AC electrokinetic manipulation of DNA

The controlled manipulation of molecules is a crucial prerequisite for the emerging field of molecular nanotechnology. AC electrokinetics provide a powerful mechanism for both positioning and inducing conformational changes in molecules. In this paper, we investigate the elongation of fluorescently-labelled DNA strands, which are covalently tethered by one end to gold microelectrodes arranged in an opposing-finger geometry, when exposed to strong ac electric fields. We found that the elongation of the DNA molecules is restricted by the geometry of the gap, and that the observed contour of the elongated DNA molecules coincides with the electric field line pattern. Further, we discuss a potential elongation mechanism and provide evidence that the major contribution to the elongation originates from the ac electrokinetic torque, which is supplemented by a small bias force provided by the electric-field-induced fluid flow. (Some figures in this article are in colour only in the electronic version) A key factor for the successful development of molecular nanotechnology will be the availability of a wide range of tools for the manipulation of molecules, molecular complexes, or particles on the molecular scale. The ability to manipulate on the molecular scale is essential to control the position and orientation, and even more importantly the conformation, of molecular complexes, and hence their efficacy. In addition, sophisticated manipulation techniques provide important tools to study fundamental properties of molecular systems. For example, the direct and controlled manipulation of single molecules of DNA has contributed enormously to the understanding of their mechanical properties [1]. A number of techniques are available for controlled manipulation at the molecular scale. Prominent tools in this field are optical [1–3] and magnetic [4] tweezers, where the molecules are labelled with an appropriate bead which is trapped by a laser beam or a magnetic field, respectively. An alternative approach is taken by scanning probe techniques [5], and in particular, atomic force microscopy (AFM) techniques, where the molecules are generally manipulated directly. This has the benefit that the labelling reaction, required for manipulation by tweezers, can be omitted. AFM techniques have been used, for example, to manipulate DNA and to study the force–extension behaviour of DNA [6]. Further, hydrodynamic forces have been employed in manipulation 3 Author to whom any correspondence should be addressed. studies, for example, to study the mechanical properties of polymers [7, 8]. Recently, ac electrokinetic manipulation techniques have received considerable attention as an alternative to the techniques mentioned above, and are becoming useful tools in molecular biology and biotechnology. The controlled manipulation of submicron particles by ac electrokinetic techniques has been demonstrated by separating and manipulating cells [9], bacteria [10], viruses [11] and submicron latex spheres [11–13]. In addition, ac electrokinetic manipulation of DNA molecules has led to novel applications such as the concentration of DNA molecules [14, 15], DNAprotein interaction studies [16], and molecular surgery of DNA [17]. However, a detailed understanding of the behaviour of DNA, and in particular of surface-immobilized DNA molecules, when exposed to high frequency ac electric fields, has to be understood in detail to capitalize fully on this technique. A number of studies have been carried out to investigate the elongation of DNA molecules using ac electrokinetic tools [14,18–20]. Here, we report on a series of experiments dedicated to the investigation of the interaction of the various forces with surface-tethered DNA when the DNA molecules are exposed to strong ac electric fields. We have immobilized four fragments of lambda-DNA of different sizes (15, 25, 35 and 48 kilobasepairs (kb)) onto specific electrodes of an 0022-3727/07/010114+05$30.00 © 2007 IOP Publishing Ltd Printed in the UK 114 AC electrokinetic manipulation of DNA Figure 1. Schematic diagram of the experimental arrangement. Fluorescently-labelled DNA molecules were tethered to the gold electrodes of an electrode array using a thiol linker [22]. The chip-package containing the wafer with the electrode array was mounted upside-down in a custom-built support. A continuous column of water was established between the wafer and the microscope lens. An ac electric potential was then applied to a chosen electrode, while the electrode directly opposite was kept at zero potential. electrode array comprising two opposing rows of individually addressable electrodes, via a terminal thiol-group. We measured the length of the elongated DNA as a function of electric field, frequency and gap separation, and we compare the elongation pattern of the DNA molecules with the electric field lines obtained from finite element calculations and with previously determined fluid flow patterns [19]. A schematic view of the experimental set-up is given in figure 1. The electrode array comprising individually addressable electrodes, 30μm wide and 15μm apart, arranged in two opposing rows, separated by either 20, 30 or 40μm, were fabricated using standard UV-photolithography. The electrodes were formed by the evaporation of an 80 nm layer of gold on top of a 20 nm adhesion layer of NiCr on Si/SiO2 wafers using standard optical lithography and lift-off techniques. The wafers were glued and bonded into a chip-package. Prior to attaching DNA to the electrodes, the wafers were cleaned in a ‘piranha’ solution (H2O2 : H2SO4, 3 : 7 ratio) for 1 h, followed by rinsing in deionized water, ethanol and deionized water again. The DNA molecules used in all experiments were either lambda-DNA (48 kb, Sigma-Aldrich), or were obtained through appropriate enzymatic restrictions from lambdaDNA (15, 25 and 35 kb). The DNA was fluorescently labelled by diluting it to 50 ngμl−1 in TE (10 mM trisHCl, 1 mM EDTA, pH 8) 1 M NaCl solution and adding the fluorescent intercalator YOYO-1 (excitation/emission wavelength 488/515 nm, Molecular Probes) at an intercalator to basepair ratio of 1 : 8. The contour length of untreated lambda-DNA is 16.5μm [21], but upon intercalation of the fluorophore YOYO-1 at the concentration used in this work, the DNA molecules lengthen and the contour length increases to approximately 20μm [2]. Similarly, the contour length of 15 kb DNA increases to 6.5μm, of 25 kb DNA to 10μm, and Figure 2. Schematic view of the electric field lines and the fluid flow pattern between two biased microelectrodes. The fluid flow pattern is taken from [19] and the field lines from [20]. Adapted and reprinted with permission from [20] Copyright 2006, American Institute of Physics. of 35 kb DNA to approximately 15μm. The DNA molecules were tethered onto the electrodes using a multi-step protocol detailed in [22]. After immobilization, the wafers were kept hydrated in deionized water at all times, and were submerged in deionized water (conductivity ≈10−5 S m−1) for all elongation experiments. The applied electric fields were generated by applying an ac potential to the particular electrode, while keeping the electrode directly opposite at ground. The electric fields referred to in this work are calculated from the amplitude of the applied potential divided by the width of the gap between the two electrodes. The elongation of the fluorescently labelled DNA molecules when exposed to the ac electric field was investigated with a fluorescence microscope (BX60, Olympus) and a laser-scanning confocal microscope (IX70, Olympus). To gain a deeper understanding of the underlying mechanism responsible for the elongation of DNA, and in particular the direct influence of the applied electric field, we modelled the electric field around the electrodes using the finite element analysis software, Femlab (Comsol). For the model, the electrodes were represented by a 100 nm-thick layer of gold, separated by gaps of 20, 30 or 40μm. The suspending medium had a conductivity of 10−5 S m−1. Figure 2 shows a schematic side-view of two opposing electrodes separated by 40μm. The left and right electrodes were set to a potential of 30 V and 0 V, respectively. We note that the electric field is highest directly at the electrode edge, but the variation is small across most of the gap and we can assume that the elongated DNA molecules experience similar field strengths over most of the gap [20]. Figure 3(a) shows the elongation length of DNA of various sizes elongated across a 40μm wide gap in an applied ac electric field of 375 kV m−1 for frequencies between 50 kHz and 1.1 MHz. The elongation is qualitatively similar for all lengths of DNA. Almost no elongation is observed at frequencies below 100 kHz, and upon increasing frequency a sharp increase in the elongation can be measured. The elongation goes through a maximum at around 250 kHz for all DNA fragments, where almost full elongation of the molecules is observed. At higher frequencies, the elongation decreases with increasing frequency, until above 1 MHz almost no elongation can be detected. These findings are in agreement with previous results for larger (500 kV m−1) electric fields on similar systems [19]. Figure 3(b) shows the elongation of 48 kb DNA (20μm contour length) for different frequencies as a function of the electric field. The behaviour is qualitatively similar for all frequencies, i.e. a steady increase in the elongation is observed with increasing ac electric fields. We