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Current Research Highlights
Multifunctional cantilever arrays: In our group we currently use multifunctional cantilever array
technology to measure with eight sensors in parallel
(see also:
monet.physik.unibas.ch
or
www.zurich.ibm.com).
The experiments we performed are all label-free and provide high-sensitivity results on molecules which are
involved in cellular processes (5, 10–12). In genomic analysis we can now detect within a complete RNA sample
whether a gene is present or not. Single nucleotide polymorphism (SNP) detection has been demonstrated earlier
(10) and we can now also measure whether a gene has been turned on (i.e. production of increased amounts of
specific mRNA) upon extra-cellular interactions of small molecules with cell membrane receptors. Such
measurements characterize a cellular system, which is first in a ‘normal’ state, and thereafter in a
transiently perturbed state. Triggering of the gene is only enabled during the time the small molecule is
present in the supernatant of the investigated cells. The induced RNA signal corresponds to a differential
nanometer deflection signal with respect to a reference cantilever placed in situ. We could show that the
measured surface stress upon hybridization of specifically bound nucleic acids correlates in a linear manner
with the concentration of the molecules present in the environment to be probed (11). The sensitivity of our
sensors currently equals that of current gene chip technology, but our key advantage is that there is no need
for amplification of the material nor for optical labeling of the molecules involved. These studies have been
conducted in collaboration with U. Certa of the Roche Medical Center of Genomics at Basel. Figure 4 shows a
scheme of the multifunc-tional cantilever arrays. We also measure transcription activation nanomechanically
by binding transcription factors to DNA targets at the interface of our cantilever sensors. In the field of
proteomics (12) our sensors were considerably improved during the last year by using special receptor
fragments such as single chain variable antibody fragments or ankyrins (13) in collaboration with
group A. Plückthun at the University of Zürich.
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Figure 4: Multifunctional cantilever allow differential label free detection of transcribed
RNA fragments within total RNA. The binding detection of transcription factors to dsDNA, soluble
protein binding to antigen activated interfaces and tracking conformational changes in membrane proteins.
Such sensors – operated in dynamic mode - are able to follow microorganism growth in real time and to
investigate antibiotic susceptibility within a couple of hours. |
The sensitivity of our new sensors is enhanced by a factor of 100 as compared to previously published
results (13) and lies now in a regime comparable to current surface plasmon resonance technology. By
using ankyrin receptor molecules we shall be able to screen libraries of precursors and optimize our
receptors for soluble proteins. Analysis on the cellular level will also involve interactions on the cell
membrane. Our membrane protein sensitized cantilevers are able to convert interactions on the cell membrane
into nanomechanically measurable signals. The beauty of nano-mechanical sensors is that these sensors are
transducing conformational changes in the membrane protein upon ligand binding into a mechanical deflection.
Furthermore, the binding of the ligand (e.g. mass change) can be tracked by measuring the shift in resonance
frequency in situ. These features are not available in parallel using competing techniques. The only parameter
that changes when investigating genomics or proteomics applications is the receptors placed on our sensor
interfaces (e.g. DNA sequences, protein epitops).
A rapid-response biosensor for the detection of microorganism growth was developed using micromechanical
oscillators coated by common nutritive layers (“shrinkage of a centimeter-sized Petri dish down to
micron-sized cantilevers”). The change in resonance frequency as a function of increasing mass on a
cantilever array represents the basis of the detection scheme. The sensor is able to detect active growth
of E. coli cells within 1 hour (14) or the growth of microfungi in ~ 4 hours (15), which is considerably
faster than in conventional assay where such tests take ~ 8–12 hours (E. coli) or up to weeks (microfungi).
Furthermore, this method allows detection of selective growth of E. coli within only two hours by adding
antibiotics to the nutritive layers (16). This new sensing method for the detection of selective bacterial
growth allows future applications in e.g. rapid antibiotic susceptibility testing.
As obvious from the above mentioned examples pursued in our group, the cantilever array technology could
provide a new approach for a new hand-held label-free nano-mechanical cantilever-diagnostics devices for
rapid parallel diagnosis of multiple pathogens causing diseases (e.g. measurement of gene activation,
binding of antigens to selected multiple target-specific synthetic ankyrin repeat proteins as sensors and
selective susceptibility detection of microorganisms). Such a novel device could provide a tool for the
comprehensive and fast disease management at the point of care. Just from the diversity of the projects
pursued in our group it is obvious that without an interdisciplinary approach such tasks could never be
tackled.
The long-term scope of our future projects will focus on merging single molecule sensitivity (achieved in
the first two types of experiments conducted in our group) with parallel measurements and multifunctional
cantilever array technology by downscaling of measurement sensors and periphery and other adaptations.
Additional information upon our activities can be found in ref (17).
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