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A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Lawrence Livermore National Laboratory

We describe a family of calcium indicators for magnetic resonance imaging (MRI), formed by combining a powerful iron oxide nanoparticle-based contrast mechanism with the versatile calcium-sensing protein calmodulin and its targets. Calcium-dependent protein-protein interactions drive particle clustering and produce up to 5-fold changes in T2 relaxivity, an indication of the sensors' potency. A variant based on conjugates of wild-type calmodulin and the peptide M13 reports concentration changes near 1 microM Ca(2+), suitable for detection of elevated intracellular calcium levels. The midpoint and cooperativity of the response can be tuned by mutating the protein domains that actuate the sensor. Robust MRI signal changes are achieved even at nanomolar particle concentrations (<1 microM in calmodulin) that are unlikely to buffer calcium levels. When combined with technologies for cellular delivery of nanoparticulate agents, these sensors and their derivatives may be useful for functional molecular imaging of biological signaling networks in live, opaque specimens.

Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin.

Acoustofluidic devices for manipulating microparticles in fluids are appealing for biological sample processing due to their gentle and high-speed capability of sorting cell-scale objects. Such devices are generally limited to moving particles toward locations at integer fractions of the fluid channel width (1/2, 1/4, 1/6, etc.). In this work, we introduce a unique approach to acoustophoretic device design that overcomes this constraint, allowing us to design the particle focusing location anywhere within the microchannel. This is achieved by fabricating a second fluid channel in parallel with the sample channel, separated from it by a thin silicon wall. The fluids in both channels participate to create the ultrasound resonance, while only one channel processes the sample, thus de-coupling the fluidic and acoustic boundaries. The wall placement and the relative widths of the adjacent channels define the particle focusing location. We investigate the operating characteristics of a range of these devices to determine the configurations that enable effective particle focusing and separation. The results show that a sufficiently thin wall negligibly affects focusing efficiency and location compared to a single channel without a wall, validating the success of this design approach without compromising separation performance. Using these principles to design and fabricate an optimized device configuration, we demonstrate high-efficiency focusing of microspheres, as well as separation of cell-free viruses from mammalian cells. These "transparent wall" acoustic devices are capable of over 90% extraction efficiency with 10 μm microspheres at 450 μL min(-1), and of separating cells (98% purity), from viral particles (70% purity) at 100 μL min(-1).

Acoustic focusing with engineered node locations for high-performance microfluidic particle separation.

The mechanical properties of ordinary materials degrade substantially with reduced density because their structural elements bend under applied load. We report a class of microarchitected materials that maintain a nearly constant stiffness per unit mass density, even at ultralow density. This performance derives from a network of nearly isotropic microscale unit cells with high structural connectivity and nanoscale features, whose structural members are designed to carry loads in tension or compression. Production of these microlattices, with polymers, metals, or ceramics as constituent materials, is made possible by projection microstereolithography (an additive micromanufacturing technique) combined with nanoscale coating and postprocessing. We found that these materials exhibit ultrastiff properties across more than three orders of magnitude in density, regardless of the constituent material.

Ultralight, ultrastiff mechanical metamaterials.

Particle sorting using acoustofluidics has enormous potential but widespread adoption has been limited by complex device designs and low throughput. Here, we report high-throughput separation of particles and T lymphocytes (600 μL min(-1)) by altering the net sonic velocity to reposition acoustic pressure nodes in a simple two-channel device. The approach is generalizable to other microfluidic platforms for rapid, high-throughput analysis.

Spatial tuning of acoustofluidic pressure nodes by altering net sonic velocity enables high-throughput, efficient cell sorting.

Although real-time PCR (RT-PCR) has become a diagnostic standard for rapid identification of bacterial species, typical methods remain time-intensive due to sample preparation and amplification cycle times. The assay described in this work incorporates on-chip dielectrophoretic capture and concentration of bacterial cells, thermal lysis, cell permeabilization, and nucleic acid denaturation and fluorescence resonance energy transfer assisted in situ hybridization (FRET-ISH) species identification. Combining these techniques leverages the benefits of all of them, allowing identification to be accomplished completely on chip less than thirty minutes after receipt of sample, compared to multiple hours required by traditional RT-PCR and its requisite sample preparation.

Microfluidic-Based Amplification-Free Bacterial DNA Detection by Dielectrophoretic Concentration and Fluorescent Resonance Energy Transfer Assisted in Situ Hybridization (FRET-ISH).

Maxim Shusteff

Materials Engineering Division

Lawrence Livermore National Laboratory

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice