Integrated microfluidic system for separation and detection of extracellular vesicles from plasma

다국어 초록 (Multilingual Abstract)

Recent research has found a correlation between the levels of immune checkpoint markers present in extracellular vesicles (EVs) and the effectiveness of immunotherapy treatments. In this work, we describe the development of an integrated microfluidic system (IMS) that allows for the separation and identification of EVs containing the immune checkpoint markers programmed death ligand 1 (PD-L1) and programmed death protein 1 (PD-1) without the contamination of lipoproteins, which are prevalent in plasma. We first created a size difference between lipoproteins and EVs by immobilizing the lipoproteins on the surface of beads within the plasma using antigen-antibody reactions. Subsequently, the pre-treated sample was introduced into a spiral microfluidic channel to separate nanometer-sized EVs from micrometer-sized lipoprotein-bead complexes. We designed a microfluidic system to connect the channel where pure EVs are separated to the detection unit so that the separated EVs can be immobilized on the electrochemical sensor surface coated with PD-L1 and PD-1 antibodies. This device can rapidly detect the levels of immune checkpoint markers present in EVs over a broad clinical concentration range (1 × 104 to 1 × 108 EVs/mL). Additionally, analysis of 28 plasma samples from 8 healthy donors and 20 lung cancer patients showed that PD-L1 and PD-1 expression on EVs could potentially improve the immunotherapy candidate selection process. This suggests that this device, when used in conjunction with traditional tissue-based PD-L1 testing, may help identify additional patients who may benefit from immunotherapy treatment. Therefore, we anticipated that our approach would contribute positively to the clinical assessment of candidates for immunotherapy.

목차 (Table of Contents)

• Contents ................................................................................................................... i
• List of Figures ........................................................................................................ iv
• List of Tables ...................................................................................................... viii
• Abstract .................................................................................................................. ix
• Chapter 1. Introduction ...................................................................................... １
• 1.1 Extracellular vesicles (EVs) .................................................................................... １
• 1.2 Enrichment of EVs using a microfluidic chip ........................................................... 3
• 1.3 Objectives of the research ......................................................................................... 6
• Chapter 2. EV separation and detection through the integration of spiral
• microfluidic channels and electrochemical sensors. ........................................... 7
• 2.1 Introduction ............................................................................................................... 7
• 2.2 Materials and methods ............................................................................................ 12
• Electrochemical sensor fabrication .................................................................... 12
• Microfluidic device fabrication and integration with electrochemical sensors . 14
• Device characterization ..................................................................................... 15
• Preparation of beads coated with antibodies ...................................................... 15
• Sample preparation ............................................................................................ 16
• Simulations of computational fluid dynamics (CFD) ........................................ 17
• Ultracentrifugation (UC) ................................................................................... 17
• Size exclusion chromatography (SEC) .............................................................. 17
• Microfluidic device ............................................................................................ 18
• Nanoparticle tracking analysis (NTA) ............................................................... 19
• Transmission electron microscopy (TEM) ........................................................ 19
• Flow cytometry .................................................................................................. 19
• Electrochemical analysis ................................................................................... 20
• 2.3 Results and discussion ............................................................................................ 23
• Strategy to separate EVs from lipoproteins in plasma ....................................... 23
• Investigation of the experimental conditions in the spiral channel using
• fluorescent beads. .............................................................................................. 26
• Assessment of the efficacy in separation of EVs from lipoproteins utilizing the
• spiral channel ..................................................................................................... 31
• Resistance design for integrating microfluidic channels with detection part .... 36
• EV detection by electrochemical technique ....................................................... 40
• Clinical sample analysis .................................................................................... 47
• Conclusion ...................................................................................................................... 53
• References ...................................................................................................................... 54
• Accomplishments ........................................................................................................... 60