NANO LETTERS
Imaging Surface Charges of Individual Biomolecules
2009 Vol. 9, No. 7 2769-2773
Carl Leung,*,† Helen Kinns,‡ Bart W. Hoogenboom,§ Stefan Howorka,‡ and Patrick Mesquida† Department of Mechanical Engineering, King’s College London, London WC2R 2LS, England, Department of Chemistry, UniVersity College London, London WC1H 0AH, England, and London Centre for Nanotechnology and Department of Physics & Astronomy, UniVersity College London, London WC1H 0AH, England Received April 23, 2009; Revised Manuscript Received June 4, 2009
ABSTRACT Surface charges play a key role in determining the structure and function of proteins, DNA, and larger biomolecular structures.1-4 Here we report on the measurement of the electrostatic surface potential of individual DNA and avidin molecules with nanometer resolution using Kelvin probe force microscopy. We also show, for the first time, the surface potential of buffer salts shielding individual DNA molecules, which would not be possible with conventional ensemble techniques.
Kelvin probe force microscopy (KPFM) is an atomic force microscopy (AFM)-based technique to simultaneously map the topography and electrostatic surface potential of a sample.5 KPFM minimizes the force that arises when an AFM tip and sample have different electrostatic surface potentials. The applied counteracting tip potential required for neutralization is recorded as signal output, thus providing a measure of the sample surface potential independent of sample topography (see Figure 1). Usually, KPFM is applied in the fields of material science and semiconductor physics, as the technique provides valuable insights into the electronic properties of a surface, such as the work function, conductivity, and the presence of surface dipoles.6-11 The KPFM technique is currentless and provides a quantitative, differential measurement of the surface potential in millivolts with sufficient sensitivity to detect potential variations between neighboring atoms on TiO2.12 The applications of KPFM to study biomolecules are much less explored, despite the ubiquity and role of surface charges in biological systems. For example, negatively charged DNA strands electrostatically interact with histone proteins, transcription factors, or polymerases thereby influencing the readout of genetic information and the development of cancer. Similarly, the central process of protein folding and protein interaction, often governed by charges, is the major factor in protein-folding diseases such as Alzheimer’s or Parkinson’s disease. In an early work, Knapp et al.,13 applied KPFM * Corresponding author,
[email protected] or
[email protected]. † Department of Mechanical Engineering, King’s College London. ‡ Department of Chemistry, University College London. § London Centre for Nanotechnology and Department of Physics & Astronomy, University College London. 10.1021/nl9012979 CCC: $40.75 Published on Web 06/11/2009
2009 American Chemical Society
to visualize light-dependent variations of the surface potential of purple membrane in humid air. Since then, most KPFM work in biology-related systems has been restricted to organic thin films, such as in the study of pulmonary surfactant films,14,15 protein structures and patterns,16,17 and DNA,18 albeit at low resolution. Recently, Sinensky et al.19 showed that KPFM can be applied as a label-free technique to distinguish three base-pair mismatches between anchored DNA strands and their corresponding target sequences within an ensemble of multiple biomolecules. However, thus far, there have been no experimental methods to spatially resolve the electrostatic surface potential of individual biological molecules. In general, the investigation of individual molecules can shed light on their dynamic behavior or on static heterogeneity which is masked in ensemble measurements. To demonstrate that KPFM can be conducted at the molecular level, we examined avidin, a robust 58 kDa tetrameric protein on a Topometrix Explorer microscope (Veeco Corp., Santa Barbara, CA). Each avidin monomer has a net positive charge, stemming from 16 basic and 11 acidic, solvent-accessible residues.20 The resulting high isoelectric point (pI) of 10.521 is expected to give rise to a positive signal in KPFM. We first established an appropriate protocol for creating dilute arrays of primarily single avidin molecules on a silicon substrate suitable for KPFM measurements, and a representative topographic image of the avidincovered surface (prepared as described in the Supporting Information) is shown in Figure 2. We can clearly distinguish elevated features of a few nanometers in height, which can be identified as avidin molecules, since no surface peaks were seen on control surfaces incubated with the same buffer
Figure 1. Schematic of KPFM setup. KPFM can measure surface charges by contactless recording of the electrostatic force between a conductive AFM tip and a biomolecule on a support. To achieve this, the AFM tip is simultaneously excited at its mechanical resonance frequency and by an electrical (ac) voltage. This periodic electrical voltage on the tip leads to a force between the tip and the charges on the biomolecule, which is recorded by means of a lock-in amplifier and nullified by the Kelvin mode feedback by applying a separate dc voltage (not shown). The polarity and magnitude of this dc voltage correspond to the local surface charge profile (in millivolts) which is recorded simultaneously with the topography of the biomolecule.
lacking avidin (not shown). The volume of individual peaks was estimated from AFM measurements with supersharp silicon tips (nominal radius
