Preparations And Applications Of Hybrid Nanoprobes Based On Lanthanide-doped Upconversion Nanocrystals

Fluorescence resonance energy transfer（FRET） is an energy transfer process happened between two fluorophores, which is called donor-acceptor pair. Usually, only when the emission of the energy donor and the absorbance of energy acceptor match well, and the distance between the donor-acceptor pair is less than 10 nanometers, can the FRET process take place efficiently. Lanthanide-doped upconversion nanoparticles（UCNPs） has received increasing popularity due to the low toxicity, optical and chemical properties, large stokes shifts, near-infrared（NIR） light as an excitation source, weak auto-fluorescence backgrounds, and resistance to photo-bleaching. The emission of the lanthanide ions is usually arising from the f-f transitions, which is different from the fluorescent process of the organic dyes. So the energy transfer process when lanthanides are involved as the energy donor is generally called as luminescence resonance energy transfer（LRET）. This thesis is focused on the design and the synthesis of LRET-based hybrid nanoprobes by using UCNPs as energy donor, as well as their applications in the detection.1) It is greatly important to seek a fast and sensitive method for the detection of Cu²⁺ ions because of their vital roles in human body. Fluorescent probes, especially the rhodamine derivatives, have been considered as promising candidates for detection of Cu²⁺ ion due to their attractive feature. However, one problem frequently encountered in rhodamine-based fluorescent probes is that some of these probes could not distinguish Cu²⁺ from Hg²⁺, which limits their application in biological samples. In this work, a new rhodamine B derivative（RBH） was presented and then grafted to mesoporous silica coated upconversion nanoparticles to fabricate an organic-inorganic hybrid nanoprobe. On the addition of Cu²⁺ ion, an emission band at about 580 nm appeared while the intensity of the green emission at about 545 nm of the nanoparticles decreased upon the excitation of a 980 nm laser, implying a LRET from the core-shell upconversion nanoparticles（CS-UCNP） to the RBH-Cu²⁺ complex. The obtained LRET hybrid nanoprobe could detect Cu²⁺ exclusively even at the existence of Hg²⁺ with a detection limit 0.82 μM in ethanol solution. Furthermore, this nanoprobe can be used for monitoring subcellular distribution of Cu²⁺ in living cells based on upconversion luminescence and downconversion fluorescence.2) A novel luminescence “Turn-On” nanoplatform based on LRET from aptamer ss DNA（5’-NH2-TCTCAATGGCTGCCTCCC-3’） functionalized hydrophilic upconversion nanoparticles（Cit-UCNPs-ss DNA, energy donor） to single-walled carbon nanohorns（SWCNHs, energy acceptor） was prepared for sensitive detection of PML/RARα fusion gene. In the presence of the target DNA, a PML/RARα fusion gene of acute promyelocytic leukemia（APL）, the Ï€-Ï€ stacking interaction between the energy donor Cit-UCNPs-ss DNA and energy acceptor SWCNHs weakened and their distance enlarged. Therefore, the luminescence of Cit-UCNPs-ss DNA would be recovered（turn on） due to the inhibition of the LRET process. Upon the addition of single-base mismatch DNA and non-complementary DNA, the luminescence intensities of Cit-UCNPs-ss DNA were nearly not changed. These results demonstrated the high specificity of the nanobiosensor systems towards PML/RARα fusion gene. Based on this fact, a sensitive method was developed for the turn-on detection of APL with a detection limit as low as 0.28 n M. To the best of our knowledge, this is the first time that upconversion nanoparticles and SWCNHs were used as a donor-acceptor pair to detect PML/RARα fusion gene sequences through a LRET process.3) Two kinds of luminescent nanoprobes were developed for Pb²⁺ detection based on LRET. UCNPs functionalized with sodium citrate were covalently grafted with the Pb²⁺ aptamer p DNA（5’-NH2-（CH2）6-GGGTGGGTGGGTGGGT-3’） as the energy donor（Cit-UCNPs-p DNA） while SWCNHs or GO as the energy acceptor. By using the SWCNHs（or GO） as matrix, the final Cit-UCNPs-p DNA-SWCNHs（or Cit-UCNPs-p DNA-GO） nanoprobe can be obtained, in which the LRET process happened and the upconversion luminescence of the probe was quenched. With the addition of Pb²⁺, a stable G-quadruplex-Pb²⁺ complex was formed, which resulted in the separation between Cit-UCNPs-p DNA nanoparticles and SWCNHs（or GO） with the recovery of the upconversion luminescence. The two turn-on luminescence probes could detect Pb²⁺ ion distinguishing from other ions with high sensitivity and selectivity in aqueous solution. To the best of our knowledge, this is the first example of using UCNPs and SWCNHs（or GO） as a donor-acceptor pair for detecting Pb²⁺ ion through a LRET process.