Poster Presentation The 46th Lorne Conference on Protein Structure and Function 2021

Fast-responding electrochemical protein biosensors for novel diagnostic applications (#207)

Cagla Ergun Ayva 1 , Guo Zhong 1 2 , Selvakumar Edwardraja 3 , Patricia Walden 1 2 , Kirill Alexandrov 1 2
  1. School of Biology and Environmental Science, Centre for Agriculture and the Bioeconomy, Queensland University of Technology, Brisbane, QLD, Australia
  2. CSIRO-QUT Synthetic Biology Alliance, ARC Centre of Excellence in Synthetic Biology, Institute of Health and Biomedical Innovation, Institute for Future Environments, Queensland University of Technology, Brisbane, QLD, Australia
  3. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australia

Therapeutic monitoring of immunosuppressant drugs is essential in organ transplantation to achieve extended graft survival. Current methods used in organ transplant management are immunochemistry and mass spectrometry analysis which are complex and time-consuming1. Biosensors are an emerging alternative to these methods and Guo et al. recently reported the first generation of artificial allosteric biosensors for immunosuppressants including rapamycin, cyclosporin A, and tacrolimus2,3. Furthermore, these biosensors can be used to create electrochemical Point-of-Care tests similar to glucometers since they are based on engineered glucose dehydrogenase (GDH)4. The main objective of this project is to optimize the response time of the core switch module of such biosensors: GDH-Calmodulin chimera (GDH-CaM). Current response time of GDH-CaM biosensor is in the order of tenth minutes, while the ideal diagnostic device should return results seconds. By investigating the kinetics and the underlying mechanisms of allosteric switch activation, we aim to design GDH-CaM-based biosensors with faster reaction kinetics. For this purpose, we generated a focused library of GDH-CaM chimeras by inserting CaM into several sites on GDH. Additionally, a comprehensive search of structural databases revealed 29 different structures of CaM binding peptides in complex with Calmodulin. We performed a medium-throughput enzymatic activity screening of generated GDH-CaM chimeras against different peptides (total 348 combinations of peptide-biosensor). As a result, we have identified two different insertion sites and two different peptides (BP2 and BP29) which improves the reaction rate (kobs) and/or response time. One combination resulted in 2-times higher kobs and 4 times faster response time than previously best variant, reducing the response time from 20 minutes to 5 minutes. This work lays ground for construction of fast responding GDH based biosensors.

  1. Freudenberger K, Hilbig U, Gauglitz G. Recent advances in therapeutic drug monitoring of immunosuppressive drugs. TrAC Trends Anal Chem 2016; 79: 257–268.
  2. Guo Z, Murphy L, Stein V, Johnston WA, Alcala-Perez S, Alexandrov K. Engineered PQQ-Glucose Dehydrogenase as a Universal Biosensor Platform. J Am Chem Soc 2016; 138: 10108–10111.
  3. Guo Z, Johnston WA, Whitfield J, Walden P, Cui Z, Wijker E et al. Generalizable Protein Biosensors Based on Synthetic Switch Modules. J Am Chem Soc 2019; 141: 8128–8135.
  4. Yoo EH, Lee SY. Glucose biosensors: An overview of use in clinical practice. Sensors 2010; 10: 4558–4576.