Engineers at UK-based Cambridge Consultants (CCL) have developed a new system for the phase imaging of Surface Plasmon Resonance (SPR), enabling the high-throughput array-based analysis of molecular binding events, label-free.
SPR has become widely used to characterise single biomolecular binding events, but there has been a real need for array-based analytical methods that can be used to detect such interactions without a requirement for molecular labelling. This is especially true in the case of protein-protein interactions where labelling can interfere with protein function, but where high-throughput is required.
CCL’s new proprietary sensing technique provides a solution for this problem, exploiting the intrinsic high sensitivity of SPR imaging and interferometry in a single robust unit to enable the array-based, phase imaging analysis of biomolecular interactions, label free.
A typical SPR instrument involves the reflection of light at a thin metallic film to which ligand molecules are attached. The intensity and phase of the reflected light, when observed using SPR, depend on the refractive index of the medium in contact with the film. Molecular binding between the ligands attached to the surface of the film and the proteins in a secondary fluid gives rise to such refractive index variations.
Unlike conventional intensity-based SPR instruments, the basis of the new system is the interferometric measurement of the spatial variation of the reflected phase as a function of the refractive index.
The spatial variation of this phase change is measured using a new design of interferometer which is inherently stable and can be of monolithic construction, resulting in very low phase noise (10-6 RIU at 1.5Hz) and reductions in temperature, strain and vibration sensitivity, thus enabling the high-throughput of sample arrays.
Phase extraction is currently performed using a Fourier transform technique requiring no moving parts. The potential to use higher resolution phase measurement techniques exists within the basic framework of the system.
Dr. Robert Jones, who led the development of the new system, believes that this new phase based technique is better suited to the measurement of multiple, small-binding sites than current intensity based methods.
‘Existing intensity based methods fall broadly into two categories,’ he explains. ‘Either they employ differential imaging using plane wave illumination, or intensity minimum methods whereby the position of the dark line, observed when a divergent light field is incident on the resonant surface, is located. But both of these are constrained in their ability to image and measure multiple small binding sites.’
Julian Pieters, Product Engineering Group Leader at CCL adds: ‘Our new technique enables the highly parallel measurement of multiple small binding sites using high density arrays, speeding up analysis by a significant factor. We have successfully imaged patterned substrates with a grid spacing of 250µm and achieved low levels of background noise without environmental screening or temperature control. This has been achieved in real-time and with all the benefits of the high-sensitivity intrinsic to SPR.’
CCL’s patented design is expected to enable high-density arrays with greater than 103 parallel measurements, label free detection, real time capability for kinetics, high sensitivity, high stability, and a compatibility with flow-through operation.
A PCT application has been filed, and the technology is now available for development through contract and licensing agreements with interested organisations.