In this work, we are focussed on molecular mechanics using micro-sensor technology. We have developed the lateral molecular-force microscope (LMFM) with a patented detection system and this technique is already demonstrating to be a unique tool for measuring and manipulating bio-molecular systems with nanometer resolution and femtoNewton sensitivity. Reducing the sensor size to the nanoscale, as pioneered in Bristol, will disclose an unprecedented level of detail of bio-molecular structures and their dynamic behaviour in vivo.The next strategic objective is to combine mechanical measurements of bio-molecules with other physical properties, in particular: electron transfer, polarisability and optical spectroscopy. The technology to access these combined measurements is not currently available, and we are in a unique position to integrate these new functionalities in our current nanosensors. We will develop, in an initial proof-of-concept stage, the methods and the procedures necessary to produce and use force sensors with the ability to measure extremely small currents or capacitance in a liquid environment. The following stage will require high-level interdisciplinary collaborations to address crucial questions in bio-molecular science and explore commercialisation avenues of these nanodevices.
Comparison of ultra-soft cantilever with commercial contact mode AFM cantilevers (MLCT from Veeco, Santa Barbara, CA, USA) (scale bar is 200 mm).
Experimental set up of the LMFM
Processive stepping of a single kinesin molecule. Time course of a single kinesin molecule as it moves along an MT in 8.0 ± 0.4 nm steps at 1 mM ATP concentration. The superimposed parallel lines are 8.0 nm apart. The molecule binds to the MT after 0.12 s and it reaches a distance of 110 ± 5 nm from the centre position, before stalling at a force of 4.0 ± 0.2 pN. (Inset) The last short step (arrow) is enlarged and confirms that the system can clearly measure events lasting ≈3 ms. Data were recorded at 64 kHz (grey curve) and interpolated using cubic spline interpolation (black curve). The inset shows raw data.
The processive stepping of a single kinesin molecule
The images below show atomic force microscopy analysis of Mx bacteria. (A) A sinusoidal sideways movement is imparted to the sample stage in order to gently push a single adsorbed bacterium (blue sphere; pale-blue outer layer represents surface adhesins) against the cantilever. The change in the sample stage position (Δx1) required to achieve contact is measured upon addition of either ligand (CEACAM1 or Fn) or buffer/control protein. The graphs on the right show representative data measured for the change in the contact point position before (in red) and after (in green) the addition of (B) control, (C) CEACAM1, and (D) Fn. The red and green lines represent the mean value of the red and green points, respectively.
