Exosomes, first isolated 30 years ago, are nanoscale vesicles shed by most types of cells. The nucleic acid rich content of these nanoparticles, floating in virtually all bodily fluids, has great potential for non-invasive molecular diagnostics and may represent a novel therapeutic delivery system. However, current isolation techniques such as ultracentrifugation are not convenient and do not result in high purity isolation. This represents an interesting challenge for microfluidic technologies, from a cost-effective perspective as well as for enhanced purity capabilities, and point-of-care acquisition and diagnosis. In this frontier review, we present the current challenges, comment the first microfluidic advances in this new field and propose a roadmap for future developments. This review enables biologists and clinicians familiar with exosome enrichment to assess the performance of novel microfluidic devices and, equally, enables microfluidic engineers to educate themselves about this new class of promising biomarker-rich particles and the challenges arising from their clinical use.

Plasma is a rich mine of various biomarkers including proteins, metabolites and circulating nucleic acids. The diagnostic and therapeutic potential of these analytes has been quite recently uncovered, and the number of plasma biomarkers will still be growing in the coming years. A significant part of the blood plasma preparation is still handled manually, off-chip, via centrifugation or filtration. These batch methods have variable waiting times, and are often performed under non-reproducible conditions that may impair the collection of analytes of interest, with variable degradation. The development of miniaturised modules capable of automated and reproducible blood plasma separation would aid in the translation of lab-on-a-chip devices to the clinical market. Here we propose a systematic review of major plasma analytes and target applications, alongside existing solutions for micro-scale blood plasma extraction, focusing on the approaches that have been biologically validated for specific applications.

Circulating biomarkers are on the verge of becoming powerful diagnostic tools for various human diseases. However, the complex sample composition makes it difficult to detect biomarkers directly from blood at the bench or at the point-of-care. Blood cells are often a source of variability of the biomarker signal. While the interference of hemoglobin is a long known source of variability, the release of nucleic acids and other cellular components from hemocytes is a new concern for measurement and detection of circulating extracellular markers. Research into miniaturised blood plasma separation has been thriving in the last 10 years (2006-2016). Most point-of-care systems need microscale blood plasma separation, but developed solutions differ in complexity and sample volume range. But could blood plasma separation be avoided completely? This focused review weights the advantages and limits of miniaturised blood plasma separation and highlights the most interesting advances in direct capture as well as smart blood plasma separation.

The separation of red blood cells from plasma flowing in microchannels is possible by biophysical effects such as the Zweifach-Fung bifurcation law. In the present study, daughter channels are placed alongside a main channel such that cells and plasma are collected separately. The device is aimed to be a versatile but yet very simple module producing high-speed and high-efficiency plasma separation. The resulting lab-on-a-chip is manufactured using biocompatible materials. Purity efficiency is measured for mussel and human blood suspensions as different parameters, such as flow rate and geometries of the parent and daughter channels are varied. The issues of blood plasma separation at the microscale are discussed in relation to the different regimes of flow. Results are compared with those obtained by other researchers in the field of micro-separation of blood.

A microfluidic system was developed for blood plasma separation at high flow rate. This system uses only hydrodynamic forces to separate plasma from whole blood. The microfluidic network features a series of constrictions and bifurcations to enhance the product yield and purity. A maximum purity efficiency of 100% is obtained on blood with entrance hematocrit level up to 30% with a flow rate of 2 mL h(-1). Flow cytometry was performed on the extracted plasma to evaluate the separation efficiency and to assess cell damage. A core target of this study was the detection of cell-free DNA from the on-chip extracted plasma. To this effect, PCR was successfully carried out off-chip on the cell-free DNA present in the plasma extracted on-chip. A house-keeping gene sequence (GAPDH) was amplified without the need for a purification after the separation, thereby showing the high quality of the plasma sample. The resulting data suggests that the system can be used as a preliminary module of a total analysis system for cell-free DNA detection in human plasma.

Recent advances in microparticle separation in continuous flow are presented. It is intended for scientists in the field of separation science in biology, chemistry and microsystems engineering. Recent techniques of micron-sized particle separation within microsystems are described with emphasis on five different categories: optical, magnetic, fluidic-only, electrical and minor separation methods. Examples from the growing literature are explained with insights on separation efficiency and microengineering challenges. Current applications of the techniques are discussed.

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