Lab on a Chip Blog

By developing an ion-sensitive field-effect transistor with small gate dimensions, scientists at the University of Applied Sciences Kaiserslautern in Germany were able to measure cell-substrate adhesion on the single cell scale.

To survive, most mammalian cells attach to other cells and the extracellular environment in order to regulate their growth, proliferation, and migration. Electrical impedance spectroscopy is one way to quantitatively monitor cell-substrate interactions. The strength of cellular adhesion to a substrate with integrated electrodes can be measured by comparing the ratio of the readout voltage to the applied alternating current. Yet this method is limited groups of many cells as the size of the microelectrode must be larger than 100 μm in diameter. Smaller features are subject to greater interface impedance between the electrode and liquid media and this background impedance overwhelms the desired cell-substrate measurements. Suslorapova and colleagues thus used an ion-sensitive field-effect transistor (ISFET) with small gate dimensions to overcome this limitation. The group was able measure the effects of enzymatic digestion with trypsin and an apoptosis-inducing drug on single cell detachment using the ISFET devices with a 16 by 2 square micron gate.

The authors create an equivalent circuit model to interpret recorded impedance spectra from their single cell and small cell groups grown in contact with the field-effect transistor devices. The seal resistance and membrane capacitance parameters which can be extracted from the measured transistor transfer function (TTF) provide measures of cell shape and adhesion to the substrate. Changes in TTF correspond to adhesion of individual cells on top of the ISFET gates. This platform and the model developed to interpret TTF signal opens exciting avenues to monitoring cell adhesion in high throughput yet still at single cell resolution.

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Electrical cell-substrate impedance sensing with field-effect transistors is able to unravel cellular adhesion and detachment processes on a single cell level
A. Susloparova , D. Koppenhöfer , J. K. Y. Law , X. T. Vu and S. Ingebrandt. Lab Chip, 2015, 15, 668-679. DOI: 10.1039/C4LC00593G

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Groups collaborating across Sweden, Denmark, and Korea develop a chip-integrated acoustic focusing technique to precisely arrange particles for fast sizing and counting using impedance analysis.

The size and number of particles in a mixture can be quickly determined using a Coulter counter. Changes in resistance across a Coulter counter orifice through which particles pass correspond to the volume particles occupy as they displace the ionic carrier fluid (impedance spectroscopy). As fabrication methods transition to planar electrode formats to facilitate device development, the precise position of particles in the orifice becomes crucial to obtaining accurate results. Using planar electrodes on the channel bottom, the electric field across the orifice varies and thus sizing information from amplitude changes in impedance depend on consistent particle positioning. Previous methods using fluid flow focusing require complex fabrication steps and suffer from ion diffusion between virtual channel boundaries (fluid-fluid interfaces). Thus, Carl Grenvall in the Biomedical Engineering department in Lund University and his colleagues developed an acoustic actuation method to focus particles into the middle of the channel before they pass into the sensing aperture containing planar electrodes.

The team used two different frequencies to form standing waves in horizontal and vertical directions of the ‘prefocusing channel’ to guide particles to the center of the aperture where impedance was analyzed. Concentration studies helped determine the optimal density of particles to enable rapid sample analysis yet prevent formation of doublets. Confocal imaging confirmed simulation results to show distribution of focused particles and narrow confinement – 2.04% coefficient of variation after removing doublets, which is on par with other experimental and commercial cytometry platforms. The group was able to discriminate particle sizes from 3, 5, and 7 μm as well as separate 7 μm beads in a diluted blood sample. This demonstration of efficient particle focusing in two dimensions is an exciting development to create integrated simple-to-manufacture microchip impedance microscopy platforms. Standing wave acoustophoresis is gentle on cells as several studies even reporting in-field cell culturing¹, thus suggesting further opportunities for integration of microscale cytometers into microscale experimental platforms.

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Two-dimensional acoustic particle focusing enables sheathless chip Coulter counter with planar electrode configuration
Carl Grenvall, Christian Antfolk, Christer Zoffmann Bisgaard, and Thomas Laurell. Lab Chip, 2014, 14, 4629 – 4637.
DOI: 10.1039/c4lc00982g

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[1] M. A. Burguillos, C. Magnusson, M. Nordin, A. Lenshof, P. Augustsson, M. J. Hansson, E. Elmer, H. Lilja, P. Brundin and T. Laurell, PloS one, 2013, 8, e64233.

Collaborators across the University of Augsburg, Harvard University, and the University of Glasgow create a fluorescence-activated cell sorter relying on acoustofluidics to guide particles to their final location.

Traditional fluorescence-activated cell and droplet sorting (FACS, FADS) machines are expensive and require considerable time for analysis as well as maintenance (i.e., rinsing and cleaning of tubing to prepare for RNase-free processing). Cheap and disposable microfluidic devices can alleviate the expense and maintenance required, but still lag in particle sorting speed because they depend on fluidic, dielectric, and magnetic actuation to direct particles after fluorescence interrogation.

Lothar Schmid, David Weitz, and Thomas Franke overcame these issues by using traveling surface acoustic waves (SAWs) to drive particles into select channels based on readout of a fluorescent signal. The group oscillated PDMS structures from below by embedded interdigitated transducers to achieve focused acoustic radiation forces which gently moved droplets and cells via acoustic streaming.

The group was able to achieve sorting independent of cell size and compressibility on the order of 3000 particles/second into multiple outlet channels. This fast separation of particles given fluorescence signal readout enables efficient sorting of populations which vary widely in shape and volume. Further, the particles did not have to be first encapsulated into drops. This simplification avoids biohazard aerosol formation, provides higher signal to noise on the fluorescent signal interrogation, and streamlines the separation process. The group demonstrated gentle sorting of melanoma cells in a single fluid based on metabolic activity and membrane integrity. It will be exciting to see how acoustic streaming can further be used to direct particles to aid rare cell separations and cell isolations from complex samples.

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Sorting drops and cells with acoustics: acoustic microfluidic fluorescence-activated cell sorter
Lothar Schmid, David A. Weitz, and Thomas Franke. Lab Chip, 2014, 14, 3710-3718.
DOI: 10.1039/C4LC00588K

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Researchers at Virginia Tech create an elegant device to perform DNA amplification starting from whole cells by taking advantage of diffusivity differences in PCR components.

Diffusion can be friend or foe in the microscale regime, depending on the application. For active mixing, relying on diffusion can lengthen reaction time and thereby decrease reaction efficiency. But for separating reaction products, low ratios of convection to diffusion (Péclet number) enable control over elements based on their diffusivity^[1]. Professors Luke Achenie and Chang Lu from the chemical engineering department at Virginia Tech took advantage of this diffusion-enabled control to combine cell lysis and PCR reactions in ‘one pot’ with temporal separation of how components add to the chamber due to diffusivity differences. Separation of cell lysis and DNA amplification steps in PCR is important as many traditional chemical reagents for cell lysis inhibit polymerases used in PCR and Phusion polymerases tolerant to surfactant lysis reagents are incompatible with downstream SYBR green dyes.

The device consists of a single reaction chamber connected on both sides to two separate loading chambers. A hydration line ensures minimal evaporation during the PCR cycle in the main chamber. The loading chambers are opened in sequence to let molecules into the reaction chamber via two-layer control valves. The substantial difference in reagent diffusivity in the lysis and amplification processes allow diffusion gradients to drive molecules from new solutions contacting the reaction chamber and replace reagents from previous steps without disturbing the DNA of interest. Taq polymerase and proteins are two orders of magnitude larger in diffusivity than typical (50 kb) DNA fragments, while primers, dNTPs, and lysis buffers are three orders smaller. Relying solely on diffusion to deliver reagents to the main chamber increases the time of the reaction, but this can be addressed by elevating the temperature or increasing concentration of starting reagents in the loading chambers.

The authors showed the functionality of their device with purified human genomic DNA as well as single cells. This work opens up new capabilities to perform multi-step preparation and amplification assays for DNA in a single chamber starting directly from few cells to a single cell.

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Diffusion-based microfluidic PCR for “one-pot” analysis of cells

Sai Ma, Despina Nelie Loufakis, Zhenning Cao, Yiwen Chang, Luke E Achenie and Chang Lu
DOI:10.1039/C4LC00498A

References: [1] T. M. Squires and S. R. Quake, Reviews of Modern Physics, 2005, 77, 977.

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About the Webwriter

Sasha is a PhD student in bioengineering working with Professor Beth Pruitt’s Microsystems lab at Stanford University. Her research focuses on evaluating relationships between cell geometry, intracellular structure, and force generation (contractility) in heart muscle cells. Outside the lab, Sasha enjoys hiking, kickboxing, and interactive science outreach.