Manipulating biomolecules
The controlled manipulation of small particles, e.g. biomolecules, on the molecular scale is important not only for molecular assembly, but has great impact on biotechnology in general and is a crucial pre-requisite for the emerging field of nanotechnology. In particular, for many applications the ability to manipulate molecules, control their position and orientation at this level is essential. Experts believe that nanotechnology will enable visualisation of biological processes at the cellular and molecular level, fostering the development of new applications in medicine, diagnostics and therapeutics, such as tracing the very early stages of diseases. Furthermore, molecular level manipulation techniques provide an important tool to study fundamental properties of molecular systems, e.g. the direct and controlled manipulation of single molecules of DNA has contributed substantially to the understanding of the mechanical properties of DNA¹.

Figure 1: Christoph Wälti in front of FV1000

AC electrokinetic manipulation of surface-tethered DNA
The manipulation of small particles using dielectrophoresis and ac electrokinetic techniques has recently received considerable attention as an alternative to optical tweezers or scanning probe techniques, and is becoming a useful tool in molecular biology and biotechnology. For example, dielectrophoretic forces have been employed to separate and manipulate cells^2,3, bacteria^4,5 and viruses⁶, inter alia. Other studies have investigated the dielectrophoretic manipulation of DNA molecules as they have many potential applications in molecular nanotechnology owing to their exceptional self-assembly properties^7–10. These studies have led to novel applications such as the concentration of DNA molecules, DNA-protein interaction studies and molecular surgery of DNA¹¹. However, a detailed understanding of the behaviour of surfaceimmobilised DNA molecules when exposed to high frequency ac electric fields, and in particular the orientation and elongation of DNA as a result of electrokinetic force and torque, has to be understood in detail to capitalise fully on this technique. In addition, precisely controlled positioning of DNA at the single-molecule level is required. Therefore, the underlying science and engineering of this phenomenon is of great interest to us and we are pursuing an active research programme dedicated to this area^9,10,12.

Figure 2: Microscope setup – IX81 with FV1000 confocal scanning unit

We performed detailed investigations of the electrokinetic elongation of surface-tethered DNA molecules when exposed to an ac electric field for a number of different frequencies, electric fields, DNA lengths and across different gap separations. A detailed understanding of the elongation mechanism and interplay between the various forces was achieved by performing three-dimensional imaging using confocal microscopy6. Fluorescently labelled DNA strands were covalently tethered by one end to gold microelectrodes and subjected to strong ac electric fields. This was investigated using an Olympus BX60 fluorescent microscope and Olympus FV1000 FluoView laser scanning confocal microscope (LSCM) with the inverted Olympus IX81 microscope using a 60x water immersion lens (see figures 1 and 2). We then compared the observed threedimensional elongation patterns with the previously determined fluid flow patterns and with the electric field lines obtained from numerical simulations (see figure 3). Furthermore, the addition of a secondary laser scanner (SIM) scanner and in particular the advanced objective-based TIRFM module provided us with a system that also allows us to carry out advanced fluorescence studies, including FRET, advanced bleaching and time-resolved measurements.

Figure 3: Cross section of a three-dimensional image of surfaceimmobilised DNA elongated in an ac electric field. The blue lines indicate the calculated electric field lines and the red arrow the observed induced fluid flow.

Conclusions
We have investigated the elongation of DNA by ac electric fields using 3D optical imaging techniques. We have revealed substantial support for a mechanism for the elongation of surface immobilised DNA, where the DNA molecules are elongated by a combination of the electrokinetic torque acting on the DNA, and an additional bias force pulling on the free end of the DNA^12–14. When exposed to an electric field, a dipole is induced along the DNA, and the resulting electrokinetic torque that acts on these induced dipoles aligns the DNA locally with the electric field. However, the local alignment can be parallel or anti-parallel. In order to achieve elongation, an additional bias force is required so that the forward, parallel local alignment of the DNA is favoured^12–14. We are currently expanding this programme to investigate the dynamics of the elongation using FRET microscopy, and to investigate the behaviour of other biological materials.

References
^1. C. Bustamante, Z. Bryant, S. B. Smith, Ten years of tension: singlemolecule DNA mechanics, Nature 421, 423-427 (2003).
^2. J. Gimsa, A comprehensive approach to electro-orientation, electrodeformation, dielectrophoresis, and electrorotation of ellipsoidal particles and biological cells, Bioelectrochemistry 54, 23-31 (2001).
^3. J. Suehiro, R. Pethig, The dielectrophoretic movement and positioning of a biological cell using a three-dimensional grid electrode system, J. Phys. D 31, 3298-3305 (1998).
^4. Y. Huang, K. L. Ewalt, M. Tirado, R. Haigis, A. Forster, D. Ackley, M. J. Heller, J. P. O‘Connell, M. Krihak, Electric manipulation of bioparticles and macromolecules on microfabricated electrodes, Anal. Chem. 73, 1549-1559 (2001).
^5. G. H. Markx, P. A. Dyda, R. Pethig, Dielectrophoretic separation of bacteria using a conductivity gradient, J. Biotechnol. 51, 175-180 (1996).
^6. H. Morgan, N. G. Green, Dielectrophoretic manipulation of rod-shaped viral particles, J. Electrostat. 42, 279-293 (1997).
^7. C. L. Asbury, A. H. Diercks, G. Van der Engh, Trapping of DNA by dielectrophoresis, Electrophoresis 23, 2658-2666 (2002).
^8. Suzuki S, Yamanashi T, Tazawa S, Kurosawa O, and Washizu M, Quantitative analysis of DNA orientation in stationary AC electric fields
using fluorescence anisotropy, IEEE Trans. Ind. Appl. 34, 75 (1998).
^9. Germishuizen WA, Wälti CP, Wirtz R, Johnston MB, Pepper M, Davies AG and Middelberg APJ. Selective dielectrophoretic manipulation of surface-immobilzed DNA molecules. Nanotechnology 2003. 14: 896-902.
^10. Germishuizen WA, Tosch PA, Middelberg APJ, Wälti CP, Davies AG, Wirtz R and Pepper M. Influence of alternating current electrokinetic forces and torque on the elongation of immobilized DNA. Journal of Applied Physics 2005. 97: 014702/1–7.
^11. M. Washizu, O. Kurosawa, I. Arai, S. Suzuki, N. Shimamoto, Applications of electrostatic stretch-and-positioning of DNA, IEEE Trans. Ind. Appl. 31, 447-456 (1995).
^12. Wälti CP, Tosch PA, Davies AG, Germishuizen WA and Kaminski CF. Establishment of the ac electrokinetic elongation mechanism of DNA by three-dimensional fluorescent imaging. Applied Physics Letters 2006. 88, (15): 153901/1-3.
^13. Wälti CP, Germishuizen WA, Tosch P, Kaminski CF and Davies AG. AC electrokinetic manipulation of DNA. Journal of Physics D: Applied Physics 2007. 40: 114-118.
^14. A. E. Cohen, Nanoscale Mechanics (PhD thesis, University of Cambridge, 2003).