AIBN Seminar Series HDR Seminar

Sophie Kenny

Title
Tuning the target-binder interface with PCR-based phage-target sensing and electrohydrodynamic mixing on microfluidic devices to improve molecular detection and molecular library panning

Abstract
The characterisation of molecular interactions and fouling properties of surfaces is vital for biological technologies such as microfluidics platforms, medical devices, and molecular library biopanning. However, traditional approaches can be relatively inflexible and challenging to apply directly to a desired device. Our lab has previously developed a single-round molecular library biopanning platform, which makes use of electrohydrodynamic mixing to increase molecular collisions and shear forces near functionalised surfaces. Using this approach combined with an in-house sequencing pipeline, a novel phage clone that bound to Dengue 3 non-structural protein 1 was isolated. However, the level of physical enrichment of binding phage clones was never validated, and the system itself was not explored in depth. In this talk, I will discuss how various aspects of the platform, including electrohydrodynamic mixing, surface functionalisation, nonspecific adsorption and specific adsorption of library-displaying phage have been further investigated. I will also discuss a simple and fast technique that has been developed for quantifying and probing molecular behaviour at the solid-liquid interface, in any system of choice. This approach has enabled us to explore the behaviour of molecules in our system with more depth than was previously possible and could be used to understand the behaviours of other desired systems where surface fouling and optimal surface functionalisation is required. Using this system in our technology, we successfully quantified more than a 100-fold reduction of the ratio of a binding phage spiked into a non-binding phage, with very high retention of the spiked-in binding phage against our target. We have also gained greater insight into how the EHD mixing in our device affects target binding and off-target effects.

Bio

Sophie is a PhD Student in the Trau group at AIBN UQ. She is researching novel microfluidic platforms for molecular library biopanning and has particular interest in accurately measuring molecular interactions at surfaces and in solution. Current applications of her research include deepening the understanding of phenomena such as Electrohydrodynamic Mixing in microfluidic devices, to increase molecular interactions and particle collision in fluids. She is also applying her work to molecular library panning, to improve the stringency of each biopanning round in phage library panning to reduce the number of biopanning rounds required and therefore reduce time to novel binder isolation.

Karthik Balaji Shanmugasundaram

Title
Phospho PD-L1 in extracellular vesicles as a proxy for tumour PD-L1 expression in lung cancer

Abstract
Immune checkpoint inhibitors (ICI) targeting programmed cell death ligand 1 (PD-L1), or its receptor, PD-1 have improved survival in patients with non-small cell lung cancer (NSCLC). Assessment of PD-L1 expression is currently used to identify patients most likely respond to single agent anti PD-1/PD-L1 therapy. This requires a tissue biopsy or fine needle aspiration with sufficient cells to generate a tissue proportion score (TPS) >50%. Acquiring cancer tissue from patients with NSCLC is often challenging due to the inaccessible location of the tumour. As such a liquid biopsy-based technique to identify the PD-L1 expression of the tumour is desirable. The current work examined the phosphorylation status of PD-L1 and quantified levels of phosphorylated PD-L1 in plasma extracellular vesicles (EV pPD-L1) from patients with NSCLC. To determine the EV pPD-L1 levels, an ultrasensitive interfacial biosensing platform was used that includes nano-mesoporous gold electrodes. The 3-dimensional mesoporous network of the electrodes provides a high surface area, high signal-to-noise ratio and a superior electro-conductive framework, which supported sensitive analysis of EV pPD-L1. Applied to test and validation cohorts of plasma samples from NSCLC patients, the assay detected… 64% of the patients studied had detectable levels of EVphosphoPD-L1 (EV pPD-L1) in the plasma. The EV pPD-L1 status had a linear correlation (R2=0.9755) with the tumour PD-L1 TPS assessed by immunohistochemistry irrespective of the tumour stage. The test and validation cohorts confirmed the reproducibility of assay. In contrast to the EV results, soluble serum PD-L1 levels, analysed with ELISA, did not correlate with the tumour PD-L1 TPS score nor with plasma EV pPD-L1 levels. The results indicate that plasma EV pPD-L1 levels could provide a non-invasive means to determine tumour PD-L1 expression and hence identify those patients in the clinic for whom single agent ICI therapy is appropriate and, potentially, patients with early stage disease most likely to benefit from neoadjuvant therapy.

Bio

Karthik Balaji Shanmugasundaram is currently a Ph.D. candidate at the Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland (UQ). Previously, he was a Research Assistant at the Center for Personalised Nanomedicine. He did his bachelor’s (Hons) in Biomedical Engineering in India. Karthik’s research interest focuses on the development of plasmonic nanostructures-based biosensors for point-of-care diagnostics and surface enhanced Raman spectroscopy analysis for liquid biopsy biomarker detection.