Jiaul Islam1, 2, Martina Jones2, Stephen Mahler2, Simon Corrie1, 2*


1Department of Chemical Engineering, Monash University, Clayton, Australia, ARC Centre of Excellence in Convergent BioNano Science and Technology
Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St Lucia, Australia


Fluorescence-based affinity probes have many advantages over time consuming, laborious and multistep conventional bioassays. While a range of biosensors have been reported, “reagentless” biosensors that use antibody fragments (e.g. single chain variable fragment, scFv) as the biorecognition element to detect proteins using simple fluorescence readout are in their infancy. [1] To date, none have demonstrated detection of large biomolecules (e.g. proteins) nor has immobilisation on a solid surface for biosensor development been attempted to our knowledge. 

One of the major challenges in fluorescence-based biosensor development is the site-specific fluorescence labelling of target proteins without disturbing the structure and functionality. Traditional labelling strategies such as maleimide chemistry, fluorescent fusion proteins and short peptide tags have suffered limitations in terms of site specificity, structural perturbation, reduced functionality, cross-reactivity and disoriented immobilisation. Fluorescence labelling via unnatural amino acids (UAAs) is a relatively new and highly efficient method of site-specific labelling, and can be genetically incorporated into any permissible site during protein synthesis. [2, 3] Here we report incorporation of an environmentally-sensitive fluorescent UAA (Anap) into various position of αEGFR scFv for the detection of a target antigen. We demonstrate that incorporation of Anap can be used for detection of local conformational changes upon target binding or denaturation, by monitoring the wavelength and/or intensity changes in emission spectra. This probe can be potentially integrated into a wide range of assay platforms (e.g. homogenous or solid-phase) for one-step protein detection.


1.    Abe, R., et al., "Quenchbodies": Quench-Based Antibody Probes That Show Antigen-Dependent Fluorescence. Journal of the American Chemical Society, 2011. 133(43): p. 17386-17394.
2.    Chatterjee, A., et al., A Genetically Encoded Fluorescent Probe in Mammalian Cells. Journal of the American Chemical Society, 2013. 135(34): p. 12540-12543.
3.    Lee, H.S., et al., Genetic Incorporation of a Small, Environmentally Sensitive, Fluorescent Probe into Proteins in Saccharomyces cerevisiae. Journal of the American Chemical Society, 2009. 131(36): p. 12921-+.

Biographic Details
Name: Jiaul Islam
Title: Mr.
Affiliation, Country: Australia
Phone: +61 412 828 643, E-mail: jiaul.islam@monash.edu  
Research interests: Antibody and protein engineering, immunodiagnostics, fluorescence biosensors, nanobiosensor, nanotechnology   


AEB 313