A biomimetic bitter receptor-based biosensor with high efficiency immobilization and purification using self-assembled aptamers

Abstract

It is of substantial interest to mimic mechanisms of biological sensing systems for the development of novel biosensors. This paper presents a novel biomimetic bitter receptor-based biosensor for the detection of specific bitter substances, in which bitter receptors were used as sensitive elements for the first time. A simple and practical self-assembled aptamer-based strategy was proposed for functional immobilization and purification of bitter receptors. A human bitter receptor, T2R4, was expressed on the plasma membrane of HEK-293 cells and fused with a His₆-tag on its C-terminal. The membrane fractions containing the expressed T2R4 were extracted and immobilized on the gold surface of a quartz crystal microbalance (QCM) pretreated with a monolayer of self-assembled aptamers that can specifically recognize and capture biomolecules labeled with His₆-tags. The QCM device was used to monitor the responses of T2R4 to various bitter stimuli. The results indicate that this biosensor can detect denatonium with high sensitivity and specificity, which is the specific target of T2R4. In addition, this biosensor shows dose-dependent responses to a certain concentration range of denatonium. The sensitivity of bitter receptor-based biosensors prepared by an aptamer-based method is 1.21 kHz mM⁻¹, which is 2 times higher than that prepared by a SAM-based method. The major advances on bitter receptor immobilization and purification presented in this work could substantially be very useful for developing other membrane receptor-based biosensors and molecular sensor arrays. This bitter receptor-based biosensor has great potential to be used as a valuable tool for bitter detection as well as for the research of taste signal transduction.