Research Of Nucleic Acid Probes And ?-cyclodextrin Polymers For Fluorescent Detection Of Biomolecules

The essential information about small molecules and nucleic acids in biological samples obtained accurately and sensitively has great significance for biological medicine study, clinical diagnosis and therapy. In recent years, Functional fluorescent nucleic acid probes have received more and more research interests in the field of biological analysis. People have developed a series of novel assays by using functional fluorescent nucleic acid probes to obtain more information of our life processes.Cyclodextrin polymer has many advantages such as special molecular recognition capability, good biocompatibilities, excellent stable performance, big solubility and multivalent binding effect. It is reported that ?-cyclodextrin polymer has super fluorescence amplifer caplity to increase the fluorescence of pyrene. This amplifer strategy can combine with other detection method to develop more analytical detection strategy with good sensitivity. In this paper, we constructed three fluorescent methods to detect biomolecules by using nucleic acid probes, ?-cyclodextrin polymer and enzymes. The details are summarized as follows:1. A novel method for DNA detection has been developed, based on the super fluorescence amplifer caplity of ?-cyclodextrin polymer to increase the fluorescence of pyrene combined with catalyzed hairpin assembly. A metastable pyrene-labeled hairpin DNA probe ?probe H1? and a metastable unlabeled hairpin DNA probe ?probeH2? were elaborately designed, which were kinetically handicapped from cross-opening in the absence of target DNA. Pyrene labled at the 5'-termini of single-stranded stem of probe H1 would be easily trapped in to the hydrophobic cavity of ?-cyclodextrin polymer because of weak sterichindrance, leading to significant fluorescence enhancement. Once the dynamic assembly was catalyzed by target DNA, a hybridized DNA duplex H1/H2 would be created continuously. In this state, it is difficult for pyrene to enter the cavityof ?-cyclodextrin polymer due to sterichindrance, leading to a weak fluorescent signal. Thus, target DNA could be detected by this simple mix-and-detect amplification method. It avoided complex design of probe and experimental process. Under the optimized conditions, the proposed assay allows sensitive and selective detection of DNA with a detection limit of 10 pmol L-1. Meanwhile, the proposed method was further successfully applied to detect DNA in celllysate samples, showing great potential for target detection from complex fluids.2. A simple assay for adenosine detection has been developed, based on the super fluorescence amplifer caplity of ?-cyclodextrin polymer to increase the fluorescence of pyrene combined with catalyzed hairpin assembly. Two hairpin probes ?probe H1 and probe H2? and aptamer-trigger/inhibitor duplex probe are employed in the system and the pyrene-labeled probe H1 was chosen as the signal unit. In the absence of adensine, Aptamer-trigger is inhibited by inhibitor strand. Hairpin probes were in the closed hairpin formation without activation of trigger strand. Pyrene labled at the 5'-termini of single-stranded stem of probe H1 would be easily trapped into the hydrophobic cavity of ?-cyclodextrin polymer because of weak steric hindrance, leading to significant fluorescence enhancement. Once the catalyzed hairpin assembly was catalyzed by the adenosine-aptamer binding event, a hybridized DNA duplex H1/H2 would be created continuously. Pyrene labled at the 5'-termini of DNA duplex H1/H2 is difficult to enter the cavity of p-cyclodextrin polymer, leading to a weak fluorescent signal. Target could be detected by this simple mix-and-detect amplification method without the need of expensive and perishable protein enzymes. As low as 42 nmol L-1 of adenosine was detected and the proposed method was further successfully applied to detect adenosine in human serum samples.3. A sensitive assay for DNA detection has been developed, based on host-guest interaction between ?-cyclodextrin polymer ??-CDP? and pyrene combined with exonuclease ?-aided signal amplification. Pyrene is labeled on the nucleobase, which is located in the middle of the hairpin probe. In the presence of target DNA, it can hybridize with the hairpin probe to form a double-stranded structure, and exonuclease III catalyzes the stepwise removal of pyrene-labeled mononucleotides from the hairpin probe. Then the pyrene-labeled mononucleotides can easily enter the cavity of P-cyclodextrin polymer, and causes the remarkable increase of fluorescence signal. At the same time, target DNA can be released and hybridize with another hairpin probe, resulting in fluorescent signal amplification of the system. Compared to conventional molecular beacons, this assay eliminates the need for quencher. Furthermore, the novel method is sensitive and selective, with the detection limit of 18 pmol L^-1.