Research Of Biomolecule Analysis Based On Cyclodextrin Polymers And Nucleic Acids Probes

Polymer is macromolecular compound built up from one or numerous kind of monomers. Common materials in production and living such as protein, nucleic acid, starch, fiber, plastic and rubber etc. are included in this category. Owning to the unique properties besides the properties of monomers, polymer has been extensively used in analysis field as fundamental material: Some polymer interacts with target molecule through intermolecular forces（hydrogen bond, hydrophilic and hydrophobic function, and Van der Waals’ force etc.）, then target recognition is realized; Some polymer can realize signal transformation and amplification by transforming the molecular recognition to easily recognizable optical, electrical and magnetic signal. Other polymer has been used as framework of probes, therefore the recognition capacity of probes are increased owning to multivalence with many recognition units or multifunction devices are formed by mang function units attached. In this dissertation, owning to the excellent advantages such as good biocompatibilities, easy fabrication and stable performance, b-cyclodextrin polymer（polyb-CD） and nucleic acid were employed as the signal transformation and target recognition components for the detection of enzymes and DNA. The researches of this dissertation are summarized as follows.1.We developed a strategy for T4 polynucleotide kinase activity（T4 PNK） detection based on polyb-CD and exonuclease reaction. The fluorescence of pyrene enhanced more than 10 times in the presence of polyb-CD, and a single pyrene-labeled ds DNA（ds DNA-pyrene） acted as the probe. In the presence of T4 PNK, the ds DNA-pyrene was phosphorylated at 5′-terminal by T4 PNK, and the resulting 5′-phosphoryl termini product was then digested by l exonuclease（l exo）, yielding pyrene-labeled mononucleotides. The pyrene attached on mononucleotides could easily enter the hydrophobic cavity of polyb-CD, accompanied with significant fluorescence enhancement. Sensitive detection of T4 PNK activity was achieved with a limit detection of 0.02 U/m L. It avoided double-labeling and complex design of DNA probe, worked well in cell lysis buffers. In addition, 50 % inhibiting concentration of ADP, Na₂HPO₄ and（NH₄）₂SO₄ was in accordance with the reported methods, which proved the potential of this method for screening of T4 PNK inhibitors.2. We developed a strategy for sensitive alkaline phosphatase（ALP） fluorescent sensing based on steric hindrance regulated supramolecular assembly between polyb-CD and pyrene. The 5′-phosphorylated ds DNA with one pyrene labeled could be cleaved by l exo, yielding pyrene attached on mononucleotides. Pyrene attached on mononucleotides could easily enter the cavity of polyb-CD, resulting in fluorescence enhancement. When ALP was introduced, it could remove 5′-phosphate groups from the ds DNA and then prevented the cleavage of ds DNA. Pyrene attached on ds DNA was difficult to enter the cavity of polyb-CD because of steric hindrance. Then the fluorescence of pyrene kept weak. Excellent performance of the assay method was achieved for ALP with a detection limit of 0.04 U /m L. The detection limit was superior or comparable with the reported methods. Besides, this method was simple in design, avoiding double-labeling of probe. In addition, 50 % inhibiting concentration of Na3VO4 was in accordance with the reported methods, which proved the potential of this method for screening of ALP inhibitors.3.We developed a strategy for sensitive methyltransferase activity based on pyrene fluorescence enhancement by polyb-CD and nuclease. In the presence of DNA adenine methylation（Dam） methyltransferase, the hairpin probe H1 with pyrene attached on 5′-terminal was methylated and cleaved by methylation-sensitive restriction endonuclease Dpn I. The pyrene attached on one of produced short ss DNA could easily enter the cavity of polyb-CD, resulting in significant fluorescence enhancement. Sensitive detection of Dam methyltransferase was achieved with a limit detection of 1 U/m L. In order to further improve the sensitivity of the system, we also introduced hairpin probe H2 with pyrene labeled on the nucleobase, which is located in the middle of H2, and exonuclease III. H2 could hybridized with one of the produced ss DNA and then digested by exonuclease III. The product of cleavage reaction, pyrene-nucleotide, could easily enter the cavity of polyb-CD and emitted strong fluorescence. In addition, 50 % inhibiting concentration of gentamicin and penicillin was in accordance with the reported methods, which proved the potential of this method for screening of methyltransferase inhibitors.4.We developed a strategy for detection of DNA by using cationic polyb-CD and hybridization chain reaction（HCR）. Two hairpin probes H1/H2 were designed and H2 was labeled with pyrene labeled on 5′-terminal. Because of the branched amino-group, the cationic polyb-CD characterized positive electrical charge at neutral aqueous solution. Electrostatic interactions between cationic polyb-CD and negatively charged H2 can assist the pyrene labeling on oligonucleotide to enter the cavity of b-CD and emitted strong fluorescence. However, when the target DNA was added and triggered HCR between H1 and H2, with pyrene inserted in the produced long ds DNA, Pyrene was difficult to enter the cavity of b-CD because of the hindrance of long ds DNA. The proposed detection of DNA exhibits a detection limit of 0.1 n M and presents good selectivity to mismatched DNA.6. We developed a label-free and non-enzymatic amplification fluorescence method for detection of DNA by using HCR and ds DNA-templated copper nanoparticles（Cu NP）. First, the biotinylated capture DNA probes were immobilized on the streptavidin-modified beads through the interaction of biotin and streptavidin. Then, target DNA hybridized with the capture DNA probes, which formed a hybridized DNA with sticky end. The sticky end triggered the HCR process and formation of ds DNA polymers while two hairpin probes coexisted. Subsequently, the ds DNA polymers were employed as template for synthesis of Cu NP with excellent fluorescent properties, which provided a label-free, non-enzymatic signal response. Meanwhile, the fluorescence sensing depended on the target DNA triggered HCR, which render this method a high selectivity against single-base mismatch sequences. We also introduced Cd Te quantum dots to improve the sensitivity of this system, which could be efficiently quenched by Cu²⁺, and the detection limit was reduced to 90 pM.