Aggregation-based DNA Detection in Microseparators
Date Issued
2012
Date
2012
Author(s)
Liu, Yen-Cheng
Abstract
A novel aggregation-based method to execute oligonucleotide detection in a PFF (pinched flow fractionation) microseparator is developed in this thesis. The label-free target DNA hybridizes with the probe DNA on the surface of microspheres and causes the polymeric aggregate, thus enlarging the average size of the aggregated particles. Depending on the aggregated particle sizes, the microparticles were separated through a PFF microseparator, enabling subsequent concentration analysis of the target DNA .
Streptavdin-coated polystyrene microspheres is conjugated respectively with two kinds of probe DNA modified with biotin, and the efficiency of labeling is analyzed by UV-Visible spectrum. The aggregation caused by mixing probe-labeled beads with the target DNA whose two ends is respectively complementary with the two DNA probes is analyzed by dynamic light scattering (DLS) particle size analysis and the fluorescence image. A PFF microseparator (20 and 1000 μm respectively for the pinched and broadening segments) is used to analyze the size of the aggregated particles.
The result shows that the size of the aggregated microparticles varied with the concentration of the target DNA (Ctarget). The PFF microseparator was used to determine Ctarget, that is, the size of the aggregated microparticles. The size of aggregated particles can be evaluated by analyzing the intensity through the broadening segment, thus obtaining the concentration of target DNA. In addition, an asymmetric outlet design is used to conduct rapid screening of the DNA detection.
The contribution of this study is the numerous features of this method such as simplicity, low cost and potential to detect other biochemical samples with distinct aggregation behaviors. Also, it is capable of performing SNP/mutation detection. Because of its high specificity, this method can also be used to conduct multi-target detection on a chip.
Subjects
microseparator
microreactor
DNA detection
PFF, particle aggregation
Type
thesis
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