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.
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