Mark S. Talary

Development of biofactory-on-a-chip technology using excimer laser micromachining

Multilayer microelectrode structures, with 10 m feature sizes, have been fabricated using excimer laser ablation techniques. These structures will be incorporated into devices for the electro-manipulation and characterization of cells, microorganisms and other particles. The AC electrokinetic phenomena of dielectrophoresis, electrorotation and travelling electric field effects are utilized, all of which are dependent on the dielectric properties of the bioparticles. Examples are presented of a travelling wave junction and selective particle trap to be incorporated into a prototype biofactory-on-a-chip device. A technique for profiling the edges of via-holes has been developed to facilitate robust electrical connections through polyimide films in these multilayer devices.

Enhancing traveling-wave dielectrophoresis with signal superposition

In this article, a new method for cell separation and characterization and for monitoring cell physiological changes was described. In this approach termed superposition-TWD (travelling-wave dielectrophoresis), one or more DEP and TWD signals are applied together. The effect of such superposition of signals is to change the levitation height of the particles above the electrode plane, and in so doing to alter the range of frequencies over which TWD occurs. Appropriate choices of TWD signal strengths and frequencies, as well as the senses of the applied quadrature phase sequences, can result in cells of different type or physiological state traveling in opposite directions. This provides significant advantages over previously described TWD methods and can result in improved levels of attainable sensitivity and purity of cell separations on shorter electrode tracks.

Dielectrophoretic studies of the activation of human T lymphocytes using a newly developed cell profiling system

Human T lymphocytes were stimulated using phorbol myristate acetate and ionomycin. Twenty‐four hours post‐activation the cells were harvested for DNA content and for measurements using a newly developed cell profiling system employing dielectrophoresis. This system provides individual cell size and dielectrophoresis data for statistically relevant numbers of control and activated cells. From this it was determined that the mean membrane specific capacitance decreased from 13.49 (± 4.72) mF/m2 to 10.62 (± 5.13) mF/m2. This can be related to a 21.3% reduction in the effective membrane surface area associated with membrane topography (e.g. reduction of membrane associated microvilli, blebs and folding), or to other changes of membrane architecture, following cell activation. From cytometric determinations of DNA content, it was concluded that these effects were related to a 3.0‐fold decrease of cells in S‐phase, and a 1.5‐fold increase in G1 cells. This work demonstrates the powerful potential of using dielectrophoresis as a noninvasive tool to follow physiological changes that accompany transmembrane signaling events.

Future trends in diagnosis using laboratory-on-a-chip technologies.

There has been an enormous growth in the development of biotechnological applications, where advances in the techniques of microelectronic fabrication and the technologies of miniaturization and integration in semiconductor industries are being applied to the production of Laboratory-on-a-Chip devices. The aim of this development is to create devices that will perform the same processes that are currently carried out in the laboratory in reduced timescales, at a lower cost, requiring less reagents, and with a greater resolution of detection and specificity. The expectations of this Laboratory-on-a-Chip revolution is that this technology will facilitate rapid advances in gene discovery, genetic mapping and gene expression with broader applications ranging from infectious diseases and cancer diagnostics to food quality and environmental testing. A review of the current state of development in this field reveals the scale of the ongoing revolution and serves to highlight the advances that can be perceived in the development of Laboratory-on-a-Chip technologies. Since miniaturization can be applied to such a wide range of laboratory processes, some of the sub-units that can be used as building blocks in these devices are described, with a brief description of some of the fabrication processes that can be used to create them.

Cell Physiometry Tools Based on Dielectrophoresis

Dielectrophoresis is a technique for moving cells and other particles using radiofrequency electric fields. The usefulness of this method depends on the ability to generate highly non-uniform electric fields using microelectrodes, and also on the intrinsic dielectric properties of the cells and their surrounding medium. Selective cell isolation or concentration can be achieved without the need for biochemical labels, dyes or other markers and tags, and the cells remain viable after this process. Changes in cell state, such as those associated with activation, apoptosis, differentiation, necrosis, as well as responses to chemical and physical agents for example, can be monitored by observing changes in dielectrophoretic behavior. The basic theories and experimental techniques of dielectrophoresis are described in this chapter, and a summary is given of our present understanding of how the dielectrophoretic behavior of cells relate to their physiological and physico-chemical properties.

Microelectrode devices for manipulating and analysing bioparticles

Micro-fabrication techniques, such as photo- and electron beam lithography and etching processes, are currently being used to develop microelectrode structures and new devices for applications in biomedicine and biotechnology. With these devices various types of bioparticle can be subjected to strong inhomogeneous electric fields, rotating fields or travelling fields. These fields can be generated over a wide range of electrical frequencies, so that the characteristic dielectric properties of the bioparticles can be tharaughly diagnosed or exploited. This article describes how the AC electrokinetic techniques of dielectrophoresis (motion of particles in inhomogeneous fields) and electrorotation can be used to detect or selectively manipulate cells and micro-organisms. New results, employing travelling field dielectrophoresis, of the separation of red and white blood cells and of the selective separation and concentration of viable yeast cells, are also presented.

Manufacture of miniature bioparticle electromanipulators by excimer laser ablation

Multilevel microelectrode structures have been produced using excimer laser ablation techniques to obtain devices for the electro-manipulation of bioparticles using traveling electric field dielectrophoresis effects. The system used to make these devices operates with a krypton fluoride excimer laser at a wavelength of 248 nm and with a repetition rate of 100 Hz. The laser illuminates a chrome-on-quartz mask which contains the patterns for the particular electrode structure being made. The mask is imaged by a high- resolution lens onto the sample. Large areas of the mask pattern are transferred to the sample by using synchronized scanning of the mask and workpiece with sub-micron precision. Electrode structures with typical sizes of approximately 10 micrometers are produced and a multi-level device is built up by ablation of electrode patterns and layered insulators. To produce a traveling electric field suitable for the manipulation of bioparticles, a linear array of 10 micrometers by 200 micrometers microelectrodes, placed at 20 micrometers intervals, is used. The electric field is created by energizing each electrode with a sinusoidal voltage 90 degree(s) out of phase with that applied to the adjacent electrode. On exposure to the traveling electric field, bioparticles become electrically polarized and experience a linear force and so move along the length of the linear electrode array. The speed and direction of the particles is controlled by the magnitude and frequency of the energizing signals. Such electromanipulation devices have potential uses in a wide range of biotechnological diagnostic and processing applications. Details of the overall laser projection system are presented together with data on the devices which have been manufactured so far.

Development of biofactory-on-a-chip technology

The miniaturised Biofactory-on-a-Chip devices described here are integrated systems capable of the rapid analysis of small volume particulate samples and have applications in areas such as medical and biological cell diagnostics, chemical detection and water quality control. The devices use the A.C. electrokinetic phenomena of dielectrophoresis, travelling wave dielectrophoresis and electrorotation to manipulate, separate and characterise particle systems by exploiting their dielectric properties. Biofactory fabrication makes use of conventional photolithographic processes along with precision excimer laser ablation based micromachining. Using this combination of technologies, a wide range of manufacturing issues have been addressed and are discussed here. For instance, reliable interconnection of multilayer electrodes has been achieved using laser machining of via- holes between lithographically produced electrodues. Also, accurate fluidic microchannel systems with varying curved cross-sections that allow the smooth transport of a sample through the device whilst eliminating problems of particle trapping have been developed using excimer laser machining. Although the biofactory devices presented here have been applied to the fractionation of micro-organisms such as E. coli from red blood cells, the flexibility of design allows these devices to perform a wide range of complex bioprocessing function in a single, low-cost and miniaturised package.