17 February 2015
SWISS MED. WEEKLY: CANCER DETECTION by NANOSENSORS.
Review article | Published 9 February 2015, doi:10.4414/smw.2015.14092
Cite this as: Swiss Med Wkly. 2015;145:w14092
Cite this as: Swiss Med Wkly. 2015;145:w14092
Nanosensors for cancer detection
a SNI, Swiss Nano Institute, Institute of Physics, University of Basel, Basel, Switzerland
b Ludwig Center for Cancer Research of the University of Lausanne, Épalinges VD, Switzerland
b Ludwig Center for Cancer Research of the University of Lausanne, Épalinges VD, Switzerland
Summary
Cancer is a major burden in today’s
society and one of the leading causes of death in industrialised
countries. Various avenues for the detection of cancer exist, most of
which rely on standard methods, such as histology, ELISA, and PCR. Here
we put the focus on nanomechanical biosensors derived from atomic force
microscopy cantilevers. The versatility of this novel technology has
been demonstrated in different applications and in some ways surpasses
current technologies, such as microarray, quartz crystal microbalance
and surface plasmon resonance. The technology enables label free
biomarker detection without the necessity of target amplification in a
total cellular background, such as BRAF mutation analysis in malignant
melanoma. A unique application of the cantilever array format is the
analysis of conformational dynamics of membrane proteins associated to
surface stress changes. Another development is characterisation of
exhaled breath which allows assessment of a patient's condition in a
non-invasive manner.
Key words: microcantilever;
nanotechnology; cancer therapy; personalised healthcare; diagnostics;
proteomics; genomics; metabolomics; siRNA, transcription; lab-on-a-chip
Figure 1
Two frequently used modes of cantilever sensor operation. The first one (A) shows the static mode where surface stress results in cantilever bending. The second mode (B)
shows the operation in dynamic mode. Here the cantilever is actuated
and vibrates at its specific resonance frequency. Upon binding of a
ligand to the surface the resonance frequency changes and the adsorbed
mass can be calculated from that shift. The two modes can also be used
simultaneously. (C) Shows an electron microscope image of an array with eight 750 µm long cantilevers. (D)
Illustrates the liquid handling system and the beam deflection read out
of a cantilever setup. A total of 8 Vertical Cavity Surface Emitting
Lasers (VCSEL) are used to sequentially determine cantilever
deflections. The laser beam is deflected at the end of the cantilevers
to a Position Sensitive Detector (PSD). Below the cantilever array is a
peltier element which produces a heat pulse to test the mechanical
properties of the cantilevers. Syringe pump and multi valve systems
allow a constant flow of samples to the cantilever measurement chamber.
Figure 2
(A) The example
of DNA hybridisation to immobilised oligonucleotides illustrates the
molecular crowding, which creates a surface stress resulting in
cantilever bending. A differential deflection (Δx) can be measured with
the help of a reference cantilever. (B) Measurement of
total RNA from a BRAF mutated melanoma cell line (red) and from a cell
line (black) that does not contain the BRAF mutation. Injection is
indicated with a red and black bar at the bottom of the graph. The
measurement device is washed with buffer before and after injection. The
two cell lines can be clearly distinguished.
Figure 3
This figure illustrates a future
application for cantilever arrays, where cantilevers in a single array
are functionalised with a variety of biomolecules, ranging from
antibodies to double strand DNA to single strand DNA. In such a
configuration, it will be possible to investigate multiple markers with
one cantilever array.
Figure 4
(A) Principal
Component Analysis plot showing three distinct clusters (indicated with
ellipses) that represent healthy control persons, head and neck squamous
cell carcinoma (HNSCC) patients before surgery and HNSCC patients after
surgery, i.e. after removal of the tumour by surgery. The points of the
HNSCC patients after surgery are at a similar location in the PCA plot
as those from the healthy persons and differ clearly from the points of
the HNSCC patients before surgery, indicating that the removal of the
tumour has been successful. (B) Portable USB-powered
setup for mobile characterisation of patients' breath samples using
polymer-coated membrane surface stress sensors. From left to right:
Measurement system containing chamber with piezo membrane sensor array
and pumps for aspiration of sample; breath sample bags; netbook with
operating and analysis software.
The use of nanomechanical cantilevers goes back to the development of the atomic force microscope (AFM) [1],
where a cantilever with a sharp tip at one end is utilised to image
surfaces on the molecular and atomic scale. The surface of a sample is
probed mechanically whereby the motion of the tip is monitored by
recording the bending of the cantilever, by a laser beam reflected at
the cantilever surface. In a recent development, high speed AFM has
entered the time domain of chemical processes monitoring the cellular
machinery at the nanoscale and millisecond resolution, for example
visualisation of the movement of myosin on an actin filament in real
time [2]. Complementary to imaging, biomolecular interactions on the surface of a cantilever can be studied (fig. 1).
Such binding processes are analysed either by cantilever bending down
to the nanoscale due to changes in surface stress based on molecular
interactions on the cantilever surface (static mode, fig. 1A) [3],
or exploiting changes in resonance frequencies of a vibrating
cantilever due to increase in mass upon binding of biomolecules (dynamic
mode, fig. 1B) [4]. The dynamic mode is similar to the operation principle of a quartz crystal microbalance [5].
In recent years, arrays of cantilevers made of silicon or silicon nitride have been batch-fabricated (fig. 1C). Various methods to measure cantilever bending or vibration have been developed, among them laser beam deflection (fig. 1D) [6], interferometry [7] and the use of piezo resistive cantilevers [8].
Functionalisation by gold-thiol chemistry allows the formation of a
self-assembled monolayer (SAM) as a sensing layer. The bending of the
cantilever due to adsorption and molecular interactions between the SAM
and complementary molecules (fig. 2A) in solution is in the range of only a few nanometres. This was shown for the first time in DNA hybridisation experiments [9, 10],
where the binding of a 12 base long oligonucleotide at a concentration
of 80 nM to its complement immobilised on the cantilever surface was
studied with single base-pair specificity [9].
Further investigations focused on antibody antigen interactions on
cantilevers. These experiments showed sensitivity in the higher
nanomolar to micromolar range, when using whole antibodies (Ab)
functionalised on the surface [11].
A further improvement in antigen detection sensitivity was achieved by
using oriented single chain antibody fragments of the variable domain
(scFv), which increased the sensitivity about 500 times to low nanomolar
concentrations due to their smaller size and uniform orientation on the
cantilever surface [12].
Numerous applications lie in the study of structural changes in
proteins. This domain is particularly suited for static mode
measurements. Structural changes are often needed for protein activity,
for example to perform certain functions like enzymatic conversion or
binding of ligands. These changes can increase the surface stress in a
monolayer producing bending of a cantilever [13]. A combination of DNA and protein detection was used to investigate the binding of transcription factors [14].
Here a thiolated double stranded hairpin structured oligonucleotide
(dsOligo) was used to functionalise the gold surface of a cantilever.
The dsOligos contained binding sites specific for the transcription
factor NF-κB or SP1. Both transcription factors are important in
mediating various cellular responses and thus can play a role both in
cancer development [15] and in tumour response to treatment [16].
Cantilever bending occurred when a transcription factor recognised and
bound to its target sequence on the cantilever. The dynamic mode can be
applied to study virus adsorption [17].
Viruses can bind to surface immobilised ligands, resulting in a change
of resonance frequency, indicating a change of mass on the cantilever
surface.
An important step towards simplifying
cancer diagnostics with nanomechanical cantilevers was achieved recently
by analysing single point mutations in total RNA from melanoma cells [18] (fig. 2B). Malignant melanoma, the deadliest form of skin cancer, is characterised by a prevalent single point mutation in the BRAF
gene occurring in 50% of all cases. Highly specific drugs like
vemurafenib are now available that target this mutation, and therefore
patients have to be screened to determine their treatment eligibility.
This is important as these new drugs can cause severe side effects. BRAF is one of three RAF
genes (rapidly accelerated fibrosarcoma A, B and C) encoding cytoplasmic
protein serine/threonine kinases belonging to the mitogen-activated
protein kinase (MAPK) signal transduction cascade, a pathway controlling
various cellular processes such as proliferation, migration and
survival. There are different methods to detect the mutation, among them
is the long established procedure of polymerase chain reaction (PCR)
amplification coupled with Sanger sequencing of the product. The standard
test currently used to analyse patients' biopsies before initiation of
vemurafenib treatment relies on a real-time PCR-based assay, the so
called COBAS test [19].
Both approaches rely on amplification
and labelling of RNA or DNA extracted from melanoma cells. This is a
potential shortcoming because every additional modification, by
labelling or amplification, may disturb the original content of the
sample and consume additional time. Contrary to these procedures, total
RNA samples used for cantilevers do not have to be labelled and
amplification is not necessary, leaving the original sample undisturbed.
In Huber et al. [18]
different cantilevers were coated with thiol DNA oligonucleotides
representing the mutated gene, a reference sequence and the wild type
gene. The wild type gene serves as an additional reference as the
melanoma cells express the mutant as well as the wild type gene albeit
usually at a lower level. Thiol oligonucleotides covering the cantilever
are able to form a heteroduplex with the corresponding RNA in the
sample and thereby identify the BRAF mutation. The high sensitivity of
cantilevers always makes it necessary to include a reference cantilever
to avoid false positive responses. This can also be accomplished by
differentially coating the upper and the lower side of cantilevers [20]. The method described in Huber et al. [18]
was capable of distinguishing BRAF wild type cells from BRAF mutated
cells. In further experiments we applied total RNA from different
melanoma cell lines to evaluate the robustness of the method. Some of
these samples contained the prevalent BRAF mutation while others did not
carry the mutation or carried a different mutation in the same gene. We
also applied the method to investigate interferon treatment by
analysing mRNA from interferon treated melanoma cells [21]
in collaboration with U. Certa's group of the Roche Centre for Medical
Genomics. Interferon has powerful anti-proliferative properties and is
used in treatment of viral diseases and cancer. However, interferon
treatment also has severe side effects, which together with the
occurrence of resistance reduces its usefulness in cancer treatment [22].
A better insight in the molecular mechanisms behind these effects could
result in a better understanding of resistance to drugs and the
development of improved cancer treatments. For this purpose, they have
applied DNA microarrays with a set of probes to more than 11,000 human
transcripts to study the expression of interferon inducible genes in a
sensitive and resistant melanoma cell line. It was found that only few
genes were either up or down regulated and could be used as potential
markers to indicate tumour cell sensitivity or resistance to interferon.
The approach relied on the amplification and labelling of the RNA
extracted from the melanoma cells. As discussed previously this can be a
potential shortcoming. Samples used for cantilevers do not have to be
modified with flourophores for detection and in some cases, PCR
amplification is also not necessary, leaving the sample in its original
state. In Zhang et al. [21]
different cantilevers were coated with thiol oligonucleotides
representing one of the interferon induced genes, a so-called
housekeeping gene and a reference sequence. The housekeeping genes serve
as a positive control as this type of gene is necessary for the
survival of all cells and therefore are usually expressed at constant
levels. Zhang et al. [21]
clearly showed the induction of expression of an interferon inducible
gene, without sample labelling, though some amplification was still
necessary due to low levels of expression. In further experiments with
total cellular RNA the housekeeping gene, Aldolase A was detected
without amplification, labelling or modification of the cellular sample
at all. The latest development described in Huber et al. [18] above, shows that amplification is not necessary either.
The
sequencing of the entire human genome has revealed that about 22,000
genes encode proteins. This corresponds to about 1.5% of the whole
genome. As recently discovered, a small part of the 98.5% of the
non-coding DNA is involved in regulation of mRNA translation through the
expression of non-coding RNA sequences, including small interfering
RNAs (siRNAs). These species of RNA are able to interfere with mRNA,
thereby suppressing the translation or enhancing the degradation process
of specific mRNAs. The siRNAs are short RNA molecules of approximately
20 bases and should therefore be amenable to analysis with cantilevers.
Specific sets of siRNA are down or up-regulated in cancer cells, and the
use of siRNA in cancer therapy has been recently suggested [23]. Cantilever array technology could provide a possible way to monitor the efficiency of such therapies.
From the
many thousand genes analysed in microarray-based systems, a limited set
is often sufficient to define a particular subtype of cancer or is used
to guide therapeutic decisions. Cantilever arrays can provide a
snapshot of clinically relevant gene expression. For the future, we
foresee cantilever arrays functionalised with appropriate sets of
cancer-relevant genes to quickly and reliably analyse patient samples.
While the cantilever arrays highlighted in this review are one
dimensional and contain only 8 cantilevers, in some developments two
dimensional arrays of 8 × 8 cantilevers [24], lab-on-a-disc systems with many 100 cantilevers [25] are used. Current production technologies can produce larger arrays of 100 × 100 cantilevers [26], although so far these are used for data storage purposes and not for biosensensors.
Cancer is a complex disease and while
analysing differences in expression levels is important, these data
alone are not enough to follow the effectiveness of a therapy for
example, and the analysis of other cellular components and extracellular
markers is mandatory. The technology we describe in this review is able
to monitor cellular antigens (with the help of immobilised antibodies),
transcription factors (using as bait relevant DNA consensus sequences)
or RNAs (using specific complementary sequences). A study conducted by
Wu et al. [27]
using antibody functionalised cantilevers has shown that prostate
specific antigen (PSA) can be detected at clinically relevant
concentrations. PSA is an important marker in diagnosing prostate
cancer. This example and others described above show that a combination
of DNA, RNA and antibodies on a single array would be desirable (fig. 3). This way the genome and the proteome could be analysed with a single cantilever array.
The examples mentioned above show that
nanomechanical sensors are well in the sensitivity range for clinical
applications, such as the detection of PSA which is in the range of 0.2
ng/ml to 60 µg/ml [27], the identification of other medically relevant biomarkers [11, 12] down to 1 nM and the test for specific mutations in cancers [18]
down to 10 pM. Specifically the last example gives us an indication
that biopsies from patients can be investigated, for example the
distinction of the BRAFV600E mutation from wild type BRAF in melanoma
biopsies. Distinguishing BRAFV600E from BRAF also shows the high
specificity of our technology, because the BRAFV600E differs in a single
base (a T to A transversion at position 1799 of the gene) and we
accomplish this in total RNA extracted from cells.
Cantilever arrays can be applied not
only to liquid samples but also to gaseous samples, such as breath
samples of cancer patients. In this application a non-invasive
characterisation of breath samples can be achieved [28],
through the detection of volatile metabolites. Here cantilevers are
coated with different polymers that upon exposure to gaseous mixtures
are able to absorb volatile metabolites in a characteristic way. This
process will lead to polymer swelling and thereby produce a surface
stress creating a distinct response pattern for each sensor in the
array. Physicians have known for centuries that diseases indeed leave
traces of specific volatile organic compounds (VOC) in a patient's
breath [29].
Analysing these components with cantilever array technology can allow
the identification of various cancers, for example head and neck cancer
in a non-invasive way (fig. 4A).
Novel piezo-resistive membrane-type sensor arrays for cancer diagnosis
have led to a smaller compact system that is portable and powered by a
laptop computer (fig. 4B).
Indeed a clinical study conducted recently with the membrane type
sensors was very promising in distinguishing healthy, sick and treated
individuals [30].
The results obtained through the
cantilever sensor technology are robust in signal response, but can
sometimes vary by a factor of 2 due to alignment of the lasers on the
cantilevers. Along that line we think that major improvements will come
from progress in device and software development, especially for easier
handling of the cantilever arrays and the device. Eventually we expect
the technology to be cheaper than current methods such as the COBAS
test, once volume production of devices and arrays increases. The next
important step for medical applications of nanomechanical cantilever
biosensors lies in further miniaturisation of the technology. In
particular the liquid handling system can be reduced down to nanolitre
or picolitre volumes resulting in so-called microfluidic networks. While
reducing the dimensions of the cantilevers is one possibility to
increase sensitivity, especially in dynamic mode, further improvements
can be achieved by either increasing the density of the probe molecules
on the cantilever surface or bringing the interaction sites closer to
the cantilever surface, where the surface stress is generated. Further
miniaturisation of the whole cantilever system would allow the
development of small portable devices and the use of very small sample
volumes with the possibility of using cantilever arrays in miniaturised
total analysis systems (µTAS) [31]
or lab on a chip. We envision a small and compact biosensor system
based on cantilever technology which is capable of analysing a patient's
health at different levels by interrogating its genome, proteome or
metabolome. Furthermore, such devices may be used to monitor the
efficacy of a given cancer treatments in real time, so that appropriate
changes in treatment regimen can be rapidly implemented. All these
developments could contribute to personalised healthcare in cancer
treatment.
Acknowledgment: We
thank the Swiss Nano Institute (SNI), the NanoTera Program, the Cleven
Foundation and the Swiss National Science Foundation for financial
support. We acknowledge the support from M. Despont and U. Drechsler
(IBM Research GmbH, Rüschlikon, Switzerland) for providing cantilever
arrays, F. Loizeau from Nico de Rooij's lab at the EPFL for the surface
stress sensors and U. Certa from the Roche Centre for Medical Genomics
for material support. Furthermore the work would not have been possible
without continuous technical support from J.-P. Ramseyer, A. Tonin and
R. Maffiolini from the electronic workshop and S. Martin and his crew
from the mechanical workshop of the department of physics, University of
Basel.
Funding: NanoTera, Swiss National Science Foundation, Swiss Nano Science Institute, Cleven Stiftung.
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