2D-IR spectroscopy for serum diagnostics

Biochemical analysis of biofluids, such as blood serum, is an important source of information in the healthcare environment. Biofluids are obtained quickly and with minimal patient discomfort, while levels of proteins, lipids, sugars and other metabolites fluctuate in response to body chemistry, providing early warning signals of deterioration in health before specific symptoms become apparent. The protein content of blood serum alone is an ideal substrate for holistic analysis. Human serum contains ~70 mg/mL of proteins, composed of albumin (~35-50 mg/mL) and the globulins (~25-35 mg/mL). Diagnostically, measurement of the albumin to globulin ratio (AGR) is useful because changes in the AGR are linked to an inflammatory response. The globulins are potentially even more informative because they encompass a huge number of proteins. The bulk are so-called gamma-globulins, but levels of specific globulin proteins such as immunoglobulin-G (IgG, ~ 80% of the gamma-globulins), IgA (~13%) and IgM (~6%) are associated with specific health-related issues.
Spectroscopic analysis of serum using infrared (IR) spectroscopy is potentially transformative. The measurements provide the broad chemical fingerprint of the sample that makes for effective triage of samples, determining the need for and then guiding subsequent in-depth diagnosis. A single spectroscopic measurement would also be faster and more economical than a panel of antibody-based assays, each targeting a single biofluid component.
IR methods have yet to progress to clinical applications however, because biofluids are aqueous and water obscures signals from the proteins that comprise the bulk of serum samples and which are therefore a major diagnostic marker.
Here, we will develop an advanced spectroscopy technique, ultrafast 2D-IR spectroscopy as a tool for quantitative, label-free analysis of blood serum. Our preliminary data (Chem Sci doi: 10.1039/C9SC01590F) shows that the 2D-IR signal, which derives from a series of ultrashort (100 fs-duration) laser pulses, completely suppress the water background relative to the protein response, allowing measurements to be made in transmission on unprocessed, wet, serum samples. Using the characteristic 2D spectral signature of a protein that arises from the vibrational couplings inherent in its secondary structure, we have spectrally resolved signals from albumin and globulin protein fractions in serum and measured the biomedically-important albumin to globulin ratio with an accuracy of +/- 4% across a clinically-relevant range. We have also demonstrated that 2D-IR spectroscopy can differentiate signals from the structurally similar globulin proteins IgG, IgA and IgM, opening up a straightforward spectroscopic approach to measuring levels of serum proteins that are currently only accessible via biomedical laboratory testing.
In this proposal, we will go beyond this proof of concept and lay the scientific groundwork to drive future translation of 2D-IR technology into healthcare-related serum diagnostics applications. We will develop the sample handling, data collection, processing and analysis protocols needed to use 2D-IR analytically. We will develop internal calibration approaches needed to measure the concentration of six key serum proteins to an accuracy of +/- 1%. We will use the rich molecular information content of 2D-IR spectroscopy to measure low molecular weight fractions of serum such as sugars phospholipids and nucleic acids, delivering a broad biomedical fingerprint of a serum sample. Ultimately, we will use these methods to screen patient serum samples in order to differentiate between healthy and diseased samples.

This proposal seeks to establish the scientific principles for performing biofluid diagnostics on blood serum and we envisage that this will have downstream impact in the clinical setting as a new Healthcare Technology. It is important to stress that the work proposed is entirely novel, no other group is attempting this worldwide. As a result of this, the level of readiness for translation into the Healthcare environment is consistent with just TRL1 or 2 at this stage. It is intended that by the end of the project we will have established TRL2-3 by progressing into work with patient samples. We provide plans to advance this beyond the lifetime of the grant in the Pathways to Impact document, but in the short term, we forsee impact from our work in terms of knowledge, the economy, people and society that will be separate from the main thrust of the project:
Knowledge:
We will take significant steps forwards in applying 2D-IR spectroscopy to quantifying protein concentration and for unravelling the content of mixtures of proteins in a water-based solvent. This will be an important advance, with impact in both the physical and biological sciences, where spectroscopic studies of proteins in their natural solvent will be possible. Locally to the PI, this will have relevance for work ongoing in the York Structural Biology Laboratory but interest in the role of dynamics in biomolecule behaviour is growing globally and we expect these advances to have significant impact.
Our data analysis methods and routines are expected to have significant academic value and we expect these to be applicable far beyond the scope of the project. As such we anticipate adoption of the new data analysis methods with resulting academic impact.
Economy:
The ability to study proteins and protein mixtures in water will have significant economic impact in areas separate to Healthcare Technologies. For example, in the pharmaceutical sector. Structural studies of proteins under solution-phase conditions at room temperature will provide new information on protein-drug interactions, while the ability to unravel information about mixtures will enable the study of protein-protein complexes, which will be relevant to understanding biochemical pathways and the interruption or promotion of these via small molecules.
The ability to work in water will have impact on commercial concerns which produce biologics or biosimilar molecules on an industrial scale. The realistic possibility of harnessing our newly-developed methods to emerging high speed laser technology means that the extra information provided by 2D-IR could be used for in-line diagnostics of industrial biochemical processes in the future.
The impact of the spectroscopy would be vastly reduced without the associated data handling and analysis methods that we will develop and so we anticipate that both aspects of the project outcomes will contribute to economic impact.
People:
The work contained in the project is inherently multidisciplinary with involvement from experimental physics and physical chemistry as well as data analysis and biological chemistry. This will offer a novel training opportunity for the PDRAs involved as they will participate in all aspects of the work. Ultimately, this will impact upon the staff themselves but also on the wider scientific community as people with the new skills become the next generation of principal investigators and industrial scientists.
Society:
The cross-disciplinary nature of the work and the aims to show how the technology developed can target important healthcare-related problems will have impact on a wider, non-scientific audience. The most obvious route is through downstream adoption of the technology into the healthcare sector, but the impact that the work will have on knowledge, the economy and people will ultimately lead to societal impact, possibly in areas unanticipated at this stage.