Study Of Protein Detection Methods Based On Multiple Aptamer And Fluorescence Resonance Energy Transfer Technology

Fluorescence resonance energy transfer(FRET) is energy transfer phenomenon between two fluorescent molecules whose distance closely enough. When emission spectrum of the donor fluorescence molecules and absorption spectrum of the acceptor fluorescence molecules overlaps, and two molecular distances within 10 nm scope, a non-radioactive energy transfer can happen, namely FRET phenomenon. At the same time, the fluorescence of the donor molecule quenched and the acceptor molecule fluorescence enhanced. In recent years, people committed to the practice of this new technology, which is widely used in biosensors, molecular detection, biomedical field. Graphene oxide(GO), as an energy acceptor,can efficiently quench fluorescence of various quantum dots(QDs) and organic dye. Owing to its excellent electrical properties, GO is widely applied to biosensor based on the principles of FRET. It is worth noting that, most of GO-based fluorescence resonance energy transfer sensors are used a single aptamer to detect target proteins. However, multi-aptamer GO-FRET protein detection technology has not been reported yet. Microfluidic chip electrophoresis has many advantages, such as less intelligent injection, injection mode intelligent, and shorter time separation. Two-dimensional separation system has a higher peak capacity, with greater research potential than the one-dimensional separation. And complex microfluidic chip is easier to achieve a two-dimensional or multi-dimensional separation. Therefore, the present paper used multi-aptamers to construct FRET sensors combining efficient separation ability of microfluidic chip electrophoresis to finish a high-sensitivity multi-target detection. In this thesis, following researches was carried out:(1) Designed a biosensor based on graphene oxide-fluorescence resonance energy transfer phenomenon. Use of fluorophore labeled nucleic acid aptamer as multiple fluorescence donor molecules, graphene oxide(GO) as a fluorescent acceptor molecule. First fluorescently labeled aptamers are adsorpted by GO, and then labeled aptamers fluorescence quenching. When added the sample contains multiaptamers-protein into the system, proteins with multiple aptamers can bind and induce this oligonucleotides conformational change, thereby detached from the GO, causing resume fluorescence. In this thesis, by optimizing the concentration of multi-aptamers and GO concentration, we got the appropriate GO-FRET biosensors experimental conditions. This paper compare the detection limit of multi-aptamers with single aptamer, and initial display multiple aptamer technology significantly improving the detection sensitivity.(2) The establishment of GO-multiaptamers FRET biosensors to achieve a concentration determination method of rat urine samples of actual lysozyme. First, we added different concentration lysozyme in urine to examine the linear relationship between fluorescence recovery and lysozyme concentration, and then measure the lysozyme concentration in urine sample. This experiment shows that the method can be applied to the lysozyme measurement in the actual sample. This method has lower detection limit than routine clinical method, more conducive to clinical disease tracking and forecasting.(3) Apoptotic cells will be applied based on GO-aptamer FRET biosensor technology to detect the dynamic release of cytochrome C. Using human neuroblastoma cells to established apoptosis model in order to examine the process of apoptosis cytosolic Cyt C changes. The use of GO as an energy acceptor, fluorescently labeled aptamers as a donor to research the dynamic release of cytochrome C, and compared with classical western blot method. Also investigated the method detection limit and quantitative recovery.(4) Designed and established a microfluidic chip electrophoresis separation method based on GO-multiaptamers FRET biosensors, achieving a high sensitivity and simultaneously detect thrombin, cytochrome C and lysozyme. After fluorescence recovery used GO-aptamers FRET phenomenon, utilize the first dimension isoelectric focusing of the microfluidic chip electrophoretic to separate the three proteins and their aptamers-complexes, achieving simultaneously detect three proteins.