Microcantilevers to lift biomolecules
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three-dimensional origami scaffold. Each scaffold featured custom patterns
of single-stranded DNA tails that were complementary to the 9-base strands on the polymer. The two-dimensional rectangular scaffold, or ‘breadboard’, was endowed with tails that created tacking points for several one-dimensional paths, including a line, an L-shape, a U-shape, a wave, a staircase, and a circle. Direct imaging with AFM revealed that the DNA-labelled PPV polymer self-assembled successfully onto all of these patterns, but the yield of assembly decreased with increased degree of curvature of the target path, presumably due to the energetic penalty of creating bends on paths with high curvature such as the wave-like or staircase paths. The three-dimensional scaffold
had a cylindrical shape, with a height of 100 nm and a diameter of 60 nm. Again, single-stranded DNA tacking points were included at custom positions and were used to template a 360° right-handed helical path for the polymer. To reveal the correct assembly of the polymer into the helical path, the team used a modality of super-resolution fluorescence microscopy called DNA PAINT.
The high-density modification of the PPV polymer variant with DNA labels may affect its physical properties such as
its electroluminescence. Although Gothelf
and colleagues did not directly study
these properties, they do provide data that
suggest that the polymer retains some
of its interesting physical properties. For
example, surface potential measurements
with electrostatic force microscopy indicated
that the capability of charge transfer at the
interface of polymer/DNA is significantly
better than for DNA alone. Furthermore,
when the researchers attached a single
Alexa 647 acceptor dye molecule on one
of the origami staple strands that binds
to the polymer on the two-dimensional
DNA rectangle with the U-like shape, a
20× enhancement of the emission of the
acceptor was observed, which may be a
consequence of near-field enhancement
produced by the conjugated polymer.
For future use in electronics or other
applications, several challenges will have to
be addressed. Stacking multiple modified
DNA origami scaffolds in a layer-by-layer
fashion3, each with its particular polymer
routing and potentially including other
active components, may be a way to create
three-dimensional integrated nanoscale
circuits (Fig. 1, left). Important progress has
been made in placing DNA objects with
user-defined long-range order onto solid-
state surfaces4, which is a necessary step for
integrating such components into a device
context. But will DNA nanostructures retain
their shape when taken out of solution and
into a dry electronics environment? Some
type of petrification protocol may need to
be developed. Furthermore, Gothelf and
colleagues note that degradation of the
polymer by cleavage into shorter pieces
occurred during purification of their
synthesis products, and therefore it was
not possible to prepare polymers with
monodispersity. This finding hints at an
increased fragility of the DNA-modified PPV
variant, and this will be an important issue to
resolve if applications are to emerge. ?
Hendrik Dietz is in the Physik Department and
Walter Schottky Institute, Technische Universit?t
München, Am Coulombwall 4a, Garching near
Munich 85748, Germany.
e-mail: dietz@tum.de
References
1. Knudsen, J. B. et al.Nature Nanotech. 10, 892–898 (2015).
2. Rothemund, P. W. K. Nature440, 297–302 (2006).
3. Gerling, T., Wagenbauer, K. F., Neuner, A. M. & Dietz, H. Science
347, 1446–1452 (2015).
4. Gopinath, A. & Rothemund, P. W. K. ACS Nano
8, 12030–12040 (2014).
BIOSENSORS
Microcantilevers to lift biomolecules
Nanomechanical sensors can now detect femtomolar concentrations of analytes within minutes without the need to passivate the underlying cantilever surface.
Gajendra S. Shekhawat and Vinayak P. Dravid
A rchimedes, the Greek mathematician,
is once believed to have said “Give
me a lever long enough and a place to stand, I will lift the earth.” Today, this famous quote could be paraphrased
as ‘Give me a lever small enough, and
I will ‘weigh’ molecules.’ Microscale versions of Archimedes levers, called microcantilevers, have been used in several forms to lift or strain molecular-scale structures by invoking nanomechanical signal transduction. Specific binding of analytes to their complementary partners on the cantilever surface induces lateral surface stress, resulting in bending of the free end of the cantilever. The sensitivity
of the assay is measured by how tiny a bend one can detect using lasers, whereas the selectivity is governed by specificity
of the complementary partner for the analyte. Microcantilevers have been used
to study various biological interactions1–6,
for quantifying antibiotics in blood serum7
and for detecting bacterial resistance to
antibiotics8 with excellent specificity and
sensitivity. However, cantilevers are limited
by non-specific binding at the bottom
surface, which may negate the specific
binding that occurs at the top surface.
Passivation may resolve these issues but it
requires tedious optimization. Writing in
Nature Nanotechnology, Joseph Ndieyira
and colleagues at University College
London, Imperial College London and
Jomo Kenyatta University of Agriculture and
Technology now show that by controlling
the concentration of the receptor footprint
on the cantilever surface, it is possible to
overcome competing stresses from opposing
cantilever surfaces9. This approach allows
direct capture of molecules at femtomolar
concentrations within minutes without
the need to passivate the underlying
cantilever surface.
Human immunodeficiency virus (HIV)
mutates very easily resulting in many
different strains even within the body of
a single infected person. Several methods
have been proposed for the detection of
HIV but they are typically complicated
and not amenable to point-of-care
diagnostics. Ndieyira and co-workers used
microcantilevers to nanomechanically detect
HIV. The cantilevers were functionalized
with single-domain antibodies that are
specific to HIV. Binding of HIV to the
antibodies on the cantilever induces a lateral
surface stress and this causes the cantilever
to bend. The assay was shown to reliably
detect HIV down to about 500 fM, which is
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almost five orders of magnitude better than
surface plasmon resonance techniques.
One of their noteworthy findings is
that the cantilevers show minimal bending
signals when the receptor footprint on
the cantilever surface is about 30%.
However, when the receptor concentration
is increased to 100%, the surface density
of the receptor is maximized and this
yields the highest surface stress signal.
This means that although the number of
ligand–receptor interactions increases with
surface coverage, there is a threshold in
the surface footprint that needs to be met
to generate a mechanical response. Their
approach decouples the binding kinetics of
the surface reactions by tuning the capture
ligand–receptor concentration. These results
provide a new framework for understanding
and engineering the mechanical response
to biochemical interactions without the
need to passivate the underlying surface of
the microcantilevers.
Detection of microcantilever bending
using conventional lasers and photodiodes
has limitations for point-of-care use and
wide deployment in rural areas. To solve this,
all-electronic detection methods based on
piezoresistive, capacitive and metal–oxide–
semiconductor field-effect transistors10 are
currently being developed (Fig. 1). These
methods are more suitable for point-of-care
use because they are readily amenable to
massively parallel and multiplexed sensing,
and are compatible with microfluidics.
Furthermore, these methods enjoy all the
advantages of the classical microelectronics platform — such as remote addressability, networking capabilities, clocking the measurements at high frequencies and built-in photovoltaic or other similar power supply — thereby allowing the sensor systems to be miniaturized.
One of the benefits of these nanomechanical assays is that each (or
an array of) microcantilever(s) can be functionalized with separate capture receptors (Fig. 1). This would improve specificity and allow multiplexed detection of clinically relevant analytes in a single sensor system. These developments are promising for personalized medicine and point-of-care diagnosis because specific mutants and gene-specific biomarkers may be rapidly detected using patient samples. Furthermore, advances in on-chip readout technology4,10 mean that parallel sensing of multiple mutations may be possible. The microcantilever assay technology can undoubtedly simplify and speed up the identification of diseases so they can be managed earlier and more effectively. Regardless of the nature of probe–target binding and signal transduction, there are fundamental challenges to any
‘substrate-based’ sensor systems. They
require highly selective receptors for
specific binding to target analytes, which
is not always easy to obtain. Furthermore,
sample handling (for example, splicing of
cells and concentrators) and downstream
signal detection, display and power supply
make integration of the overall system
challenging under the constraints of cost,
weight, power requirements and related
practical deployment issues. Moreover,
these systems must be amenable to sensing
in physiological fluidic environments,
where flow rate, viscosity and turbidity of
fluids may compromise the consistency
of the data. Clearly, as shown by Ndieyira
and colleagues for the specific cases of
HIV and vancomycin, improved specificity
of receptors, appropriate immobilization
approaches and the introduction of
identical cantilevers to ensure reproducible
responses are obtained would further
enhance selectivity.
Given the rapid advances in
microfabrication and miniaturization, it may
be possible to utilize such nanomechanical
systems for in vivo monitoring of essential
biomarkers coupled to timed therapeutic
release — all in a potentially single
integrated implanted system. Archimedes
would have been proud to see the cantilever
and mechanics put to good use for
human health. ?
Gajendra S. Shekhawat and Vinayak P. Dravid
are in the Department of Material Science and
Engineering and the NUANCE Center, Northwestern
University, Evanston, Illinois 60208, USA.
e-mail: g-shekhawat@https://www.wendangku.net/doc/c0944408.html,;
v-dravid@https://www.wendangku.net/doc/c0944408.html,
References
1. Fritz, J. et al.Science288, 316–318 (2000).
2. Wu, G. et al.Proc. Natl Acad. Sci. USA 98, 1560–1564 (2001).
3. Zhang, J. et al.Nature Nanotech. 1, 214–220 (2006).
4. Shekhawat, G. S., Tark, S. & Dravid, V. P. Science
311, 1592–1595 (2006).
5. Huber, F., Lang, H. P., Backmann, N., Rimoldi, D. & Gerber, C.
Nature Nanotech. 8, 125–129 (2013).
6. Kosaka, P. M. et al.Nature Nanotech. 9, 1047–1053 (2014).
7. Ndieyira, J. W. et al.Nature Nanotech. 9, 225–232 (2014).
8. Ndieyira, J. W. et al.Nature Nanotech. 3, 691–696 (2008).
9. Patil, S. B. et al. Nature Nanotech. 10, 899–907 (2015).
10. Mustafa, S. IEEE Electron Dev. Lett.32, 408–410 (2011).
Published online:
17 August 2015
Figure 1 | Illustration of multiplexed cantilever system with on-chip readout technology. Parallel arrays
of cantilevers with embedded metal–oxide–semiconductor field-effect transistors (MOSFET s, black and green regions) are functionalized with different receptors (shown as brown squiggly lines and purple
‘Y’ shapes). Next to each functionalized cantilever is a reference cantilever that is not functionalized. When complementary ligand partners bind to the receptors, surface stress causes the cantilever to bend. The deflection, which is typically tens of nanometres, is measured using electronic readout from stress-induced changes in the MOSFET drain current. This integrated approach offers very low noise, high sensitivity and multiplexed direct readout. Such a tool could be used in personalized healthcare and deployed as a point-of-care diagnostic4.