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Cantilever Sensors:

very attractive. Biosensing technologies


based on cantilever arrays have the
potential of satisfying this need for multi-
target detection with high sensitivity and
selectivity using very small volumes of
sample.

Nanomechanical Microcantilevers are micromechanical


beams that are anchored at one end, such
as diving spring boards, that can be read-
ily fabricated on silicon wafers and other

Tools for materials. Their typical dimensions are


approximately 100 microns long, 20
microns wide, and 1 micron thick. The

Diagnostics
microcantilever sensors are physical sen-
sors that respond to surface stress changes
due to chemical or biological processes.1
When fabricated with very small force
constants, they can measure forces and
Ram Datar, Seonghwan Kim, Sangmin Jeon, stresses with extremely high sensitivity.
Peter Hesketh, Scott Manalis, Anja Boisen, The very small force constant (less than 0.1
N/m) of a cantilever allows detection of
and Thomas Thundat surface stress variation due to the adsorp-
tion (or specific surface-receptor interac-
tion) of molecules. Adsorption of
molecules on one of the surfaces of the
Abstract
typically bimaterial cantilevers (silicon or
Cantilever sensors have attracted considerable attention over the last decade
silicon nitride cantilevers with a thin gold
because of their potential as a highly sensitive sensor platform for high throughput and
layer on one side) results in a differential
multiplexed detection of proteins and nucleic acids. A micromachined cantilever platform
surface stress due to adsorption-induced
integrates nanoscale science and microfabrication technology for the label-free detec-
forces, which manifests as a deflection. In
tion of biological molecules, allowing miniaturization. Molecular adsorption, when addition to cantilever bending, the reso-
restricted to a single side of a deformable cantilever beam, results in measurable bend- nance frequency of the cantilever can vary
ing of the cantilever. This nanoscale deflection is caused by a variation in the cantilever due to mass loading. These two signals,
surface stress due to biomolecular interactions and can be measured by optical or elec- adsorption-induced cantilever bending
trical means, thereby reporting on the presence of biomolecules. Biological specificity in when adsorption is confined to one side of
detection is typically achieved by immobilizing selective receptors or probe molecules the cantilever and adsorption-induced fre-
on one side of the cantilever using surface functionalization processes. When target quency change due to mass loading, can
molecules are injected into the fluid bathing the cantilever, the cantilever bends as a be monitored simultaneously.2
function of the number of molecules bound to the probe molecules on its surface. Mass-
produced, miniature silicon and silicon nitride microcantilever arrays offer a clear path to Mass Detection Using Variation in
the development of miniature sensors with unprecedented sensitivity for biodetection Resonance Frequency
applications, such as toxin detection, DNA hybridization, and selective detection of As described earlier, the resonance fre-
pathogens through immunological techniques. This article discusses applications of quency, f, of a cantilever varies sensitively
cantilever sensors in cancer diagnosis. as a function of mass loading (m),
according to:

1 k
f= . (1)
Introduction 2 m * + m
The detection of multiple target mole- disease process. Detection of a single bio-
cules in a small volume of sample has marker has only limited specificity and The spring constant of the cantilever is
immediate relevance in the early detec- therefore cannot be sufficiently informa- k, m* is the effective mass of the can-
tion of diseases, such as cancer. It is well tive. Hence, cancer diagnostics have been tilever, and is a numerical constant.
known that many cancers can be cura- shifting from traditional monitoring of Increasing the surface area of a cantilever
tively treated if diagnosed early when the single biomarkers to the detection of mul- by nanopatterning can lead to higher
tumors are still small and localized. tiple markers. Detection of multiple bio- adsorbed mass and higher sensitivity of
However, the unfortunate reality is that a markers is particularly important in detection. Lee et al. has demonstrated a
significant proportion of cancers are diag- screening for cancers of low prevalence, cantilever with nanofabricated holes for
nosed only after the tumors have spread such as ovarian cancer. Ideally, these increasing the adsorbed mass.3 Although
distally through blood or lymphatic fluid measurements would be done in a single mass detection using the cantilever reso-
(metastasized) to multiple locations. Since readout with samples that are readily nance frequency is well suited for measur-
cancer is a complex disease, its diagnosis available and minimally invasive, such as ing mass in vacuum and air, its mass
will require monitoring for alterations in blood serum. Therefore, a technique that resolution is very poor when operated
multiple parameters at molecular, cellu- can detect multiple biomarkers simulta- under solution.4 Resonance frequency
lar, and tissue levels to provide a compre- neously using a single sensor platform variations, therefore, are generally not
hensive picture of the extent of the and minimal sample volume would be used for the highly sensitive detection of

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Cantilever Sensors: Nanomechanical Tools for Diagnostics

adsorbed mass in liquid environments. piezoelectricity,11,12 embedded MOSFET tilever beam has been developed where the
Detecting surface stress variations is (metal oxide semiconductor field-effect doped silicon layer is only on one side of
therefore a method of choice when bio- transistor),13 capacitance,14 and electron the neutral axis of the cantilever beam and
markers have to be detected in body flu- tunneling.15 In optical beam deflection, the shows variation in its resistance as a func-
ids, such as serum. However, Burg et al. cantilever motion is detected by reflecting tion of the extent of the deflection.17 These
recently demonstrated a hollow cantilever a focused beam of light from the tip of a cantilevers are composed of several layers
concept called the suspended microchan- cantilever beam into a position-sensitive of materials at different regions where sin-
nel resonator (SMR) that is capable of detector. In the piezoresistive technique, gle crystal silicon serves as an active func-
detecting biological interactions in liquids the resistance of an asymmetrically doped tional element. The active silicon region is
with unprecedented sensitivity.5 (See the cantilever varies sensitively as a function sandwiched between silicon nitride and
section on Suspended Microchannel of bending. The piezoelectric technique silicon dioxide layers for insulation and
Resonators later in this article.) involves coating the cantilevers with protection, Figure 1a. These piezoresistive
piezoelectric materials, which develop a cantilevers are fabricated using predoped
Mechanics of Cantilever measurable charge due to cantilever silicon with insulating layers of silicon
Deflection bending. In the embedded MOSFET read- nitride on both sides. The thickness of the
Adsorption of molecules on a surface out method, a field-effect transistor is insulating layers (silicon nitride and silicon
results in a decrease in surface free energy. embedded at the base of the cantilever. oxide) is adjusted in such a way that the
If the adsorption of molecules on a surface The stress from the bending of the can- neutral axis of the bending cantilever is
is restricted mostly to one side, for exam- tilever changes the carrier mobility and outside of the doped silicon. The can-
ple, by making the opposite surface inert, drain current. In the capacitance method, tilevers fabricated using this method have
a differential surface stress is generated the capacitance between a bending can- a higher signal-to-noise ratio, less can-
between the two surfaces of a cantilever tilever and a fixed substrate varies as a tilever drift, and increased sensitivity, as
beam. Surface stress, g, and surface free function of cantilever bending. The elec- compared to piezoresistive cantilevers fab-
energy, , can be related using the tron tunneling method is extremely sensi- ricated, where the neutral axis lies at
Shuttleworth equation: tive, and it is based on the tunneling of the boundary between the doped and
electrons between a cantilever and a fixed undoped regions of the silicon. Since the
dg , electrode.16 silicon nitride/silicon oxide layer is insulat-
g=g+ (2)
de ing, these piezoresistive cantilevers can be
Piezoresistive Cantilever Array used in liquid environments.
where the surface stress, epsilon, is defined Fabrication For the cantilevers fabricated, there are
as the ratio of the change in surface area to Piezoresistive materials, such as doped two parallel silicon stripes (20 m wide)
the total area. Since the bending of the silicon, show piezoresistivity where the in each cantilever (see Figure 1). Two
cantilever is very small compared to the resistance of the material varies as a func- gold/titanium metal leads connect silicon
length of the cantilever, the strain contri- tion of applied stress. A multilayer can- stripes at their base and end at the contact
bution is often neglected. However, this is
a subject under active discussion in the lit-
a
erature.68
The differential surface stress created by
molecular adsorption results in cantilever
bending. Stoneys equation relates the dif-
ference in surface stress g between the L
chemically modified surface and the
untreated surface with the cantilever
deflection, h:
LR
Eh t 2,
g = (3)
4(1n) L
w
h
where, is the Poissons ratio of the mate-
rial, E is Youngs (elastic) modulus of the
b
cantilever material, and t and L are the
thickness and the length of the cantilever, c
respectively. The surface stress also can be Sensing Layer
thought of as a change in surface energy
density or a change in surface tension. It is Si3N4
clear that the longer the cantilever, the Si
more sensitive the cantilever to measure
surface stresses. SiO2
Si3N4
Modalities of Cantilever Deflection-
Based Sensing
The motion of the cantilever response
can be sensitively monitored using a vari- Figure 1. (a) Schematic diagram of a microcantilever indicating dimensions length, L,
ety of techniques, such as variations in resistor length, LR, width, w, and thickness, h. (b) An array of piezoresistive cantilevers.
optical beam deflection,9 piezoresistivity,10 (c) Cross-sectional diagram through the layers of the microcantilever sensors.

450 MRS BULLETIN VOLUME 34 JUNE 2009 www.mrs.org/bulletin


Cantilever Sensors: Nanomechanical Tools for Diagnostics

pads at the edge of the chip. To complete a


the circuit, the silicon stripes are electri-
cally shorted by a layer of gold at the free
end. The resistivity of the silicon was
designed at 3.4 103 cm. The doping
concentration was 2.2 1019 cm3. The can-
tilever bending due to residual stresses in
the buried oxide layer can be eliminated
by depositing silicon nitride film on the b
back of the beam to create tensile
stresses.18 The typical resistance of a 26Jun05 x250 200 um

piezoresistive cantilever is around a few


kilo-ohms (1 and 5 k depending upon
the length).
The cantilever is shown in Figure 2. For
piezoresistive cantilevers, higher sensitiv-
ity can be obtained when thickness of the
cantilever is reduced.18 The piezoresistive
coefficient of silicon is a function of the
doping level and dopant type. Unlike a
tip-loaded piezoresitive atomic force
microscope cantilever, where p-type 26Jun05 x25 2 mm
dopant is standard, with microcantilever
sensors, a higher gauge factor (normal-
ized change in resistance per unit stress) is Figure 2. (a) Piezoresistive microcantilever and (b) 10-cantilever array. Resonant frequency
achieved with n-type dopant.18 ~20 kHz, spring constant ~0.003 N/m. (Image courtesy of P. Heskth, Georgia Institute of
Technology.)
Suspended Microchannel
Resonators
As mentioned earlier, SMRs are can- the suspended microchannel. Various gold film. Organosilane coatings also are
tilevers with microchannels fabricated methods for trapping an individual cell of the order of a monolayer but can
inside the cantilever. Figure 3 shows a within the SMR so that the cell mass can become multilayered upon extended
scanning electron micrograph of a SMR. be monitored as a function of time are exposure to the solution. Regardless of the
Although the SMR cantilever is placed currently under development. Such meth-
inside a vacuum for obtaining a high- ods may be used to investigate how cell
quality factor (Q is defined as a ratio of growth relates to progression through the
resonance frequency and the full width at division cycle and if the response of cancer
half maximum of the resonance ampli- cells to pathway-directed therapeutics can
tude) of resonance, liquid analytes can be be classified according to subtle changes
pumped through the cantilever. This in growth.
allows measuring masses of liquid and
suspended biomaterials such as cells and Receptor Immobilization
cancer markers. At present, the SMR is Many approaches can be used to immo-
fabricated in such a way that the hollow bilize the molecular recognition agents to
cantilever is vacuum sealed inside a the microcantilever sensor, depending
chamber with an optical window for opti- upon the final application. Generally, can-
cal beam-based cantilever motion detec- tilevers are coated on one side with 23
tion. Since the liquid is inside the nm of chromium then with 2530 nm
cantilever, which is vibrating in a vacuum, of gold using an e-beam evaporator.
a Q factor of 15,000 can be achieved for Chromium acts as an adhesion layer for
this device. High Q values enable mass to the gold. Both the silicon side of the can-
be measured with femtogram resolution tilever (using silane chemistry) and the
in a 1 Hz bandwidth.5 The ability to circu- gold side of the cantilever (using thiol
late liquid through the cantilever allows chemistry) have been utilized depending
for the monitoring of biological interac- on the final application for the molecular
tions between immobilized receptors recognition assay. For thiol self-assembled
inside the hollow cantilever and passing monolayers (SAMs) and organosilane
analytes. For example, changes in reso- modification, dip coating is the preferred
nance frequency induced by the adsorp- method for functionalization to allow for
tion of cancer marker molecules and high density immobilization on the can- Figure 3. A scanning electron
micrograph of a suspended channel
immobilized receptors can be used as a tilever surface; all reactive surfaces of the microresonator. The channels can be
selective and sensitive method for moni- cantilever and substrate that are exposed seen inside the cantilever. The hollow
toring the presence of cancer markers in to the modifying solution(s) will have a cantilever vibrates inside a vacuum.
the passing sample. Additionally, single coating. Thiol SAMs are self-limited to (Image courtesy of S. Manalis,
cells can be weighed as they pass through coverages of a monolayer of the thiol on a Massachusetts Institute of Technology.)

MRS BULLETIN VOLUME 34 JUNE 2009 www.mrs.org/bulletin 451


Cantilever Sensors: Nanomechanical Tools for Diagnostics

coating chemistry employed, typically all Figure 4 shows the surface stress variation PSA. PSA is a serine protease secreted by
experimental surfaces are freshly pre- of a piezoresistive cantilever as a function prostatic luminal epithelial cells. When
pared no more than 48 hours prior to time due to ssDNA (thiol link) immobiliza- used in population screening for the
assay. Stability studies to determine the tion and subsequent hybridization with detection of elevated serum, PSA is cred-
effects of aging on the prepared surfaces complementary ssDNA. The specific bind- ited with dramatic advances in the early
remain to be done. ing between the complementary DNA diagnosis and management of men with
strands on the cantilever results in a sur- prostatic carcinoma. The majority of the
Biomolecular Detection Assays face stress variation of 3040 mN/m. Wu recently marketed assays are based on the
Antigen-antibody interactions are a et al. investigated the origins of cantilever commonly used reference range (<4 ng of
class of highly specific protein-protein deflection due to biomolecular interactions PSA in a ml of blood), and almost all of
binding that play a critical role in molecu- and found that the deflection resulted from them employ some variation of the tech-
lar biology. Since cantilever bending origi- a change in free energy of one cantilever nique of enzyme-linked immunosorbent
nates from the free energy change induced surface.22 The interplay between the ener- assay (ELISA).
by specific biomolecular binding, this bio- getic and entropic contributions deter- Figure 5 shows the potential of micro-
molecular detection assay offers a com- mined the direction of cantilever motion. cantilevers as a platform for developing a
mon platform for high-throughput, Although both DNA hybridization and sensitive and specific assay for PSA using
multiplexed label-free analysis of biomol- protein-protein (antigen-antibody) bind- the optical beam deflection method for
ecules, such as protein-protein binding, ing can be detected using cantilever cantilever bending. The cantilevers were
DNA hybridization, and DNA-protein deflections, what remained unclear for a immobilized with polyclonal anti-PSA
interactions.1927 When antibody mole- while was whether this technique had suf- antibodies. Binding of PSA on the immo-
cules are immobilized on one surface of a ficient specificity and sensitivity to be bilized polyclonal antibody resulted in a
cantilever, specific binding between anti- used for the detection of disease-related deflection of the cantilever. Furthermore,
bodies and antigens produces cantilever proteins at clinically relevant conditions changes in surface stress were related
deflection. Similarly, the cantilever under- and concentrations. To address this tech- quantitatively to the concentration of PSA.
goes bending when single-stranded DNA nologically critical issue, sensitive and Results in Figure 5 are from a model sol-
(ssDNA) probes are immobilized on the specific detection of a prostate cancer vent system prepared with phosphate-
cantilever hybridized with complemen- marker, prostate specific antigen (PSA), buffered saline and shows steady-state
tary ssDNA (target) molecules in the solu- was conducted as an example of both cantilever deflection as a function of PSA
tion. Such specific deflection was not seen protein-protein binding in general and of concentration against a much higher back-
when the incoming DNA strands were a cancer diagnostic tumor marker detec- ground of bovine serum albumin. Similar
noncomplementary due to the absence of tion in particular.21 Prostate cancer has tests were performed against high back-
hybridization. Therefore, it is possible to emerged as the most common nonskin grounds of human serum albumin and
design ssDNA probes specifically to cancer and the second leading cause of human plasminogen, both of which are
detect mutations in the DNA sequence of cancer death in men in North America and found abundantly in human sera. Of note
target DNA responsible for many cancers. Europe (www.cancer.gov). While transrec- was the finding that PSA concentrations
The following section briefly describes tal ultrasonography and digital rectal can be detected below 4 ng/ml, the clini-
development of this assay. examination are common clinical exami- cal threshold for prostate cancer. In fact,
nations, the most widely used biochemical concentrations down to 0.2 ng/ml were
Detection of Specific DNA test involves analyzing the presence of detected. Since for the same PSA concen-
Sequences trations cantilever deflections varied with
Several groups have shown can- their geometry, it is important to stan-
60
tilever deflections due to adsorption of dardize these measurements in terms of
ssDNA on virgin cantilevers as well surface stress rather than cantilever
as hybridization of complementary 30 deflections using Stoneys formula. The
Stress (mN/m)

sequences.19,20,22,23,27 ssDNA can be immo- technique is simpler and potentially more


bilized on one side of a cantilever by coat- cost-effective than ELISA, the current
ing that side with gold and using a thiol 0 gold standard assay for PSA detection,
linker at one end of ssDNA. It has been because it does not require labeling and
experimentally found that adsorption of can be performed in a single reaction
ssDNA on a cantilever results in a surface 30 without additional reagents.
stress variation of 3050 mN/m. Note that
the surface stress variation is directly Challenges of Cantilever-Based
induced by the adsorption of sulfur atoms 60 Detection
0 500 1000 1500
on the thiol chain on the gold substrate. Despite the unprecedented sensitivity
The ssDNA bound to the cantilever acts as Time (sec) demonstrated using microcantilever sen-
a probe (or receptor) molecule for the tar- sors, the selectivity performance and
get complementary strands. The addition Figure 4. A plot of the surface stress robustness are not consistent, and the full
of noncomplementary ssDNA into the variations of a piezoresistive cantilever potential remains to be developed and val-
solution with immobilized ssDNA probes as a function of time due to the immo- idated. There are a number of challenges to
bilization (red) and hybridization with
produces no mechanical signals. The can- overcome before cantilever sensors come
complementary ssDNA (blue). The im-
tilever bending signal also can be used for mobilization and hybridization response into widespread use. It is possible to
detecting mutations in the DNA sequence curves are superimposed to show the achieve higher selectivity, sensitivity, and
(single nucleotide polymorphism [SNP]), response direction. Inset schematics robustness through optimization of can-
where a single nucleotide of noncomple- show configurations of ssDNA (below) tilever geometries, immobilization tech-
mentary nature appears in the sequence.20 and double stranded DNA (above).22 niques, and analyte delivery schemes.

452 MRS BULLETIN VOLUME 34 JUNE 2009 www.mrs.org/bulletin


Cantilever Sensors: Nanomechanical Tools for Diagnostics

for microcantilevers, which are a universal


platform to base electromechanical sen-
sors for selective and sensitive detection of
cancer markers.

Future Trends and Summary


The trend in miniaturization of sensor
arrays for multiplexed detection couples
very well with the versatility of cantilever
arrays. Currently available microfabrica-
tion technologies could be used to make
multitarget sensor arrays involving
multiple cantilevers, electronic process-
ing, and even local telemetry on a single
chip. The technology for designing
and simulating electronic chips is well
advanced. Integration of electronic,
mechanical, and fluidic designs, however,
is still in its infancy. Additional receptors
Figure 5. Steady state cantilever deflection (measured using optical beam deflection) as a and immobilization methods will need to
function of the concentration of free prostate specific antigen (fPSA) in the solution. The be developed and added to the libraries.
responses from five different concentrations are shown. BSA, bovine serum albumin.
These could include improvements such
Please note the width of the cantilever is 20 m.
as the application of aptamers or molecu-
lar imprinting polymers as surface-bound
As mentioned earlier, the cantilevers molecules, many of them at much higher capture receptors. The stability of immo-
can be fabricated in such a way as to concentrations than the target analyte, bilized receptors is an issue that poten-
increase their detection sensitivity. For there is a likelihood of false positives due tially limits shelf life and long-term
sensing methods based on adsorption- to binding of the immobililzed antibody reliability of the sensors and will need to
induced cantilever deflection, longer and to a nontarget molecule that has a similar be addressed. Here, the advances could
thinner cantilevers with small force con- structural motif. The number of false pos- come in the form of regenerating re-
stants show higher sensitivity. However, itives may increase further if polyclonal ceptors. Using multiple cantilevers for
as in the case of all surface adsorption- antibodies are used for capture. Another single target detection will lower noise,
based sensors, larger area cantilevers potential limitation for an optical readout greatly increase selectivity, and increase
show faster detection time for low concen- sensor is the turbidity of serum. robustness.
trations of target molecules. Therefore, the Some of the ways to alleviate these Cantilever arrays have the potential of
optimal cantilever dimension will depend problems include use of capture satisfying the need for multitarget detec-
on the dimension of the cantilever cham- reagents with significantly higher speci- tion necessary in cancer diagnostics with
ber and the analyte delivery system. Since ficity (such as single chain antibodies or high sensitivity and selectivity using very
most measurements are carried out using aptamers), allowance for multiple wash small volumes of sample and not requir-
a reference cantilever, the common mode steps similar to an ELISA procedure, or ing repeated body fluid sampling.
rejection (differential measurement with preconcentrating the serum for the tar- Because cantilever sensors report on the
respect to a reference cantilever) basically get analyte by reducing the concentra- existence of biomarkers in a label-free
improves sensitivity. New designs of tion of the abundant nontarget proteins manner, they can be employed in a rela-
piezoresistive cantilevers show less drift in serum. The latter, for example, can be tively inexpensive assay format, requiring
and improved signal-to-noise ratios.28 achieved by affinity chromatography to fewer manipulative steps compared to the
Godin et al. demonstrated that the bend- remove as many as 12 abundant protein currently available diagnostic platforms
ing of a cantilever beam strongly depends species such as serum albumin, actin, or such as ELISA assays for proteins or
on the surface roughness of the gold immunoglobulins. microarrays for nucleic acids. Also, since
film.29 Vacuum-deposited gold films with One of the challenges in translating can- the turnaround times for assays can be
larger grain sizes on the cantilever show tilevers as practical sensors for biological shortened due to multiplexing, substan-
increased bending sensitivity. The sensi- applications is the sensor reproducibility. tial savings are possible in diagnostic
tivity of bending also depends on the uni- Since selectivity is achieved by coating the workup schedules. Ultimately, all of these
formity of the immobilization layer and cantilever with selective receptor coatings advantages, including early detection,
cleanliness of the sensing surface.30 such as antibodies, peptides, DNA, or will have significant implications in
Selectivity of detection in complex sam- enzymes, the specificity of the receptor- reducing the assay costs and hence costs
ples still remains to be solved. It must be target interaction controls the selectivity to the patient and healthcare providers.
noted here that while PSA detection, and sensitivity. The cantilever response
described previously, yielded a clinically also depends on the uniformity of the Acknowledgments
relevant level of sensitivity when tested in coating on the cantilever surface. Often, We would like to thank our colleagues
a model protein-containing buffer solu- coverage of surface immobilized receptor and collaborators cited in this review for
tion system, its sensitivity was much molecules can vary from cantilever to can- their contributions. R. Datar, S. Kim, and
lower in actual human serum. This tilever due to contamination, resulting in T. Thundat would like to thank DOE BER
decreased sensitivity may be a result of irreproducible responses. Consequently, for its support. Oak Ridge National
one or more of the following factors. Since more work is urgently needed to develop Laboratory is managed by UT-Battelle
serum contains thousands of various bio- more reliable immobilization techniques under contract No. DE-AC05-000R227255.

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Cantilever Sensors: Nanomechanical Tools for Diagnostics

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