Study On New Strategies And Methods For Signal Amplification And DNA Detection

With the completion of the Human Genome Project, we have entered the era of genetic revolution. Because sequence-specific nucleic acid detection is essential to the application from a simple diagnosis to major medical evaluation. A series of DNA detection devices with simple, sensitive and portable are extremly needed. These devices can not only work in the analytical laboratory work, but also play an important role in clinical diagnosis, monitoring of infections, genetic diseases and environmental and forensic applications. This thesis focuses on exploring new methods to analyze and detect nucleic acid, and it contains three parts:1. Visual Scanometric Detection of DNA through Silver Enhancement Regulated by Gold-Nanoparticle AggregationA convenient and label-free scanometric approach for DNA assay was designed by integrating a metal-ion-mediated conformational molecular beacon (MB) and silver-signal amplification regulated by gold-nanoparticle (AuNP) aggregation. The strategy was based on displacing the interaction between the target DNA sequence and a competitor Hg2+ion with a link DNA sequence. In the absence of the target DNA sequence, a link DNA sequence interacted with the Hg2+ions, thus forming an inactive cyclic conformation of the MB. This result led to the poor aggregation of polyadenosine-functionalized AuNPs (A-AuNP). In the presence of a target DNA sequence with a stronger affinity than that of the competitor, hybridization between the link DNA and target DNA sequences turned on the trigger. The polythymidine end of the resulting linear duplex structure could react with A-AuNP, thus leading to a cross-linking aggregation. This aggregation weakened AuNP-catalyzed silver enhancement on a spot substrate. Further, by using scanometric detection, the concentration of the target DNA sequence could be conveniently read out within a linear range from1.0to30nM. Interestingly, in the same amount of Hg2+ions, one base mismatched DNA showed only22%of the relative gray-scale intensity for the target DNA sequence at the same concentration, thus indicating good specificity. The designed approach, with the help of the ion-mediated conformational MB, was simple, cost effective, adaptable, and convenient and provided significant potential applications in clinical analysis.2. Ultrasensitive Electrochemical Detection of Nucleic Acids with Template Enhanced Hybridization and Rolling Circle AmplificationAn ultrasensitive strategy for electrochemical detection of DNA is designed by using quantum dots (QDs) as signal tag with template enhanced hybridization process (TEHP) and rolling circle amplification (RCA). Upon the recognition of the target DNA, assistant DNA, target DNA and molecular beacon (MB) can hybridize to form a ternary "Y-junction". The resulting structure can be dissociated by the reaction of nicking endonucleases with a TEHP. Thus the fragmented MB can act as the primer of the RCA reaction. With the help of the oligonucleotide functionalized QDs attached on the RCA product, the electrochemical signal can be easily read out by square-wave voltammetry (SWV). Because of the triplex signal amplification strategy and the little nonspecific adsorption provided by affinity interaction of biotin-strepavidin system, this designed system produced an ultrasensitive electrochemical methods for detection of DNA down to attomolar level (11aM) with a linear range of6orders of magnitude (from1x10-17to1x10-11M) and can discriminate perfect matched target DNA from mismatched DNA with high selectivity, The high sensitivity and specificity make this method a great potential for early diagnosis in gene-related diseases.3. Enzymatic Signal Amplification for sensitive Detection of Telomerase Activity in Cancer CellsA sensitive strategy for fluorescence detection of telomerase activity in Hela cells was designed by enzymatic amplification coupled with recognition molecular beacon. Based on the extension reaction of a telomerase substrate primer in the presence of the telomerase, this method can detect telomerase activity of in10-1000Hela cells, which shows a convenient and simple process for telomerase detection.