Label-free spatially-resolved molecular analysis of lipid bilayers by Raman spectroscopy: Going beyond the diffraction limit

Lead Research Organisation: University of Nottingham
Department Name: Sch of Physics & Astronomy


During the last two decades, it has become evident that the lipid bilayer forming the membranes of the cells plays a key role in many biological processes (cell signaling, cell death, etc) as well as pathological conditions (Alzheimer's disease, cardiovascular disorders, etc). Understanding the properties of the bilayers at a molecular level and correlating the molecular mechanisms with the functionality of the cells is crucial for advancing our understanding of the cell biology as well as developing new targeted therapies for many diseases.

Despite increasing efforts in this field, many physical and chemical properties of the membranes and their interactions with other cellular molecules (e.g. membrane receptors) are still not well understood, especially at the nanoscale. Supported lipid bilayers provide a biologically relevant model for cell membranes, which provide control on the composition and environment of the bilayers in order to give a simplified representation of the membranes. However, understaning the molecular properties of such nanometric structures require advanced tools with a high level of sensitivity, ability to provide detailed molecular information without disturbing the system and ideally with a nanometric spatial resolution. Non-invasive techniques would also be advantageous as they allow measurements of dynamic molecular events. However, the above requirements raise huge challanges for the tools currently available to life scientists.

The rechnique proposed in this project, tip-enhanced Raman spectroscopy (TERS), combines the chemical specificity of Raman laser spectroscopy with the high sensitivity and nanoscale spatial resolution of scanning probe microscopy to enable aquisition of spatially-resolved information from regions of the membranes where various constituents segregate. TERS has been applied recently to investigations of biological materials, but a key limitation of TERS has emerged: reproducibility. Difficulties in obtaining TERS tips with predictable performance have limited the availability of TERS to only few groups worldwide. It is also often that results reported by one group cannot be confirmed by other groups, especially for biological samples.

A new and innovative approach is required to release the potential of TERS for the benefit of the wide ranging fundamental and applied research community in biosciences. Our aim is to make TERS a 'standard' technique and not the reserve of a few specialist laboratories.
To achieve this aim, we have proposed a step-change approach to engineer TERS-tips by replacing the conventional off-line fabrication methods with an in-situ method, which promises a higher level of control, optimisation and reproducibility (in-situ techniques reduce the risk of contamination and damage during handling). Compared to the thermal evaporation off-line methods, our preliminary experiments show that the properties of the tips can be control by adjusting several experimental parameters. While we have demonstrated the ability to use this novel technique to measure TERS spectra of biological nanomaterials (~10,000 fold increase in sensitivity and ~20nm spatial resolution), the factors which determine the properties of the TERS tips still require optimisation. In this project we will optimise the tip fabrication methods and then show the feasability of using this new technique to study molecular properties of supported lipid bilayers. We will focus on label-free mapping of lipid molecules in phase-separated domains in lipid bilayers consisting of two and three lipid types.

While the current project focuses on lipid bilayers, the proposed technique may be used to address a broad range of applications in biosciences, such as self-assembly of peptide nanotubes, amyloid-like fibrils, tubular proteins or virus shaft protein

Technical Summary

The main objective of this project is to develop a novel method for in-situ engineering of optimised probes for tip-enhanced Raman spectroscopy (TERS) to enable label-free non-invasive investigations of the molecular properties of supported lipid bilayers with nanometer scale spatial resolution. The in-situ technique has several advantages compared to the conventional off-line fabrication methods: i) it has the potential to optimise the shape and size of the tip geometry, ii) the TERS activity of the tip can be monitored during the fabrication process; iii) it is rapid (~1 minutes) and requires no other specialised equipment; iv) increases considerable the yield of TERS active tips as the risks of oxidation, contamination, electrostatic and mechanical damage are eliminated.

The first task of the project is to optimise the experimental parameters used for the fabrication of the TERS tips. Our preliminary results show that Ag nanoparticles can be grown at the tip apex and that the sensitivity and spatial resolution of the TERS tips is suitable for investigations of biological nanostructures. However, the orientation of the nanorods at the tip apex is rather random, therefore the TERS enhancement is not consistent between tips. While the selection of the laser wavelength and power can control the shape of the nanoparticle, Ag nanoparticles can grow at random positions on the tip and into spherical shapes because the availability of Ag+ ions is approximately equal over the laser spot. In this proposal we plan to use a non-uniform electric field (apply a voltage between the tip and the substrate) and polarised laser excitation light to preferentially direct the Ag+ ions towards the apex of the tip and thus preferentially support the downwards growth of a Ag nanorod at the apex of the tip. Tips with a single vertical Ag nanorod (~20nm radius of curvature) at its apex would represent ideal TERS tips: high enhancement, high spatial resolution and high reproducibility.

Planned Impact

Recent research has shown that lipid microdomains are important in a wide range of biological processes, including signal transduction, membrane trafficking and sorting, and can be the entry points for viruses, bacteria or other toxins. Rafts have also been shown to play significant roles in many diseases, such as Alzheimer's disease and cardiovascular disorders. Despite sustained efforts during the last decade, many properties of cellular membranes still remain unanswered. For example, how lipid molecules interact with membrane proteins and how proteins affect the organisation of the lipids? how microdomains affect protein trafficing and receptor signalling? what is the role of various lipid molecules in pathological conditions? Anwers to such fundamental questions would advance our understaning of the cell membrane and underpin developments of new therapies to many diseases. TERS could help find out answers to these questions as detailed label-free molecular analysis could be performed on model supported lipid bilayers to determine the spatial distribution of various molecular species and detect changes in vibrational frequencies caused by molecular interactions.

While the focus of this project is on optimising TERS for studying lipid membranes, this novel technique can also be applied to a wide range of biological nanostructures. We have showed the ability to measure TERS spectra of self-assembly peptide nanotubes, which have a high relevance to ineurodegenerative diseases (e.g. Alzheimer disease). However, similar studies can be expanded to other similar nanostructures, such as amyloid-like fibrils, tubular proteins or virus shaft proteins.

A wide range of biological nanomaterials have also been proposed for various biotechnology applications, such as drug delivery, tissue engineering, biosensing, etc. However, to achieve the ultimate functionality of such materials, a better understanding of the relationship between their physical properties as well as molecular structure is therefore important. TERS could have a big impact in this field as the avalability of analytical tools able to measure the molecular properties of individual nanostructures is currently limited. The worldwide market for nanotechnologies is predicted to be $1trillion by 2015. However, the lack of appropriate tools, in particular for characterisation of biological nanomaterials, has been highlighted by most reports as one of the key obstacles in maximising the exploitation of nanotechnology (for example Taylor report 2002, " New Dimensions for Manufacturing: A UK Strategy for Nanotechnology"; the Royal Society and Royal Academy of Engineering 2004 report "Nanoscience and Nanotechnologies: Opportunities and Uncertainties"). The recent topical review "The European nanometrology landscape" (Nanotechnology 22 (2011) 062001) also highlights that the applications of nanotechnology in life sciences and health raises unique challenges related to controlling the properties of the nanomaterials as well as limiting the potential risks ("Characterising the potential risks posed by engineered nanoparticles" UK Government DEFRA Report, 2006). Medical devices/implants (e.g. biosensors) and medicines based on biocompatible nanomaterials are expected to have a huge impact in the health care technologies over the next two decades, therefore the development of reproducible analytical tools able to investigate nanomaterials at a nanoscale represents a key part for accelerating the translation of nanotechnology and benefit the society.
Description We have developed a new method for fabrication of probes for tip-enhanced Raman spectroscopy. Dielectrophoresis was used to fabricate in-situ probes for tip-enhanced Raman spectroscopy (TERS) based on Au nanoparticles. A typical conductive atomic force microscope (AFM) was used to functionalize iridium-coated conductive silicon probes with Au nanoparticles of 10-nm diameter. Suitable TERS probes were produced, and the ability to measure TERS of organic molecules was demonstrated.
Exploitation Route Further development is required, using the optimised equipment in the PI's laboratory. Methods have been uptake by other research groups, leading to two publications:
Leiterer C. et al., "Dielectrophoretic positioning of single nanoparticles on atomic force microscope tips for tip-enhanced Raman spectroscopy," Electrophoresis. 36, , 1142 -1148 (2015). 0173-0835
Leiterer C. et al., "High precision attachment of silver nanoparticles on AFM tips by dielectrophoresis," Anal. Bioanal. Chem.. 408, , 3625 -3631 (2016). 1618-2642
Sectors Chemicals,Creative Economy,Electronics,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description Diagnosis of tumours during tissue conserving surgery by multimodal spectral imaging
Amount £1,394,000 (GBP)
Funding ID EP/L025620/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 09/2019