Determination of the size distribution of blood microparticles directly in plasma using atomic force microscopy and microfluidics

Abstract

Microparticles, also known as microvesicles, found in blood plasma, urine, and most other body fluids, may serve as valuable biomarkers of diseases such as cardiovascular diseases, systemic inflammatory disease, thrombosis, and cancer. Unfortunately, the detection and quantification of microparticles are hampered by the microscopic size of these particles and their relatively low abundance in blood plasma. The use of a combination of microfluidics and atomic force microscopy to detect microparticles in blood plasma circumvents both problems. In this study, capture of a specific subset of microparticles directly from blood plasma on antibody-coated mica surface is demonstrated. The described method excludes isolation and washing steps to prepare microparticles, improves the detection sensitivity, and yields the size distribution of the captured particles. The majority of the captured particles have a size ranging from 30 to 90 nm, which is in good agreement with prior results obtained with microparticles immediately isolated from fresh plasma. Furthermore, the qualitative shape of the size distribution of microparticles is shown not to be affected by high-speed centrifugation or the use of the microfluidic circuit, demonstrating the relative stable nature of microparticles ex vivo.

Fig. 1

Flow cell setup. (a) Open PDMS flow cell. (b) Microfluidic flow cell setup with schematic side view. The holder system (4,5,6) is used to press the PDMS chip (3) onto the mica surface (1). The glass capillary tubes (7) are guided through holes in the holder top plate (5) to reach the connection chambers in the chip (3). The metal disc (2) is used as a support for the mica surface. In the photo (c), the middle channel is connected and filled with a dark blue solution; the glass capillary tubes are bent towards the side using scotch tape. The blue solution and scotch tape are for illustration purposes and are not used in experiments

Fig. 2

Schematic overview of experiments. Collected blood plasma is centrifuged twice to acquire PPP. In some experiments blood plasma proteins are removed by use of high-speed centrifugation (a). Using an antibody coated mica surface, a fresh PDMS chip and a holder system a microfluidic setup is build, and the PPP is run over a small surface area (b). Finally, mica surface is removed and imaged using AFM, followed by automated image analysis (c)

Fig. 3

AFM image quantification. Original AFM image, intensity represent height, see scale bar on the right side (a). This image is flattened using the standard linear regression background subtraction (b). White squares show all the particles that are found on the image from image a (c). The background is subtracted, corrected for the shadowed regions, and the particles are correctly sized (d). The bottom row (e, f, g, h) shows an enlarged region of (a, b, c, d) respectively, scale is 500 nm. The measured particles are indicated with red ellipses (d, h). The size distribution graph of particles detected from this image (100 μm2) is depicted (i)

Fig. 4

Relationship between the MP concentration in the sample and the number of CD41-positive MPs detected by AFM. MPs isolated from frozen-thawed citrate PPP are diluted from 50% to 2.5% (100% is undiluted reconstituted-isolated MPs) in Hepes buffer and run through the microfluidics device (a). These experiments were done on two different days using the same plasma pool of one healthy volunteer. The size distribution from a single dilution (3.8%) is based on three images (b). A normalized size distribution of all dilutions averages is weighted equally (c). Scale bars represent the standard error of the mean