Anisotropy Resolved Multidimensional Emission Spectroscopy (ARMES) as a tool for biophysical analysis

Abstract

The development of analytical tools for the biophysical analysis of biological samples is an area of tremendous application and potential. In this work the use and efficacy of an analytical methodology, known as anisotropy resolved multidimensional emission spectroscopy (ARMES) [1, 2], as a tool for biophysical analysis is explored. ARMES comprises of a 4-D measurement method used in combination with multi-way data analysis. The ARMES methodology is being developed with the aim of addressing some of the challenges surrounding the analysis of biological therapeutics. At present, many available methods used in biological therapeutic manufacture are either destructive, time-consuming, or alter the sample [3]. Protein analysis by intrinsic fluorescence is attractive as it is fast, sensitive, inexpensive and non-invasive, with good robustness, high sample throughput, ease of use and low cost required for use as a process analytical technology (PAT) tools [4]. Proteins are generally multi-fluorophore systems, with overlapping emission from the aromatic amino acids (Trp, Tyr and Phe) which makes multidimensional fluorescence spectroscopy (MDF) measurement techniques like excitation-emission matrix (EEM) and total synchronous fluorescence scan (TSFS) potentially beneficial [5-7]. These MDF methods can be further developed by coupling with factor based chemometric methods to resolve the individual fluorophore contributions of the intrinsic emission. In ARMES, an additional dimension of anisotropy (r) is collected, which is related to rotational speeds, hydrodynamic volumes, and thus molecular size, and adds further information to the MDF measurement. This ARMES methodology forms the foundation for this research [2, 8]. In the first project presented in this thesis work, the application of ARMES in the analysis of Förster resonance energy transfer (FRET) is investigated (Chapter 3 & 4) [9]. FRET is a widely used technique to study the structure and dynamics of biomolecular systems, and also causes the non-linear fluorescence response observed in multi-fluorophore proteins, so accurate FRET analysis is critical. Here, a model system of human serum albumin (HSA) as a FRET donor and 1,8-anilinonaphathalene sulfonate as a FRET acceptor was used. The results of this work found ARMES enabled resolution of the fluorescence emission into its constituent fluorophore emission and facilitated a more accurate analysis of the interactions and photophysical processes occurring in the HSA-ANS system. This enabled a new way of calculating biophysical parameters including quenching constants and FRET efficiencies using the multi-dimensional emission of individual donor fluorophores, and a significant increase in the FRET efficiency values recovered using the ARMES method was observed. In addition, ARMES enabled the extraction of the emission arising from indirect excitation via FRET, which is of significance in understanding the effects of FRET on MDF spectra. In the second project of my thesis research, the use of ARMES in investigating protein-liposome interactions is explored (Chapter 5 & 6). Studying the interaction between plasma proteins and liposomes is critical for many different scientific applications, particularly in their use as drug delivery systems (DDS) [10, 11], such as those used in COVID-19 vaccines [12]. Here, a model system of HSA and DMPC liposomes was used, and their interactions were monitored in three different aqueous environments: water (pH ~7.9), NH4HCO3 (ABC) (50 mM, pH ~7.8), and phosphate buffered saline (PBS) (10 mM, pH ~7.4). Interestingly, a dramatically different interaction mechanism was observed in each environment with HSA observed to penetrate the lipid bilayer in water and ABC, but not in the case of PBS. Here, ARMES enabled the resolution of fluorescence emission into interacting populations of HSA which had penetrated the lipid bilayer from populations of HSA which were surface bound or free in aqueous solution and provided an informative approach for monitoring protein-liposome interactions. In conclusion, the application of ARMES methodology for biophysical analysis on two different molecular systems is demonstrated in this thesis work. ARMES facilitated a more detailed analysis of the photophysical of FRET, providing a new means of calculating biochemical parameters (FRET efficiencies and quenching constants) and provided a new way of assessing protein-liposome interactions using intrinsic protein emission. The studies show ARMES has tremendous potential as a tool of biophysical analysis of interacting molecular systems.