AVS2009 Session AS2+BI-ThM: Scanning Probe Studies of Biological Materials

The discovery of penicillin in 1928 marked the beginning of a remarkable new era of antibiotic ‘wonder drugs’, saving millions of lives across the world. However the widespread and often indiscriminate use of antibiotics has fuelled the alarming growth of antibiotic resistant superbugs, including methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococci (VRE). To remain one step ahead of the superbugs, there is now an urgent need to develop new antibiotics and yet the drug pipeline is severely limited. We recently reported the nanomechanical detection of vancomycin-cell wall peptide interactions on cantilever arrays and discriminated between vancomycin-sensitive and vancomycin-resistant phenotypes.¹ In this talk I will present our new work which exploits this technology for the search of new superdrugs active against VRE. We have investigated a series of vancomycin derivatives and detect a dramatic enhancement in surface binding affinities compared to homogeneous solution measurements. We identify a glycopeptide which binds 11,000 more strongly to resistant peptide analogues. Our findings reveal fundamental new insights into the mechanism of antibiotic bindings to a model bacterial cell wall peptides using nanosensors, which not only has important implications on the design of new devices with significantly improved antibiotic detection sensitivity but will also impact on our understanding of the mode of action of antibiotics on intact bacteria. These findings highlight the potential of BioMEMs devices for application in pharmaceutical industry and will accelerate the discovery of new antibiotics.

[1] ‘Nanomechanical detection of antibiotic mucopeptide binding in a model for superbug drug resistance.’ Ndieyira, W.N, Watari, M., Donoso-Barrera, A., Batchelor, M., Zhou, D., Vogtli, M., Bactchelor, M., Cooper, M., Strunz, T., Abell, C.A., Rayment, T., Aeppli, G. & McKendry R.A. Nature Nanotechnology 3, 691 - 696 (2008). Also featured in Nature Nanotechnology News and Views, BBC, New Scientist, Physics Today, Chemistry World, UK T&I.

Over the past decade, considerable interest has focussed on the ability of atomic force microscopy (AFM) and related techniques to record forces on or between single biological molecules. Many elegant examples are evident within the literature where such approaches have been employed, for example, for studies of force induced protein and RNA unfolding processes, and the dissociation of a wide-range of biomolecular complexes, including those involved in cell adhesion. Despite these elegant examples, and the considerable advantages of performing measurements at the single molecule level, such measurements are still far from routine. Indeed, even in the most experienced hands the experiments can suffer various problems which can arise from poor control of the chemistries employed for biomolecular surface attachment e.g. such as non-specific binding, difficulties in single molecule pickup and variability between experiments.

This presentation will provide an overview of the approaches we have employed in recent studies to address such issues. This for example, will include our studies of the mechanical properties of long molecules of DNA (>1000 base pairs) in which we have demonstrated the advantages of the addition specific terminal functionalities for surface attachment [1]. The advantages will be highlighted through our investigations of the effects on DNA mechanical properties of a range of different DNA binding molecules (e.g. drugs and proteins involved in DNA replication [1,2]). For studies of the dissociation of biomolecular complexes, we will highlight the advantages of an alternative approach, in which we have exploited the unique properties of dendron functionalized surfaces. Developed in collaboration with the group of Professor J.W. Park (Pohang University of Science and Technology (POSTECH), we have recently employed such surfaces for studies of DNA hybridization [3], and interactions between intracellular signal transduction proteins [4].

[1] W. Zhang, R. Barbagallo, C. Madden, C.J. Roberts, A. Woolford, S. Allen. (2005) Nanotechnology 16 2325-2333

[2] W. Zhang, C. Machon, A. Orta, N. Phillips, C.J. Roberts, S. Allen, P. Soultanas (2008) Journal of Molecular Biology 377, 706-714

[3] J. Yung, B.J. Hong, W. Zhang, S.J.B. Tendler, P.M. Williams, S. Allen and J.W. Park (2007). Journal of the American Chemical Society, 129(30), 9349-9355.

[4] I.H. Kim, H.Y. Lee, H.D. Lee, Y.J. Jung, S.J.B. Tendler, P.M. Williams, S. Allen, S.H. Ryu, and J.W. Park (2009) Analytical Chemistry 81(9), 3276-3284

The use of nanoarrayed surfaces in the field of biomolecular recognition is very promising for the improvement of bio-detection performance. Atomic Force microscope (AFM)  is widely used to produce and characterize nanoarrayed surfaces and to carry out studies in the biological field. This work presents the study and characterization by AFM measurements of the fabrication steps of nanoarrayed surfaces based on organothiols (carboxylic and Polyethylene oxide) and the study of an immunoassay performed on these surfaces. The immunoassay was based on the bio-recognition between Human IgG/anti-Human IgG.

The nanoarray was fabricated by plasma colloidal lithography following the procedure developed in our lab. AFM studies of the process of nanoarray fabrication showed that during lithographic process, the etching step is crucial for the final the characteristics of the surface and that the process originates a good chemical nano-contrast.

The AFM analysis of the bio-interaction was performed after Human IgG immobilisation and anti-Human IgG recognition steps. The nanoarray was incubated with Human IgG solution resulting in an increase of height on the nano areas.  The AFM image demonstrates that IgG molecules are adsorbed mainly on the border between the two organothiols. The preferential disposition of proteins on the borders of the two different materials with hydrophobic and hydrophilic groups was already reported and associated to the fact that the proteins tend to adsorb where they can find better accommodation. In our case, this effect can be explained by the fact that having the carboxylic spots with hydrophilic character the IgG hydrophobic groups are better accommodated on the border between the two materials, leading to higher adsorption on the boundaries. After the Human IgG incubation, the surface was blocked with BSA and the following step consisted in the immobilisation of anti-Human IgG. After this step, a height increase on the border of the COOH nano area is detected by AFM. The increase is about the double of the initial with Human IgG. This is an indication that the anti Human IgG binds specifically with the Human IgG already on the surface. The distribution on the borders of the nano-area can explain the better efficiency of the nano-patterns in biomolecular recognition as already described on different studies. The preferential proteins immobilisation on the nano areas boundaries seems to improve the binding efficiency of the immobilised Human IgG bio-detector by a better availability of the binding sites on the surface and reduction of steric hindrance.

Scanning Probe Microscopy techniques provide a wealth of information on nanoscale interactions. The rapid emergence of spectroscopic imaging techniques in which the response to local force, bias, or temperature is measured at each spatial location necessitates the development of data interpretation and visualization techniques for 3- or higher dimensional data sets.

In this presentation, we summarize recent advances in applications of neural network based artificial intelligence methods in scanning probe microscopy. The examples will include biological identification based on the dynamic of the electromechanical response, direct mapping of dynamic disorder in ferroelectric relaxors, and reconstruction of random bond-random field Ising model parameters in ferroelectric capacitors. The future prospects for smart multispectral SPMs are discussed.

Research was supported by the U.S. Department of Energy Office of Basic Energy Sciences Division of Scientific User Facilities and was performed at Oak Ridge National Laboratory which is operated by UT-Battelle, LLC.