Introduction

Increasing efforts are being directed towards applying the technologies of microelectronics, such as photolithography and microfabrication, to the development of micro-devices for applications in a wide range of medical and biologically-related technologies. Our contributions to this subject are directed towards developing Biofactory-on-a-Chip (BFC) devices capable of performing a wide range of complex diagnostic tasks in a single, miniaturized, low-cost package. Such devices have the advantage of being automated systems capable of the rapid analysis of small volume samples and have applications in such areas as medical and single-cell diagnostics, chemical detection and water quality control. An important aspect is that these devices need not employ microfluidic systems. The bioparticles can either be suspended in a stationary fluid and various forms of motion imparted on them by the application of A.C. electric fields, or travelling electric fields can be used to pump fluids in selected channels.

The phenomena exploited in BFC devices utilise the dielectric properties of particles, and in particular on the following ponderomotive responses to externally applied time-varying electrical fields:

• Dielectrophoresis - the translational motion induced when particles are exposed to stationary non-uniform electric fields.
• Travelling wave dielectrophoresis - the translational motion induced when particles are exposed to travelling electric fields.
• Electrorotation – resulting from the torque exerted on particles in rotating electric fields

Reviews of recent progress, and perceived biotechnological applications of these A.C. electrokinetic phenomena, are available (Pethig, 1996; Pethig & Markx, 1997). In this presentation we will describe the basic modular design, construction and operation principles of the BFC technology.

Biochip Sub-Units

i) Travelling wave Dielectrophoresis (TWD) Conveyor Tracks

The main sub-unit in our devices is the TWD conveyor track, an example of which is shown in Figure 1. Depending on their dielectric properties, and over a relatively narrow frequency range, particles exposed to a travelling field may experience a force which constantly pulls them along the TWD track. By controlling the magnitude and frequency of the energising voltages the speed and direction of particles can be controlled. This can either facilitate the spatial separation of different bioparticles along the track, or be employed to selectively move bioparticles around the biochip.

Figure 1 A TWD track. The travelling waves are generated by energising the electrode elements with quadrature (90^o phase separated) sinusoidal voltages. In this multilayer design, four bus-bars can be used to energise tracks composed of a hundred or more electrode elements. Over a narrow frequency range particles can be levitated above the tracks by negative dielectrophoresis, and be induced to move along the track in response to the travelling wave.

The tracks can be coated with an insulating film, and a channel cut into for containing the suspending fluid and for bonding onto a top cover plate. Alternatively, a channel can be cut directly into the gold plated tracks. Multilayer TWD devices have already been constructed and tested for their bioparticle manipulation and separation characteristics (e.g. Talary et al, 1996; Morgan et al, 1997).

ii) Selective and Non-Selective Dielectrophoresis Traps

Some of the operations of the biochip will involve holding or trapping particles at specific locations. The non-selective trap functions in a similar manner to the TWD conveyor track principle. A section of the conveyor track consists of castellated, interdigitated, electrodes designed to create regions of highly inhomogeneous fields. When used as a conventional TWD track, the particles are levitated by the action of negative dielectrophoresis and are propelled over the electrodes. However, by changing the frequency and phases of the applied voltage, a stationary positive dielectrophoretic force can be generated to attract and immobilise the particles at the electrodes. By reverting to a TWD-type field on the electrodes, the particles (or a subset of them) can be made to resume their movement along the track. Dielectrophoretic trapping of this form has been demonstrated for various types of cells and microorganisms, including for example its use in the selective enrichment of CD34⁺ cells from peripheral stem cell harvests (Stephens et al, 1996). Further examples are detailed in two recent reviews (Pethig, 1996; Pethig & Markx, 1997).

iii) Directional TWD Junctions

The ability to effectively separate sub-populations of particles from a heterogeneous sample offers many advantages and applications. Both separation and combination of sub-populations can be carried out using a directional TWD junction of the form shown in figure 2. Depending on the direction of particle travel, the junctions can be used to either separate particle types, or to allow different particle types to be combined into a mixture. At the entrance of the junction two independent travelling wave fields are applied to particles in the channel. The field frequency on the main track is chosen to move the majority of the particles, whilst that on the spur track is optimised for a particular sub-population of particles. As they travel along the track the particles are gradually attracted to either side of the channel. At the junction, each side of the channel is exposed to a different travelling field, which causes the particles to follow the appropriate spur. The separation of particle types can be enhanced by the application of weak dielectrophoretic forces superimposed on the travelling fields, with the purpose of attracting the desired particles types towards the correct side of the channel.

Figure 2 A TWD junction, for the separation or mixing together of different particle types. The electrode elements are typically 5 to 10 µm in width. The tracks are energised to produce two travelling fields of different frequency, each chosen according to the dielectric properties of the particle types to be separated (or mixed together for controlled chemical reactions).

iv) Electrorotation Units

These rotation analysers are used to monitor the dielectric properties of particles, e.g. microorganisms, to quantify factors such as their type or viability, or to monitor the binding of an antigen to an antibody coated bead (e.g. Burt et al, 1996). Particles are directed along a TWD conveyor track to a chamber structure consisting of four electrodes orthogonal to each other. These electrodes are then energised with quadrature sinusoidal voltages (0^o, 90^o, 180^o, 270^o). This produces a rotating electric field in the chamber. As the particles interact with the electric field they experience a torque, the magnitude and polarity of which can be related to the structure and properties of the particles. Observation of the rotation can be carried out using either digital image processing techniques, or direct CCD analysis. Having quantified the properties of the particles they are then attracted to the output side of the chamber, where they experience a TWD field in the output conveyor track. This draws the particles out of the chamber leaving it ready for the next sample to be studied.

A monolayer device, depicted in figure 3, that combines TWD with ROT has been constructed and tested for concentrating and the viability assay of Crpytosporidium oocysts (Goater et al, 1997). By energising the four electrodes with quadrature signals, a travelling wave is produced over the spiral electrodes. This can be used to selectively direct and concentrate particles into the central region of the microelectrode structure, where they are then subjected to a rotating electric field. The viability of a cell is then obtained by observation of the sense of induced rotation.

Figure 3 Schematic outline of a micro-device that combines the effects of travelling wave dielectrophoresis and electrorotation. The device is energised by applying sinusoidal voltages, of relative phases indicated, to the four spiral elements. One application has been in the concentration and viability determination of water-borne toxic parasites (Goater et al, 1997)

Excimer Laser Ablation

The BFC devices are constructed as multilayer, thin film, devices and excimer laser ablation is proving to be an ideal method for their fabrication. Thin-metal films can be patterned directly by laser ablation without the need for the multi-step processes involved in photolithography. Furthermore, the modular design of the BFC’s makes it possible to fabricate one chrome-on-quartz mask with all of the patterns necessary to manufacture each module, and then to ‘stitch’ together each of the modules as required during the laser patterning process. The CNC system, which controls the patterning action of the laser, can be programmed so as to index each of the mask patterns in the required sequence. The system used for this work is the Exitech Series 8000 microfabrication workstation, incorporating a krypton fluoride excimer laser operating at 248nm and 100 Hz. The main XY stage provides for 200 x 200mm travel at 0.1 µm resolution. A rotation stage, using off-axis aligner software, enables layer-by-layer registration to be accomplished. The stages are controlled by two PC-based motion controllers, one of which is coupled to a laser firing card. The system can operate in either a serial write mode (where the workpiece or mask moves) or laser beam scanning mode.

This work is supported by the Biotechnology and Biological Research Council (grant No: TO6337), Severn Trent Water Ltd., Genera Technologies Ltd, and P&B (Microtech) Ltd.

References

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• Goater, A.D., Burt, J.P.H., and Pethig, R. 1997. Combined travelling wave dielectrophoresis and electrorotation device: Applied to the concentration and viability determination of Cryptosporidium. J. Phys.D.: Appl. Phys. 30: L65-70
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• Pethig, R. 1996. Dielectrophoresis: using inhomogeneous AC electrical fields to separate and manipulate cells. Crit. Rev. Biotechnol. 16: 331-348.
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• Talary, M.S., Burt, J.P.H., Tame, J.A., and Pethig, R. 1996. Electro manipulation and separation of cells using travelling electric fields. J. Phys.D.: Appl. Phys. 29: 2198-2203.
• Zhou, X-F., Markx, G.H., and Pethig, R. 1996. Effect of biocide treatment on electrorotation spectra of yeast cells. Biochim. Biophys. Acta 1281: 60-64.