Download Nanomechanics of superbugs and superdrugs

Bionanotechnology III: from Biomolecular Assembly to Applications
Nanomechanics of superbugs and superdrugs:
new frontiers in nanomedicine
Rachel A. McKendry1
London Centre for Nanotechnology and Division of Medicine, University College London, 17–19 Gordon Street, London WC1H 0AH, U.K.
Abstract
The alarming rise in drug-resistant hospital ‘superbugs’ and the associated increase in fatalities is
driving the development of technologies to search for new antibiotics and improve disease diagnostics.
One of the most successful drug targets is the bacterial cell wall, an evolutionary feature of virtually all
prokaryotes and vital for their survival by providing mechanical strength. The recent discovery of bacterial
cytoskeletal proteins analogous to the key force-bearing machinery in eukaryotes also provides new
opportunities for drug discovery, but little is known about their mechanical role in bacteria. In the present
short article, I review recent developments in the field of nanotechnology to investigate the mechanical
mechanisms of action of potent antibiotics on cell wall and cytoskeletal targets with unprecedented spatial,
temporal and force resolution and the development of a new generation of nanomechanical devices to
detect pathogens for point-of-care diagnostics.
Introduction
Bacteria represent some of the most genetically diverse
and mechanically robust organisms on the planet and are
able to survive in a remarkable range of harsh physical
environments. They play a vital role in the global ecosystem
and offer a valuable source of new medicines. However,
bacteria also pose some of the greatest threats to human
health. New infections can emerge unexpectedly and spread
rapidly, while old enemies, including tuberculosis, are a major
source of morbidity and mortality across the world. Fleming’s
serendipitous discovery of penicillin and its subsequent
development into a ‘wonder drug’ by Florey, Chain and coworkers is rightly considered to be one of the most important
breakthroughs of modern science and has contributed to
the enormous gains in life expectancy over the last century.
However, the widespread emergence of drug-resistance looks
set to challenge these advances [1]. The alarming rise in
drug-resistance has been fuelled by the indiscriminate use
of these precious medicines which has forced bacteria to
selectively evolve resistance via increasingly sophisticated
mechanisms. Today, the emergence of new infections and
the re-emergence of old enemies with acquired resistance, including MRSA (methicillin-resistant Staphylococcus
aureus), VRE (vancomycin-resistant Enterococcus), NDM-1
(New Delhi metallo-β-lactamase-1) and Escherichia coli,
extensively drug-resistant tuberculosis and, most recently,
totally resistant Mycobacterium tuberculosis, is a major global
healthcare problem, and the pipeline of potential drugs
that might combat infections has dried to a trickle, with
only a couple of new classes of antibiotics discovered over
Key words: antibiotic-resistance, atomic force microscopy (AFM), drug-resistance, methicillinresistant Staphylococcus aureus (MRSA), nanomechanics, nanomedicine, superbug, superdrug.
Abbreviations used: AFM, atomic force microscope; MRSA, methicillin-resistant Staphylococcus
aureus; VRE, vancomycin-resistant Enterococcus.
1
email [email protected]
Biochem. Soc. Trans. (2012) 40, 603–608; doi:10.1042/BST20120082
the last 40 years [2]. This has sparked renewed interest
in understanding how drugs work on targets such as the
bacterial cell wall, a vital evolutionarily conserved feature
of virtually all bacteria, which confers mechanical strength
and is key to their survival. The cell wall is a crosslinked peptidoglycan matrix that protects bacteria from harsh
external forces and high internal osmotic pressures, and,
importantly, it is not found in humans, making it an ideal
drug target. However, there remains much to be learnt about
its structure and mechanical properties and the mechanism of
action of drugs on this complex surface.
Several major antibiotics target the cell wall. Penicillin,
the first known antibiotic, targets the final stage of cell
wall cross-linking, transpeptidation, whereas vancomycin,
currently the last line of treatment for MRSA, complexes
to nascent cell wall peptides. Although the chemical basis of
these interactions is well known, much less is known about
the mechanics; essentially, these antibiotics, and others, work
by engineering mechanical ‘defects’ in the cell wall. Yet the
mechanism by which these local ‘surface defects’ collectively
lead to the cell weakening and death is not understood [3].
This is important, since it is the mechanical failure induced
by these drugs that actually kills the bacteria and combats
the infection. Moreover, the recent astonishing discovery
of bacterial cytoskeleton proteins [4] analogous to the key
force-bearing machinery in eukaryotic cells highlights the
need for an improved understanding of bacterial mechanics,
work which could pave the way for new antibiotics. However,
progress in our understanding of the mechanics of bacteria
has been limited by the lack of physical tools to study
their properties at submicron length scales. Indeed, the
mechanics of bacteria are much less well understood than
their eukaryotic counterparts [5]. Optical imaging is limited
by the wavelength of light, and cryo-electron microscopy is
powerful but requires specialized sample preparations and
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fixing steps, but provides little information on mechanical
properties.
In the present short article, I review some of the recent
advances in the field of nanotechnology that offer new capabilities to probe the structure and mechanics of bacteria and
the molecular workings of antibiotics with unprecedented
spatial, temporal and force resolution. Nanotechnology, the
science of the small, is a rapidly advancing field that offers
tremendous opportunities for revolutionalizing healthcare,
energy and information technology. Although much of this
trillion-dollar industry is driven by the miniaturization of
microelectronic processing chips, the powerful impact of this
technology in the field of medicine is particularly exciting.
In the present article, I focus on advances in mechanical
tools based on the AFM (atomic force microscope) to
probe pathogens and antibiotics focusing on three key
areas: (i) advanced imaging of pathogens at the nanoscale;
(ii) nanomechanical mapping of single bacteria; and (iii)
nanomechanical biosensors for diagnostics applications. It
should be noted that the present article is not intended to
be an exhaustive review of the field, but instead provides a
snapshot of recent developments in the field and highlights
important future challenges.
Advanced imaging of pathogens at the
nanoscale
The AFM offers a unique tool to image the dynamics of
live bacteria cells with nanometre-scale lateral resolution
under ambient conditions (liquid or air). It works much
like an old-fashioned record player by scanning a sharp tip
across a surface, mapping three-dimensional topographical
features and nanomechanical stiffness simultaneously [6].
AFM has been applied to study live cells and cell fragments
[7], resolved nanoscale features in cell wall and membrane
organization [8,9], and single spores [10]. Until recently, one
of the bottlenecks has been the long acquisition time: AFM
typically takes several minutes to capture a scan, which is slow
compared with the life cycle of some bacteria. However, the
recent breakthrough in the development of high-speed AFM
using miniaturized cantilevers and multi-harmonic methods
to image live bacteria with nanometre resolution and large
enough scan sizes to image several cells simultaneously. In
2010, Belcher and co-workers demonstrated the first use of
this technology to directly track the kinetics of drug action of
antimicrobial peptides on E. coli [11]. These studies revealed
drug activity within 1 min, but considerable heterogeneity
even on cells cloned from single colony, and identified a
two-step killing process: (i) a time-variable incubation phase
(which takes seconds to minutes), and (ii) a more rapid
execution phase. Multi-harmonic imaging methods recently
developed by Contera and co-workers can probe local
properties of cells using the zeroth, first and second harmonic
components of the Fourier spectrum of the AFM cantilevers
interacting with the cell surface, deriving local stiffness,
stiffness gradient and the viscoelastic dissipation of live
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compared with quasi-static AFM [12]. The advantage of
the latter approach over miniaturized cantilevers is that it
is amenable to conventional AFMs and therefore likely to
become more widely available in the near future.
Nanomechanical mapping of single
bacteria
In the force-spectroscopy mode of the AFM, cantilever
deflection is monitored as a function of the vertical
displacement of the sample, i.e. the tip is pushed towards
and away from the sample. The indentation part of the
curve provides a direct measure of the local stiffness of
the bacteria. Nanomechanical measurements of Myxococcus
xanthus, for example, have shown that cell wall stiffness
varies significantly across the cell and extracellular polymeric
substances [13]. Another mode of force spectroscopy
measures the characteristic adhesion between the AFM tip
and the sample to map chemical groups and receptor sites
on the surface of bacteria with single-molecule sensitivity.
For example, Dufrene and co-workers used AFM tips
functionalized with heparin-binding haemagglutinin adhesin
to measure specific binding forces of individual adhesins
and map their distribution on the surface of Mycobacterium
smegmatis, and found that the adhesins are concentrated
into nanodomains [14]. Dufrene and co-workers have also
developed AFM tips coated with the antibiotic vancomycin
to map individual receptor sites on the division septum of
living Lactococcus lactis bacteria [15].
To date, most force-mapping studies have focused on
the outer cell wall; exciting recent work by Shaevitz and
co-workers revealed for the first time the mechanical role
of internal cytoskeletal filaments [16]. Using force-sensing
methods based on optical traps, they investigated the
mechanical role of the actin analogue MreB using the inhibitor
A22, and found that MreB contributes up to 50 % of a
cell’s global stiffness [16], findings which are set to challenge
the long-accepted view that the ‘rigid shell’ of the cell wall
primarily determines bacterial mechanics.
Nanomechanical biosensors
In recent years, the key force-transducing element of the
AFM, the cantilever, has evolved beyond the well-established
realm of imaging to innovative point-of-care nanosensor
applications for biomedical analysis and disease diagnosis
(Figure 1). By tailoring the cantilever with a capture layer, it is
possible to study reactions with different analytes in solution
by measuring changes in surface stress and/or resonance
[17–36]. The major advantage of cantilever sensors is that
they are label-free sensors that do not require complex
sample labelling or amplification steps, which are timeconsuming and costly, and can affect the conformation
of biomolecules, thereby allowing rapid sensitive detection
with high specificity using in situ reference cantilevers.
Bionanotechnology III: from Biomolecular Assembly to Applications
Figure 1 Nanomechanical detection of vancomycin–cell wall peptide interactions on multiple cantilevers
(a) Schematic diagram of cantilevers coated with lysine-d-alanine-d-alanine, lysine-d-alanine-d-lactate or PEG [poly(ethylene
glycol)] alkanethiol monolayers. Vancomycin is injected into solution and binds specifically to the cell wall analogues,
causing the cantilever to bend downwards owing to a compressive stress. (b) The chemical interaction between vancomycin
and the bacterial cell wall analogue. It is known from solution data [37] that the specificity of this complex arises due to (i) the
interaction of the C-terminus free carboxy group of the peptide with the three amide bonds in the vancomycin backbone; (ii)
the formation of two C=O···H-N hydrogen bonds; and (iii) hydrophobic interactions of alanine methyl groups with aromatic
residues of vancomycin. The broken lines represent five intermolecular hydrogen bonds. The yellow broken line represents
the hydrogen bond associated with bacterial resistance. (c) The deletion of a single hydrogen bond in mutated d-lactate
cell wall peptides gives rise to drug-resistance. The binding pocket of vancomycin is represented schematically and the
grey broken line represents the deleted hydrogen bond and electrostatic repulsion between oxygen lone pairs of electrons.
Reprinted by permission from Macmillan Publishers Ltd: Nature Nanotechnology, Ndieyira, J.W., Watari, M., Barrera, A.D.,
Zhou, D., Vogtli, M., Batchelor, M., Cooper, M.A., Strunz, T., Horton, M.A., Abell, C., Rayment, T., Aeppli, G. and McKendry, R.A.
(2008) Nanomechanical detection of antibiotic mucopeptide binding in a model for superbug drug resistance, 3, 691–696.
c 2008.
Cantilever fabrication is also immediately compatible with
silicon-processing methods and device integration for lowcost nanosensor applications. Moreover, cantilever sensors
are uniquely able to measure changes in the mechanics of
adsorbed species, in contrast with competing technologies
such as surface plasmon resonance, and are therefore uniquely
suited to study the mechanics of bacteria and the workings of
antibiotics [3].
There are essentially two main modes of operation: (i)
the static mode which measures changes in bending due to
surface stress (see below), and (ii) the dynamic mode where
the cantilever is driven to oscillate close to its resonance
frequency to serve as a microbalance. Early studies of bacteria
on cantilevers were confined to a vacuum [23], but in
2005 Hegner and co-workers demonstrated the power of
cantilever mass sensors to detect live bacteria using arrays
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coated with selective-growth medium to test susceptibility
to different antibiotics in real-time with the sensitivity to
detect thousands of cells [26]. This approach offers major
advantages in terms of speed and sensitivity compared with
conventional microbiological methods that requires more
than 24 h for sufficient colonies of bacteria to grow on Petri
dishes in specialist laboratories with trained staff. Tamayo
and co-workers later showed that the dynamic cantilever
signal is convoluted by changes in added mass and stiffness
of adsorbates [27], whereas Hoogenboom, together with my
group at UCL, have shown that we can decouple these effects
by nanopatterning the cantilever, which could provide future
opportunities to study the mechanics of bacteria in response
to different antibiotics [28].
Boisen and Thundat [24] and Roukes and co-workers [25]
have shown that the sensitivity of these devices is dependent
on the dimensions and material of the cantilever itself, but,
in liquid, the dynamic signal is heavily damped, resulting in
low quality factors. To overcome this limitation, Manalis and
co-workers have developed novel types of hollow cantilever
sensors where cantilevers have an embedded microfluidic
channel inside the beam, allowing the device to be operated
in a vacuum with high-quality factors [28]. Remarkably, they
have shown the sensitivity to weigh individual E. coli cells
(110 fg) and Bacillus subtilis (150 fg) bacteria in aqueous
environments [29]. This approach shows enormous promise
for both basic and applied research.
The surface-stress mode of operation has been the most
sensitive way to detect small-molecule interactions, and my
team at UCL have exploited this inherent sensitivity to
probe a range of biomolecular interactions, including DNA
and RNA detection with Gerber and co-workers [18,30],
plus molecular motors [31], self-assembled monolayers [32–
36], bacteria, HIV and cell detection. One of the key
breakthroughs of our work has been the development of
a unified multiscale multidimensional model to predict the
direction and magnitude of surface-stress signals on cantilever
sensors, work which spans across the traditionally distinct
modelling areas of materials science, solid-state physics,
organic chemistry and soft matter theory, and we find
remarkably strong agreement between experiment and theory
[33]. In the present paper, I highlight our work on vancomycin
[3], one of the most powerful drugs in the battle against
superbugs such as MRSA, where the deceptively simple
alteration of the cell wall structure from an amide to an ester
linkage confers vancomycin-resistance (Figure 1). Indeed,
VRE is the second leading superbug in U.S. hospitals and
is responsible for urinary tract, heart, wound and blood
infections, underlining the urgent need for new antibiotics
[37].
We are using multiple arrays of eight silicon cantilever
‘diving boards’ to examine the process which takes place
in the body when vancomycin binds to the outer bacterial
cell wall. Using cantilever arrays coated with mucopeptides
that mimic the cell wall in VSE (vancomycin-susceptible
Enterococcus) and VRE, we found that, as the antibiotic
attaches itself to the cell wall mucopeptides, it generates
C The
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Authors Journal compilation a surface stress which can be detected by a tiny bending
movement of the levers (illustrated in Figure 1). By measuring
the surface drug-target binding constants on the cantilever
arrays, we see that even tiny changes in drug-resistant cell
wall structures make it approximately 800-fold harder for
the antibiotic to attach itself on to VRE analogues, leaving it
much less able to disrupt the bacterial cell’s structure and
thus therapeutically ineffective. The relationship between
cantilever bending and the cell wall peptide surface density
was also investigated, and from this, a new surface-stress
model, based on percolation, was derived, which is dependent
on two factors: a chemical factor, and a geometric factor.
By varying the density of the underlying peptide film, a
critical threshold at a peptide coverage of ∼10 %, below
which there is no signal and above which surface stress
increased as a function of peptide density, was found. This
means is that the stress transduction is actually a collective
phenomenon, requiring a relatively large fraction of surface to
be covered, so as to establish chemical connectivity between
different regions where there are peptides bound to the
vancomycin. The surface stress build-up is therefore the
product of the connectivity of the chemically transformed
localized region and the interactions between nodes of the
entire network (Figure 2). These findings are leading to a
dramatically improved sensitivity of cantilever devices for
applications in functional drug discovery. The study also
led to the idea that this surface stress contributes to the
disruption of bacterial cell wall and the eventual breakdown
of the bacteria themselves [3]. Our current efforts focus on
investigating the mechanical mechanism of action of some of
the most promising new drugs in development with activity
against clinically problematic pathogens. We are testing the
hypothesis that vancomycin slowly weakens the cell over
24 h by forming point-defects, whereas dimeric drugs such
as oritavancin self-assemble into dimers forming extended
line-defects across bacteria, likened to a ‘San Andreas fault’,
leading to rapid cell death within 2 h (Figure 2).
Conclusions and future perspectives
The nanotechnology ‘toolkit’ offers new capabilities to
study the mechanical mechanism of action of antibiotics
on pathogens with nanometre spatial, piconewton force and
microsecond temporal resolution, and new frameworks based
on percolation are emerging to understand how these drugs
mechanically kill bacteria. The first mechanical investigations
of the role of bacterial cytoskeletal proteins are revealing the
major role of internal proteins on the global stiffness of cells,
challenging the long accepted view that bacteria are simply
‘empty sacks’ where the cell wall alone imparts mechanical
strength. More work is needed to study the spatiotemporal
dynamic interplay of the cell wall and cytoskeleton and
its role on local and global cell stiffness during the cell
cycle. As this powerful technology becomes more amenable
to microbiologists, research will move beyond proof-ofprinciple experiments on model bacteria and antibiotics to
detailed understanding of the systems biology of clinically
Bionanotechnology III: from Biomolecular Assembly to Applications
Figure 2 The concept of percolation on (left) cantilevers and (right) bacteria
Below the critical threshold, pc , bound complexes are isolated and no stress is detected. However, above pc , sites become
connected, leading to large-scale stressed network connections on cantilevers. We speculate that this percolation concept may
also aid our understanding of the mode of action of antibiotics on real bacteria where molecules ‘team up’ to mechanically
weaken bacteria, forming point-defects or extended line-defects, leading to cell rupture and combating infections. Reprinted
by permission from Macmillan Publishers Ltd: Nature Nanotechnology, Ndieyira, J.W., Watari, M., Barrera, A.D., Zhou, D.,
Vogtli, M., Batchelor, M., Cooper, M.A., Strunz, T., Horton, M.A., Abell, C., Rayment, T., Aeppli, G. and McKendry, R.A. (2008)
c 2008.
Nanomechanical detection of antibiotic mucopeptide binding in a model for superbug drug resistance, 3, 691–696. problematic pathogens, using well-established antibiotics as
tools to unravel the complexity of bacteria and testing
the mechanisms of action of new antibiotics currently in
development. In turn, through a closer collaboration between
the microbiology and physical science communities, new
nanotools are likely to emerge; for example, the integration
of high-speed AFM with fluorescence imaging will open
exciting opportunities to track the spatiotemporal dynamics
of cytoskeletal proteins, dynamic cell wall biosynthesis
and local mechanics, and the development of mechanical
devices and microfluidics to routinely study the effects of
external mechanical stresses on bacteria and coherent X-ray
diffraction to map three-dimensional strains [36]. Moreover,
the development of low-cost devices to detect pathogens
with such high sensitivity may lead to early-warning disease
diagnosis, resulting in better stratified treatments, improving
patient quality of life and preventing the spread of infection
in our society.
Funding
This work was funded by the Engineering and Physical Sciences Research Council [grant numbers EP/G062064/1, EP/D505925/1 and
EP/G068437/1], Biotechnology and Biological Sciences Research
Council, Interdisciplinary Research Collaboration in Nanotechnology
(Cambridge, Bristol and UCL) and the Royal Society.
References
1 Hopwood, D.A. (2007) A call to arms. Nat. Rev. Drug Discovery 6, 8–12
2 Cooper, M.A. and Shlaes, D. (2011) Fix the antibiotics pipeline. Nature
472, 32
3 Ndieyira, J.W., Watari, M., Barrera, A.D., Zhou, D., Vogtli, M., Batchelor,
M., Cooper, M.A., Strunz, T., Horton, M.A., Abell, C. et al. (2008)
Nanomechanical detection of antibiotic mucopeptide binding in a model
for superbug drug resistance. Nat. Nanotechnol. 3, 691–696
4 Jones, L.J.F., Carballido-Lopez, R. and Errington, J. (2001) Control of cell
shape in bacteria: helical, actin-like filaments in Bacillus subtilis. Cell
104, 913–922
5 Fletcher, D.A. and Mullins, R.D. (2010) Cell mechanics and the
cytoskeleton. Nature 463, 485–492
6 Binnig, G.K., Quate, C.F. and Gerber, C. (1986) Atomic force microscope.
Phys. Rev. Lett. 56, 930–933
7 Dufrene, Y.F. (2008) Towards nanomicrobiology using atomic force
microscopy. Nat. Rev. Microbiol. 6, 674–680
8 Muller, D.J., Baumeister, W. and Engel, A. (1999) Controlled unzipping of
a bacterial surface layer with atomic force microscopy. Proc. Natl. Acad.
Sci. U.S.A. 96, 13170–13174
9 Hayhurst, E.J., Kailas, L., Hobbs, J.K. and Foster, S.J. (2008) Cell wall
peptidoglycan architecture in Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A.
105, 14603–14608
10 Plomp, M., Leighton, T.J., Wheeler, K.E., Hill, H.D. and Malkin, A.J. (2007)
In vitro high-resolution structural dynamics of single germinating
bacterial spores. Proc. Natl. Acad. Sci. U.S.A. 104, 9644–9649
11 Fantner, G.E., Gray, D.S. and Belcher, A.M. (2010) Kinetics of
antimicrobial peptide activity measured on individual bacterial
cells using high-speed atomic force microscopy. Nat. Nanotechnol. 5,
280–285
12 Raman, A., Trigueros, S., Cartagena, A., Stevenson, A.P., Susilo, M.,
Nauman, E. and Contera, S.A. (2011) Mapping nanomechanical
properties of live cells using multi-harmonic atomic force microscopy.
Nat. Nanotechnol. 6, 809–814
13 Pelling, A.E., Li, Y.N., Shi, W.Y. and Gimzewski, J.K. (2005) Nanoscale
visualization and characterization of Myxococcus xanthus cells with
atomic force microscopy. Proc. Natl. Acad. Sci. U.S.A. 102, 6484–6489
14 Dupres, V., Menozzi, F.D., Locht, C., Clare, B.H., Abbott, N.L., Cuenot, S.,
Bompard, C., Raze, D. and Dufrene, Y.F. (2005) Nanoscale mapping and
functional analysis of individual adhesins on living bacteria. Nat.
Methods 2, 515–520
15 Gilbert, Y., Deghorain, M., Wang, L., Xu, B., Pollheimer, P.D., Gruber, H.J.,
Errington, J., Hallet, B., Haulot, X., Verbelen, C. et al. (2007)
Single-molecule force spectroscopy and imaging of the
vancomycin/D-Ala-D-Ala interaction. Nano Lett. 7, 796–801
C The
C 2012 Biochemical Society
Authors Journal compilation 607
608
Biochemical Society Transactions (2012) Volume 40, part 4
16 Wang, S.Y., Arellano-Santoyo, H., Combs, P.A. and Shaevitz, J.W. (2010)
Actin-like cytoskeleton filaments contribute to cell mechanics in bacteria.
Proc. Natl. Acad. Sci. U.S.A. 107, 9182–9185
17 Fritz, J., Baller, M.K., Lang, H.P., Rothuizen, H., Vettiger, P., Meyer, E.,
Guntherodt, H.J., Gerber, C. and Gimzewski, J.K. (2000) Translating
biomolecular recognition into nanomechanics. Science 288, 316–318
18 McKendry, R., Zhang, J.Y., Arntz, Y., Strunz, T., Hegner, M., Lang, H.P.,
Baller, M.K., Certa, U., Meyer, E., Guntherodt, H.J. and Gerber, C. (2002)
Multiple label-free biodetection and quantitative DNA-binding assays on
a nanomechanical cantilever array. Proc. Natl. Acad. Sci. U.S.A. 99,
9783–9788
19 Braun, T., Ghatkesar, M.K., Backmann, N., Grange, W., Boulanger, P.,
Letellier, L., Lang, H.P., Bietsch, A., Gerber, C. and Hegner, M. (2009)
Quantitative time-resolved measurement of membrane protein–ligand
interactions using microcantilever array sensors. Nat. Nanotechnol. 4,
179–185
20 Backmann, N., Zahnd, C., Huber, F., Bietsch, A., Plückthun, A., Lang, H.P.,
Güntherodt, H.J., Hegner, M. and Gerber, C. (2005) A label-free
immunosensor array using single-chain antibody fragments. Proc. Natl.
Acad. Sci. U.S.A. 102, 14857–14592
21 Ghatkesar, M.K., Barwich, V., Braun, T., Ramseyer, J.P., Gerber, C.,
Hegner, M., Lang, H.P., Drechsler, U. and Despont, M. (2007) Higher
modes of vibration increase mass sensitivity in nanomechanical
microcantilevers. Nanotechnology 18, 445502
22 Mertens, J., Rogero, C., Calleja, M., Ramos, D., Martin-Gago, J.A., Briones,
C. and Tamayo, J. (2008) Label-free detection of DNA hybridization
based on hydration-induced tension in nucleic acid films. Nat.
Nanotechnol. 3, 301–307
23 Ilic, B., Czaplewski, D., Zalalutdinov, M., Craighead, H.G., Neuzil, P.,
Campagnolo, C. and Batt, C. (2001) Single cell detection with
micromechanical oscillators. J. Vac. Sci. Technol. B 19, 2825–2828
24 Boisen, A. and Thundat, T. (2009) Design and fabrication of cantilever
array biosensors. Mater. Today 12, 32–38
25 Arlett, J.L., Myers, E.B. and Roukes, M.L. (2011) Comparative advantages
of mechanical biosensors. Nat. Nanotechnol. 6, 203–215
26 Gfeller, K.Y., Nugaeva, N. and Hegner, M. (2005) Micromechanical
oscillators as rapid biosensor for the detection of active growth of
Escherichia coli. Biosens. Bioelectron. 21, 528–533
27 Ramos, D., Tamayo, J., Mertens, J., Calleja, M., Villanueva, L.G. and
Zaballos, A. (2008) Detection of bacteria based on the
thermomechanical noise of a nanomechanical resonator: origin of the
response and detection limits. Nanotechnology 19, 035503
C The
C 2012 Biochemical Society
Authors Journal compilation 28 Gruter, R.R., Khan, Z., Paxman, R., Ndieyira, J.W., Dueck, B., Bircher, B.A.,
Yang, J.L., Drechsler, U., Despont, M., McKendry, R.A. and Hoogenboom,
B.W. (2010) Disentangling mechanical and mass effects on
nanomechanical resonators. Appl. Phys. Lett. 96, 023113
29 Burg, T.P., Knudsen, S.M., Shen, W., Carlson, G., Foster, J.S., Babcock, K.
and Manalis, S.R. (2007) Weighing of biomolecules, single cells and
single nanoparticles in fluid. Nature 446, 1066–1069
30 Zhang, J., Lang, H.P., Huber, F., Bietsch, A., Grange, W., Certa, U.,
McKendry, R., Hegner, M. and Gerber, C. (2006) Rapid and label-free
nanomechanical detection of biomarker transcripts in human RNA. Nat.
Nanotechnol. 1, 214–220
31 Shu, W.M., Liu, D.S., Watari, M., Riener, C.K., Strunz, T., Welland, M.E.,
Balasubramanian, S. and McKendry, R.A. (2005) DNA molecular motor
driven micromechanical cantilever arrays. J. Am. Chem. Soc. 127,
17054–17060
32 Watari, M., Galbraith, J., Lang, H.P., Sousa, M., Hegner, M., Gerber, C.,
Horton, M.A. and McKendry, R.A. (2007) Investigating the molecular
mechanisms of in-plane mechanochemistry on cantilever arrays. J. Am.
Chem. Soc. 129, 601–609
33 Sushko, M.L., Harding, J.H., Shluger, A.L., McKendry, R.A. and Watari, M.
(2008) Physics of nanomechanical biosensing on cantilever arrays. Adv.
Mater. 20, 3848
34 Watari, M., Ndieyira, J.W. and McKendry, R.A. (2010) Chemically
programmed nanomechanical motion of multiple cantilever arrays.
Langmuir 26, 4623–4626
35 Ransley, J.H.T., Watari, M., Sukumaran, D., McKendry, R.A. and Seshia,
A.A. (2006) SU8 bio-chemical sensor microarrays. Microelectron. Eng.
83, 1621–1625
36 Watari, M., McKendry, R.A., Vogtli, M., Aeppli, G., Soh, Y.A., Shi, X.W.,
Xiong, G., Huang, X.J. and Robinson, I.K. (2011) Differential stress
induced by thiol adsorption on facetted nanocrystals. Nat. Mater. 10,
862–866
37 Williams, D.H. and Bardsley, B. (1999) The vancomycin group of
antibiotics and the fight against resistant bacteria. Angew. Chem. Int. Ed.
38, 1173–1193
Received 14 March 2012
doi:10.1042/BST20120082