Flow Homogenization Enables a Massively Parallel Fluidic Design for High-throughput and Multiplexed Cell Isolation.

Microfluidic devices are widely used for applications such as cell isolation. Currently, the most common method to improve throughput for microfluidic devices involves fabrication of multiple, identical channels in parallel. However, this 'numbering up' only occurs in one dimension, thereby limiting gains in volumetric throughput. In contrast, macro-fluidic devices permit high volumetric flow-rates but lack the finer control of microfluidics. Here, we demonstrate how a micro-pore array design enables flow homogenization across a magnetic cell capture device, thus creating a massively parallel series of micro-scale flow channels with consistent fluidic and magnetic properties, regardless of spatial location. This design enables scaling in 2-dimensions, allowing flow-rates exceeding 100 mL/hr while maintaining >90% capture efficiencies of spiked lung cancer cells from blood in a simulated circulating tumor cell system. Additionally, this design facilitates modularity in operation, which we demonstrate by combining two different devices in tandem for multiplexed cell separation in a single pass with no additional cell losses from processing.

A nanotweezer system for evanescent wave excited surface enhanced Raman spectroscopy (SERS) of single nanoparticles.

We experimentally demonstrate the integration of near-field optical tweezers with surface enhanced Raman scattering (SERS) spectroscopy by using the optical evanescent wave from a silicon nitride waveguide to trap single shell-isolated metallic nanoparticles (NPs) and simultaneously excite SERS signals of Raman reporter molecules adsorbed on the surface of the trapped metallic NPs. Both evanescent wave excited Stokes and anti-Stokes SERS spectra of waveguide trapped single silver (Ag) NPs were acquired, which were compared to their far-field SERS spectra. We investigated the trapping of bare and shell-isolated metallic NPs and determined that the addition of a shell to the metallic NPs minimized particle-induced laser damage to the waveguide, which allowed for the stable acquisition of the SERS spectra. This work realizes a new nanophotonic approach, which we refer to as near-field light scattering Raman (NLS-Raman), for simultaneous near-field optical trapping and SERS characterization of single metallic NPs.

Isolation and mutational analysis of circulating tumor cells from lung cancer patients with magnetic sifters and biochips.

Detection and characterization of circulating tumor cells (CTCs) may reveal insights into the diagnosis and treatment of malignant disease. Technologies for isolating CTCs developed thus far suffer from one or more limitations, such as low throughput, inability to release captured cells, and reliance on expensive instrumentation for enrichment or subsequent characterization. We report a continuing development of a magnetic separation device, the magnetic sifter, which is a miniature microfluidic chip with a dense array of magnetic pores. It offers high efficiency capture of tumor cells, labeled with magnetic nanoparticles, from whole blood with high throughput and efficient release of captured cells. For subsequent characterization of CTCs, an assay, using a protein chip with giant magnetoresistive nanosensors, has been implemented for mutational analysis of CTCs enriched with the magnetic sifter. The use of these magnetic technologies, which are separate devices, may lead the way to routine preparation and characterization of "liquid biopsies" from cancer patients.

Effect of Magnetic Field Gradient on Effectiveness of the Magnetic Sifter for Cell Purification.

In our experiments with NCI-H1650 lung cancer cell lines labeled with magnetic nanoparticles via the Epithelial Cell Adhesion Molecule (EpCAM) antigen, we demonstrate capture efficiencies above 90% even at sample flow rates of 5 ml/h through our microfabricated magnetic sifter. We also improve the elution efficiencies from between 50% and 60% to close to 90% via optimization of the permanent magnet size and position used to magnetize the sifter. We then explain our observations via the use of finite element software for magnetic field and field gradient distributions, and a particle tracing algorithm, illustrating the impact of magnetic field gradients on the performance of the magnetic sifter. The high capture and elution efficiencies observed here is especially significant for magnetic separation of biologically interesting but rare moieties such as cancer stem cells for downstream analysis.

Rapid Characterization of Magnetic Moment of Cells for Magnetic Separation.

NCI-H1650 lung cancer cell lines labeled with magnetic nanoparticles via the Epithelial Cell Adhesion Molecule (EpCAM) antigen were previously shown to be captured at high efficiencies by a microfabricated magnetic sifter. If fine control and optimization of the magnetic separation process is to be achieved, it is vital to be able to characterize the labeled cells' magnetic moment rapidly. We have thus adapted a rapid prototyping method to obtain the saturation magnetic moment of these cells. This method utilizes a cross-correlation algorithm to analyze the cells' motion in a simple fluidic channel to obtain their magnetophoretic velocity, and is effective even when the magnetic moments of cells are small. This rapid characterization is proven useful in optimizing our microfabricated magnetic sifter procedures for magnetic cell capture.

Silane-based functionalization of synthetic antiferromagnetic nanoparticles for biomedical applications.

Synthetic antiferromagnetic nanoparticles (SAFNPs) have been successfully coated with two different kinds of silanes, 3-aminopropyltrimethoxysilane and 2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane. The morphology of SAF particles is characterized by scanning electron microscopy and magnetic properties by alternating gradient magnetometry. The attachment of silane molecules is verified by Fourier-transform infrared spectroscopy and colloidal stability is studied using dynamic light scattering. These two silanes change the surface chemical properties of SAFNPs dramatically in different ways, which in turn affects the stability of these particles.

Structural and magnetic characterizations of high moment synthetic antiferromagnetic nanoparticles fabricated using self-assembled stamps.

High-moment synthetic antiferromagnetic (SAF) nanoparticles were produced using 4 in. diameter stamps made by self-assembly and nanosphere lithography of latex nanospheres. This leads to a significant increase in particle yield over a pre-existing technique which utilizes a 1 cm(2) stamp patterned using e-beam lithography. Changes in nanopillar dimensions from the self-assembled stamps and variations in the associated processing conditions can lead to the fabrication of particles with different dimensions. We demonstrate that it is possible to produce reasonably uniformly sized SAFs with diameters from 70 nm upward using self-assembled stamps. The particles exhibit low remanence at low externally applied magnetic fields, and that the saturation magnetization more than double that for conventional iron oxide nanoparticles.

Synthetic antiferromagnetic nanoparticles with tunable susceptibilities.

High-moment monodisperse disk-shaped Co-Fe magnetic nanoparticles, stable in aqueous solution, were physically fabricated by using nanoimprinted templates and vacuum deposition techniques. These multilayer synthetic antiferromagnetic nanoparticles exhibit nearly zero magnetic remanence and coercivity, and susceptibilities which can be tuned by exploiting interlayer magnetic interactions. In addition, a low cost method of scaling up the production of sub-100 nm synthetic antiferromagnetic nanoparticles is demonstrated.

Formation and properties of magnetic chains for 100 nm nanoparticles used in separations of molecules and cells.

Optical observations of 100 nm metallic magnetic nanoparticles are used to study their magnetic field induced self assembly. Chains with lengths of tens of microns are observed to form within minutes at nanoparticle concentrations of 10(10) per mL. Chain rotation and magnetophoresis are readily observed, and SEM reveals that long chains are not simple single particle filaments. Similar chains are detected for several 100 nm commercial bio-separation nanoparticles. We demonstrate the staged magnetic condensation of different types of nanoparticles into composite structures and show that magnetic chains bind to immunomagnetically labeled cells, serving as temporary handles which allow novel magnetic cell manipulations.

Microfabricated magnetic sifter for high-throughput and high-gradient magnetic separation.

A microfabricated magnetic sifter has been designed and fabricated for applications in biological sample preparation. The device enables high-throughput, high-gradient magnetic separation of magnetic nanoparticles by utilizing columnar fluid flow through a dense array (~5000/mm(2)) of micropatterned slots in a magnetically soft membrane. The potential of the sifter for separation of magnetic nanoparticles conjugated with capture antibodies is demonstrated through quantitative separation experiments with CD138-labelled MACS nanoparticles. Capture efficiencies ranging from 28-37% and elution efficiencies greater than 73% were measured for a single pass through the sifter.