Immobilization and detection of platelet-derived extracellular vesicles on functionalized silicon substrate: cytometric and spectrometric approach

Abstract

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 abstractHuman 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). Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-016-0036-5) contains supplementary material, which is available to authorized users.

Using a lactadherin-immobilized silicon surface for capturing and monitoring plasma microvesicles as a foundation for diagnostic device development

“…The ability of ToF-SIMS to differentiate membrane lipids combined with the novel method of surface immobilization of PMVs and their components provides a unique opportunity to examine the phospholipids characteristic to PMVs [ 14 ]. Enrichment in PS, which is commonly observed in PMVs, was confirmed for both types of LACT-functionalized surfaces (Fig.…”

“…Given that the binding of MVs to the LACT-immobilized surface occurs through PS, our platform is universal compared with the surface described in our previous study [ 14 ]. Moreover, our platform is capable of capturing MVs originating from various body fluids or cell culture media.…”

“…In the last years, lab-on-a-chip (LOC) technologies have attracted much attention, as they have the potential to provide devices based on different approaches (immunoaffinity, size exclusion, or flow-induced) for automated separation and analysis of different populations of extracellular vesicles (EVs) including MVs [ 13 ]. In a previous study, we showed that PMVs can be immobilized on a modified silicon substrate functionalized with a specific IgM murine monoclonal antibody against human glycoprotein IIb/IIIa complex (PAC-1) [ 14 ]. This approach, followed by different analytical techniques, can be developed to replace antibodies with a glycoprotein lactadherin (LACT), also known as Milk Fat Globule-EGF Factor 8 (MFG-E8), that has an affinity to the components present on the MV surface, e.g.…”

Platelet-Derived Extracellular Vesicles as Target of Antiplatelet Agents. What Is the Evidence?

“…Atomic force microscopy can be used to study EVs immobilized on a functionalized surface avoiding interference from abundant proteins (fibrinogen, albumin, immunoglobulins, etc.) and can be coupled to other techniques such as surface plasmon resonance to detect different subpopulations of EVs over a wide concentration range (Gajos et al, 2017; Obeid et al, 2017).…”

Dendron-Functionalized Surface: Efficient Strategy for Enhancing the Capture of Microvesicles

“…Its exposure to the extracellular leaflet is often considered as an early sign of programmed cell death, and its presence in MV outer membranes is also well documented (Owens and Mackman, 2011, Segawa and Nagata, 2015, Nagata et al., 2016, Iba and Ogura, 2018, van Engeland et al., 1998). MV capture devices using annexin V have been published since 1997 and have been improved over the years (Obeid et al., 2017, Gajos et al., 2017). However, this MV capture process depends on Ca 2+ in the sample milieu by the concentration of millimoles (van Engeland et al., 1998).…”