Fluorescent and colorimetric biosensors based on DNAzymes and DNA aptamers.

摘要

Since the invention of combinatorial selection of functional nucleic acids, a large number of catalytic DNA (DNAzymes) and aptamers have been isolated, many of which possess high analyte specificity. As a result, these functional nucleic acids can be used for sensing and diagnostic applications. However, the number of successful sensors made from these nucleic acids is limited. On the other hand, technology for sequence-selective DNA detection has advanced remarkably. Two primary examples are molecular beacon-based fluorescent detection and nanoparticle-based colorimetric detection. Therefore, combining the two fields opens new avenues to obtain highly sensitive and selective biosensors for a broad range of analytes in applications such as consumer markets, environmental monitoring, homeland security, clinical and electronic industries. A catalytic molecular beacon Pb2+ sensor based on a Pb2+-specific DNAzyme was designed previously. The sensor was highly sensitive and selective at 4 °C; while high background fluorescence was observed at room temperature. To suppress background, a dual quencher labeling method was developed. The signal-to-background ratio increased by 10-fold with this modification. With the improved design, miniaturization of the sensor was realized by performing detections in microfluidic devices. To pursue the possibility of metal sensing by conformation change of the DNAzyme, fluorescence resonance energy transfer (FRET) was used to study folding of the DNAzyme. A new method of multiple fluorophore FRET was developed to study branched biomolecules. In complementary to highly sensitive fluorescent sensors, colorimetric sensors were also developed to make on-site and real-time detection easier, which took advantage of the recent development on DNA-directed assembly of gold nanoparticles. Upon assembly, color of gold nanoparticles changes from red to blue. There are two basic approaches to design DNAzyme-based colorimetric sensors: Pb2+-inhibited assembly of nanoparticles and Pb 2+-induced disassembly of nanoparticle aggregates. Both approaches were accomplished in the thesis work. DNAzymes are ideal for metal sensor design, because most DNAzymes require metal ions as cofactors for their activities. However, few DNAzymes are known to employ non-metal cofactors. To apply the nanoparticle-based detection method to detect analytes beyond metal ions, colorimetric sensors based on aptamers and allosteric DNAzymes (aptazymes) have also been demonstrated.