Multi-purpose instrument for advanced Raman spectroscopy techniques

Lead Research Organisation: University of Manchester
Department Name: Chemistry

Abstract

The characterisation of biological systems from whole cells to their component parts is important so that we can learn how the cell functions and how it responds to interactions with other substances (be they artificial or natural). Many of the parts that drive the cell are enzymes, large molecules that speed up the reactions that make life possible. Once we understand how enzymes function and how we may improve their functionality (that is, how they turn one substrate into a specific product and how quickly they do it), we can exploit these for industrial purposes, leading to more efficient 'greener' processes. These could be just like the biological washing powders, that use enzymes to break down fats, cellulose, proteins and starch; they could be enzymes that are used to generate high-value industrial products and medicines; they may be used in biological sensors, like the blood sugar monitors used by diabetics. Some enzymes are also being used to directly convert the chemical energy in substances like alcohol into electricity, or to take surplus electricity to convert carbon dioxide into chemical starting materials.

The problem is that whilst some methods do exist to characterise enzymes, they are laborious and destroy or compromise the sample. By contrast, we have been developing and exploiting a method based on the interaction of light with matter that will enable a very powerful and non-invasive analysis. One process that happens when light is shone at a substance is that it is scattered and sometimes this light is scatter at a different wavelength, an effect named Raman scattering. This generated Raman light gives us very specific molecular information about the structure of the system under analysis.

The aim of this grant is to acquire a new Raman instrument that can be used both: (i) for protein and enzyme characterisations where a technique called resonance Raman allows us to follow very fast reactions in real time; and (ii) to separate features that are just a 10,000th of a millimeter apart from each other - about the size of switches inside computers or the membrane on the outside of cells - when mapping cells and cell-component system.

Technical Summary

We propose a unique, multi-purpose Raman instrument with applications in many areas of bioscience research. The instrument will contain multiple laser lines to allow studies of a wide array of samples and will have a broad range of capabilities, including Raman atomic force microscopy (AFM), tip-enhanced Raman scattering (TERS), resonance Raman (RR), polarised Raman, anti-Stokes Raman and time-resolved Raman. This equipment will be truly unique to the UK bioscience community and will provide significant infrastructure for many research groups at Manchester and will be made available to BBSRC-funded researchers from other academic institutes as well as industrial scientists.

All applicants from University of Manchester are well funded (mainly BBSRC) and engaged in biophysical analysis of a range of systems including biotransformations, protein/enzyme structure-function studies, post-translational modifications of proteins, whole cellular systems (microorganisms and their biomolecular products, as well as animal and plant biomaterials) and biomolecular interactions with graphene.

The system will comprise multiple laser lines (405 nm, 532 nm and 633 nm with the potential for more in the future) for probing a wide range of biological samples (e.g. for RR applications) and will contain the following accessories: microscope with a computer controlled motorised stage, fully integrated AFM accessory for TERS, notch filters to allow anti-Stokes and measurements close to the laser line, polarisers to enable polarised Raman to provide information about ordering and orientation in macromolecular materials, a variable-temperature cell to determine thermodynamic parameters and for protein denaturation studies, and a stopped-flow cell to allow kinetic analyses.

Once the system is optimised not only will researchers at the University of Manchester benefit but also BBSRC-funded scientists in other institutes as well as industrial researchers.

Planned Impact

The requested multi-purpose instrument for advanced Raman spectroscopy will be able to generate a variety of Raman measurements including normal Raman spectra, resonance Raman spectra, Raman-AFM and TERS. These will include anti-Stokes as well as Stokes Raman measurements; these can be recorded close to the Rayleigh/laser line, and shall also include polarised Raman data. Raman readings can be from ambient conditions, within a temperature control unit, or within a stopped-flow device.

There is currently no other Raman platform with the same broad range of capabilities available to either: (i) the investigators from The University of Manchester; or (ii) the UK bioscience community, be they academic or industrial. Therefore the scientific impact will be immense as there is an urgent need to establish resonance Raman and Raman-AFM capabilities for the entire UK bioscience community.

Therefore the research that will be enabled by the acquisition of this Raman instrumentation will be far-reaching and would have potential benefit to: (i) academic researchers who are utilising Raman spectroscopy across the remits of BBSRC's committees; (ii) governmental and private sector scientists who conduct applied analytical chemistry studies; (iii) private sector scientists from the Raman spectroscopy (and software) industries, (iv) international organisations.

In order to allow the instrumentation to be fully exploited we would ensure that the equipment was fully accessible to all UK academics, who would also benefit from the high level of expertise and Raman spectroscopy-specific training that will be available at Manchester. Access to both internal and external users would be managed via a steering committee comprising at least thee academic staff members (Goodacre, Munro, Blanch) with Heyes and Ellis (who would provide hands-on help and training to the various project).

The impact of the requested Raman instrumentation would be maximised by: (i) organising a project/facility specific workshop; (ii) generation of a web-portal for work conducted and opportunities; (iii) including details of the spectrometer on the N8 Shared Research Equipment Inventory to network with industry and via equipment.data.ac.uk; (iii) continuing our industrial collaborations; (iv) presentations to the scientific community via conferences and publications; (v) public engagement.

We will measure the success of our impact activities by the number of: (i) registered users of our community website; (ii) applications to use the Raman facility; (iii) scientific publications that refer to our Raman facility; (iv) participants that attend our workshop.

Publications

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Description This grant allowed us to established novel Raman and Tip enhanced Raman spectroscopy (SERS).
The facility has been used and has contributed in the following areas, many of which have lead to good publications:
1. Assessment of biocatalytic activity of enzymes.
2. TERS for the assessment of glycosylation of single protein molecules.
3. We were awarded a Dean's fund for an incubator that allows us to grow eukaryotic cells and look at metabolism and drug perturbations
4. We have coupled metabolomics through isotope labelling of single bacterial cells with an aim to assess primary carbon sources in environmental conditions.
5. We have used this for high throughput medium quantification of multiple determinands - including metabolites glucose and lactic acid as well as protein products
6. Unfolding and aggregation of proteins using heating within the sampling device.
7. Food security for assessment of adulterants.

This equipment has been a real benefit to many PDRAs and PhD students, collaborators and the PIs involved in the proposal.
Exploitation Route This is really articulated above.

As this was an equipment grant the impact is more direct for the investigators involved.
Sectors Agriculture, Food and Drink,Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology