Development of High Throughput Screening Platforms for Biotechnology

UoM administered thesis: Phd

  • Authors:
  • Emily Kempa

Abstract

High throughput screening can be transformative in the discovery of new pharmaceutical products, improving enzyme variants and for cell differentiation. Methods that can screen upwards of 10^4 samples per day are mostly reliant on inherent or incorporated fluorescent labels as the detectable species, which can restrict analyte scope. Label-free analytical platforms based on mass spectrometry (MS) measurements provide an alternative route, however, the throughput of current commercially available platforms is limited by their sample infusion robotics. The ability to screen reaction mixtures in their crude form is desirable, as sample purification methods for biological systems are often lengthy to limit contamination and improve the sensitivity of the mass spectrometry method employed. This thesis focuses on the use and development of two mass spectrometry-based sample introduction methods namely desorption electrospray ionisation (DESI)-MS and microfluidics and applies these to the study of model analytes and enzymatic reactions. In Chapter 2 I show how DESI-MS can be applied to directly monitor biotransformations, termed DiBT-MS. This screening methodology is successfully applied to industrially relevant biocatalysts and the application of the workflow to a directed evolution experiment allowed for the activity enhancement of a phenylalanine ammonia lyase (PAL) enzyme towards electron-rich cinnamic acid derivatives. The output from this approach provides mass-selected heat-maps of the product(s) in a format analogous to 96-well plates and is semi-quantitative when compared with NMR and HPLC. DiBT-MS screening of these samples permitted throughputs equivalent to ~40 seconds per sample. Commercial droplet microfluidics coupled MS platforms are still in their infancy, and in Chapter 3 I show how we have designed and interfaced microfluidic chips to four different commercially available mass spectrometers showing how the fluidics and chips can be adapted to different electrospray ionisation (ESI) ion sources. Hardware and software developments allow throughputs of 33 samples per second to be reached on an ion mobility mass spectrometer, with particular emphasis put upon establishing a platform suited to biotechnology workflows. The advantages and disadvantages of the different ion sources and mass spectrometers are explored, and the designs allowing MS coupling presented. Looking forward, the application of the methods and means described here will be significant to the development of successful commercial droplet microfluidics-MS apparatus and the Final Chapter (4) describes recent work wherein droplet reinjection is demonstrated, which will further the applications of coupling MS with uHTS microfluidics.

Details

Original languageEnglish
Awarding Institution
Supervisors/Advisors
Award date1 Aug 2021