Studies of biofluid analysis using 2D-IR spectroscopy

Student thesis: Doctoral Thesis

Abstract

In this thesis, the application of two-dimensional infrared (2D-IR) spectroscopy is evaluated to provide a quantitative analysis of the protein content of blood serum. The foremost challenge is obtaining protein measurements in physiologically relevant solvents as the most informative infrared peak relating to protein studies, the amide I band, overlaps with the bending mode of H2O, making label-free detection of the protein content in serum challenging. This project demonstrates that 2D-IR can surmount the major obstacle to serum protein analysis as the 2D-IR amide I signature of proteins is shown to dominate that of water. Furthermore the link between protein secondary structure and the 2D-IR amide I lineshape allows differentiation of protein signals in serum leading to clinically relevant measurements of the biomedically important proteins. Detection limits for 2D-IR are also established allowing projection of the sensitivity of 2D-IR for future applications. Quantification of proteins is key for diagnosis and prognosis outcomes, however using 2D-IR, standardisation of measurement protocols needs to be addressed in order to achieve this.A new method is demonstrated for 2D-IR spectroscopy to internally normalise spectral signals, allowing normalisation of the protein response to the thermal response of water which is temporally separate from the protein signal, reducing the impact of measurement fluctuations on the data. Furthermore, normalisation of sample signals enables calibration curves of serum albumin to be produced allowing absolute protein concentrations to be obtained using 2D-IR spectroscopy. The application of 2D-IR is explored further and attempts to detect drug-binding at clinically relevant levels to serum albumin are made. Changes in the secondary structure of albuminare detected upon drug-binding however the complex nature of serum makes assigning changes observed using 2D-IR challenging.
Date of Award23 Sep 2020
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SponsorsEPSRC (Engineering and Physical Sciences Research Council)
SupervisorNeil Hunt (Supervisor) & Matthew Baker (Supervisor)

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