University of Twente Student Theses


Proteome secretion analysis from PCa cell lines using optical and electrochemical measuring methods

Andreoli, D.S. (2024) Proteome secretion analysis from PCa cell lines using optical and electrochemical measuring methods.

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Embargo date:13 May 2026
Abstract:Prostate Cancer is a leading malignancy affecting men worldwide, necessitating accurate diagnosis and effective treatment strategies. This study aims to evaluate the proteome secretion of PCa cells based on their cell cycle phases and to develop a multiplexing platform for identifying potential biomarkers. Moreover, it compares the efficacy of optical and electrochemical measurement setups in analyzing proteome secretion. A comprehensive methodology was employed, starting with the identification of potential biomarkers using proteome profiling arrays. PCa cell lines (LnCAP, PC-3, and 22Rv1) were cultured, and their supernatants analyzed to identify distinct protein secretion profiles. Immunocytochemistry (ICC) staining was used to validate intracellular protein levels and visualize PSA secretion. Fluorescence-Activated Cell Sorting (FACS) was utilized to sort cells into G1, and G2/M phases, followed by analysis of PSA secretion using the ELISpot assay and staining of PVDF membranes. A spotting device was further developed to multiplex multiple antibodies onto a single membrane. In parallel, Poly-L-Lysine (PLL) derivatives were synthesized and characterized using Nuclear Magnetic Resonance (NMR) spectroscopy. Their functionality was validated through Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) measurements. Electrochemical sensing using Cyclic Voltammetry (CV) was performed to detect the attachment of EpCAM proteins on functionalized Pt-plated electrodes, with Methylene Blue as a redox mediator. The results revealed significant heterogeneity in PSA secretion across different cell cycle phases, with only the G1 phase secreting PSA. The proteome array identified multiple potential biomarkers, including PSA, suggesting that multiplexing provides a comprehensive understanding of PCa proteome secretion. The spotting device concept was proven feasible, though further optimization is required. Synchronization of cells to the G1 phase remains a challenge needing refinement. Comparing optical and electrochemical methods, optical measurement provides a comprehensive and sensitive multiplexing platform but is complex and time-consuming. Electrochemical measurement offers real-time monitoring of binding events and requires less preparation but is limited to single biomarker detection. The societal impact of this research lies in its potential to improve early detection and personalized treatment of PCa. By identifying heterogeneity in PSA secretion across different cell cycle phases, the findings underscore the need for accurate diagnostic tools that consider this variability. The development of a multiplexing platform to identify multiple potential biomarkers could pave the way for better understanding tumor heterogeneity, leading to more effective and personalized therapeutic strategies. Additionally, liquid biopsy, being less invasive and easier to perform, may enhance patient compliance and outcomes.
Item Type:Essay (Master)
Faculty:TNW: Science and Technology
Subject:42 biology, 50 technical science in general, 53 electrotechnology
Programme:Biomedical Engineering MSc (66226)
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