Studies On New Signal Amplification Methods For Fluorescence Detection Of Nucleic Acids And Proteins

The highly sensitive detection of nucleic acids and proteins has attracted considerable interest in clinical medicine, bioengineering, judicial expertise and environmental monitoring. Fluorescence spectrophotometry is a powerful technique for nucleic acids and proteins detection due to its simple equipment, extremely high sensitivity and selectivity. Since the amount of some nucleic acids and proteins is usually low in the body fluid or tissues with the high interfering substances, the development of ultrasensitive protocols for these nucleic acids and proteins is surely of desirable. The main method for improving sensitivity is signal amplification technology. This thesis focus on the combination of signal amplification with fluorescence analysis have developed some quantitative analysis methods of nucleic acids and proteins with high sensitivity. The main researches included in this dissertation are presented as follows:1. A highly sensitive and multiplexed DNA detection assay has been developed. It was demonstrated with the ebola virus and human immunodeficiency virus nucleotide sequences as a model. The capability to discriminate single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) structure coupled with the extraordinary fluorescence quenching of graphene oxide (GO) allows the proposed strategy to simultaneously detect several DNAs, while the polymerase aided amplification endows the detection method with high sensitivity. When the target DNAs were absent, primer extension reaction would not happen, and the fluorescence was quenched due to the probes were absorbed on the surface of GO. With the addition of the target, the hairpin structure was opened and a primer extension reaction happened. Simultaneously, the target was displaced by the polymerization, and a duplex with full length was synthesized. The displaced target subsequently bound to a new hairpin probe, and a new extension cycle occurred. Afterwards a strong fluorescent emission was observed due to the weaker interaction between the formed duplex helix and GO. Thus, the simultaneous detections of two targets can be realized by synchronous fluorescence analysis.2. A sensitive protein detection method has been developed based on the advantages of aptamer such as good stability and strong affinity. This method employed two hairpin species (H1and H2). H1, which is modified at its5'ends with a fluorophore, consists of the aptamer sequence of thrombin. Meanwhile, H2is partially complementary to H1. Upon the addition of the target, it can facilitate the opening of the hairpin structure of H1and thus accelerate the hybridization between H1and H2, resulting in the hybridization between H1-H2and Blocker-DNA. The formation of H1-H2and Blocker-DNA can not be absorbed on the surface of GO and the fluorescence can not be quenched. Compared with the bimolecular beacons, the flurescence signal was increased by self-assembling of three-stranded DNA. Moreover the introduction of GO not only reduced the background, but also did not need to modified DNA with any quencher, which reduced the cost.3. While effective sensing has been achieved by GO, nonspecific probe displacement might occur to produce a false positive signal. Herein, a novel immuno-sensor have developed applied to protein detection based on exonuclease ? (Exo ?)-induced signal amplification. On one hand, the sensitivity has been raised. On the other hand, the background has been reduced because of the capability to discriminate ssDNA and mononucleotide coupled with the extraordinary fluorescence quenching of GO. This proposed method can be applied in detecting the target in serum sample, and the GO do not have to covalently link any DNA or antibody with it. This biosensor could be severed as universal platform for the detection of other proteins.4. The method based on nucleic acid molecular light switch and quantum dots (QDs) can be used to differentiate between dsDNA and ssDNA, while it cannot be used to detect specific sequence ssDNA. In order to solve this problem, we combine hybridization chain reaction (HCR) with QDs and light switch molecular coupled with magnetic beads separation technique. The target DNA and the capture DNA hybridizes the HCR products link to the magnetic beads. Then, a great amount of light switch molecular can intercalate into the HCR products and restore the fluorescence of the QDs. Based on the variation of fluorescence signals of the QDs, the target DNA is determined. This biosensor greatly extends application areas of light switch molecular and QDs in nucleic acid. We can detect special sequence DNA using QDs and ruthenium complex. There is no obvious difference between the fluorescence intensities obtained from serum sample and those in buffer solution because of the introduction of magnetic beads separation technique.5. For further extending the application areas of nucleic acid molecular light switch and QDs in bioanalysis, we introduce the strepavidin-modified magnetic beads in this work. On one hand, it combines HCR and immunoassay. On the other hand, the streptavidin-modified magnetic beads combine with a large amount of biotin-DNAs, which we employed as a amplification strategy. Results show that this developed strategy not only has high selectivity, but also can be applied to detect the target protein in human serum samples. Moreover, this sensitive sensing platform presented herein could be modified and extended to the detection of the other proteins.