Development of an electrochemical aptasensor for indirect detection of target protein

Abstract

An aptasensor is a biosensor that makes use of aptamers as a recognition element to detect target analytes. Aptamers are sequences of single-stranded DNA (ssDNA) or RNA that can selectively bind to a wide range of target analytes. A spectroelectrochemical based technique called electrochemical surface enhanced Raman spectroscopy (EC-SERS) is an ideal method for aptamer-based target detection based on vibrational spectroscopy. While EC-SERS has been successfully used to develop an aptasensor for detection of heme-protein (cytochrome c), direct detection of non-heme protein such as Human Immunoglobulin E (IgE) remains a challenging task. Therefore, an indirect method of detection is explored with IgE as the target protein for this research. The anti-Ig E aptamer has a stem and loop configuration with a double-stranded DNA (dsDNA) stem, which is considered to become an open-loop configuration in the presence of IgE. This change of configuration from dsDNA to ssDNA can be utilized in aptasensor development. The aim of the current research is to develop an EC- SERS based aptasensor to detect non-heme protein by employing Raman reporter molecules that are selective towards dsDNA. For this purpose, five different potential Raman reporter molecules were evaluated, namely methylene blue, proflavine, doxorubicin, ethidium bromide and bisbenzimide H 33258 using SERS, EC-SERS and electrochemical methods to detect DNA hybridization . However, the Raman reporters showed SERS signals for both ssDNA and dsDNA, and hence, hybridization could not be detected. This was likely due to non-specific binding of the reporters to the substrate. A control study suggested the presence of defects in the fabrication of alkanethiol monolayer, which remained a key challenge for this project. Three different methods to reduce defects were explored including mixed monolayer formation by sequential immersion in 11-mercapto-1-undecanol and 6-mercaptohexanol. Such alkanethiol fabrication seemed to be promising to eliminate any defects present in the monolayer and hence, reduce any non-specific binding in future studies.