Research Of Nucleic Acid Probes And Tool Enzymes For Fluorescent Detection Of Biomolecules

In recent years, nucleic acid probes have received more and more researchinterests in the field of biological analysis. In order to build more sensitive andconvenient methods to get various information in the process of life, people havedeveloped a series of novel assays by using nucleic acid probes combined with thecatalytic capability of tool enzymes. Due to many advantages such as flexible design，simple operation and the diversity of signal conversion means, tool enzymes-basednucleic acid probes have been widely applied in bioengineering, clinical diagnosis,food industry, environmental protection and other fields. In this paper, we constructedthree fluorescent methods to detect biomolecules by using nucleic acid probes andtool enzymes. The details are summarized as follows:1. A novel method for the detection of platelet derived growth factor-BB(PDGF-BB) has been developed using double-strand DNA-copper nanoparticles(dsDNA-CuNPs) as fluorescent markers. A nucleic acid probe is designed whichcontains the aptemer sequence of PDGF-BB. In the presence of PDGF-BB, theaptamer-based probe undergoes a conformational change upon binding with targetprotein, and subsequently triggers polymerization reaction to generate dsDNA. Then,the resultant dsDNA can be used as a template for the formation of CuNPs with highfluorescence. Under the optimized conditions, the proposed assay allows sensitive andselective detection of PDGF-BB with a detection limit of4nmol/L. This possiblymakes it an attractive platform for the detection of a variety of biomolecules whoseaptamers undergo similar conformational change.2. A simple assay for adenosine detection has been developed, based on the superfluorescence amplifer caplity of-cyclodextrin polymer to increase the fluorescenceof pyrene combined with exonuclease I. An adenosine aptamer is labeled with pyreneat its3′-end as the probe, which can be stepwise digested by exonuclease I to generatemononucleotides containing pyrene (mononucleotide-pyrene) in the absence ofadenosine. Due to the small steric hindrance, mononucleotide-pyrene can easily enterthe lipophilic cavity of-cyclodextrin polymer and the fluorescence intensity ofpyrene is amplifered remarkably. In the other hand, in the presence of adenosine, theformed probe-adenosine complex is hardly cleaved by exonuclease I. The pyrenelocated in the probe-adenosine complex is hindered to reside in their lipophiliccavities. Therefore, only weak fluorescence of the system can be detected. This assay is simple in design and need only single pyrene-labeled probe. The detection limit is9μmol/L.3. A sensitive assay for DNA detection has been developed, based on the superfluorescence amplifer caplity of-cyclodextrin polymer to increase the fluorescenceof pyrene combined with exonuclease III-aided signal amplification. Pyrene is labeledon the nucleobase, which is located in the middle of the hairpin probe. In the presenceof target DNA, it can hybridize with the hairpin probe to form a double-strandedstructure, and exonuclease III catalyzes the stepwise removal of pyrene-labeledmononucleotides from the hairpin probe. Then the pyrene-labeled mononucleotidescan easily enter the cavity of-cyclodextrin polymer, and causes the remarkableincrease of fluorescence signal. At the same time, target DNA can be released andhybridize with another hairpin probe, resulting in fluorescent signal amplification ofthe system. Compared to conventional molecular beacons, this assay eliminates theneed for quencher. Furthermore, the novel method is sensitive and selective, with thedetection limit of22pmol/L.