What happens to proteins when they interact with particles with nanometre-scale dimensions like quantum dots, carbon nanotubes and graphene? Tethered biomacromolecules like enzymes and antibodies are at the heart of assays (e.g., the antibodies used in ELISA), biosensors (e.g., glucose oxidase in third-generation glucose biosensors),[1] and emerging therapeutic technologies (e.g., magnetic nanoparticles for hyperthermal therapy).[2] However, surface texture and chemistry, electrostatics, ionic strength and neighbouring biomolecules will affect the proteins’ form and function. The aim of this project is to develop combinations of spectroscopic techniques to establish foundational biophysical rules for how proteins change when they are bound to nanomaterials. The findings from this work will be applied to existing technologies and related to how nanomaterials interact with more complex biological systems.
This project will use common, copper-containing metalloproteins such as azurin and bilirubin oxidase. These proteins have high-resolution X-ray structures, known patterns of activity, and have been extensively characterised spectroscopically, so we have clear points of comparison to their nano hybrid form. You will use pulsed and continuous-wave electron paramagnetic resonance (EPR) spectroscopy to probe the copper(II) ions in the proteins to detect changes in ligand geometry and to measure how ions from neighbouring proteins interact with each other. You will apply circular dichroism (CD) to measure changes in the proteins’ secondary and tertiary structure.[3]
Bulk spectroscopy of bio–nano hybrids is challenging. First, a solution or suspension of bio–nano hybrids will always be more dilute than the biomolecule on its own, so we will be pushing the detection limits of techniques like EPR spectroscopy and may need to design new analysis cells. Second, the nanomaterials scatter and absorb light, complicating optical analyses like CD and Raman spectroscopy, so we will build on existing models to compensate for effects like peak flattening.[4]
The project will be based in the MIB and at the EPSRC EPR National Research Facility in the Photon Science Institute (PSI). The nanomaterials used in the project will either be made by established methods or be provided by collaborators. You will use EPR spectrometers in the MIB and PSI. You will carry out CD measurements in the MIB or on the B23 beamline at the Diamond Light Source.[5] Quartz-crystal microbalance (QCM) measurements to quantify protein binding to nanomaterials, spectroelectrochemical titration to measure changes in the coppers’ potential, and electrochemical and solution assays to measure changes in catalytic activity will all be done in the MIB. Bulk measurements will be supported by analyses of individual hybrids using transmission electron microscopy (TEM) in the School of Materials or the Faculty of Life Sciences and a new tip-enhanced Raman spectroscopy (TERS) system in the Manchester Institute of Biotechnology (MIB).
For more information on our groups’ research, please see:
• http://www.manchester.ac.uk/research/christopher.blanford/
• http://www.chemistry.manchester.ac.uk/people/staff/profile/?ea=alistair.fielding
• http://www.manchester.ac.uk/research/stephen.rigby/