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Direct Detection of Extracellular Ve...

Lopez Baltazar, Jesus,

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Direct Detection of Extracellular Vesicle Transmembrane Protein - Antibody Interactions Using Surface Plasmon Resonance Biosensors /

Record Type:	Electronic resources : Monograph/item
Title/Author:	Direct Detection of Extracellular Vesicle Transmembrane Protein - Antibody Interactions Using Surface Plasmon Resonance Biosensors // Jesus Lopez Baltazar.
Author:	Lopez Baltazar, Jesus,
Description:	1 electronic resource (193 pages)
Notes:	Source: Dissertations Abstracts International, Volume: 86-03, Section: B.
Contained By:	Dissertations Abstracts International86-03B.
Subject:	Chemical engineering. -
Online resource:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=31488418
ISBN:	9798384052937

Direct Detection of Extracellular Vesicle Transmembrane Protein - Antibody Interactions Using Surface Plasmon Resonance Biosensors /
Lopez Baltazar, Jesus,

Direct Detection of Extracellular Vesicle Transmembrane Protein - Antibody Interactions Using Surface Plasmon Resonance Biosensors /Jesus Lopez Baltazar. - 1 electronic resource (193 pages)

Source: Dissertations Abstracts International, Volume: 86-03, Section: B.

Extracellular vesicles (EVs) are membrane-bound particles released by all cells for intracellular communication, and they carry biomarkers that play important roles in disease progression. Surface plasmon resonance (SPR) is a powerful label-free, real-time optical detection method that has recently emerged for the detection of EVs via interactions of transmembrane proteins (TMPs) with antibodies. Effective development of SPR-based immunoassays is imperative to advance EV-based diagnostic platforms that could be used in clinical settings. In this thesis, we focused on: 1) the development of SPR-based immunoassays for the detection of EVs derived from mouse monocyte cells, 2) the implementation of a dual-antibody surface functionalization strategy to co-capture EVs via tetraspanin markers, and 3) the proteomic engineering of EV samples and their detection via TMPs using SPR biosensors.First, we developed a series of immunoassays for the detection of EVs derived from mouse monocyte cells (RAW 264.7) using surface plasmon resonance (SPR) biosensors. We chemically immobilized antibodies onto mixed self-assembled monolayers of oligo ethylene glycol (OEG) alkane thiolates with carboxylic and hydroxylic terminal groups. The effects of antibody clonality (monoclonal vs. polyclonal) and antibody surface coverage in targeting EVs via CD81 tetraspanins were investigated. We determined binding kinetic parameters, establishing trends from steric hindrance effects and epitope recognition properties of antibodies. Next, we used the optimal surface chemistry conditions to co-target EV tetraspanin markers CD81, CD63, and CD9 using a dual antibody functionalization strategy providing a critical comparison against single antibody EV capture. We used dual-Ab surfaces to co-target tetraspanins on tetraspanin-enriched microdomains (TEMs), and we further investigated EV capture with non-tetraspanin protein partners LFA-1, ICAM-1, and VCAM-1 associated with CD9 and CD81 in TEMs. Finally, we engineered EVs by prompting the expression of cancer-associated HER-2 and PD-L1 TMPs on their surface and show that overexpression of these markers can be assessed using SPR-based immunoassays. Our work addresses key challenges into immunoassay development for EV detection using SPR biosensors that can further advance EV-based diagnostic platforms.

English

ISBN: 9798384052937Subjects--Topical Terms:

560457
Chemical engineering.
Subjects--Index Terms:

Biosensors

Direct Detection of Extracellular Vesicle Transmembrane Protein - Antibody Interactions Using Surface Plasmon Resonance Biosensors /
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520 $a Extracellular vesicles (EVs) are membrane-bound particles released by all cells for intracellular communication, and they carry biomarkers that play important roles in disease progression. Surface plasmon resonance (SPR) is a powerful label-free, real-time optical detection method that has recently emerged for the detection of EVs via interactions of transmembrane proteins (TMPs) with antibodies. Effective development of SPR-based immunoassays is imperative to advance EV-based diagnostic platforms that could be used in clinical settings. In this thesis, we focused on: 1) the development of SPR-based immunoassays for the detection of EVs derived from mouse monocyte cells, 2) the implementation of a dual-antibody surface functionalization strategy to co-capture EVs via tetraspanin markers, and 3) the proteomic engineering of EV samples and their detection via TMPs using SPR biosensors.First, we developed a series of immunoassays for the detection of EVs derived from mouse monocyte cells (RAW 264.7) using surface plasmon resonance (SPR) biosensors. We chemically immobilized antibodies onto mixed self-assembled monolayers of oligo ethylene glycol (OEG) alkane thiolates with carboxylic and hydroxylic terminal groups. The effects of antibody clonality (monoclonal vs. polyclonal) and antibody surface coverage in targeting EVs via CD81 tetraspanins were investigated. We determined binding kinetic parameters, establishing trends from steric hindrance effects and epitope recognition properties of antibodies. Next, we used the optimal surface chemistry conditions to co-target EV tetraspanin markers CD81, CD63, and CD9 using a dual antibody functionalization strategy providing a critical comparison against single antibody EV capture. We used dual-Ab surfaces to co-target tetraspanins on tetraspanin-enriched microdomains (TEMs), and we further investigated EV capture with non-tetraspanin protein partners LFA-1, ICAM-1, and VCAM-1 associated with CD9 and CD81 in TEMs. Finally, we engineered EVs by prompting the expression of cancer-associated HER-2 and PD-L1 TMPs on their surface and show that overexpression of these markers can be assessed using SPR-based immunoassays. Our work addresses key challenges into immunoassay development for EV detection using SPR biosensors that can further advance EV-based diagnostic platforms.
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653 $a Surface plasmon resonance
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