to apply them in bioimaging. The main contents are as follows:1. We fabricated a SWUCNPs, in which most emitting ions are confined on or near the surface, thereby within the range of effective energy transfer. It is too challenging to achieve such controlled distribution of ions in traditional homogeneous UCNPs. Therefore, we deposited a layer of NaYF4:Yb,Er doped with emitting ions on an inert NaYF4 matrix core. To protect the emitters from environmental quenching, another NaYF4 shell is further deposited on the surface of the inner shell to form a type of SWUCNPs (NaYF4@NaYF4:Yb,Er@NaYF4). To examine the energy transfer efficiency of SWUCNPs, an organic dye was selected as the energy acceptor, which can be adsorbed onto the surface of SWUCNPs for FRET via electrostatic attraction. Compared to homogeneous UCNPs, the energy transfer efficiency of SWUCNPs are obviously enhanced and can be optimized by tuning the thickness of the shell layers.2. To investigate the feasibility of constructing a highly sensitive UC-FRET-based probe with SWUCNPs as the energy donors, we used a commercial calcium probe (Fluo-4) as the energy acceptor and the specific receptor for Ca2+ to fabricate a NIR Ca2+ nanoprobe. Firstly, the surface ligands of SWUCNPs are removed via acid treatment to acquire bared SWUCNPs. Fluo-4 is directly linked to bared-SWUCNPs via the chelation between the four carboxyl groups in the Ca2+ recognition motif and the exposed Ln3+ ions on SWUCNPs, quenching the luminescence from the donor. The absence of extra ligands on bared SWUCNPs enables direct contact with the acceptor, minimizing the distance between donor-acceptor pair and improving the energy transfer efficiency. The introduction of Ca2+ leads to recovery of the luminescence of SWUCNPs, because the stronger affinity of Fluo-4 toward Ca2+ causes the binding between Fluo-4 and Ca2+, and the detachment of Fluo-4 from the surface of SWUCNPs, which thus pulls the donor-acceptor pair apart and inhibits the FRET process. Ca2+ can be quantitated by the degree of emission recovery of SWUCNPs. This probe affords significantly improved FRET efficiency and ultrahigh sensitivity with good stability and biocompatibility. It is able to detect [Ca2+] in solution and monitor Ca2+ content in living cells and in tissues.3. To further expand the application area of SWUCNPs-based upconversion nanoprobe, we constructed UC-FRET-based nanoprobe to solve a practical issue in biomedicine. An azo dye (mOG) responsive to OH was designed and syntheized as the ligand and energy acceptor to establish OH fluorescence probe based on UC-FRET, which showed intense light absorption and was suitable to be the energy acceptor to SWUCNPs. mOG was loaded on the bared surface of SWUCNPs via the coordination between the carboxyl groups of mOG and the exposed lanthanide ions. The absorption band of mOG matches well with the emission of the SWUCNPs, resulting in quenching of the upconversion luminescence (UCL) by up to 90% or more and ensuring the sensitivity of the nanoprobe. Upon the reaction of mOG with OH, the azo bond is broken and the absorption spectrum is changed. The light absorption of mOG is therefore altered, inhibiting the energy transfer from SWUCNPs to mOG. As a consequence, the UCL of SWUCNPs is recovered, which allows quantitative detection of OH. Thanks to the high resistance of SWUCNPs to interference and the high energy transfer efficiency, the nanoprobe exhibits an excellent sensitivity and is the first probe capable of detecting the subtle variation of OH in tissues.4. Thanks to the advantages of UC-FRET probe in bioimaging, we applied it at subcellular level, diversed the biological application of SWUCNPs probe, and minimized photobleaching compared to small molecule probes. A carbazole-based hemicyanine dye (Caz-Hcy) was designed as an energy acceptor of SWUCNPs, and used to construct an upconversion nanoprobe targeting ClO- in mitochondria. The probe was assembled through the chelation between the carboxyl groups of Caz-Hcy and the exposed lanthanide ions on the surface of bared SWUCNPs. Disruption of the ?-conjugated system of Caz-Hcy by reacting with ClO- can shift the dye's absorption spectrum, blocking the energy transfer from SWUCNPs to Caz-Hcy. Furthermore, a positively charged nitrogen heterocyclic moiety in Caz-Hcy endows the nanoprobe with specific mitochondria targeting, enriching it in mitochondria and detect the [ClO-] level in mitochondria. |
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