A blood-based, low cost alternative to radiation intensive CT and PET imaging is critically needed for cancer prognosis and management of its treatment. "Liquid biopsies" of circulating tumor cells (CTCs) from a relatively non-invasive blood draw are particularly ideal, as they can be repeated regularly to provide up to date molecular information about the cancer, which would also open up key opportunities for personalized therapies. Beyond solely diagnostic applications, CTCs are also a subject of interest for drug development and cancer research. In this paper, we adapt a technology previously introduced, combining the use of micro-scale vortices and inertial focusing, specifically for the high-purity extraction of CTCs from blood samples. First, we systematically varied parameters including channel dimensions and flow rates to arrive at an optimal device for maximum trapping efficiency and purity. Second, we validated the final device for capture of cancer cell lines in blood, considering several factors, including the effect of blood dilution, red blood cell lysis and cell deformability, while demonstrating cell viability and independence on EpCAM expression. Finally, as a proof-of-concept, CTCs were successfully extracted and enumerated from the blood of patients with breast (N = 4, 25-51 CTCs per 7.5 mL) and lung cancer (N = 8, 23-317 CTCs per 7.5 mL). Importantly, samples were highly pure with limited leukocyte contamination (purity 57-94%). This Vortex approach offers significant advantages over existing technologies, especially in terms of processing time (20 min for 7.5 mL of whole blood), sample concentration (collecting cells in a small volume down to 300 μL), applicability to various cancer types, cell integrity and purity. We anticipate that its simplicity will aid widespread adoption by clinicians and biologists who desire to not only enumerate CTCs, but also uncover new CTC biology, such as unique gene mutations, vesicle secretion and roles in metastatic processes.

Multiple methods of fabrication exist for microfluidic devices, with different advantages depending on the end goal of industrial mass production or rapid prototyping for the research laboratory. Polydimethylsiloxane (PDMS) has been the mainstay for rapid prototyping in the academic microfluidics community, because of its low cost, robustness and straightforward fabrication, which are particularly advantageous in the exploratory stages of research. However, despite its many advantages and its broad use in academic laboratories, its low elastic modulus becomes a significant issue for high pressure operation as it leads to a large alteration of channel geometry. Among other consequences, such deformation makes it difficult to accurately predict the flow rates in complex microfluidic networks, change flow speed quickly for applications in stop-flow lithography, or to have predictable inertial focusing positions for cytometry applications where an accurate alignment of the optical system is critical. Recently, other polymers have been identified as complementary to PDMS, with similar fabrication procedures being characteristic of rapid prototyping but with higher rigidity and better resistance to solvents; Thermoset Polyester (TPE), Polyurethane Methacrylate (PUMA) and Norland Adhesive 81 (NOA81). In this review, we assess these different polymer alternatives to PDMS for rapid prototyping, especially in view of high pressure injections with the specific example of inertial flow conditions. These materials are compared to PDMS, for which magnitudes of deformation and dynamic characteristics are also characterized. We provide a complete and systematic analysis of these materials with side-by-side experiments conducted in our lab that also evaluate other properties, such as biocompatibility, solvent compatibility, and ease of fabrication. We emphasize that these polymer alternatives, TPE, PUMA and NOA, have some considerable strengths for rapid prototyping when bond strength, predictable operation at high pressure, or transitioning to commercialization are considered important for the application.

Optical microscopy is one of the most widely used diagnostic methods in scientific, industrial, and biomedical applications. However, while useful for detailed examination of a small number (< 10,000) of microscopic entities, conventional optical microscopy is incapable of statistically relevant screening of large populations (> 100,000,000) with high precision due to its low throughput and limited digital memory size. We present an automated flow-through single-particle optical microscope that overcomes this limitation by performing sensitive blur-free image acquisition and nonstop real-time image-recording and classification of microparticles during high-speed flow. This is made possible by integrating ultrafast optical imaging technology, self-focusing microfluidic technology, optoelectronic communication technology, and information technology. To show the system’s utility, we demonstrate high-throughput image-based screening of budding yeast and rare breast cancer cells in blood with an unprecedented throughput of 100,000 particles/s and a record false positive rate of one in a million.

Controlling the shape of fluid streams is important across scales: from industrial processing to control of biomolecular interactions. Previous approaches to control fluid streams have focused mainly on creating chaotic flows to enhance mixing. Here we develop an approach to apply order using sequences of fluid transformations rather than enhancing chaos. We investigate the inertial flow deformations around a library of single cylindrical pillars within a microfluidic channel and assemble these net fluid transformations to engineer fluid streams. As these transformations provide a deterministic mapping of fluid elements from upstream to downstream of a pillar, we can sequentially arrange pillars to apply the associated nested maps and, therefore, create complex fluid structures without additional numerical simulation. To show the range of capabilities, we present sequences that sculpt the cross-sectional shape of a stream into complex geometries, move and split a fluid stream, perform solution exchange and achieve particle separation. A general strategy to engineer fluid streams into a broad class of defined configurations in which the complexity of the nonlinear equations of fluid motion are abstracted from the user is a first step to programming streams of any desired shape, which would be useful for biological, chemical and materials automation.

An effective approach to separating shaped particles is needed to isolate disease-causing cells for diagnostics or to aid in purifying nonspherical particles in applications ranging from food science to drug delivery. However, the separation of shaped particles is generally challenging, since nonspherical particles can freely rotate and present different faces while being sorted. We experimentally and numerically show that inertial fluid-dynamic effects allow for shape-dependent separation of flowing particles. (Spheres and rods with aspect ratios of 3:1 and 5:1 have all been separable.) Particle rotation around a conserved axis following Jeffery orbits is found to be a necessary component in producing different equilibrium positions across the channel that depend on particle rotational diameter. These differences are large enough to enable passive, continuous, high-purity, high-throughput, and shape-based separation downstream. Furthermore, we show that this shape-based separation can be applied to a large range of particle sizes and types, including small, artificially made 3-m particles as well as bioparticles such as yeast. This practical approach for sorting particles by a previously inaccessible geometric parameter opens up a new capability that should find use in a range of fields.

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 tumor cells (CTCs) are emerging as rare but clinically significant non-invasive cellular biomarkers for cancer patient prognosis, treatment selection, and treatment monitoring. Current CTC isolation approaches, such as immunoaffinity, filtration, or size-based techniques, are often limited by throughput, purity, large output volumes, or inability to obtain viable cells for downstream analysis. For all technologies, traditional immunofluorescent staining alone has been employed to distinguish and confirm the presence of isolated CTCs among contaminating blood cells, although cells isolated by size may express vastly different phenotypes. Consequently, CTC definitions have been non-trivial, researcher-dependent, and evolving. Here we describe a complete set of objective criteria, leveraging well-established cytomorphological features of malignancy, by which we identify large CTCs. We apply the criteria to CTCs enriched from stage IV lung and breast cancer patient blood samples using the High Throughput Vortex Chip (Vortex HT), an improved microfluidic technology for the label-free, size-based enrichment and concentration of rare cells. We achieve improved capture efficiency (up to 83%), high speed of processing (8 mL/min of 10x diluted blood, or 800 μL/min of whole blood), and high purity (avg. background of 28.8±23.6 white blood cells per mL of whole blood). We show markedly improved performance of CTC capture (84% positive test rate) in comparison to previous Vortex designs and the current FDA-approved gold standard CellSearch assay. The results demonstrate the ability to quickly collect viable and pure populations of abnormal large circulating cells unbiased by molecular characteristics, which helps uncover further heterogeneity in these cells.

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