Real-Time Detection and Control of Microchannel Resonance Frequency in Acoustic Trapping Systems by Monitoring Amplifier Supply Currents.

The utilization of bulk acoustic waves from a piezoelectric transducer for selective particle trapping under flow in a microchannel is limited by the high sensitivity of the resonance frequency to tolerances in device geometry, drift during trapping, and variations in the local flow or sample conditions in each channel. This is addressed by detecting the resonance condition based on the impedance minimum obtained by monitoring the amplitude of the stimulation voltage across the piezo transducer and utilizing real-time feedback to control the stimulation frequency. However, this requires an overlap in the frequency bandwidth of the detection and the stimulation system and is also limited by the decline in the acoustic trapping power when the stimulation and resonance frequency measurement are conducted simultaneously. Instead, we present a novel circuit implementation for on-chip real-time resonance frequency measurement and feedback control based on monitoring the current drawn from the amplifier used to stimulate the piezo transducer, since the need for high measurement sensitivity in this mode does not lower the power available for stimulation of the transducer. The enhanced level of control of acoustic trapping utilizing this current mode is validated for various localized channel perturbations, including drift, wash steps, and buffer swaps, as well as for selective sperm cell trapping from a heterogeneous sample that includes lysed epithelial cells.

Self-aligned microfluidic contactless dielectrophoresis device fabricated by single-layer imprinting on cyclic olefin copolymer.

The trapping and deflection of biological cells by dielectrophoresis (DEP) at field non-uniformities in a microfluidic device is often conducted in a contactless dielectrophoresis (cDEP) mode, wherein the electrode channel is in a different layer than the sample channel, so that field penetration through the interceding barrier causes DEP above critical cut-off frequencies. In this manner, through physical separation of the electrode and sample channels, it is possible to spatially modulate electric fields with no electrode-induced damage to biological cells in the sample channel. However, since this device requires interlayer alignment of the electrode to sample channel and needs to maintain a thin interceding barrier (~ 15 µm) over the entire length over which DEP is needed (~ 1 cm), variations in alignment and microstructure fidelity cause wide variations in cDEP trapping level and frequency response across devices. We present a strategy to eliminate interlayer alignment by fabricating self-aligned electrode and sample channels, simultaneously with the interceding barrier layer (14-µm width and 50-µm depth), using a single-layer imprint and bond process on cyclic olefin copolymer. Specifically, by designing support structures, we preserve fidelity of the high aspect ratio insulating posts in the sample channel and the interceding barrier between the sample and electrode channels over the entire device footprint (~ 1 cm). The device operation is validated based on impedance measurements to quantify field penetration through the interceding barrier and by DEP trapping measurements. The presented fabrication strategy can eventually improve cDEP device manufacturing protocols to enable more reproducible DEP performance. Graphical abstract.

High-throughput dynamical analysis of dielectrophoretic frequency dispersion of single cells based on deflected flow streamlines.

Phenotypic quantification of cells based on their plasma membrane capacitance and cytoplasmic conductivity, as determined by their dielectrophoretic frequency dispersion, is often used as a marker for their biological function. However, due to the prevalence of phenotypic heterogeneity in many biological systems of interest, there is a need for methods capable of determining the dielectrophoretic dispersion of single cells at high throughput and without the need for sample dilution. We present a microfluidic device methodology wherein localized constrictions in the microchannel are used to enhance the field delivered by adjoining planar electrodes, so that the dielectrophoresis level and direction on flow-focused cells can be determined on each traversing cell in a high-throughput manner based on their deflected flow streamlines. Using a sample of human red blood cells diluted to 2.25 × 108 cells/mL, the dielectrophoretic translation of single cells traversing at a flow rate of 1.68 µL/min is measured at a throughput of 1.1 × 105 cells/min, to distinguish positive versus negative dielectrophoresis and determine their crossover frequency in media of differing conductivity for validation of the computed membrane capacitance to that from prior methods. We envision application of this dynamic dielectrophoresis (Dy-DEP) method towards high-throughput measurement of the dielectric dispersion of single cells to stratify phenotypic heterogeneity of a particular sample based on their DEP crossover frequency, without the need for significant sample dilution. Grapical abstract.

On-Chip Impedance for Quantifying Parasitic Voltages During AC Electrokinetic Trapping.

OBJECTIVE: Assessing the effectiveness of microfluidic device structures for enabling electrokinetic or acoustic trapping requires imaging of model particles within each device under the requisite force fields. To avoid the need for extensive microscopy, the use of valuable biological samples, and reliance on a trained operator to assess efficacy of trapping, we explore electrical means to identify device geometry variations that are responsible for the poor trapping. RESULTS: Using the example of AC electrokinetic trapping over an insulated channel in a contact-less dielectrophoresis mode, we present an on-chip method to acquire impedance spectra on the microfluidic device for quantifying the parasitic voltage drops. CONCLUSION: Based on the parasitic voltage drops, device geometries can be designed to maximize fraction of the applied voltage that is available for dielectrophoretic manipulation and the measured on-chip impedance can rapidly inform downstream decisions on particle manipulation.

Conductance-Based Biophysical Distinction and Microfluidic Enrichment of Nanovesicles Derived from Pancreatic Tumor Cells of Varying Invasiveness.

Diagnostics based on exosomes and other extracellular vesicles (EVs) are emerging as strategies for informing cancer progression and therapies, since the lipid content and macromolecular cargo of EVs can provide key phenotypic and genotypic information on the parent tumor cell and its microenvironment. We show that EVs derived from more invasive pancreatic tumor cells that express high levels of tumor-specific surface proteins and are composed of highly unsaturated lipids that increase membrane fluidity, exhibit significantly higher conductance versus those derived from less invasive tumor cells, based on dielectrophoresis measurements. Furthermore, through specific binding of the EVs to gold nanoparticle-conjugated antibodies, we show that these conductance differences can be modulated in proportion to the type as well as level of expressed tumor-specific antigens, thereby presenting methods for selective microfluidic enrichment and cytometry-based quantification of EVs based on invasiveness of their parent cell.

Real Time Electronic Feedback for Improved Acoustic Trapping of Micron-Scale Particles.

Acoustic differential extraction has been previously reported as a viable alternative to the repetitive manual pipetting and centrifugation steps for isolating sperm cells from female epithelial cells in sexual assault sample evidence. However, the efficiency of sperm cell isolation can be compromised in samples containing an extremely large number of epithelial cells. When highly concentrated samples are lysed, changes to the physicochemical nature of the medium surrounding the cells impacts the acoustic frequency needed for optimal trapping. Previous work has demonstrated successful, automated adjustment of acoustic frequency to account for changes in temperature and buffer properties in various samples. Here we show that, during acoustic trapping, real-time monitoring of voltage measurements across the piezoelectric transducer correlates with sample-dependent changes in the medium. This is achieved with a wideband peak detector circuit, which identifies the resonant frequency with minimal disruption to the applied voltage. We further demonstrate that immediate, corresponding adjustments to acoustic trapping frequency provides retention of sperm cells from high epithelial cell-containing mock sexual assault samples.

Review: Microbial analysis in dielectrophoretic microfluidic systems.

Infections caused by various known and emerging pathogenic microorganisms, including antibiotic-resistant strains, are a major threat to global health and well-being. This highlights the urgent need for detection systems for microbial identification, quantification and characterization towards assessing infections, prescribing therapies and understanding the dynamic cellular modifications. Current state-of-the-art microbial detection systems exhibit a trade-off between sensitivity and assay time, which could be alleviated by selective and label-free microbial capture onto the sensor surface from dilute samples. AC electrokinetic methods, such as dielectrophoresis, enable frequency-selective capture of viable microbial cells and spores due to polarization based on their distinguishing size, shape and sub-cellular compositional characteristics, for downstream coupling to various detection modalities. Following elucidation of the polarization mechanisms that distinguish bacterial cells from each other, as well as from mammalian cells, this review compares the microfluidic platforms for dielectrophoretic manipulation of microbials and their coupling to various detection modalities, including immuno-capture, impedance measurement, Raman spectroscopy and nucleic acid amplification methods, as well as for phenotypic assessment of microbial viability and antibiotic susceptibility. Based on the urgent need within point-of-care diagnostics towards reducing assay times and enhancing capture of the target organism, as well as the emerging interest in isolating intact microbials based on their phenotype and subcellular features, we envision widespread adoption of these label-free and selective electrokinetic techniques.

A wide-bandwidth power amplifier for frequency-selective insulator-based dielectrophoresis.

Insulator-based dielectrophoresis enables contact-less separation and analysis of biosystems, but it is unable to operate effectively in the MHz frequency range, which is necessary for the manipulation of biological cells based on the characteristic electrophysiology of their cytoplasm or biomolecular preconcentration based on their unique conformation. To address the steep drop in output power and the rise of signal distortions within conventional amplifiers at MHz frequencies due to slew rate limitations, we present the design principles for a wideband amplifier. This is validated by demonstrating the absence of harmonic distortions and parasitic DC within the amplifier output up to 15 MHz, thereby enabling analysis of cytoplasmic alterations on oocysts of Cryptosporidium parvum, due to constant force dispersion in the MHz range.