Fabio Fachin

Microfluidic, marker-free isolation of circulating tumor cells from blood samples

The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen–independent microfluidic CTC-iChip technology. The CTC-iChip uses deterministic lateral displacement, inertial focusing and magnetophoresis to sort up to 107 cells/s. By using two-stage magnetophoresis and depletion antibodies against leukocytes, we achieve 3.8-log depletion of white blood cells and a 97% yield of rare cells with a sample processing rate of 8 ml of whole blood/h. The CTC-iChip is compatible with standard cytopathological and RNA-based characterization methods. This protocol describes device production, assembly, blood sample preparation, system setup and the CTC isolation process. Sorting 8 ml of blood sample requires 2 h including setup time, and chip production requires 2–5 d.

Nanoporous Elements in Microfluidics for Multiscale Manipulation of Bioparticles

Solid materials, such as silicon, glass, and polymers, dominate as structural elements in microsystems including microfluidics. Porous elements have been limited to membranes sandwiched between microchannel layers or polymer monoliths. This paper reports the use of micropatterned carbon-nanotube forests confined inside microfluidic channels for mechanically and/or chemically capturing particles ranging over three orders of magnitude in size. Nanoparticles below the internanotube spacing (80 nm) of the forest can penetrate inside the forest and interact with the large surface area created by individual nanotubes. For larger particles (>80 nm), the ultrahigh porosity of the nanotube elements reduces the fluid boundary layer and enhances particle-structure interactions on the outer surface of the patterned nanoporous elements. Specific biomolecular recognition is demonstrated using cells (≈10 μm), bacteria (≈1 μm), and viral-sized particles (≈40 nm) using both effects. This technology can provide unprecedented control of bioseparation processes to access bioparticles of interest, opening new pathways for both research and point-of-care diagnostics.

Microfluidic Isolation of Circulating Tumor Cell Clusters by Size and Asymmetry

Circulating tumor cell clusters (CTC clusters) are potent initiators of metastasis and potentially useful clinical markers for patients with cancer. Although there are numerous devices developed to isolate individual circulating tumor cells from blood, these devices are ineffective at capturing CTC clusters, incapable of separating clusters from single cells and/or cause cluster damage or dissociation during processing. The only device currently able to specifically isolate CTC clusters from single CTCs and blood cells relies on the batch immobilization of clusters onto micropillars which necessitates long residence times and causes damage to clusters during release. Here, we present a two-stage continuous microfluidic chip that isolates and recovers viable CTC clusters from blood. This approach uses deterministic lateral displacement to sort clusters by capitalizing on two geometric properties: size and asymmetry. Cultured breast cancer CTC clusters containing between 2–100 + cells were recovered from whole blood using this integrated two-stage device with minimal cluster dissociation, 99% recovery of large clusters, cell viabilities over 87% and greater than five-log depletion of red blood cells. This continuous-flow cluster chip will enable further studies examining CTC clusters in research and clinical applications.

Analytical extraction of residual stresses and gradients in MEMS structures with application to CMOS-layered materials

Accurate thin-film characterization is a key requirement in the MEMS industry. Residual stresses determine both the final shape and the functionality of released micromachined structures, and should therefore be accurately assessed. To date, a number of techniques to characterize thin-film materials have been developed, from substrate curvature measurement to methods that exploit the post-release deformation of test structures. These techniques have some major drawbacks, from high implementation costs to accuracy limitations due to improper boundary condition modeling. Here, we present a new technique for the characterization of multilayered, composite MEMS structures that uses easily accessible experimental information on the post-release deformation of microbridges only, with no need for multiple beam lengths. The method is based on an analytical solution of the (post-)buckling problem of microbridges, including the effect of residual stresses (both mean and gradient) and non-ideal clamping (boundary flexibility). The method allows simultaneous characterization of both the mean and the gradient residual stress components, as well as the effective boundary condition associated with the fabrication process, yielding approximately one order of magnitude improvement in resolution compared to extant methods using the same type and number of test structures. The higher resolution is largely attributable to proper accounting for boundary flexibility by our method, with the boundary condition for the structures in this work being ~90% as stiff in bending relative to the commonly assumed perfectly clamped condition. Additional enhancement can be achieved with post-release deformation measurements of simple cantilevers in addition to the microbridges. The method is useful as it ensures very low stress extraction uncertainty using a limited number of microbridge test structures, and it is transferrable to package-stress characterization. The analytical approach can also be extended to device design, quantifying the effect of residual stresses and boundary flexibility on a structures post-release state.

Monolithic Chip for High-throughput Blood Cell Depletion to Sort Rare Circulating Tumor Cells

Circulating tumor cells (CTCs) are a treasure trove of information regarding the location, type and stage of cancer and are being pursued as both a diagnostic target and a means of guiding personalized treatment. Most isolation technologies utilize properties of the CTCs themselves such as surface antigens (e.g., epithelial cell adhesion molecule or EpCAM) or size to separate them from blood cell populations. We present an automated monolithic chip with 128 multiplexed deterministic lateral displacement devices containing ~1.5 million microfabricated features (12 µm–50 µm) used to first deplete red blood cells and platelets. The outputs from these devices are serially integrated with an inertial focusing system to line up all nucleated cells for multi-stage magnetophoresis to remove magnetically-labeled white blood cells. The monolithic CTC-iChip enables debulking of blood samples at 15–20 million cells per second while yielding an output of highly purified CTCs. We quantified the size and EpCAM expression of over 2,500 CTCs from 38 patient samples obtained from breast, prostate, lung cancers, and melanoma. The results show significant heterogeneity between and within single patients. Unbiased, rapid, and automated isolation of CTCs using monolithic CTC-iChip will enable the detailed measurement of their physicochemical and biological properties and their role in metastasis.

Flexible sensor for blood pressure measurement

A new approach for the design and fabrication of a highly flexible blood pressure sensor is introduced in this paper. The goal is to measure the pressure within an aneurysm sac for post-endovascular aneurysms repair (EVAR) surveillance. Biocompatible polydimethylsiloxane (PDMS) membranes with embedded aligned carbon nanotubes (CNTs) are used to build the conductive elements of the pressure sensitive capacitor and the inductor for telemetry. Inductive coupling will be used to measure the internal capacitive variations. Fabricated test sensors validate the approach and demonstrate that CNTs/PDMS technology can be used to build highly flexible pressure sensors.

Integration of vertically-aligned carbon nanotube forests in microfluidic devices for multiscale isolation of bioparticles

Presently, an estimated 35 million people are living with HIV, 300 million with Hepatitis C (HCV), with thousands of human fatalities registered ever day due to these and similar infectious diseases. Efficient, reliable, inexpensive medical solutions are therefore needed to tackle these issues. Identification of HIV and HCV is however not easy. Being significantly smaller than cells and bacteria, these viruses escape the isolation capabilities of both macroscopic and microscopic (MEMS) medical instrumentation. Allowing access to sub-micron species such as viruses and cancer cells, integration of nanotechnologies in medical devices has the potential to revolutionize the field of biomedicine. In this work, we explore the potential of nanoporous, patterned forests of vertically-aligned carbon nanotubes (VACNTs) for bioparticle isolation, demonstrating their ability to access particles over several orders of magnitude in size, from viruses (∼40nm) to cells (∼10µm). Modifying the flow field inside microfluidic channels, CNT-enhanced biodevices result in a seven-fold increase in capture efficiency compared to a nonporous design, as well as the ability to simultaneously isolate multiple distinct biospecies both inside and on the outer surface of the VACNT features. Our technology represents a versatile, highly efficient approach to biological isolation, with applications ranging from point-of-care diagnostics to subsequent therapeutic modalities in both infectious diseases as well as cancer applications.

Mechanics of Out-of-Plane MEMS via Postbuckling: Model-Experiment Demonstration Using CMOS

A novel approach to out-of-plane microelectromechanical systems (MEMS) is demonstrated where elements are designed in the postbuckling regime, exploiting buckling phenomena and residual-stress control to create functional elements that extend significantly out of the wafer plane. An analytical tool for out-of-plane MEMS design is presented, based on nonlinear postbuckling of layered structures, including boundary nonideality. The analytical design tool is applied to several MEMS designs where low-order elements (e.g., beams) are controllably formed into out-of-plane shapes. Various architectures are experimentally demonstrated using CMOS processes, including one that could find application in three-axis single-heater thermal accelerometers. The on chip approach is compatible with several MEMS fabrication techniques (e.g., CMOS and micromachining), thus providing a new extension of state-of-the-art microfabrication techniques to out-of-plane elements.