Study On Energy Acceptors Of Upconversion Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET), which is highly sensitive and selective homogeneous bioassay technique, has been widely used in the fields of bio-analytical and biomedical. Considerable achievements have made in the field of FRET technique, however some inherent disadvantages of conventional FRET technique are still exist, especially when the technique is applied to complex biological samples or matrices. In conventional FRET systems, conventional down conversion fluorescent materials such as organic dyes, quantum dots (QDs) and fluorescent proteins are adopted as energy donors, where the energy donor is excited with ultraviolet or visible light, autofluorescence or scattering light always arises from biomolecules upon excitation of the energy donor, and the energy acceptor is often excited directly because of the spectra overlap, which limits the use of fluorescence in the assay of biomolecules, especially in complicated biological matrixes.Upconversion nanoparticles (UCNPs), which is a new class of rare-ion-doped inorganic luminescent, can be excitable with near-infrared (NIR) light to give emission in visible region. The NIR-excitation nature endows UCNPs with the ability to eliminate interferences associated with autofluorescence and/or scattered light. Besides, UCNPs also feature large anti-Stokes shift, narrow emission peak and favourable chemical stability and photostability. Hence, UCNPs can be promising energy donors in FRET-based assays, since the direct excitation of energy acceptor is thoroughly avoided, precluding possible false positive signals. The application of the UCNPs-based FRET technique in the assay of various targets has been well documented and the significance of using the near-infrared light-excitable UCNPs as energy donors in bioassays has been proven in the literatures. Nevertheless, the detection sensitivity of UCNPs based FRET assays is still restricted with the relatively low fluorescence quenching efficiency of the energy donor, which is due to the structural character of up-converting materials. That is to say, only the emitters (doped rare earth ions) at or near the surface of the nanocrystals can be efficiently quenched. In the previous reports on homogeneous FRET assays using UCNPs and organic dyes as energy donor-acceptor pairs, the fluorescence quenching rates were normally lower than30%. As known, larger quenching rates generally results in higher sensitivity because of the increased signal-to-background ratios, In order to obtain better fluorescence quenching and thus to get improved sensitivity, it is of great importance to new energy acceptors and new strategies to acquire improved quenching efficiencies as well as analytical performances of UCNPs-based FRET methods. In order to solve the above mentioned problem, in this dissertation, several biosensors based on upconvertion fluorescence resonance energy transfer have been constructed and used to the detection of targets in complicated biological matrixes. The main contents are as follows:1. Molecular beacon (MB) sensors using UCNPs as the energy donor and organic dye TAMRA as the energy acceptor was constructed, inspired by the fact that the donor-to-acceptor distance in the case of the MB mode is reduced to molecular size, i.e., less than1nm, which is the closest distance in all FRET models available. Two MB sensors were designed for ssDNA recognition and protein determination, respectively. The acceptor was tagged to the3'end of the sequence, UCNPs was covalently link to5'end, which formed a stem-closed structure through the hybridization between the five base pairs at two ends in natural state, and the donor and acceptor were taken into close proximity, the up-converting fluorescence of UCNPs would be quenched by TAMRA. In the presence of the target, the stem-closed hairpin structure was destroyed. The energy donor and the acceptor are thus separated blocking the FRET process, and therefore the fluorescence of the donor restores. The energy-transfer efficiency from up-converting phosphors was effectively enhanced by employing UCNPs as the energy donors of MB sensors, and increased sensitivities in both DNA recognition and protein quantification were thus obtained. Moreover, the MB sensor was competent for target detection in a complicated biological sample matrix due to the NIR-excitation nature. Through rational design of the loop region of the beacon, other UCNPs-TAMRA sensors with improved performances can be expected2. An ultrathin MnCO2nanosheet is established as a label-free two-dimensional nanoplatform for homogeneous biosensing. First, an ssDNA chain was used as the probe for sensing of OTA. The OTA aptamers tagged to UCNPs are spontaneously assembled on the flat surface of MnO2, resulting in fluorescence quenching of UCNPs. The interaction of the aptamers with OTA molecules changes the conformation of the aptamers. The physisorption of the aptamers on MnO2surface is weakened. Because of the separation of the donor-acceptor pair, the emission of the UCNPs is recovered enabling the recognition and quantification of the target. Then another FRET sensor used a peptide chain as the probe for sensing of Cat D, which is based on the specific cleavage of the peptide by Cat D. A polypeptide chain with specific sequence was designed linked to UCNPs. Similar to the ssDNA probe, the peptide was assembled on the surface of Mno2due to the van der Waals interaction between the aromatic residues and the nanosheet, resulting in the quenching of the UCNPs fluorescence. With the introduction of Cat D, which specifically cleaves the substrate leading to the detachment of UCNPs from the nanosheet, the fluorescence intensity of UCNPs is gradually restored. The sensors constructed on this nanoplatform can be applied in not only aqueous solutions but also complicated sample matrixes including red wine and human serum with favourable sensing performances, which is expected to be applied in biosensing target in real sample.3. Biosensing platform based UCNPs as energy donor and MoS2nanosheets as energy acceptor is demonstrated for the sensing of tumor biomarker VEGF165. The VEGF]65aptamers were covalently modified with UCNPs. The fluorescence of the UCNPs was quenched by MoS2nanosheet due to the physisorption of the probes on the nanosheet which takes UCNPs and the MoS2nanosheet into close proximity or molecular contact. Subsequently, upon the introduction of sensing target VEGF165, which binds with the VEGF165aptamers and alter their molecular conformation leading to weakened physisorption because the nucleobases are shielded by the phosphate backbone resulting in retention of the fluorescence of UCNPs. As a result, the fluorescence of the probe is expected to provide a quantitative readout of the target VEGF165. MoS2nanosheet possesses the advantage of high fluorescence quenching efficiency, label-free, good water solubility, in combination with the NIR-excited nature of UCNPs, the proposed UC-FRET biosensor has been successfully applied in the sensitive detection of VEGF165in human serum in a homogeneous format with satisfactory results obtained, which is valuable for clinical diagnosis and related biological research of tumor.4. Homogeneous bioassay for tumor biomarker carcinoembryonic antigen (CEA) detemination based on the FRET from UCNPs to label-free energy acceptor WS2nanosheet is developed. WS2nanosheets could adsorb UCNPs-labeled CEA aptamers probes via the van der Waals force between the nucleobases and the basal plane of WS2nanosheet which cause the distance between UCNPs and WS2nanosheet into close proximity and then quench the fluorescence of UCNPs. In the presence of CEA, the formation of UCNPs-aptamers/CEA complex together with UCPs-aptamers and CEA, weakened the interaction between UCNPs-aptamer and WS2nanosheet, which causes the recovery of UCNPs fluorescence, offering the quantitative determination of CEA. It turns out that the constructed sensor was competent for CEA assay in complicated biological sample matrix due to the NIR-excited nature of the UCNPs donor. And this method is highly sensitive and selective. What's more, the flexible construction of the sensor also provides the possibility to extend for detection of other biomolecules through changing the types of aptamers or substrates for different analytes.