Rosette-induced separation of T cells by acoustophoresis.

Breakthrough cell therapies for the treatment of cancers require the separation of specific cells, such as T cells, from the patient's blood. Current cell therapy processes rely on magnetic separation, which adds clinical risk and requires elevated manufacturing controls due to the added foreign material that constitutes the magnetic beads. Acoustophoresis, a method that uses ultrasound for cell separation, has demonstrated label-free enrichment of T cells from blood, but residual other lymphocytes limit the ultimate purity of the output T cell product. Here, to increase the specificity of acoustophoresis, we use affinity reagents to conjugate red blood cells with undesired white blood cells, resulting in a cell-cell complex (rosette) of increased acoustic mobility. We achieve up to 99% purity of T cells from blood products, comparable to current standards of magnetic separation, yet without the addition of separation particles.

Effect of elastic modulus on inertial displacement of cell-like particles in microchannels.

Label-free microfluidic-based cell sorters leverage innate differences among cells (e.g., size and stiffness), to separate one cell type from another. This sorting step is crucial for many cell-based applications. Polystyrene-based microparticles (MPs) are the current gold standard for calibrating flow-based cell sorters and analyzers; however, the deformation behavior of these rigid materials is drastically different from that of living cells. Given this discrepancy in stiffness, an alternative calibration particle that better reflects cell elasticity is needed for the optimization of new and existing microfluidic devices. Here, we describe the fabrication of cell-like, mechanically tunable MPs and demonstrate their utility in quantifying differences in inertial displacement within a microfluidic constriction device as a function of particle elastic modulus, for the first time. Monodisperse, fluorescent, cell-like microparticles that replicate the size and modulus of living cells were fabricated from polyacrylamide within a microfluidic droplet generator and characterized via optical and atomic force microscopy. Trajectories of our cell-like MPs were mapped within the constriction device to predict where living cells of similar size/modulus would move. Calibration of the device with our MPs showed that inertial displacement depends on both particle size and modulus, with large/soft MPs migrating further toward the channel centerline than small/stiff MPs. The mapped trajectories also indicated that MP modulus contributed proportionally more to particle displacement than size, for the physiologically relevant ranges tested. The large shift in focusing position quantified here emphasizes the need for physiologically relevant, deformable MPs for calibrating and optimizing microfluidic separation platforms.

Scalable high-throughput acoustophoresis in arrayed plastic microchannels.

Microfluidic acoustophoresis is a label-free technique that isolates a purified product from a complex mixture of cells. This technique is well-studied but thus far has lacked the throughput and device manufacturability needed for many medical and industrial uses. Scale-up of acoustofluidic devices can be more challenging than in other microfluidic systems because the channel walls are integral to the resonant behavior and coupling to neighboring channels can inhibit performance. Additionally, the increased device area needed for parallel channels becomes less practical in the silicon or glass materials usually used for acoustofluidic devices. Here, we report an acoustic separator with 12 parallel channels made entirely from polystyrene that achieves blood cell separation at a flow rate greater than 1 ml/min. We discuss the design and optimization of the device and the electrical drive parameters and compare the separation performance using channels of two different designs. To demonstrate the utility of the device, we test its ability to purify lymphocytes from apheresis product, a process that is critical to new immunotherapies used to treat blood cancers. We process a leukapheresis sample with a volume greater than 100 ml in less than 2 h in a single pass without interruption, achieving greater than 90% purity of lymphocytes, without any prepurification steps. These advances suggest that acoustophoresis could in the future aid in cell therapy bioprocessing and that further scale-up is possible.

Acoustic separation in plastic microfluidics for rapid detection of bacteria in blood using engineered bacteriophage.

A more effective treatment of bacteremia requires a diagnostic platform that is both sensitive, accurate and rapid. Currently, clinical laboratory techniques require growth of bacteria prior to diagnosis, take days to complete, and leave empiric therapy and broad spectrum antibiotics as the only option at the onset of treatment. In order to bypass this growth requirement, we engineered a system that purifies bacteria from blood to improve performance in a bacteriophage-based luminescence assay. To perform the purification, we used acoustophoresis in plastic microfluidic chips, enabling future development into a low cost point-of-care system. Acoustophoresis achieves differential separation on the basis of size differences between bacteria and blood cells. We show isolation of three known pathogen species, including members of both Gram-negative and positive-bacteria from blood, and show isolation at clinically relevant concentrations. Using the device as a preparation step prior to the bacteriophage-based luminescence assay, we demonstrate a 33-fold improvement in limit of detection, compared with the unpurified sample, achieving a limit of detection of 6 bacteria.

Rapid prototyping and parametric optimization of plastic acoustofluidic devices for blood-bacteria separation.

Acoustic manipulation has emerged as a versatile method for microfluidic separation and concentration of particles and cells. Most recent demonstrations of the technology use piezoelectric actuators to excite resonant modes in silicon or glass microchannels. Here, we focus on acoustic manipulation in disposable, plastic microchannels in order to enable a low-cost processing tool for point-of-care diagnostics. Unfortunately, the performance of resonant acoustofluidic devices in plastic is hampered by a lack of a predictive model. In this paper, we build and test a plastic blood-bacteria separation device informed by a design of experiments approach, parametric rapid prototyping, and screening by image-processing. We demonstrate that the new device geometry can separate bacteria from blood while operating at 275% greater flow rate as well as reduce the power requirement by 82%, while maintaining equivalent separation performance and resolution when compared to the previously published plastic acoustofluidic separation device.

Dispersion of a Nanoliter Bolus in Microfluidic Co-Flow.

Microfluidic systems enable reactions and assays on the scale of nanoliters. However, at this scale nonuniformities in sample delivery become significant. To determine the fundamental minimum sample volume required for a particular device, a detailed understanding of mass transport is required. Co-flowing laminar streams are widely used in many devices, but typically only in the steady-state. Because establishing the co-flow steady-state consumes excess sample volume and time, there is a benefit to operating devices in the transient state, which predominates as the volume of the co-flow reactor decreases. Analysis of the co-flow transient has been neglected thus far. In this work we describe the fabrication of a pneumatically controlled microfluidic injector constructed to inject a discrete 50nL bolus into one side of a two-stream co-flow reactor. Using dye for image analysis, injections were performed at a range of flow rates from 0.5-10µL/min, and for comparison we collected the co-flow steady-state data for this range. The results of the image analysis were also compared against theory and simulations for device validation. For evaluation, we established a metric that indicates how well the mass distribution in the bolus injection approximates steady-state co-flow. Using such analysis, transient-state injections can approximate steady-state conditions within predefined errors, allowing straight forward measurements to be performed with reduced reagent consumption.

Local drug delivery with a self-contained, programmable, microfluidic system.

The development and optimization of many new drug therapies requires long-term local delivery with controlled, but variable dosage. Current methods for chronic drug delivery have limited utility because they either cannot deliver drugs locally to a specific organ or tissue, do not permit changes in delivery rate in situ, or cannot be used in clinical trials in an untethered, wearable configuration. Here, we describe a small, self-contained system for liquid-phase drug delivery. This system enables studies lasting several months and infusion rates can be programmed and modified remotely. A commercial miniature pump is integrated with microfabricated components to generate ultralow flow rates and stroke volumes. Solutions are delivered in pulses as small as 370 nL, with pulses delivered at any interval of 1 min or longer. A unique feature of the system is the ability to infuse and immediately withdraw liquid, resulting in zero net volume transfer while compounds are exchanged by mixing and diffusion with endogenous fluid. We present in vitro results demonstrating repeatability of the delivered pulse volume for nearly 3 months. Furthermore, we present in vivo results in an otology application, infusing into the cochlea of a guinea pig a glutamate receptor antagonist, which causes localized and reversible changes in auditory sensitivity.

High-density flexible interconnect for two-dimensional ultrasound arrays.

We present a method for fabricating flexible multilayer circuits for interconnection to 2-D array ultrasound transducers. In addition, we describe four 2-D arrays in which such flexible interconnect is implemented, including transthoracic arrays with 438 channels operating at up to 7 MHz and intracardiac catheter arrays with 70 channels operating at up to 7 MHz. We employ thin and thick film microfabrication techniques to batch produce the interconnect circuits with minimum dimensions of 12-mum lines, 40-mum vias, and 150-mum array pitch. The arrays show 50-Omega insertion loss of -60 to -84 dB and a fractional bandwidth of 27 to 67%. The arrays are used to obtain real time, in vivo volumetric scans.

Progress in two-dimensional arrays for real-time volumetric imaging.

The design, fabrication, and evaluation of two dimensional array transducers for real-time volumetric imaging are described. The transducers we have previously described operated at frequencies below 3 MHz and were unwieldy to the operator because of the interconnect schemes used in connecting to the transducer handle. Several new transducers have been developed using new connection technology. A 40 x 40 = 1,600 element, 3.5 MHz array was fabricated with 256 transmit and 256 receive elements. A 60 x 60 = 3,600 element 5.0 MHz array was constructed with 248 transmit and 256 receive elements. An 80 x 80 = 6,400 element, 2.5 MHz array was fabricated with 256 transmit and 208receive elements. 2-D transducer arrays were also developed for volumetric scanning in an intra cardiac catheter, a 10 x 10 = 100 element 5.0 MHz forward-looking array and an 11 x 13 = 143 element 5.0 MHz side-scanning array. The-6dB fractional bandwidths for the different arrays varied from 50% to 63%, and the 50 omega insertion loss for all the transducers was about-64 dB. The transducers were used to generate real-time volumetric images in phantoms and in vivo using the Duke University real time volumetric imaging system, which is capable of generating multiple planes at any desired angle and depth within the pyramidal volume.