Immobilization and detection of platelet-derived extracellular vesicles on functionalized silicon substrate: cytometric and spectrometric approach.
Among the various biomarkers that are used to diagnose or monitor disease, extracellular vesicles (EVs) represent one of the most promising targets in the development of new therapeutic strategies and the application of new diagnostic methods. The detection of circulating platelet-derived microvesicles (PMVs) is a considerable challenge for laboratory diagnostics, especially in the preliminary phase of a disease. In this study, we present a multistep approach to immobilizing and detecting PMVs in biological samples (microvesicles generated from activated platelets and human platelet-poor plasma) on functionalized silicon substrate. We describe the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and spectroscopic ellipsometry methods to the detection of immobilized PMVs in the context of a novel imaging flow cytometry (ISX) technique and atomic force microscopy (AFM). This novel approach allowed us to confirm the presence of the abundant microvesicle phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) on a surface with immobilized PMVs. Phosphatidylcholine groups (C5H12N+; C5H15PNO4+) were also detected. Moreover, we were able to show that ellipsometry permitted the immobilization of PMVs on a functionalized surface to be evaluated. The sensitivity of the ISX technique depends on the size and refractive index of the analyzed microvesicles. Graphical abstract Human platelets activated with thrombin (in concentration 1IU/mL) generate population of PMVs (platelet derived microvesicles), which can be detected and enumerated with fluorescent-label method (imaging cytometry). Alternatively, PMVs can be immobilized on the modified silicon substrate which is functionalized with a specific IgM murine monoclonal antibody against human glycoprotein IIb/IIIa complex (PAC-1). Immobilized PMVs can be subjected to label-free analyses by means ellipsometry, atomic force microscopy (AFM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS).
Department of Advanced Materials Engineering, M. Smoluchowski Institute of Physics, Jagiellonian University, 11 Łojasiewicza Street, 30-348, Krakow, Poland.
Katarzyna
K
Gajos
Department of Medical Physics, M. Smoluchowski Institute of Physics, Jagiellonian University, ul. S. Łojasiewicza 11, 30-348, Krakow, Poland.
Agnieszka
A
Kamińska
Department of Advanced Materials Engineering, M. Smoluchowski Institute of Physics, Jagiellonian University, 11 Łojasiewicza Street, 30-348, Krakow, Poland.
Kamil
K
Awsiuk
Department of Advanced Materials Engineering, M. Smoluchowski Institute of Physics, Jagiellonian University, 11 Łojasiewicza Street, 30-348, Krakow, Poland.
Adrianna
A
Bajor
Department of Molecular Diagnostics, Holycross Cancer Center, 3 Stefana Artwińskiego Street, 25-734, Kielce, Poland.
Krzysztof
K
Gruszczyński
Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 7 Gronostajowa Street, 30-387, Krakow, Poland.
Anna
A
Pawlak
Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 7 Gronostajowa Street, 30-387, Krakow, Poland.
Andrzej
A
Żądło
Department of Molecular Diagnostics, Holycross Cancer Center, 3 Stefana Artwińskiego Street, 25-734, Kielce, Poland.
Artur
A
Kowalik
Department of Advanced Materials Engineering, M. Smoluchowski Institute of Physics, Jagiellonian University, 11 Łojasiewicza Street, 30-348, Krakow, Poland.
Andrzej
A
Budkowski
Department of Medical Physics, M. Smoluchowski Institute of Physics, Jagiellonian University, ul. S. Łojasiewicza 11, 30-348, Krakow, Poland. e.stepien@uj.edu.pl.
Ewa
E
Stępień
D001792:Blood Platelets; D000067128:Extracellular Vesicles / Q000737:chemistry*; D005434:Flow Cytometry; D006801:Humans; D008081:Liposomes; D018625:Microscopy, Atomic Force; D012825:Silicon / Q000737:chemistry*; D013057:Spectrum Analysis / Q000379:methods*; D013499:Surface Properties
D008081:Liposomes; D012825:Silicon
Biomarkers; Extracellular vesicles; Flow cytometry; Nanoparticles/nanotechnology; Time-of-flight secondary ion mass spectrometry