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A novel broadband impedance method for detection of cell-derived microparticles.

ABSTRACT: A novel label-free method is presented to detect and quantify cell-derived microparticles (MPs) by the electrochemical potential-modulated electrochemical impedance spectroscopy (EIS). MPs are present in elevated concentrations during pathological conditions and play a major role in the establishment and pathogenesis of many diseases. Considering this, accurate detection and quantification of MPs is very important in clinical diagnostics and therapeutics. A combination of bulk solution electrokinetic sorting and interfacial impedance responses allows achieving detection limits as low as several MPs per ?L. By fitting resulting EIS spectra with an equivalent electrical circuit, the bulk solution electrokinetic and interfacial impedance responses were characterized. In the bulk solution two major relaxations were prominent-?-relaxation in low MHz region due to the MP capacitive membrane bridging, and ?-relaxation at ?10 kHz due to counter ions diffusion. At low frequencies (10-0.1 Hz) at electrochemical potentials exceeding -100 mV, a facile interfacial Faradaic process of oxidation in MPs coupled with diffusion and non-Faradaic double layer charging dominate, probably due to oxidation of phospholipids and/or proteins on the MP surface and MP lysis. Buffer influence on the MP detection demonstrated that a relatively low conductivity Tyrode's buffer background solution is preferential for the MP electrokinetic separation and characterization. This study also demonstrated that standard laboratory methods such as flow cytometry underestimate MP concentrations, especially those with smaller average sizes, by as much as a factor of 2-40.

SUBMITTER: Lvovich V

PROVIDER: S-EPMC2946439 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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A novel broadband impedance method for detection of cell-derived microparticles.

Lvovich Vadim V Srikanthan Sowmya S Silverstein Roy L RL

Biosensors & bioelectronics 20100803 2

A novel label-free method is presented to detect and quantify cell-derived microparticles (MPs) by the electrochemical potential-modulated electrochemical impedance spectroscopy (EIS). MPs are present in elevated concentrations during pathological conditions and play a major role in the establishment and pathogenesis of many diseases. Considering this, accurate detection and quantification of MPs is very important in clinical diagnostics and therapeutics. A combination of bulk solution electroki ...[more]

PMID: 20729061

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Project description:BackgroundMicroparticles (MPs), also called microvesicles (MVs) are plasma membrane-derived fragments with sizes ranging from 0.1 to 1?m. Characterization of these MPs is often performed by flow cytometry but there is no consensus on the appropriate negative control to use that can lead to false positive results.Materials and methodsWe analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL) B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets). Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM) and Scanning Electron microscopy (SEM).ResultsAnnexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies) was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins).ConclusionWe observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls.

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A novel broadband impedance method for detection of cell-derived microparticles.

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A novel broadband impedance method for detection of cell-derived microparticles.

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