Shady Gawad

Impedance spectroscopy flow cytometry: On-chip label-free cell differentiation

The microfabricated impedance spectroscopy flow cytometer used in this study permits rapid dielectric characterization of a cell population with a simple microfluidic channel. Impedance measurements over a wide frequency range provide information on cell size, membrane capacitance, and cytoplasm conductivity as a function of frequency. The amplitude, opacity, and phase information can be used for discrimination between different cell populations without the use of cell markers.

Single cell dielectric spectroscopy

Over the last century a number of techniques have been developed which allow the measurement of the dielectric properties of biological particles in fluid suspension. The majority of these techniques are limited by the fact that they only provide an average value for the dielectric properties of a collection of particles. More recently, with the advent of microfabrication techniques and the Lab-on-a-chip, it has been possible to perform dielectric spectroscopic experiments on single biological particles suspended in physiological media. In this paper we review current methods for single cell dielectric spectroscopy. We also discuss alternative single cell dielectric measurement techniques, specifically the ac electrokinetic methods of dielectrophoresis and electrorotation. Single cell electrical impedance spectroscopy is also discussed with relevance to a microfabricated flow cytometer. We compare impedance spectroscopy data obtained from measurements made using a microfabricated flow cytometer with simulation data obtained using an equivalent circuit model for the device.

Substrate arrays of iridium oxide microelectrodes for in vitro neuronal interfacing.

The design of novel bidirectional interfaces for in vivo and in vitro nervous systems is an important step towards future functional neuroprosthetics. Small electrodes, structures and devices are necessary to achieve high-resolution and target-selectivity during stimulation and recording of neuronal networks, while significant charge transfer and large signal-to-noise ratio are required for accurate time resolution. In addition, the physical properties of the interface should remain stable across time, especially when chronic in vivo applications or in vitro long-term studies are considered, unless a procedure to actively compensate for degradation is provided. In this short report, we describe the use and fabrication of arrays of 120 planar microelectrodes (MEAs) of sputtered Iridium Oxide (IrOx). The effective surface area of individual microelectrodes is significantly increased using electrochemical activation, a procedure that may also be employed to restore the properties of the electrodes as required. The electrode activation results in a very low interface impedance, especially in the lower frequency domain, which was characterized by impedance spectroscopy. The increase in the roughness of the microelectrodes surface was imaged using digital holographic microscopy and electron microscopy. Aging of the activated electrodes was also investigated, comparing storage in saline with storage in air. Demonstration of concept was achieved by recording multiple single-unit spike activity in acute brain slice preparations of rat neocortex. Data suggests that extracellular recording of action potentials can be achieved with planar IrOx MEAs with good signal-to-noise ratios.

Broadband single cell impedance spectroscopy using maximum length sequences: theoretical analysis and practical considerations

Measurements of the dielectric (or impedance) properties of cells can be used as a general characterization and diagnostic tool. In this paper, we describe a novel impedance spectroscopy technique for the analysis of single biological cells in suspension. The technique uses maximum length sequences (MLS) for periodic excitation signal in a microfluidic impedance cytometer. The method allows multi-frequency single cell impedance measurements to be made in a short time period (ms). Spectral information is obtained in the frequency domain by applying a fast M-sequence transform (FMT) and fast Fourier transform (FFT) to the time domain response. Theoretically, the impedance is determined from the transfer function of the system when the MLS is a current excitation. The order of the MLS and sampling rate of A/D conversion are two factors that determine the bandwidth and spectral accuracy of the technique. Experimentally, the applicability of the technique is demonstrated by characterizing the impedance spectrum of red blood cells (RBCs) in a microfluidic cytometer. The impedance is measured within 1 ms at 512 discrete frequencies, evenly distributed in the range from 976.56 Hz to 500 kHz. The measured spectrum shows good agreement with simulations.

Dielectric spectroscopy of single cells: time domain analysis using Maxwell’s mixture equation

Dielectric spectroscopy is a powerful tool for investigating the dielectric properties of biological particles in suspension. For low volume fractions, the dielectric properties of the particles are related to the measured properties of the suspension by Maxwell’s mixture equation. A number of different techniques can be used to measure the dielectric spectrum in the frequency domain or the time domain. In time domain dielectric spectroscopy, data can be converted into the frequency domain using convolution or Fourier transform, prior to data analysis. In this paper, we present a general method for transforming Maxwell’s mixture equation from the frequency domain to the time domain allowing analysis of cell dielectric properties directly in the time domain. The derivation is based on the Laplace transform of the single shell model for a spherical particle, and can be extended to the multi-shell model. For a single shelled cell two characteristic relaxation time constants are derived. The results are compared with published analytical models. We show that the original frequency dependent mixture equation can be recovered by Fourier transform back to the frequency domain. As a result, a general relationship for the dielectric response of a mixture of particles is presented which links the frequency and time domains.

Impedance spectroscopy using maximum length sequences: application to single cell analysis.

A maximum length sequence (MLS) is used to perform broadband impedance spectroscopy on a dielectric sample. The method has a number of advantages over other pulse-based or frequency sweep techniques. It requires the application of a very short sequence of voltage steps in the microsecond range and therefore allows the measurement of time-dependent impedance of a sample with high temporal resolution over a large bandwidth. The technique is demonstrated using a time-invariant passive RC network. The impedance of single biological cell flowing in a microfluidic channel is also measured, showing that MLS is an ideal method for high speed impedance analysis.

The Application of Microfluidics in Biology

Recent advances in the bio- and nanotechnologies have led to the development of novel microsystems for bio-particle separation and analysis. Microsystems are already revolutionising the way we do science and have led to the development of a number of ultrasensitive bioanalytical devices capable of analysing complex biological samples. These devices have application in a number of diverse areas such as pollution monitoring, clinical diagnostics, drug discovery and biohazard detection. In this chapter we give an overview of the physical principles governing the behaviour of fluids and particles at the micron scale, which are relevant to the operation of microfluidic devices. We briefly discuss some of the fabrication technologies used in the production of microfluidic systems and then present a number of examples of devices and applications relevant to the biological and life sciences.

Microfluidic impedance spectroscopy flow cytometer: particle size calibration

This paper presents a microfabricated impedance spectroscopy flow cytometer which permits rapid and easy dielectric characterization of cells as they flow in a microchannel. Both a simple theoretical model and a finite element simulation show that the effects of particle size dominate the entire spectrum. Dielectrophoretic forces are used to focus polystyrene beads along the centerline of the channel and decrease the spread in signal amplitude. Measurements of the beads show that size differences of less than 1 /spl mu/m diameter can be discriminated.

Impedance Spectroscopy Cell Analysis in Microchannels

This paper presents a micromachined high throughput flow-cytometer capable of discriminating cells using their electrical parameters as measured by impedance spectroscopy. The system is based on a set of electrodes integrated in a microchannel to measure the differential impedance in two successive segments as the cell passes consecutively through each. This analyzer is intended to drive a sorting actuator realising a subsequent cell separation. Size reduction and integration of functions are essential in achieving precise measurements and high throughput.

Leukocytes Discrimination by Impedance Spectroscopy Flow Cytometry

A device is presented which allows precise measurement and control of single cells using electrical means in a flow-through microchannel. The manipulation is based on the negative dielectrophoresis principle and the measurement is done by impedance spectroscopy. New results show the improvements obtained from cell pre-focussing on the measurement reproducibility as well as a fast sorting geometry. An application of the device to the discrimination of leukocytes sub-populations has been demonstrated.