The role of polarization-induced reorientation of DNA strands on organic field-effect

transistor-based biosensors sensitivity at high ionic strength

S. Lai, M. Barbaro, and A. Bonfiglio

Citation: Appl. Phys. Lett. 107, 103301 (2015); doi: 10.1063/1.4930303

View online: https://doi.org/10.1063/1.4930303
View Table of Contents: http://aip.scitation.org/toc/apl/107/10
Published by the American Institute of Physics

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 APPLIED PHYSICS LETTERS 107, 103301 (2015)

The role of polarization-induced reorientation of DNA strands on organic

field-effect transistor-based biosensors sensitivity at high ionic strength
S. Lai,1,a) M. Barbaro,1 and A. Bonfiglio1,2
1
Department of Electrical and Electronic Engineering, University of Cagliari, Piazza d’Armi, 09123 Cagliari,
Italy
2
CNR–Institute of Nanoscience, S3 Centre, Via Campi 213A, 41125 Modena, Italy
(Received 20 June 2015; accepted 21 August 2015; published online 9 September 2015)
The detection of the intrinsic charge of biochemical molecules is a promising strategy for the
fabrication of field-effect transistor (FET)-based sensors for direct, non-destructive detection of
several biochemical reactions. Nevertheless, the high ionic concentration of standard environments
for biochemical species represents a significant limitation to this sensing strategy. Here, an investi-
gation on the physical mechanisms behind the ability of an organic FET-based sensor to detect
DNA hybridization at high ionic strengths is proposed. The capability of the device to correctly
detect single-stranded DNA oligonucleotides and their hybridization with a complementary target
sequence has been analyzed in detail. In particular, the electrical response in solutions with differ-
ent ionic strengths was investigated and put in relation with the nano-scale properties of DNA
strands employed as receptors. Fluorescence analysis shows that it is possible to electrically modify
their orientation and consequently improve the device sensitivity in conditions close to those occur-
ring during in vivo hybridization. VC 2015 AIP Publishing LLC.

[http://dx.doi.org/10.1063/1.4930303]

The possibility of an electronic readout of chemical and
biological reactions has attracted a rising interest in the last
decades as a suitable alternative to classical analytical tech-
niques. In particular, field-effect transistor-based biosensors
(bioFETs) have been widely studied for detecting and trans-
ducing several kinds of biological reactions related to a vari-
ation of the charge and/or charge distribution in the close
proximity of the sensing area of the device.
By definition, a bioFET consists of a field-effect transis-
tor integrated with a layer of electrically active biomolecules
that act as receptors. Several transduction mechanisms can
be implemented according to the way biomolecules are inte-
grated in the device and to the employed technology.
Starting from the basic working principle of the Ion-
Sensitive FET (ISFET,1), several examples of bioFETs in sil-
icon technology have been proposed.2 More recently, several
examples of bioFETs fabricated with the organic technology
have also been proposed.3 Organic technology allows the
fabrication of devices on large areas with relatively low
costs, thus resulting particularly promising for the fabrication
of disposable biosensors on flexible substrates as plastic and
paper. Moreover, in addition to the ISFET working princi-
ple,4,5 different transduction mechanisms can be imple-
mented. For instance, the direct functionalization of the
organic semiconductor with DNA single strands has been
widely employed as a sensing strategy for DNA hybridiza-
tion detection in dry6,7 and in wet conditions.8,9
Independently of the employed technology and the
implemented transduction mechanism, the performances of
bioFETs are strictly related to the features of the receptors at
the nanoscale. Biological molecules chemically grafted onto
a surface form Self-Assembled Monolayers (SAMs), whose
a)
Author to whom correspondence should be addressed. Electronic mail:
stefano.lai@diee.unica.it
properties are strongly influenced by parameters as density,
chain length, orientation, and tilt angle with respect to the
normal of the surface; as a consequence, all these features
play a significant role in the transduction mechanism of the
device. Moreover, all these characteristics have to be consid-
ered in relation with the environment where the tests are car-
ried out, especially for operations in liquids, that is a typical
condition for the majority of biological reactions. More in
detail, the detection ability of field-effect devices is heavily
affected by the ionic strength of the measurement environ-
ment:10 the higher is the ionic strength, the larger is the
screening effect on the intrinsic charge of the molecules by
the free ions in solution. The screening effect is generally
described in terms of Debye length, j1, given by
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1 e0 er k B T
j ¼ ; (1)
2NA e2 I
where e0 is the electrical permittivity of the vacuum, er is
the relative dielectric constant of the solution, kB is the
Boltzmann constant, T is the absolute temperature, NA is
the Avogadro number, e is the elementary charge, and I is
the ionic strength of the solution. According to the defini-
tion, only the molecular charge located within the Debye
length from the sensing surface of the device can contribute
to the field-effect modulation, while the charge that lies
beyond the Debye length is completely screened. As a con-
sequence, the charge associated with large macromolecules
can be electrically detected only in very low ionic strength
solutions, i.e., in conditions that are very different from
those of reactions occurring in vivo. This is therefore con-
sidered as a strong limitation to the potential of these devi-
ces for a reliable detection. As an example, for a 1:1
electrolyte with sodium chloride, Equation (1) (Ref. 11)
can be approximated by

0003-6951/2015/107(10)/103301/5/$30.00 107, 103301-1 C 2015 AIP Publishing LLC

V
103301-2 Lai, Barbaro, and Bonfiglio
0:304
j1  pffiffiffiffiffiffiffiffiffiffiffiffiffi nm: (2)
½NaCl
Thus, for a 1 M NaCl electrolyte, the Debye length is 0.304 nm,
which is approximately the distance between adjacent bases
in a DNA chain. As a consequence, only the first bases of
hybridized DNA are unscreened and only their charge could
be detected by a field-effect device. As DNA hybridization
typically occurs in solutions with salt concentration between
100 mM and 1 M,12–14 the employment of bioFETs for
hybridization detection at high ionic strengths seems not fea-
sible. On the other hand, lowering the ionic strength of the
measurement solution (after hybridization has occurred) is
not advisable as, in these conditions, a denaturation of the
hybridized double-strands may occur.15
In the recent past, we proposed a device structure for
DNA hybridization sensing based on floating gate transistors,
which can be implemented both in CMOS (Charge-Modulated
FET, CMFET16) and organic technology (Organic CMFET,
OCMFET17,18). The structure of the OCMFET is based on a
floating gate structure; a part of the floating gate, namely, the
sensing area, is used for detection in liquid environment with-
out needing of a reference electrode in solution.19 In particular,
in Lai et al.,18 a new implementation of the device capable to
operate at low voltages was presented; such a feature was
obtained thanks to an innovative, hybrid organic-inorganic
insulating film which can be fabricated with an highly reliable
process.20 The transduction mechanism of the OCMFET is
based on the modulation of the floating gate voltage due to the
variation of the charge related to biomolecules anchored onto
the sensing area. As thoroughly explained elsewhere,21 this
turns into a modulation of the threshold voltage DVTH of the
device, proportionally to the charge on the sensing area QS
according to the relationship
QS
DVTH ¼  ; (3)
CSUM
where CSUM is the sum of all the capacitances in the device.
The shift of the threshold voltage is recorded both after the
anchoring of single-stranded DNA probes (functionalization)
and after their hybridization with fully complementary target
sequences, as a consequence of the negative charge of DNA
backbones.
Such device, though based on a field-effect transistor,
reproducibly showed record values of sensitivity and se-
lectivity at relatively high ionic strengths of the measure-
ment buffer ([NaCl] ¼ 50 mM), while, according to
Equation (2), the effect of the hybridization should be, in
this case, completely screened. These results suggest that
the relationship between the device working state, the
DNA strand’s features at the nanoscale and the ionic
strength of the measurement liquid must be considered in
a more exhaustive way.
The aim of the present paper is therefore to shed light
upon the mechanisms that give rise to these characteristics,
enhancing the relation between the “macro” properties
derived by the electrical characterization of the device, and
the micro- and nano-features of the receptor layer that allow
achieving the desired chemo-sensitivity. As it will be shown
Appl. Phys. Lett. 107, 103301 (2015)
in the following, the recorded sensitivity seems to be moti-
vated by the effect of device polarization on the orientation
of DNA single strands immobilized on the sensing surface of
the device. Such a phenomenon was thoroughly investigated
by Rant and co-workers22,23 through the direct application of
a voltage drop between a functionalized electrode and the so-
lution. By switching the voltage, a persistent tilting of the ol-
igonucleotides was obtained; in addition, the distribution of
ions inside the solution, and so the screening length, was
affected by a repulsion/attraction mechanism according to
the sign of the voltage and the charge of the ions. The combi-
nations of these effects lead to an increase of the effective
Debye length on one hand, and to an increase of the portion
of the molecule included within the Debye length on the
other hand. Such effects may also be invoked for explaining
the results obtained with the OCMFET; however, in this
case, the bias effect on the receptor orientation is indirect as
both the sensing area and the solution are floating. Thus, a
direct evaluation of the effective voltage drop between them
is impossible, thus making tests and considerations manda-
tory. In the following, a key-experiment is described and
discussed.
At first, the effect of the sole ionic strength on the sensor
response was considered. In order to decouple the effect of
hybridization efficiency from the effect of ionic strength on
the effective Debye length, the tests were carried out on the
thiol-modified, single-stranded DNA (HS-ssDNA) probes
onto the sensing area. Indeed, the ionic strength of the
hybridization solution strongly affects the efficiency of the
hybridization process. Thus, by varying the ionic strength in
an experiment on double-stranded DNA (dsDNA), a varia-
tion in the sensor response could be observed as an effect of
the hybridization efficiency variation rather than, purely, to a
variation of the Debye length. A 24-base long probe
sequence was employed, P24 ¼ HS 50 -(T)6-GGT TTC CGC
CCC TTA GTG-30 ;19 beyond this length, ssDNA molecules
typically starts to fold.24
The functionalization was verified by extracting the
threshold voltage variation from the transfer characteristic
curves taken before and after the process, according to a ba-
sic model developed by Braga and Horowitz.25 In particular,
differential measurements were performed: a reference (i.e.,
not functionalized) device underwent to the same biological
and chemical treatments and to the same storing and mea-
surement conditions of the sensor, in order to evaluate and
compensate any unspecific threshold voltage variation in the
sensor response. The Debye length of the measurement buffer
was modulated by employing different NaCl concentration
([NaCl] ¼ 50 mM, 10 mM, 1 mM); consequently, estimated
Debye lengths j1 of 1.36 nm, 3.04 nm, 9.62 nm, respectively,
were obtained.
The length of P24 is about 7.9 nm, so, accordingly to
the estimated Debye length in the experimental conditions,
the sensor response has been recorded when the probe
should be almost completely screened ([NaCl] ¼ 50 mM),
when almost one half is unscreened ([NaCl] ¼ 10 mM), and
when it is totally unscreened ([NaCl] ¼ 1 mM). This sce-
nario is described in the cartoon in Figure 1(a). In Figure
1(b), the net variation of the threshold voltage recorded in
the different salt concentrations is reported. In particular,
103301-3 Lai, Barbaro, and Bonfiglio
the values have been obtained by averaging upon three dif-
ferent samples for each value of the salt concentration. It is
possible to notice that the net threshold voltage variation
due to the electrical charge of the probes decreases as the
salt concentration increases, as expected, thus demonstrat-
ing that a coherent response of the sensor can be obtained
in “quasi-static” conditions.
Subsequently, in order to further explore the mecha-
nisms that give rise to the observed sensitivity of the device
in hybridization detection, the tests carried out in Ref. 18
were here repeated by modulating the probe length for a
fixed Debye length in the measurement buffer. In particular,
three probes were employed: P24, P31, and P42. The only dif-
ference between the sequences was the initial timing spacer,
which is, respectively, 6, 13, and 24 bases long, thus setting
the distance between the first base of the hybridized segment
at 1.98 nm, 4.3 nm, and 7.92 nm from the anchoring point of
the DNA chain, respectively. The P31 sequence was the same
employed in the original tests reported in Ref. 18; conse-
quently, P24 and P42 allow considering a lower and a higher
hybridization distance. Hybridization was performed in am-
bient conditions and at room temperature.19 The electrical
response to the hybridization was measured by monitoring in
real-time the output current of the sensors. In the inset of
Figure 2, the measurement setup is shown: a constant
source-to-drain voltage drop VDS was set, while the control
gate was biased with a square-wave voltage VGS in order to
reduce the bias stress effect on the organic semiconductor
that could lead to a continuous current reduction possibly
masking the expected current increase related to the hybrid-
ization effect. The amplitude of the gate voltage was chosen
FIG. 1. (a) Schematic representation of the functionalized sensing area, with
the different employed Debye lengths in relation to the probe chains’ length;
(b) electric response of the OCMFET to the functionalization as a function
of the different employed Debye lengths.
Appl. Phys. Lett. 107, 103301 (2015)
FIG. 2. (a) Percentage variation of the output current as a response of the
device to hybridization process when chains with different spacers are con-
sidered; in the inset, the variation for P42 probe is reported with a different
scale to result visible in the plot; (b) supposed characteristics of the DNA
monolayer during the measurements; in the inset, the employed measure-
ment setup is shown.
in the different devices (that may have different threshold
voltages) in order to operate them in the same over threshold
conditions. VGS frequencies from 10 Hz to 100 Hz were cho-
sen. The efficiency of the hybridization was evaluated by
measuring the relative variation in the output current, DIDS/
IDS,baseline, where IDS,baseline is the output current before the
spotting of the target sequences. The results are reported in
Figure 2(a). By reducing the length of the spacer, hybridiza-
tion can be better detected by the sensor, as expected.
Interestingly, the hybridization signal is almost completely
lost (DIDS/IDS,baseline of about 0.1%) only with the P42 chain,
i.e., with a 24-base long spacer. According to Equation (2),
with the ionic strength chosen for this experiment, the
expected Debye length should be 1.4 nm; therefore, the
screening effect of the ions in solution should affect the de-
vice response for any chain length, not only for P42. As this
did not happen, we must assume that the dsDNA molecules
are not lying perpendicularly to the surface but are tilted to a
certain extent such that, at least for P24 and P31, a certain por-
tion of the molecule is included within the Debye length; a
plausible scenario is shown in Figure 2(b).
Tilt angles of 30 have been reported for the DNA
molecules in the monolayer formed on gold coated silicon
with a similar biochemical procedure.26 According to the
obtained results, the tilting should be substantially higher
than this value (about 80 , estimated as the arc sine of the
ratio between the Debye length and the spacer length).
Such large difference with the previously extracted value
of the tilt angle could be partially explained with the
roughness of the sensing area, which is larger than that
reported for gold on silicon as the device is entirely fabri-
cated on plastic.27 In addition, it could also be an effect of
the polarization of the sensing area on the DNA chains, as
suggested in Ref. 22. Moreover, as in this case a redistribu-
tion of the screening ions induced by the charged surface
may occur, a larger Debye length can be assumed, and an
actual tilt angle smaller than estimated could be conse-
quently derived.
103301-4 Lai, Barbaro, and Bonfiglio
A final, key-experiment was then done, in order to
prove the possible effect of the device polarization on the
specific tilt/repulsion effect observed in the previous
experiments. Variations in the DNA strands’ tilt angle due
to the device’s polarization have been investigated by
means of fluorescence quenching tests. P31 ssDNA strands
modified with Cyanine-3 fluorescent dyes were employed
in the functionalization process; the chains were later hybri-
dized using the complementary sequence according to the
previously reported procedures. Quenching was evaluated
by analyzing the photographs of the sensing area taken by
means of a digital camera directly connected to the fluores-
cence microscope. As for the hybridization measurements
previously presented, VGS was switched from 0 to 2 V
using a waveform generator. Several seconds are needed to
observe a switch of DNA in the experiments reported in
Ref. 22; in our measurements, a period of 60 s was waited
in order to allow a complete reorientation of the molecules
after switching the potential. This slow response is prob-
ably related to the fact that the voltage drop is not directly
imposed, thus being affected by the device dynamic. By
evaluating red (R), green (G), and blue (B) levels obtained
by an average on the whole picture by a custom-made
R
MatlabV script, the luminance of the image was calculated
L ¼ 0:2126 R þ 0:7152 G þ 0:0722 B: (4)
The results are reported in Figure 3: it is possible to observe
that, as a response to the negative voltages applied to the
control capacitor and to the drain, a reduction of the lumi-
nance was obtained with respect to the zero-voltage condi-
tion. Moreover, when the device was grounded again, the
original luminance was perfectly restored. In Figure 3(b), the
wavelengths in the emission band of the Cy-3 dye
(570–650 nm), which comprises red, yellow (Y), and ma-
genta (M) colors, are separately analyzed and compared with
FIG. 3. (a) Results of the fluorescence quenching tests when a polarization
is applied to the sensor in terms of relative variation of luminance; (b) corre-
spondent relative variation of red, yellow, magenta, green, and blue levels
are shown to correlate he luminance variation with the Cy-3 emission band.
In the cartoon, the correspondent quenching event within the applied voltage
is depicted.
Appl. Phys. Lett. 107, 103301 (2015)
the green and blue levels, which, on the contrary, lie outside
the emission band. It is clearly noticeable that the luminance
variation is mainly related to the variation of RYM levels
during the application of the polarization, while the G and B
levels are almost constant. These results prove a fluorescence
quenching, which occurs when Cy-3 dyes are close to a
metal surface that absorbs part of their energy, thus leading
to a reduction of the fluorescence. Consequently, the
observed quenching can be reasonably ascribed to a physical
displacement of the fluorescent sites in the DNA molecules
that occurs as a response to the voltage switching. This result
demonstrates that the conditions for inducing a tilt of the
DNA molecules are obtained in the OCMFET structure
when it is in the ON state. As a consequence, the charge to
be sensed is closer to the surface, and, at the same time, the
Debye length in the solution is increased as a consequence of
the repulsion of the positive ions from the surface.
Moreover, the attraction exerted by the surface potential on
the targets compensates the electrostatic repulsion with the
probes, determining a faster and more efficient hybridization.
The combination of these effects is therefore suggested for
explaining the capability of OCMFETs of detecting DNA
hybridization with a sensitivity that is well beyond the limits
normally accepted as due to screening effects in liquids. The
proposed analysis, never performed before for FET-based
sensors, is of general interest, suggesting that several biolog-
ical reactions, in addition to DNA hybridization, as antigen-
antibodies interactions and enzyme activity, can be detected
by field-effect devices in liquids at relatively high salt con-
centrations, i.e., in conditions comparable with an in vivo
environment.
S. Lai gratefully acknowledges Sardinia Regional
Government for the financial support of his Ph.D. scholarship
(P.O.R. Sardegna F.S.E. Operational Programme of the
Autonomous Region of Sardinia, European Social Fund
2007–2013-Axis IV Human Resources, Objective l.3, Line of
Activity l.3.1.).
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