Upconversion Fluorescence Resonance Energy Transfer In Bio-detection And Photodynamic Therapy

Fluorescence resonance energy transfer (FRET)-based analytical methods have gained considerable attention as powerful tools for biological detections because of their simplicity and high sensitivity. There are a number of conventional ?uorescent biolabels used including organic dyes and quantum dots in this system. These conventional down-conversion ?uorescent materials require ultraviolet or blue excitation wavelengths. Many biological samples show auto?uorescence under such conditions, which decreases the sensitivity of detection. For photodynamic therapy (PDT),the visible light needed to activate most photosensitizers cannot pass through a thick tissue, which resulted in the PDT effect is bad. Upconversion nanoparticles (UCNPs) appear as a breakthrough to resolve the problem. It could convert lower-energy near-infrared (NIR) light to higher-energy light through excitation, which can penetrate deep tissues without causing sample damage and avoid auto?uorescence from biological samples. Thus, upconversion materials show potential to be used for FRET based immunoassay and PDT. Around this point, the main results from our experiments are outlined as followings:(1) In order to achieve biological functions of UCNPs, NaYF4:Yb3+, Er3+ upconversion nanoparticles (UCNPs) were synthesized via the hydrothermal assisted homogeneous precipitation method and then coated with silica. The SEM image demonstrated that the samples were uniform in size distribution with ca. 25 nm, before and ca. 250 nm after silica coating, respectively. The photoluminescence spectra and lifetime measurement showed that the silica shell had hardly effect on the properties of UCNPs fluorescence. The naked eye-visible green fluorescence pattern was acquired from the sample in the PBS buffer excited by 980 nm laser. The UCNPs were linked to the antibodies. The circular dichroism (CD) spectra of pure antibody and bioconjugates were very similar to each other. Finally, the immunofluorescence assay indicated that the UCNPs-antibody bioconjugates had excellent species-specific detection ability.(2) We have developed a feasible surface ligand exchange method for getting water-soluble, small in size and amido-functionalized UCNPs. The construct based on FRET between avidin-conjugated NaYF4: Er3+, Yb3+ UCNPs as donors and biotinylated R-Phycoerythrin as acceptors were employed. A sample model of biotin detection was applied. Such approach enabled the detection and quanti?cation of the biomolecular.(3) Extensive efforts have been invested in FRET based homogeneous bioaffinity assays utilizing UCNPs as a donor. However, there are some fundamental problems related to fluorescence measurements. Self-fluorescence from biological matrixes and other sample materials and relative weak upconversion emission among the ultraviolet-visible wavelengths region could cause ambiguous or unmeasured emission. To address this problem, we have utilized NaYF4: Yb3+, Tm3+ UCNPs as an energy donor, which can emit intense (NIR) emission around 800 nm ranges, and gold nanoparticales (GNPs) as an energy acceptor, which has a surface plasmon absorption maximum at 784 nm. UCNPs and GNPs were conjugated with goat antihuman IgG and human IgG, respectively. When free human IgG is added, it competitively binds to UCNPs-goat antihuman IgG, and inhibits the FRET process. As a result, the fluorescence change effect was correlated with the concentration of human IgG.(4) Nowadays, there is a growing interest in the use of QDs in design of LRET-based aptamer luminescent reporters. However, there are concerns regarding the potential photosensitized breakage and damage of aptamer molecules due to the production of reactive oxygen intermediates (ROI) by photoactivated QDs. In this work, we have developed a novel aptamer-based alternative to QDs. Nitroblue tetrazolium (NBT) assay has ?cormned that generation of ROI by photoactivated UCNPs could be completely neglected. The feasibility of this principle in a model ATP assay has been demonstrated.(5) FRET immunoassays with QDs as energy acceptors are of particular interest because the extremely high-extinction coefficients of the QDs over a broad absorption spectrum enlarged the scope of the recognition events through the increase of the F?rster radius. Large absorptions of the QDs first appeared as a drawback, providing undesired direct excitation of the acceptor. The problem has been recently solved by demonstrating the feasibility of time resolved measurement of sensitized QDs emission in combination with long-lifetime fluorescent lanthanide labels as donors. However, these systems typically require complex experimental setup and expensive pulsed lasers. We have fabricated a FRET-based prototype of molecular recognition switch, utilizing UCNPs as a donor and QDs as an acceptor. As QDs have no absorbance at near-infrared wavelength, it enabled to eliminate problem associated with excitation light in measurement without need for temporal resolution.(6) Because NIR light can penetrate thick tissue, UCNPs which are then coated with a thin layer of silica incorporating photosensitizer were used for PDT. However, it is difficult to make uniform and thin silica coatings on UCNPs. We construct UCNPs_RPE (UCNPs_R-Phycoerythrin) bioconjugates through a simple covalent coupling. After exposed to NIR light, the UCNPs convert NIR light to visible light which activates the RPE to produce reactive singlet oxygen to kill cancer cells. The inhibition effects on the hepatocarcinoma H22 cells show that the design will provide a new way to PDT.