Sensors and Actuators B 79 (2001) 115126

Micromechanical cantilever-based biosensors

Roberto Raiteria, Massimo Grattarolaa,*, Hans-Jurgen Buttb, Petr Skladalc
a

Neural and Bioelectronic Technologies Group, Department of Biophysical and Electronic Engineering,
University of Genova, via all'Opera Pia 11a, 16145 Genova, Italy
b
Institut fur Physikalische Chemie, Universitat-Gesamthochschule Siegen, Adolf-Reichwein Strasse, 57068 Siegen, Germany
c
Department of Biochemistry, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic
Received 13 January 2001; received in revised form 6 May 2001; accepted 10 May 2001

Abstract
The merging of silicon microfabrication techniques with surface functionalization biochemistry offers new exciting opportunities in
developing microscopic biomedical analysis devices with unique characteristics. Micro-mechanical transducers for chemical and
biosensing applications represent one possibility. Microcantilevers can transduce a chemical signal into a mechanical motion with high
sensitivity. In this review we summarize how cantilever-based sensors can be operated, and their working principle is presented in few
selected biosensing experiments which have been performed recently in our groups in the study of biotinstreptavidin and antigenantibody
interactions, and specic surface charge development of organic molecules. We also discuss the advantages of this novel technique as well
as its potentials. # 2001 Elsevier Science B.V. All rights reserved.
Keywords: Microfabrication; Cantilever; Biosensor; Immunosensor; Surface functionalization

1. Introduction
Biosensors have attracted considerable interest in the last
few years since the monitoring of a specic substance is
central in many applications ranging from clinical analysis
to environmental control and for monitoring many industrial
processes.
A biosensor, as any other sensing device, can be divided
into three main components: a detector which recognizes the
signal of interest, a transducer which converts the signal into
a more useful output, typically an electronic signal, and a
read-out system which lters, amplies, displays, records, or
transmits the transduced signal.
A biosensor employs a biological or biochemical detector,
which can range from single proteins and enzymes up to
whole cells and microorganisms.
Biosensors can be classied according to different
schemes: the detector type, e.g. immunosensors or enzymatic sensors, the transduction principle, e.g. amperometric
or piezoelectric, and the application, e.g. clinical sensors or
environmental sensors.
In the following we will focus on a novel mechanical
transduction mechanism. It is based on the bending of
microfabricated silicon cantilevers, caused by the adsorption
*
Corresponding author. Tel.: 39-10-353-2761; fax: 39-10-353-2133.
E-mail address: gratta@dibe.unige.it (M. Grattarola).

of molecules onto the sensor surface. It can be the basis of a

new class of miniaturized, highly sensitive biosensors.
1.1. Cantilever-based sensors
Atomic force microscopy (AFM) [1] provides a relatively
new approach to the study of specic bio-interactions (for a
recent review on the subject, see [2]). With AFM, it is
possible to measure directly avidinbiotin binding [3,4],
antigenantibody interaction [57], and hybridization of
complementary DNA strands interactions [8]. Imaging
labeled surface immunological reactions with the AFM is
also possible [9]. Recently, biosensors based on functionalized magnetic beads and a scanning AFM have been
proposed [10].
The use of microcantilevers as force sensors in AFM is,
therefore, a well-established application and it takes advantage of the silicon micromachining techniques developed for
integrated circuit (IC) process technology [11,12]. Silicon,
silicon oxide or silicon nitride cantilevers are commercially
available with different shapes, dimensions and force sensitivities. When used as probes for AFM they have an integrated sharp tip at the free end which is used to increase
lateral resolution while scanning the surface (Fig. 2a). When
cantilevers are used as sensors such a tip is not necessary.
Fig. 1a shows an image of ve rectangular tipless cantilevers
with different lengths, designed for stress measurements.

0925-4005/01/$ see front matter # 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 5 - 4 0 0 5 ( 0 1 ) 0 0 8 5 6 - 5

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R. Raiteri et al. / Sensors and Actuators B 79 (2001) 115126

Fig. 1. (a) SEM image of five microfabricated (poly)silicon rectangular

cantilevers of different lengths. They are all supported by the same silicon
chip. (b) Zoomed image of a cantilever with length L 100 mm, width
w 40 mm and thickness t 0:5 mm (images by IMM, Mainz, Germany,
reproduced with permission).

In the last decade, several research groups observed that

microcantilevers can transduce a number of different signal
domains, e.g. mass, temperature, heat, electromagnetic eld,
stress, into a mechanical deformation: either a bending or a
change in the resonance frequency, with a resolution which
is orders of magnitude higher than that achievable with
macroscopic structures as it will be described in Section 2.2.
Changes in temperature and dissipated/adsorbed heat
bend a cantilever composed by a sandwich of materials
with different thermal expansion coefcients (bimetallic
effect, Fig. 2b). A microfabricated cantilever is capable of
measuring changes in temperature as small as 10 5 K [13]
and can be used either for photothermal measurements
[1417] or as a microcalorimeter to study the heat evolution
in catalytic chemical reactions [13] and enthalpy changes at
phase transitions [18]. Bimetallic microcantilevers have
been extended to perform photothermal spectroscopy with

a sensitivity of 150 fJ [19] and sub-millisecond time resolution [20]. Theoretical estimations have shown that they are
in principle able to detect heat changes with attoJoule
sensitivity [13,21].
Changes in medium viscoelasticity or mass cause a shift
of the cantilever resonance frequency: a higher viscous
medium surrounding the cantilever as well as an added
mass will damp the cantilever oscillation lowering its fundamental resonance frequency. Cantilevers can therefore be
vibrated by a piezoelectric actuator to resonate and used
either as viscosity-meters [22] (Fig. 1c) or as microbalances
to detect changes in their mass (Fig. 1d). In the latter case, it
is important to know that during adsorptiondesorption
processes, the deposited material can change cantilever
mechanical properties, like stiffness. It was observed that
water adsorption on a gelatin coated cantilever causes an
increase of its resonance frequency [2325], which is contradiction with the effect of the mass increase (see Eq. (1)). This
crosstalk between mass and stiffness changes can be minimized when the sensing layer is concentrated at the free end
of the cantilever (Fig. 1d). In this case, the shift in resonance
frequency can be directly related to the change in mass by
the following equation:


K 1
1
Dm 2 2
(1)
4p f1 f02
where K is the cantilever spring constant and f0 and f1 are the
resonance frequencies before and after adsorption, respectively. By allowing adsorption at the apex only, however, the
adsorption area is reduced and, consequently, the sensitivity.
By using a porous material, like zeolites, as the sensing layer
one can enhance the sensitivity [26]. Theoretical estimations
based on commercially available cantilevers showed a minimum detectable mass density of 0.67 ng/cm 2. This value is
comparable to acoustic sensors like surface acoustic wave
oscillator (SAW) and quartz crystal microbalance (QCM)
and results, taking into account the active area of the
structure, into a minimum detectable mass of 10 15 g [27].
Unfortunately, when operated in liquid, both the resonance peak and its quality value shift toward much lower
values than in air due to the damping effect of the liquid [28].
This affects considerably the achievable resolution in terms
of minimum detectable mass change and makes the method
less suitable to monitor monolayer adsorption of molecules
in aqueous environment than the surface stress detection,
described in the following section.
2. The bending plate method

Fig. 2. Schematics of possible uses for a cantilever transducer (side view):

(a) AFM force sensor; (b) temperature, heat sensor; (c) medium
viscoelasticity sensor; (d) mass sensor (end load); (e) stress sensor and
(f) sensor to monitor the presence of magnetic beads on its surface (picture
reproduced with permission from Berger [104]).

A thin bar or plate can be used to measure changes in the

differential surface stress between its opposite sides. The
principle is straightforward: a uniform surface stress acting
on an isotropic material tend either to increase (compressive
stress) or decrease (tensile stress) the surface area. If this
effect is not compensated by an equal stress on the opposite

R. Raiteri et al. / Sensors and Actuators B 79 (2001) 115126

117

therefore rises from the way the new surface is formed. In

solids new surface area can be created elastically by stretching pre-existing surface or, less often, plastically by, for
example, cleaving a crystal. For liquids, the situation is
simplied since molecules are mobile and can therefore
enter or leave a strained surface (for a more detailed discussion, see, for example [3032]). Surface stress s and surface
tension g are mathematically related by the Suttleworth
equation [33]

@g 
sg
(4)
@e 
e T;mi ;j

Fig. 3. Lateral view of a thin beam of thickness t subjected to compressive

surface stress changes Ds1 and Ds2. The beam bends around a neutral
plane with a constant radius of curvature R.

side of a thin plate or beam, it will permanently bend the

whole structure with a constant radius of curvature (Fig. 3).
At the beginning of the 20th century, Stoney performed
measurements of deposition-induced bending of beams in a
electrochemical environment and related the differential
surface stress change Ds1 s2 Ds1 Ds2 between
the opposite faces of a thin beam with the resulting radius
of curvature R [29]


1
1 u
6
(2)
Ds1 Ds2
R
Et2
where t is the thickness of the beam and E and u are
mechanical parameters of its material: Young modulus
and Poisson ratio, respectively.
For a rectangular beam of length L, xed at one end and
free to move at the opposite end, it is possible to write an
approximate relation between the differential surface stress
change and the resulting change in deection Dz at its free
end
Dz

31

uL2

Ds1 Ds2
(3)
Et2
Hence, by measuring the deflection one can calculate the
difference in surface stress between the opposite sides which
generated it.
2.1. Surface stress versus surface energy
In general, adsorption of molecules to a surface causes a
change in the surface free energy, also called surface tension.
The mechanical response, in our case the bending of the
cantilever, is caused by a change in the surface stress.
Surface stress and tension are related but distinct quantities.
Surface tension is dened as the reversible work per unit area
needed to create new surface plastically, while the surface
stress is the reversible work per unit area required to create
new surface by stretching it elastically. The difference

where ee  DSe =S is the elastic surface strain, and DSe the

change in surface area S due to an elastic deformation before
and after adsorption. For liquids, the second term of Eq. (4)
is 0 and surface tension is equal to surface stress. The
dependence of g on the elastic strain ee is, in general,
complicated and depends on possible surface reconstruction.
It is therefore not straightforward even to estimate the
surface stress trend given a change in surface tension. Ibach
studied carefully surface stress on crystalline cantilevers
induced by adsorption of single atoms both experimentally
[34,35] and with finite element analysis [36]. However,
when dealing with complex molecules like proteins as it
is often the case in biochemical sensing, there are several
other possible sources of stresses than simple ion adsorption
onto a clean crystal surface. Electrostatic interaction
between neighboring adsorbates, changes in surface hydrophobicity, and conformational changes of the adsorbed
molecules can all induce stresses which may contrast with
each other and make the change in stress not directly related
to the receptorligand binding energy or the rupture force.
As an example of the complexity of the issue, Wu et al. [37]
have recently observed how adsorption of complementary
single-stranded DNA onto the cantilever surface can induce
either compressive or tensile stress depending on the ionic
strength of the buffer in which the hybridization takes place.
They interpreted this behavior as the interplay between two
opposite driving forces: a reduction of the configurational
entropy of the adsorbed DNA after hybridization which
tends to lower the compressive stress, and intermolecular
electrostatic repulsion between adsorbed DNA which tends
to increase the compressive stress.
2.2. Micro versus macro
Macroscopic cantilevers have been extensively used to
monitor surface stresses caused by crystal growth and surface reconstruction in air or vacuum in the last 30 years
[3844]. These processes induce large surface stress differences, in the order of few N/m. However, for biochemical
recognition, the expected changes in surface stress are in the
mN/m range; only by using microscopic transducers one can
achieve such resolution.
Typical dimensions of the used microcantilevers are
described in Fig. 1b. From Eq. (3), it is easy to see that

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R. Raiteri et al. / Sensors and Actuators B 79 (2001) 115126

stress sensitivity increases with the length to thickness ratio

L/t. To achieve a high sensitivity, cantilevers should be long
and thin. On the other hand, external mechanical noise
directly affects the minimum detectable deection. In general, vibrations at frequency f shaking a mechanical system
are damped by a factor which is proportional to f0/f, where f0
is the system fundamental resonance frequency. Resonant
frequency should therefore be as high as possible [45]. For
rectangular cantilevers, f0 is given by
s
E t
f0  0:16 
(5)
r L2
where r is the density of the cantilever material. High
sensitivity and at the same time low noise can be achieved
by using small cantilevers. Small cantilevers have also
the advantage to respond faster, due to the high resonance
frequencies. Microfabricated silicon cantilevers as those
shown in Fig. 1 have resonance frequency in the 30140
kHz range in air.
Microfabricated silicon cantilevers for atomic force
microscopy were used to measure changes in the surface
stress of solids [4650] with a stress resolution in the mN/m
range, enough to sense the presence of a single monolayer of
deposited molecules. The rst results showed that the surface stress detection is probably the most sensitive way to
operate cantilever-based sensors [49,50].
3. Cantilever deflection detection methods
With the introduction of scanning probe techniques like
AFM, several deection detection methods have been proposed to measure microcantilever deection with a subAngstrom resolution. They can be divided in optical and
electric methods and can be implemented in cantileverbased biosensors.
3.1. Optical lever
The most diffused method in AFM is the so-called beam
bounce or optical lever technique. The visible light from a
low power (few mW) laser diode is focused on the free apex
of the cantilever which acts as a mirror. In order to increase
reectivity, commercial cantilevers for AFM are often
coated by a thin layer of gold. The reected beam hits a
position sensitive photodetector or a split photodetector as
schematically described in Fig. 4. When the cantilever
bends, the reected laser light moves on the photodetector
surface. The distance traveled by the laser beam is proportional to the cantilever deection and linearly magnied by
the cantileverphotodetector distance as the arm of a lever. A
calibration is needed in order to obtain the deection signal
in terms of nanometers. A common way to do this is to press
the cantilever against a hard surface a known distance and
record the corresponding change in photodetector voltage

Fig. 4. Optical lever deflection detection method.

output. One should be aware that, with this method, the

cantilever bending rather than its displacement, i.e. the slope
at the point where the laser hits the cantilever, is actually
measured. Details about the deection calibration procedure
for stress measurements are described in [46,51].
3.2. Interferometry
Another implemented optical deection detection method
is based on interference of a reference laser beam with the
one reected by the cantilever. Interferometry is highly
sensitive and provides a direct and absolute measurement
of the displacement. Light has to be brought close to the
cantilever in order to get enough reected light. For this
purpose, Rugar et al. [52] positioned the cleaved end of an
optical ber few micrometers away from the free end of a
cantilever. By doing this, they could measure deections in
range. More recently, an interferometric system
the 0.01 A
based on two optical bers has been proposed for parallel
surface stress monitoring of two cantilevers only 200 mm
apart [53]. However, a few technical problems arise. Positioning the bers is a demanding task, interferometry works
well for small displacements: absolute deection is dened
only within a single wavelength, and measurements in
liquids are, in general, less sensitive.
An alternative approach is to use interdigitated cantilevers
as an optical diffraction grating [54,55]. In this case, the
reected laser light forms a diffraction pattern whose intensity is proportional to cantilever deection. This method has
already been successfully implemented for physical sensing
in a miniaturized accelerometer [56].
3.3. Capacitive sensor
Capacitive sensors measure displacement as a change in
the capacitance of a plane capacitor. Blanc et al. [57]
developed microfabricated capacitive sensors for AFM
where the cantilever is one of the two capacitor plates. This
technique is highly sensitive and can provide absolute
displacement. However, it is not suited to measure large

R. Raiteri et al. / Sensors and Actuators B 79 (2001) 115126

displacements and does not work in electrolyte solutions

because of the Faradic currents between the capacitor plates;
it is therefore of limited use in biosensing applications.
3.4. Piezoresistive/piezoelectric cantilevers
When a piezoresistive material like doped silicon is
strained, it changes its electrical conductivity. Piezoresistive
sensors are therefore ideally suited to monitor stresses.
Piezoresistive stress sensors can be integrated inside a
cantilevered structure. Their resistivity can be easily measured with a simple Wheatstone bridge [58,59]. Recent
improvements allowed the fabrication of thin and passivated
resistors into the cantilever structure [60,61]. This allows
their use in electrolyte solutions by avoiding Faradic currents. In order to compensate changes in resistivity due
thermal drifts, a symmetrical conguration has been developed where the output is a differential signal between a
sensing piezoresistive and a reference one [60,61].
Piezoresistive detection, compared to the optical one,
shows several advantages: no expensive and macroscopic
optical components and no time consuming laser alignment
are needed; read-out electronics can in principle be integrated on the same silicon chip supporting the cantilevers
using the same C-MOS fabrication technology. Optical
techniques can also be subjected to artifacts due to changes
in the optical properties of the medium surrounding the
cantilever, e.g. a change in the refracting index when
exchanging two different solutions, which can move the
laser spot on the photodetector surface. Piezoresistive detection does not suffer from this problem and can work in nontransparent solutions.
Piezoresistive cantilevers offer also the possibility to vary
the temperature of their surface by simply increasing the
electrical current owing through the resistor layer. Berger
et al. [62] showed how to perform thermogravimetric analysis using an oscillating piezoresistive cantilever in air. A
local control of the temperature could be also implemented
as a tool for breaking the ligandreceptor binding, thus
regenerating the sensing layer. On the other hand, the
cantilever cross-sectional structure is complex (the resistor
must be embedded into a dielectric layer to operate in
electrolytes) and, consequently, there are technological
limits in fabricating thin, highly sensitive, cantilevers.

119

hand, receptor molecules should have enough degrees of

freedom to freely interact with their specic ligand in the
environment. The receptor activity should last over time and
possibly stand regeneration of the sensing layer if the sensor
has to be reused several times. Most of these requirements
are common to other biosensors and, in fact, the proposed
coating techniques and procedures are shared with other
transducing principles.
Noble metals are often deposited either as a substrate to
anchor successive layers or as catalyst for gas adsorption.
Both evaporation and sputtering allow a precise control of
the thickness and distribution of the metal layer.
An easy and popular method to create ordered monolayers
is to use the self-assembling properties of alkane chain
molecules with thiol (SH) groups on gold substrates
[63,64] or silanes (SiOX) on silicon substrates [65,66].
They spontaneously form uniform, densely packed, robust
(covalent binding) monolayers. They can be synthesized
with different chain lengths and end groups with specic
chemical properties. They are therefore ideally suited to act
as cross-linkers to anchor the receptor molecules to the
substrate.
As an example, Fig. 5a shows a biotinylated silicon nitride
cantilever. One side was coated with 3 nm of chromium and
a 40 nm gold layer. The thin layer of chromium was used to
increase the adhesion of gold onto silicon nitride. The
cantilever was then incubated in a 1:9 mixture of thiols I
and II (Fig. 5b) dissolved in phosphate buffer saline (PBS)
solution (137 mM NaCl, 2.7 mM KCl and 10 mM phosphate
buffer, pH 7.4). A stable, dense, and highly structured thiol
monolayer on the freshly coated gold was then formed,
while on the opposite side, physisorbed thiols were removed
by rinsing the cantilever in ethanol. In this case to incubate
the whole cantilever structure in the thiol solution was
suitable. If deposition has to be on one side only, spraying

4. Cantilever surface functionalization

The receptor layer deposited on the cantilever surface
affects directly sensor selectivity, reproducibility and resolution. One wants to deposit a thin (to avoid changes in
mechanical properties of the cantilever) uniform (to generate a uniform stress) and compact (to avoid interactions
with the solid substrate beneath) layer of receptor molecules.
The layer should be stable and robust, hence the receptors
should be covalently anchored to the surface; on the other

Fig. 5. (a) schematic side view of a Si3N4 cantilever coated with a thin
layer of gold and a mixture of two different thiols (view is not in scale). (b)
Structure description of the short chain thiol type I and long chain
biotinylated thiol type II.

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R. Raiteri et al. / Sensors and Actuators B 79 (2001) 115126

or other multi-step procedures can be implemented like the

one described in the following.
In order to have two thiol monolayers with different endgroups on each cantilever side [51], 4 nm of chromium and
35 nm of gold were evaporated on one side only. The
cantilever was then immersed overnight in 1 mM octadecanethiol ethanolic solution. After eliminating all excess
of alkanethiols by rinsing with ethanol, an identical gold
layer (4 nm of chromium and 35 nm of gold) was evaporated
onto the opposite side. The temperature during evaporation did not rise above 1008C. The 1008C is a temperature
where thiol monolayers can stand. It is known that
above that temperature, they start to desorb [67]. The freshly
coated cantilever was then immersed into a 1 mM
ethanolic solution of thiols with different end groups. In
the case represented in Fig. 6, 3-mercaptopropionic acid
(HS(CH2)3COOH) was used. The new thiol solution could
not remove the previously bound octadecanethiol since
the thiolgold bond is relatively strong (binding energy
120 kJ/mol).
Another way to add specic functional groups to a solid
surface is to attach (graft) polymers of an appropriate
structure. To enhance polymer deposition on surfaces containing only a small number of surface reactive sites, plasma
treatment of the material can be successfully implemented
[68]. Betts et al. [69] deposited thin lms (150 nm) of
different polymers by spin coating. A focused ion beam
(FIB) mill was used to remove unwanted polymeric coating
from the opposite cantilever side.
A general method to create organic layers onto solid
substrates without the need of reactive surface sites is to
use LangmuirBlodgett (LB) deposition. LB transfers
ordered layers of amphiphilic molecules from the water
air interface to the solidair interface and allows a precise
control of multilayer formation [70].
The solgel technique can also be used for surface
functionalization. It allows creating layers of porous materials with a controlled pore size. The increase of the active

Fig. 6. Schematic view of a cantilever coated on both sides with gold and
an alkanethiol monolayer (upper side) and a carboxyl group terminated
thiol monolayer (bottom side).

area can act as a catalyst and the pore size can act as a
mechanical lter to increase the specicity of the interaction
[71].
Complementary to the development of effective coating
techniques, it is crucial to characterize the deposited layers
and, in particular, to check the specic activity of the
receptors once they are anchored onto the cantilever surface.
This can be done with different methods, depending on the
nature of the receptors. In the case of immuno-based sensors,
it is possible to use an enzyme linked immunosorbent assay
(ELISA) to quantitatively monitor the amount of active
antibodies on the cantilever surface, uorescence microscopy to get their spatial distribution, and AFM to image
their morphology [72].
5. Biosensing applications
Generally, biosensing is a more demanding task than
physical or chemical sensing because of the complexity
of the biochemical processes involved and the nature of
the operation environment. In biosensing applications,
detection is usually carried out in a liquid (aqueous) environment. Flow and mixing of the solution cause turbulence
which directly affects cantilever deection. Additional drifts
in deection have been observed. They can be due to both
slow electrochemical processes on either sides of the cantilever and to rearrangements of the sensing surface, which is
usually composed by multilayers of complex molecules like
proteins.
Despite these problems, several groups have already
showed how biosensing can be implemented using microcantilever sensors.
By measuring surface stress, one of us monitored unspecic bovine serum albumine (BSA) adsorption on an hydrophobic surface with a microcantilever [48]. More recently,
Moulin et al. [73] were able to differentiate between the
adsorption of low density lipoproteins and their oxidized
form on heparin and monitored the surface stress induced by
slow conformational changes of proteins like BSA and IgG
adsorbed onto a gold surface [74]. Fritz et al. [75] monitored
single strand DNA hybridization with two microcantilevers
in parallel. They were able to reversibly discriminate
between two 12-mer identical oligonucleotides with a single
base mismatch.
Baselt et al. [76] proposed to use microfabricated cantilevers as force transducers to detect the presence of receptor
coated magnetic beads which would specically stick onto
the functionalized cantilever surface (Fig. 1f). They showed
that it is in principle possible to detect the presence of a
single micrometer size magnetic bead sticking onto the
cantilever by applying an external magnetic eld and
measuring cantilever deection. By coating with specic
receptors, the cantilever surface and by labeling the analyte
with the magnetic beads, one could obtain an extremely
sensitive sensor.

R. Raiteri et al. / Sensors and Actuators B 79 (2001) 115126

Antonik et al. [77] proposed to sense mechanical

responses of living cells, cultured directly onto the cantilever
surface, to external chemical stimuli. With a microcantilever-based microbalance, Ilic et al. [78] could count, in air,
the number of bacteria specically adsorbed on an antibody
coated cantilever by monitoring shifts in its resonance
frequency.
5.1. Label-free bioaffinity interactions
The reported direct, label-free, transducing principles,
suitable for continuous real-time monitoring of bioafnity
interactions, include optical systems based on surface plasmon resonance [79], integrated grating couplers [80], resonant mirrors [81], interferometry [82], and reectometric
interference spectroscopy [83]. In addition, mass-sensitive
devices based on quartz crystal microbalance [84], acoustic
waves [85], and electrochemical systems employing capacitance measurements [86] were used.
In the following, we present few examples which show the
potential of microcantilever-based sensors in label-free
monitoring of specic interactions.
The results we present were obtained with a ow through
set-up: a peristaltic pump kept a constant ow through the
measuring cell for the entire experiment. Flow speeds were
in the order of 0.4 ml/min. Cantilevers were allowed to
equilibrate in a buffer solution, then the analyte was added
without stopping the ow.
5.2. Biotinstreptavidin interaction
The upper graph of Fig. 7 shows the deection response to
streptavidin addition of the biotin coated cantilever depicted
in Fig. 5. When adding 0.1 mM (6 mg/ml) streptavidin into
a continuous ow of PBS solution, the cantilever bends
away from the biotinylated surface. This corresponds to an

Fig. 7. deflection response vs. time of a biotinylated cantilever to the

addition of 0.1 mM (6 mg/ml) streptavidin (upper graph) and a 16 times
higher concentration (100 mg/ml) of BSA. After each addition, the
cantilever was rinsed in PBS.

121

increase of the compressive stress of the biotinylated side.

The same behavior is also observed during adsorption of
alkanethiols onto gold [50], DNA hybridization [75], and
antigenantibody interaction [75] and can be ascribed to
either repulsive electrostatic or steric intermolecular interactions, or to changes of the hydrophobicity of the surface.
The adsorption process shown in the upper graph of Fig. 7
can be divided into a fast and a slow process. The fast
association can be attributed to an unspecic adsorption
which is rinsed away by the buffer solution, while the slower
specic and practically irreversible interaction lasts longer
without reaching saturation in a suitable time interval. Longterm observations are, in fact, subjected to drift effects
which can be ascribed to thermal uctuation or slow reorganization processes of the adsorbed molecules as observed
by Moulin et al. [74]. To evaluate the specicity of the
interaction, we monitored the binding of BSA (Fluka,
Buchs, Switzerland) using another biotin-coated cantilever
(Fig. 7, bottom graph). BSA was chosen because its physicochemical properties at pH 7.5 are similar to those of
streptavidin: molecular weights of BSA (67 kDa) and streptavidin (60 kDa) are almost similar and the isoelectric points
are 4.74.9 and 5.66.5, respectively. The displayed results
show that binding of BSA leads to a three times smaller
deection than the deection caused by streptavidin, even
though a 16 times higher concentration of BSA is used.
Saturation of the surface is achieved within 10 min and
repeated exposure of the microcantilever to BSA solution
results only in a temporary change of signal, which is
restored after switching back to PBS. This suggests that
the binding of streptavidin is largely due to afnity
interaction with biotin and, only partially, to non-specic
adsorption.
5.3. Antigenantibody binding
As a model antibodyantigen interaction with potential
application in environmental control, we studied the binding
of 2,4-dichlorophenoxyacetic acid (2,4-D), a commercial
herbicide, to its monoclonal antibody (MAb) [87]. We
evaporated a thin gold layer (<30 nm) on one side only of
the cantilever. A monolayer was then deposited using
cysteamine. Incubation in glutaraldehyde formed a suitable
surface for adsorption of BSA conjugated with 2,4-D.
Details of the coating procedure are described in [87].
After allowing the cantilever to stabilize in buffer solution
(PBS), when adding 5 mg/ml of MAb against 2,4-D, the
cantilever bends 50 nm away from the coated side within
3 min, which corresponds to a 24 mN/m increase of compressive stress (Fig. 8). This qualitatively agrees with the
biotinstreptavidin results. Rinsing with PBS, after a transient response, probably due to mixing effects, a new steady
state is reached which is roughly 60% of the initial value.
Since the binding of an hapten to its antigen is practically
irreversible on this time scale, this decrease can be ascribed
to the desorption of unspecically bound MAb which is

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R. Raiteri et al. / Sensors and Actuators B 79 (2001) 115126

around pH 45. Increasing pH above 5 or below 4, the

cantilever bends away from the mercapto-propionic acid
side. This can be interpreted in terms of the dissociation of
the carboxyl group
COOH !

Fig. 8. deflection response vs. time of a cantilever coated with 2,4-D to the
addition of monoclonal antibody (MAb) against 2,4-D. First 5 mg/ml MAb
were added, then 25 mg/ml. Cantilever was kept in a continuous flow of
buffer.

rinsed away by PBS. When owing a higher concentration

of MAb (25 mg/ml), the cantilever bends again away from
the 2,4-D coated side by 40 nm which corresponds to a
further increase of the compressive stress of 20 mN/m.
Rinsing again with PBS does not inuence the deection.
After adding such a high concentration of MAb, probably all
the binding sites are occupied and unspecic binding does
not inuence the deection any more.
5.4. Specific protonization
Fig. 9 shows the change in surface stress as a function of
pH (connected circles) for a cantilever like the one depicted
in Fig. 6 [51]. In this case, when immersed in solution, upper
and lower cantilever sides display different thiolliquid
interfaces while the goldthiol interface is the same for
both sides. What gives the differential stress change is
therefore the thiolliquid interface (i.e. ``thiol surface'').
While continuously owing a 0.1 M KNO3 solution, a
strong base or acid is added to vary the pH, and the cantilever
deection response is monitored. Plotted values represent
the steady state deection response. When referring to the
COOH coated side, the surface stress shows a minimum

Fig. 9. Surface stress vs. pH for two different cantilevers coated on one
side with COOH thiols (circles) and OH terminated thiols, respectively.
For both cantilevers, the opposite side was coated with octadecanealkene
thiol.

COO H

For propionic acid, the dissociation has a pK of 4.87 [88].

Binding of a proton to a carboxylate group lowers the free
enthalpy of that group by DG kT logK where K is the
binding constant. With increasing pH, protons dissociate
leaving high-energy carboxylate groups on the surface. The
observed smearing of the bending over the 411 pH range
can be attributed to local mutual electrostatic interactions
among the densely packed carboxyl groups: the more negatively charged sites on the surface, the more difcult will be
for new carboxyl groups to dissociate, even at higher pH
values. A similar behavior was reported by Fritz et al. [89]
and Ji et al. [90] for cantilevers coated with thiols of different
lengths but with the same carboxyl end group. To conrm
that observed surface stress changes are due to proton
associationdissociation, other cantilevers were coated with
2-mercaptoethanol (HS(CH2)3OH).
The hydroxyl group dissociates
OH !

O H

at high pH (>10). According to this, the observed behavior

showed little change in surface stress with increasing pH
(Fig. 9, connected triangles).
With the same purpose, Fritz et al. [89] varied the number
of carboxyl-terminated thiol molecules on the cantilever
surface by coating with a mixed COOH and CH2 thiol
monolayers. They observed that, during titration, the cantilever bending decreases with decreasing percentage of
COOH terminated thiols.
5.5. Differential measurement of myoglobin
interaction with its monoclonal antibody
We and others [75,91] developed sensor prototypes to be
operated in liquid which hosts two neighboring cantilevers: a
sensing and a reference one. By monitoring the two deections in parallel, it is possible to subtract drifts, artifacts due
to mixing and changes in solution refractive index, and unspecic adsorption. As an example, two 100 mm long silicon
cantilevers like the one in Fig. 1 were coated with BSA and
anti-myoglobin monoclonal antibody, respectively, using the
same crosslinker, sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP) [72]. When
owing PBS solution both cantilevers show drifts (Fig. 10,
upper graph). In response to the introduction of 85 ng/ml of
myoglobin, both deection signals vary. However, when
considering the differential deection (sensing-reference) is
possible to cancel out the drifts effect and most of the mixing
response (Fig. 10, lower graph). A true specic adsorption
response of myoglobin to its monoclonal antibody can be
clearly observed.

R. Raiteri et al. / Sensors and Actuators B 79 (2001) 115126

123

 High sensitivity and resolution limits.

 Small quantities (few ml) of substances are needed for
analysis. This can save money with expensive reagents
and shorten analysis time.
 The possibility to create miniaturized, portable, and
potentially, implantable sensor devices.
The microfabrication process brings other advantages.

Fig. 10. Upper graph: deflection response of a BSA coated cantilever

(reference) and a anti-myoglobin MAb coated cantilever (sensing) to the
addition of 85 ng/ml myoglobin in a flow-through experiment. Lower
graph: differential deflection signal (sensing-reference).

The differential set-up can greatly increase the sensor

resolution assuming one has identical transducers for both
reference and sensing. This relies on the reproducibility of
cantilever parameters. Eq. (3) shows how cantilever sensitivity to stress changes depends on the modulus of elasticity
E of the cantilever material and the square of both the length
L and thickness t. Variations in the fabrication process can
vary these parameters. In particular, cantilever thickness can
vary not only from batch to batch, but even within the same
wafer as much as 20%. This is a main concern for some
AFM applications and several methods have been developed
to calibrate single cantilevers in terms of their spring constant [9296]. Fortunately, cantilevers on the same chip or
from neighboring chips on the same wafer can be fabricated
with an extremely high reproducibility.
In the case of Fig. 10, 85 ng/ml could be clearly detected.
This is a physiological concentration in healthy human
serum, and the ability to monitor it, allows practical clinical
applications in myoglobin detection as an early marker of
acute myocardial infarction [97].
6. Conclusions and future developments
Cantilever-based sensors are extremely versatile, they can
be operated in air, vacuum and liquid environment, they can
transduce a number of different signals, such as magnetic,
stress, electric, thermal, chemical, mass, and ow, into a
mechanical deection detected with sub-Angstrom resolution. As force sensors, they allow measurements at the level
of single molecules [3,98,99].
We showed how such a simple mechanical transduction
principle can be successfully implemented for biosensing
applications too. The micrometer size of the transducer
brings several advantages.

 Cantilevers are batch fabricated. Few hundreds of chips,

each containing a cantilever array, can be obtained from a
single 4-in. silicon wafer. This reduces cost, making
disposable sensors economically feasible.
 Arrays of cantilevers can be easily fabricated.
 They can be integrated into standard microelectronic
processing technologies like complementary metal oxide
semiconductors (CMOS). On-chip circuitry can be integrated together with fluid handling systems.
Several biomedical applications can be foreseen, such as
monitoring presence and concentration of substances in a
solution or gas, calculating specic binding energies, monitoring chemical surface reactions, studying adsorption
desorption processes of substances (isotherms and kinetics).
This can in principle be done from the single molecule scale
up to the whole cell behavior [77].
Future developments include optimization of cantilever
dimensions and shapes for maximum stress, mass, or temperature sensitivity and the use of large arrays of cantilevers
in parallel. This will allow higher signal-to-noise ratio by
averaging among identically coated cantilevers, multi-analyte analysis by coating cantilevers with different receptor
layers, and the possibility to differentiate biomolecules
which cannot be detected by a single receptor layer by
using pattern recognition algorithms. First attempts showed
the possibility to detect different vapors in air by monitoring
the deection of eight cantilevers in parallel using polymer
coatings and unspecic binding [100102].
In order to operate in parallel tens or hundreds of cantilevers which are few micrometers apart from each other, one
has to develop a simple deection detection system and a
coating technique with a high spatial resolution, i.e. capable
to deposit different layers onto different cantilevers of the
same array. The deection detection based on piezoresistive
sensors seems to be the most suitable method to monitor the
deection of many cantilevers in parallel. This method
would also allow to integrate the read-out electronics on
the same silicon chip. Depending on the nature of the coating
layer, microchannel systems where each cantilever is dipped
in a different owing solution or proximal techniques like
those used in ink jet printers could be implemented.
Further development of the technique may bring to a full
integration with uidic handling systems, other analytical
techniques, and signal extraction electronics to form a socalled ``laboratory on a chip''.
Berger et al. [103] proposed the term ``laboratory on a
tip'' to describe the possible integration of cantilever-based

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R. Raiteri et al. / Sensors and Actuators B 79 (2001) 115126

sensors with AFM where a cantilever with a tip can be scanned other a surface to position the sensor with nanometer
resolution on the spot where the biochemical analysis is
performed. The eld, although still far from being a wellestablished one, looks rather promising.
Acknowledgements
Myosin results were obtained in collaboration with
Catherine Grogan. Biotinylated thiols were a gift from
the Max Plank for Polymer Science, Mainz, Germany.
The authors would like to thank Rudiger Berger and Davide
Ricci for helpful discussions. This work was partly supported by the Italian Research Council under the MADESS
target programme and by CRUI under the Vigoni program.
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