Label-free, Real-time, Spatial-resolution (LRS) immunoassay: 2D mapping of extracellular signalling molecules

Individual cells within human and animal tissues communicate with each other through specific chemical and bio-molecular signals that are released from cells into their local environment. These signals have critical roles in controlling cellular activity including regulating cell growth, function, movement and death. Together these signals regulate how tissues form, function and are repaired. Inventing a technology that can create high definition maps of these molecular signals in both healthy and diseased tissues would provide invaluable insights into how tissues work and how they change during development, disease and during the aging process.

Conventional analytical techniques including enzymatic immune assays (ELISA) have been used in hospital diagnostics, home pregnancy detection kits and laboratory experiments for over 40 years. While this and other related techniques have been able to identify and quantify key signalling molecules in immune responses, tumour growth, development and tissue function they provide no information regarding where in the tissue these molecules are secreted, how the molecules diffuse in the tissue or indeed when. As a result, they are unable to address key mechanistic questions about how these signalling molecules actually work, when cells respond to these signals and why.

In this proposal we aim to develop, test and use a new analytical technology that will provide real-time mapping of molecular signals in multicellular tissues, enabling researchers and clinicians to answer these critical questions. The tool is based on a silicon photonic crystal nanostructure similar to those developed originally for applications in high speed telecommunications. We have shown recently that the sensitivity of the photonic crystals to the local environment can be exploited to detect very low concentrations of proteins. The detection occurs in real-time and the small size of the photonic crystal enables molecular detection with very high spatial resolution. In this proposal, we will demonstrate the potential of this new technology by addressing a key unknown question within biology regarding the role of signalling proteins called interleukins in the immune response.

The function of complex multicellular tissues is regulated by extrinsic cues secreted into the localised microenvironment. While conventional technologies for dissecting these signalling pathways are able to detect key signalling molecules, they provide no spatial or temporal information. The highly sensitive technology developed in this project will address this challenge and enable real-time, spatial mapping of specific proteins expressed by cells. The technology, termed the Label-free, Real-time, Spatial-resolution (LRS) immunoassay is based on silicon photonic crystal nanostructures that exhibit a transmission (or reflection) resonance that changes as a result of protein-protein binding or as cell functions change. The resonance shift occurs only over a single, micrometre-sized pixel enabling high resolution mapping of protein secretion. Building on our prior research, we will optimize the underpinning technology and develop surface functionalization strategies to enable integration of the photonic sensor with antibodies and cell cultures. We will initially optimize and validate the technology (detection sensitivity, spatial and temporal resolution) using immune cells, including T cells which secrete IL2 upon antigen stimulation. We will further expand these studies to analyse IL10 secretion by Th2 cells, and the homeostatic cytokine IL-7 and chemokine CCL21 by fibroreticular cells. Finally, we will demonstrate the potential of our assay through mapping IL10 secretion by T cells in tissue culture to determine if physiological IL10 secretions are directional or synaptic. While we focus here on the immunology of cytokines and chemokines, the low-cost technology has the potential to impact across a wide range of biological and clinical areas including wound healing, tumour biology, aging and neurobiology and can be applied to the development of advanced diagnostic instrumentation for the treatment of animal and human diseases.

The project will develop capabilities to map the spatial and temporal distribution of signaling molecules in multicellular tissue and thus provide new tools for understanding the biophysics of cellular communication and tissue function. The outputs of this project impact across the scientific disciplines and on a large variety of future applications and industries, ranging from healthcare (including clinical diagnostics, tissue engineering, regenerative medicine, etc.), instrument manufacturers (including microscope manufacturers and the in vitro diagnostic sector) and the environment sector (including environmental monitoring agencies, water industry etc.).

Potential stakeholders include:
1) Public sector organizations including NHS; Department of Health; Home Office; Ministry of Defense; Environment Agency/DEFRA.
2) Business/industry including Diagnostics companies; Pharmaceutical/Biotech companies; Research equipment manufacturers; suppliers for the above public sector organizations.
3) General public.
4) Academia/Research organizations.

Likely stakeholder benefits include:
Healthcare impact: The emergence of personalised and stratified medicine with their inherent requirement for sophisticated diagnostics, together with the transfer of clinical testing to point-of-care/primary care, are putting increasing pressure on current diagnostics services. The provision of highly sensitive, multiplexed protein arrays would greatly enhance the capabilities of diagnostic devices and support the identification of new disease biomarkers, discovery of new therapeutics and targeted treatments and accelerate patient profiling. For the Department of Health/NHS this will lead to improved and more cost-effective healthcare services. The general public will benefit through early diagnosis and access to clinical testing at point-of-care, such as the GP surgery, and ultimately through the delivery of targeted treatment options. In the longer term, benefits to the healthcare sector could include new diagnostic devices capable of monitoring the localised tissue microenvironments in vivo. Such devices have the potential to address a range of clinical challenges, for example in wound management.

Commercial and Economic impact: This programme will generate new capabilities that will underpin a range of new technologies and products. Notable beneficiaries in the short/medium term include manufacturers of analytical and microscopy instrumentation, providing new tools that will increase their capabilities and competitiveness. Importantly, the technology is low-cost and compatible with conventional microscopy instrumentation enabling widespread use and rapid technology take-up. Large international enterprises and SMEs in the in vitro diagnostics sector also have significant commercial interest in low-cost, multiplexed diagnostics technology and in portable diagnostic devices for primary care/point-of-care use. Diagnostics are becoming increasingly important across a range of sectors including in veterinary practice (animal screening), drug discovery (small molecule arrays), environmental monitoring (detection of pollutants), security (detection of biological/chemical agents) and agrotech (pathogen diagnostics).