Research On Sensitive Detection Of Disease-related Target Molecules Based On Strand-replacement Signal Amplification Technology

Nucleic acids and proteins are the two important macromolecular substances of an organism's and play a vital role in the organisms' life activities. The detection of these molecules plays a crucial role for the study of their functions and for the development of molecular diagnostics. Determining these biomolecules requires exquisite analytical specificity and sensitivity. Nucleic acids based amplification strategies could allow for ultrasensitive detection of analytes when intergrated with the founction enzyme and nanomaterial. The use of many enzymes in a detection assay brings into play other inconveniences such as enzyme storage and the need to find assay conditions that are suitable for all of the enzymes being used. Hence new strategy toward new signal conversion and enzyme free signal amplication. The main research contents and results can be summarized as the following two sections:1. Cyclic Up-regulation Fluorescence of Pyrene excimer for polynucleotide kinase acitivity detection based on dual amplificationA homogeneous and sensitive fluorescence assay based on a double amplification strategy by integrating target recycling signal amplification of DNA toehold strand displacement reaction (TSDR) with gamma-cyclodextrin (y-CD) enhancement of pyrene excimer has been rationally designed for accurate detection of PNK activity and inhibition. A spatially sensitive molecule was used as the signal reporter for switch-on detecting PNK activity. Hairpin probe (H1 DNA) was designed as a substrate of PNK. Hairpin oligonucleotides H2 and H3 are singly-labelled with pyrene to act as the probe of TSDR respectively. By introducing PNK,5'-hydroxyl terminal of H1 could be phosphorylated in the presence of T4 PNK and then was digested by ? exo to generate a trigger DNA (tDNA) of strand displacement reaction. The amount of generated tDNA is positively related to the activity of PNK. When tDNA generates, it can hybridize with H2 to open the hairpin structure of H3. The sticky end of opened H2 can serve as a toehold to make the unfoldment of H3 possible and lead to the displacement of the tDNA through a branch migration process. In this state, a pyrene moiety on probe H2 is brought into close proximity to a pyrene moiety on the neighboring H3 probe, leading to a significant increase in fluorescence of pyrene excimer. The released tDNA can re-hybridize with H2 to initiate cyclic strand displacement reaction. Thus, numerous pyrene excimers are formed, leading to cyclic up-regulation of pyrene excimer fluorescence for first step amplification. To further improve sensitivity, gamma-cyclodextrin (?-CD) was introduced to further enhance the fluorescence of pyrene excimer. Gamma-cyclodextrin (?-CD) can regulate the space proximity between two pyrene molecules labeled on a flat end through pyrene/cyclodextrin inclusion interaction because cyclodextrins are hydrophobic inside and hydrophilic outside. Therefore, the proposed dual amplification can sensitively detect T4 PNK activities in the range of 0.0005-5 U mL-1 with a correlation coefficient of 0.9980. The detection limit was estimated at 9.3 × 10-5 U mL-1 in terms of the rule of 3 times standard deviation over the blank response. By comparing inhibition effect of three model inhibitors, this strategy has the potential to discriminate the inhibition capacity of inhibitors as well as screen PNK inhibitors. Therefore, it is a highly sensitive, specific, reliable strategy for PNK activity and the inhibition detection.2. A cytometric bead assay for sensitive human telomerase RNA detection based on toehold strand displacementA flow cytometric bead assay has been proposed for the sensitive detection of hTR by integrated with target recycling signal amplification of DNA toehold strand displacement reaction (TSDR). A biotinylated hairpin DNA1 (H1) and FAM-labelled hairpin DNA2 (H2) are rationally designed. Firstly, biotinylated hairpin DNA1 (H1) is immobilized on streptavidin-functionalized beads (MBs) through biotin-avidin interaction. In the absence of hTR, hairpin oligonucleotides H1, H2 keep their stable hairpin conformation. The fluorescence of FAM on streptavidin-functionalized beads (MBs) is in the "off" state. When hTR is added, it serves as a trigger DNA of TSDR and drives circulatory interactions between singly FAM-labelled hairpin DNA2 (H2) and hairpin DNA1 (H1) to continuously form H1/H2 duplex, resulting in a "turn on" fluorescence signal of FAM on streptavidin-functionalized beads (MBs). Finally, the fluorescence of FAM on MBs is analyzed by flow cytometry and fluorescence microscopy image for the quantitative and specific of hTR. Thus, the TSDR-induced cyclic formation of H1/H2 duplex strategy can detect hTR sensitively with a detection limit of 0.3pM, which is superior to those of most existing approaches. Moreover, the proposed strategy can be successfully utilized to detect hTR in complex biological samples as well. This method can also be used for single-base mutation detection. Therefore, an enzyme free amplification approach is provided for simple, robust and rapid signal readout hTR detection.