PhD Oral Qualifying Examination : Development of homogeneous and continuous protease assays using liquid crystal

Speaker Mahbuba Jannat (Supervisor: A/Prof Yang Kun-Lin)

Host Department of Chemical and Biomolecular Engineering

Date/Time 07 Dec - 07 Dec, 9.00AM

Venue E5-02-32 , Faculty of Engineering, National University of Singapore

Synopsis

Proteases play very important roles in various biological processes such as food digestion, wound healing, blood coagulation and some terminal disease development. Because of their high importance, there has been a lot of research activities in development of next-generation protease assays. Recently, liquid crystal (LC) based protease assays attract huge attention in literatures because of their unique features such as naked-eye detection, label-free technique and real-time protease activity monitoring. However, further studies are still required to improve LC-based protease assays due to many existing challenges. Firstly, short oligopeptides are used as substrates in LC-based protease assays, but immobilization of these substrates on LC-compatible surfaces is difficult to control. Secondly, there is no direct comparison between LC-based protease assays and standard proteases assays. This is because LC-based protease assays are heterogenous assays whereas standard protease assays are homogenous assays. Finally, there is still no reliable way of quantifying the amount of peptides immobilized on the surface and degree of peptide cleavage. In this study, we aim to address these challenges by developing new LC-based assays which do not require immobilized oligopeptide as substrates. Moreover, we aim to exploit microfluidic devices and combine them with LC-based assays to enable continuous monitoring of protease activities.

Recently, we developed a homogenous assay in which free casein molecules are mixed with proteases in buffer solutions. In this configuration, protease activities are directly related to the amounts of peptides released from casein.  We anticipate that protease activities can be determined more precisely because both protease and substrates are in their native states. Next, LC is used as a tool to quantify concentrations of peptide fragment and give optical signal for visual detection. Finally, this technique is incorporated into a millifluidic device to develop a continuous assay in which manual sampling is minimized.

Biography