Marija Nikolic-Jaric

Microwave frequency sensor for detection of biological cells in microfluidic channels

We present details of an apparatus for capacitive detection of biomaterials in microfluidic channels operating at microwave frequencies where dielectric effects due to interfacial polarization are minimal. A circuit model is presented, which can be used to adapt this detection system for use in other microfluidic applications and to identify ones where it would not be suitable. The detection system is based on a microwave coupled transmission line resonator integrated into an interferometer. At 1.5 GHz the system is capable of detecting changes in capacitance of 650 zF with a 50 Hz bandwidth. This system is well suited to the detection of biomaterials in a variety of suspending fluids, including phosphate-buffered saline. Applications involving both model particles (polystyrene microspheres) and living cells-bakers yeast (Saccharomyces cerevisiae) and Chinese hamster ovary cells-are presented.

The changing dielectric properties of CHO cells can be used to determine early apoptotic events in a bioprocess

To ensure maximum productivity of recombinant proteins it is desirable to prolong cell viability during a mammalian cell bioprocess, and therefore important to carefully monitor cell density and viability. In this study, five different and independent methods of monitoring were applied to Chinese hamster ovary (CHO) cells grown in a batch culture in a controlled bioreactor to determine cell density and/or cell viability. They included: a particle counter, trypan blue exclusion (Cedex), an in situ bulk capacitance probe, an off‐line fluorescent flow cytometer, and a prototype dielectrophoretic (DEP) cytometer. These various techniques gave similar values during the exponential growth phase. However, beyond the exponential growth phase the viability measurements diverged. Fluorescent flow cytometry with a range of fluorescent markers was used to investigate this divergence and to establish the progress of cell apoptosis: the cell density estimates by the intermediate stage apoptosis assay agreed with those obtained by the bulk capacitance probe and the early stage apoptosis assay viability measurements correlated well with the DEP cytometer. The trypan blue assay showed higher estimates of viable cell density and viability compared to the capacitance probe or the DEP cytometer. The DEP cytometer measures the dielectric properties of individual cells and identified at least two populations of cells, each with a distinct polarizability. As verified by comparison with the Nexin assay, one population was associated with viable (non‐apoptotic) cells and the other with apoptotic cells. From the end of the exponential through the stationary and decline stages there was a gradual shift of cell count from the viable into the apoptotic population. However, the two populations maintained their individual dielectric properties throughout this shift. This leads to the conclusion that changes in bulk dielectric properties of cultures might be better modeled as shifts in cells between different dielectric sub‐populations, rather than assuming a homogeneous dielectric population. This shows that bulk dielectric probes are sensitive to the early apoptotic changes in cells. DEP cytometry offers a novel and unique technology for analyzing and characterizing mammalian cells based on their dielectric properties, and suggests a potential application of the device as a low‐cost, label‐free, electronic monitor of physiological changes in cells. Biotechnol. Bioeng. 2013;110: 2902–2914.

Differential electronic detector to monitor apoptosis using dielectrophoresis-induced translation of flowing cells (dielectrophoresis cytometry)

The instrument described here is an all-electronic dielectrophoresis (DEP) cytometer sensitive to changes in polarizability of single cells. The important novel feature of this work is the differential electrode array that allows independent detection and actuation of single cells within a short section ([Formula: see text]) of the microfluidic channel. DEP actuation modifies the altitude of the cells flowing between two altitude detection sites in proportion to cell polarizability; changes in altitude smaller than 0.25 μm can be detected electronically. Analysis of individual experimental signatures allows us to make a simple connection between the Clausius-Mossotti factor (CMF) and the amount of vertical cell deflection during actuation. This results in an all-electronic, label-free differential detector that monitors changes in physiological properties of the living cells and can be fully automated and miniaturized in order to be used in various online and offline probes and point-of-care medical applications. High sensitivity of the DEP cytometer facilitates observations of delicate changes in cell polarization that occur at the onset of apoptosis. We illustrate the application of this concept on a population of Chinese hamster ovary (CHO) cells that were followed in their rapid transition from a healthy viable to an early apoptotic state. DEP cytometer viability estimates closely match an Annexin V assay (an early apoptosis marker) on the same population of cells.

Electronic detection of dielectrophoretic forces exerted on particles flowing over interdigitated electrodes.

Dielectric particles flowing through a microfluidic channel over a set of coplanar electrodes can be simultaneously capacitively detected and dielectrophoretically (DEP) actuated when the high (1.45 GHz) and low (100 kHz-20 MHz) frequency electromagnetic fields are concurrently applied through the same set of electrodes. Assuming a simple model in which the only forces acting upon the particles are apparent gravity, hydrodynamic lift, DEP force, and fluid drag, actuated particle trajectories can be obtained as numerical solutions of the equations of motion. Numerically calculated changes of particle elevations resulting from the actuation simulated in this way agree with the corresponding elevation changes estimated from the electronic signatures generated by the experimentally actuated particles. This verifies the model and confirms the correlation between the DEP force and the electronic signature profile. It follows that the electronic signatures can be used to quantify the actuation that the dielectric particle experiences as it traverses the electrode region. Using this principle, particles with different dielectric properties can be effectively identified based exclusively on their signature profile. This approach was used to differentiate viable from non-viable yeast cells (Saccharomyces cerevisiae).

Electroporation and dielectrophoresis of single cells using a microfluidic system employing a microwave interferometric sensor

We describe a microfluidic device integrated with a microwave interferometric sensor that is able to simultaneously electroporate and measure the dielectrophoresis (DEP) response of single biological cells. The system can measure changes in dielectric properties of a cell permeabilized using a high-intensity pulsed electric field (PEF) from a few seconds after exposure to the pulses. It provides a mechanism for investigating time-dependent changes in the cell membrane and ion flux. Using this device, experiments performed on single Chinese hamster ovary (CHO) cells exposed to microsecond pulsed electric fields show significant changes in their DEP response immediately after exposure.

Microfluidic device for simultaneous pulsed electric field electroporation and dielectrophoresis studies of single biological cells

We present a microfluidic device able to electroporate and measure the resultant changes in dielectric properties of single biological cells. The device can measure the dielectrophoresis (DEP) response of the electroporated cell from a few seconds after exposure to electroporating pulses. Studying electroporation at the single cell level can provide insight on the electroporation process as well as a method of differentiating cellular state (e.g. normal and cancerous). Moreover, combining electroporation and dielectric characterization into a single device eliminates the need for post-sample analysis as required in techniques such as fluorescence cytometry. Experiments performed on Chinese hamster ovary (CHO) cells demonstrate the ability of the device to detect changes in the DEP response of individual cells immediately after being exposed to microsecond duration electroporating pulses.

Semi-automated detection of single cell signatures from a dielectrophoretic cytometer

We present a semi-automated event identification method for collecting the dielectrophoretic signatures of cells flowing through a dieletrophoretic cytometer. The marker free dielectrophoresis (DEP) cytometer presented in this study is capable of detecting electronic signatures of cells which identifies Claussius-Mossotti factor (CMF). The CMF can in turn be used to determine properties of the cell such as the viability. In past work the DEP cytometer signals were manually sorted by going through the entire recorded signals, which is very time-consuming. In the semi-automated method of collection, events are identified and displayed in the user interface to be accepted or rejected. We present results using semi-automated method on Hamster Chinese Ovary (CHO) cells in a batch culture and compared them with the manual analysis. The automated approach identified over 80% of the events identified manually and produced event histogram distributions nearly identical to the manual method.