UVM Theses and Dissertations
Format:
Print
Author:
Zheng, Yijian
Dept./Program:
Biomedical Engineering Program
Year:
2013
Degree:
M.S.
Abstract:
Pseudomonas aeruginosa (P. aeruginosa) is one of the primary causes of morbidity and mortality of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) patients. Without timely diagnosis and eradication, CF and COPD patients always suffer the antibiotic resistant chronic lung infection that may lead to the respiratory failure. The aim of this project is to develop a technique that can be used for the early identification of P. aeruginosa once it is present in the lung. As a unique volatile compound found in the P. aeruginosa culture and breath samples of CF patients colonized by P. aeruginosa, 2-aminoacetophenone (2-AA) is used as the biomarker for the identification. Meanwhile, as a detection technique, the aptamer-based fluorescence biosensor can be used to signal the presence of target molecules by generating the fluorescence signal. This type of detection probe is used as the method to detect 2-AA.
In this project, we selected three groups of aptamers binding 2-AA from a singlestranded DNA (ssDNA) library that contained a large amount of 91-base ssDNA (1.1 x 10¹⁸) using in vitro selection called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The selected aptamers were modified to the structure switching fluorescence signaling aptamers (SSFSAs). All ofthe SSFSAs were able to signal the presence of 2-AA, and Aptamer III generated the most significant fluorescence enhancement and showed the highest affinity to 2-AA. Five small molecules that were similar to 2-AA in chemical structure were used to test the specificity ofthe SSFSAs, and Aptamer II showed the highest specificity to not only 2-AA but also acetophenone (A). Additionally, the affinity ofthe aptamers to acetophenone (A) were higher than 2-AA. The result might be resulted from two factors, which included that 1) the 2-AA solution (98%) used in SELEX contained acetophenone, and 2) the intrinsic quenching effect of 2AA on fluorophore molecules caused the decrease in fluorescence. Fortunately, acetophenone (A) was also validated as another unique volatile compound generated by Pseudomonas aeruginosa. Therefore, acetophenone (A) can be used as a candidate-biomarker for the identification of Pseudomonas aeruginosa as well.
We have successfully selected the aptamers that can bind two very small molecules and designed the signaling aptamers with fluorescence signal generation ability. However, in contrast with those known fluorescence signaling aptamers, the fluorescence enhancements generated by our SSFSAs are still limited. The improvements can be done by optimizing the design ofthe fluorophore-DNA (FDNA) and the quencher-DNA (QDNA) molecules that play the most important roles in generating fluorescence signal.
In this project, we selected three groups of aptamers binding 2-AA from a singlestranded DNA (ssDNA) library that contained a large amount of 91-base ssDNA (1.1 x 10¹⁸) using in vitro selection called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The selected aptamers were modified to the structure switching fluorescence signaling aptamers (SSFSAs). All ofthe SSFSAs were able to signal the presence of 2-AA, and Aptamer III generated the most significant fluorescence enhancement and showed the highest affinity to 2-AA. Five small molecules that were similar to 2-AA in chemical structure were used to test the specificity ofthe SSFSAs, and Aptamer II showed the highest specificity to not only 2-AA but also acetophenone (A). Additionally, the affinity ofthe aptamers to acetophenone (A) were higher than 2-AA. The result might be resulted from two factors, which included that 1) the 2-AA solution (98%) used in SELEX contained acetophenone, and 2) the intrinsic quenching effect of 2AA on fluorophore molecules caused the decrease in fluorescence. Fortunately, acetophenone (A) was also validated as another unique volatile compound generated by Pseudomonas aeruginosa. Therefore, acetophenone (A) can be used as a candidate-biomarker for the identification of Pseudomonas aeruginosa as well.
We have successfully selected the aptamers that can bind two very small molecules and designed the signaling aptamers with fluorescence signal generation ability. However, in contrast with those known fluorescence signaling aptamers, the fluorescence enhancements generated by our SSFSAs are still limited. The improvements can be done by optimizing the design ofthe fluorophore-DNA (FDNA) and the quencher-DNA (QDNA) molecules that play the most important roles in generating fluorescence signal.