Allosteric transcription factors (aTFs) are a group of proteins of which DNA binding activities are regulated by the binding with their cognate ligands. They have been applied on small molecule biosensors as the recognition component of these systems.

In this thesis an aTF regulated two-cycle toehold mediated strand displacement (TMSD) circuit was developed to expand the capability of a previously published aTF-based biosensor designed for environmental antibiotic detection. This two-cycle TMSD circuit of enhanced signal amplification addressed the constraint on the performance of the aTF-based biosensor caused by the variety in equilibrium of aTF-DNA and aTF-ligand binding. Moreover, the matrix effect of real samples causing reduction in signal out-put of the biosensor was also overcom...[ Read more ]

Allosteric transcription factors (aTFs) are a group of proteins of which DNA binding activities are regulated by the binding with their cognate ligands. They have been applied on small molecule biosensors as the recognition component of these systems.

In this thesis an aTF regulated two-cycle toehold mediated strand displacement (TMSD) circuit was developed to expand the capability of a previously published aTF-based biosensor designed for environmental antibiotic detection. This two-cycle TMSD circuit of enhanced signal amplification addressed the constraint on the performance of the aTF-based biosensor caused by the variety in equilibrium of aTF-DNA and aTF-ligand binding. Moreover, the matrix effect of real samples causing reduction in signal out-put of the biosensor was also overcome through the addition of a second signal amplifying cycle. This aTF-regulated two-cycle DNA circuit was applied to the detection of uric acid (UA) which was an important metabolite whose level indicates various health issues. This design achieved improvement in sensitivity and rapidness, and showed good compatibility with saliva and serum samples.

In the first part of this thesis, the one-cycle TMSD circuit designed for environmental antibiotic detection were adapted for UA detection by applying the UA specific aTF, HucR. Due to the nature of relatively low affinity with ligand of HucR, satisfying sensitivity of UA detection was not able to be achieved. Therefore, in the second part of the thesis the two-cycle system was introduced to address this issue. Detection range that fell in normal UA concentration in saliva and was achieved and the system displayed good compatibility with saliva while retaining its semi-quantitative response to UA in serum samples. In the last part, feasibility of electrochemical application of the presented TMSD circuits would be discussed with supporting preliminary results.

To sum up, the two-cycle DNA circuit design enabled aTF-based simple, rapid and one-step detection of UA in saliva and serum and showed its potential on the detection of biological small molecules.