A new cytological tool, based on the microCoulter particle counter (microCPC) principle, aimed at diagnostic applications for cell counting and separation in haematology, oncology or toxicology is described. The device measures the spectral impedance of individual cells or particles and allows screening rates over 100 samples s(-1) on a single-cell basis. This analyzer is intended to drive a sorting actuator producing a subsequent cell separation. Size reduction and integration of functions are essential in achieving precise measurements and high throughput. 3D finite element simulations are presented to compare various electrode geometries and their influence on cell parameters estimation. The device is based on a glass-polyimide microfluidic chip with integrated channels and electrodes microfabricated at the length scale of the particles to be investigated (1-20 microm). A laminar liquid flow carries the suspended particles through the measurement area. Each particle's impedance signal is recorded by a differential pair of microelectrodes using the cell surrounding media as a reference. The micromachined chip and processing electronic circuit allow simultaneous impedance measurements at multiple frequencies, ranging from 100 kHz to 15 MHz. In this paper, we describe the microfabrication and characterisation of an on-chip flow-cytometer as the first building block of a complete cell-sorting device. We then discuss the signal conditioning technique and finally impedance measurements of cells and particles of different sizes and types to demonstrate the differentiation of subpopulations in a mixed sample.

Background: 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. Methods: Polystyrene beads, red blood cells (RBCs), ghosts, and RBCs fixed in glutaraldehyde were passed through a microfabricated flow cytometer and measured individually by using two simultaneously applied discrete frequencies. The cells were characterized at 1,000 per minute in the frequency range of 350 kHz to 20 MHz.

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.

We propose a model to determine the influence of different cell properties, such as size, membrane capacitance and cytoplasm conductivity, on the impedance spectrum as measured in a microfabricated cytometer. A dielectric sphere of equivalent complex permittivity is used as a simplified model to describe a biological cell. The measurement takes place between a pair of facing microelectrodes in a microchannel filled with a saline solution. The model incorporates various cell parameters, such as dielectric properties, size and position in the channel. A 3D finite element model is used to evaluate the magnitude of the electric field in the channel and the resultant changes in charge densities at the measurement electrode boundaries as a cell flows past. The charge density is integrated on the electrode surface to determine the displacement current and the channel impedance for the computed frequency range. The complete impedance model combines the finite element model, the electrode-electrolyte interface impedance and stray impedance, which are measured from a real device. The modeled dielectric complex spectra for various cell parameters are discussed and a measurement strategy for cell discrimination with such a system is proposed. We finally discuss the amount of noise and measurement fluctuations of the sensor.

Microfabricated impedance cytometers have been developed to measure the electrical impedance of single biological particles at high speed. A general approach to analytically solve the electric field distributions for two different designs of cytometers: parallel facing electrodes and coplanar electrodes, using the Schwarz -Christoffel Mapping method is presented. Compared to previous analytical solutions, our derivations are more systematic and solutions are more straightforward. The solutions have been validated by comparison with numerical simulations performed using the finite element method. The influences on the electric field distribution due to the variations in the geometry of the devices have been discussed. A simple method is used to determine the impedance sensitivity of the system and to compare the two electrode designs. For identical geometrical parameters, we conclude that the parallel electrodes design is more sensitive than the coplanar electrodes.