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Influence of molecular size and zeta potential in electrokinetic biosensing
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.ORCID iD: 0000-0002-6552-8426
KTH, School of Engineering Sciences (SCI), Applied Physics, Photonics.ORCID iD: 0000-0002-5077-3218
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.ORCID iD: 0000-0002-0695-5188
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2020 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 152, article id 112005Article in journal (Refereed) Published
Abstract [en]

Electrokinetic principles such as streaming current and streaming potential are extensively used for surface characterization. Recently, they have also been used in biosensors, resulting in enhanced sensitivity and simpler device architecture. Theoretical models regarding streaming current/potential studies of particle-covered surfaces have identified features such as the particle size, shape and surface charge to influence the electrokinetic signals and consequently, the sensitivity and effective operational regime of the biosensor. By using a set of well-characterized proteins with varying size and net surface charge, this article experimentally verifies the theoretical predictions about their influence on the sensor signal. Increasing protein size was shown to enhance the signal when their net surface charge was either opposite to that of the sensor surface, or close to zero, in agreement with the theoretical predictions. However, the effect gradually saturated as the protein size exceeded the coulomb screening length of the electrolyte. In contrast, the proteins containing the same type of charge as the surface showed little or no difference, except that the signal inverted. The magnitude of the surface charge was also shown to influence the signal. The sensitivity of the technique for protein detection varied over two orders of magnitude, depending upon the size and surface charge. Furthermore, the capacity of the electrokinetic method for direct electrical detection of various proteins, including those carrying little or no net electric charges, is demonstrated.

Place, publisher, year, edition, pages
Elsevier, 2020. Vol. 152, article id 112005
Keywords [en]
Biosensor, Electrokinetics, Improved sensitivity, Influence of molecular size and charge, Streaming current, Zeta potential
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-267788DOI: 10.1016/j.bios.2020.112005ISI: 000515210600008PubMedID: 32056733Scopus ID: 2-s2.0-85077917325OAI: oai:DiVA.org:kth-267788DiVA, id: diva2:1397763
Note

QC 20200227

Available from: 2020-02-27 Created: 2020-02-27 Last updated: 2022-06-26Bibliographically approved
In thesis
1. Development of Techniques for Characterization, Detection and Protein Profiling of Extracellular Vesicles
Open this publication in new window or tab >>Development of Techniques for Characterization, Detection and Protein Profiling of Extracellular Vesicles
2021 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Nanosized extracellular vesicles (EVs, ∼30-2000 nm) have emerged as important mediators of intercellular communication, offering opportunities for both diagnostics and therapeutics. In particular, small EVs generated from the endolysosomal pathway (∼30-150 nm), referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics and treatment monitoring based on minimally invasive liquid biopsies. This is because exosomes carry valuable biological information (proteins, lipids, genetic material, etc.) reflecting their cells of origin. Using EVs as biomarkers or drug delivery agents in clinical applications requires a full understanding of their cellular origin, functions, and biological relevance. However, due to their small size and very high heterogeneity in molecular and physical features, the analysis of these vesicles is challenged by the limited detection ranges and/or accuracy of the currently available techniques. To overcome some of these challenges, this thesis focuses on developing different techniques for characterization, detection and protein profiling of EVs at both bulk and single particle levels. Specifically, the three methods investigated are scanning electron microscopy, electrokinetic sensing, and combined fluorescence - atomic force microscopy. 

First, a protocol for scanning electron microscopy imaging of EVs was optimized to improve the throughput and image quality of the method while preserving the shape of the vesicles. Application of the developed protocol for analysis of EVs from human serum showed the possibility to use scanning electron microscopy for morphological analysis and high-resolution size-based profiling of EVs over their entire size range. Comparison with nanoparticle tracking analysis, a commonly used technique for EV size estimation, showed a superior sensitivity of scanning electron microscopy for particles smaller than 70-80 nm. Moreover, the study showed process steps that can generate artifacts resembling sEVs and ways to minimize them. 

Secondly, a novel label-free electrokinetic sensor based on streaming current was developed, optimized and multiplexed for EV protein analysis at a bulk level. Using multiple microcapillary sensors functionalized with antibodies, the method showed the capacity for multiplexed detection of different surface markers on small EVs from non-small-cell lung cancer cells. The device performance in the multichannel configuration remained similar to the single-channel one in terms of noise, detection sensitivity, and reproducibility. The application of the technique for analysis of EVs isolated from lung cancer patients with different genomic alterations and after different applied treatments demonstrated the prospect of using EVs from liquid biopsies as a source of biomarker for cancer monitoring. Moreover, the results held promise for the application of the developed method in clinical settings. 

Finally, to increase the understanding of EV subpopulations and heterogeneity, a platform combining fluorescence and atomic force microscopy was developed for multiparametric analysis of EVs at a single particle level. The use of a precise spot identification approach and an efficient vesicle capture protocol allowed to study and correlate for the first time the membrane protein composition, size and mechanical properties (Young modulus) on individual small EVs. The application of the technique to vesicles isolated from different cell lines identified both common and cell line-specific EV subpopulations bearing distinct distributions of the analyzed parameters. For example, a sEV population co-expressing all the three analyzed proteins in relatively high abundance, yet having average diameters of <100 nm and relatively low Young moduli was found in all cell lines. The obtained results highlighted the possibility of using the developed platform to help decipher unsolved questions regarding EV biology. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2021. p. 97
Series
TRITA-SCI-FOU ; 2021:44
Keywords
extracellular vesicles, streaming current, fluorescence microscopy, atomic force microscopy, scanning electron microscopy, protein profiling, size profiling
National Category
Nano Technology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biomedical Laboratory Science/Technology
Research subject
Physics, Biological and Biomedical Physics
Identifiers
urn:nbn:se:kth:diva-304800 (URN)978-91-8040-069-5 (ISBN)
Public defence
2021-12-10, Room Ångdomen and via Zoom: https://kth-se.zoom.us/j/68480621469, Osquars backe 31, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2021-11-15 Created: 2021-11-12 Last updated: 2022-09-21Bibliographically approved

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Stiller, ChristianeCavallaro, SaraEriksson Karlström, AmelieLinnros, Jan

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