Article

pubs.acs.org/ac

Part per Trillion Label-Free Electronic Bioanalytical Detection

Maria Magliulo,*, Antonia Mallardi, Roberto Gristina, Francesca Ridi,, Luigia Sabbatini,
Nicola Cio, Gerardo Palazzo,, and Luisa Torsi

Dipartimento di Chimica, Universita degli Studi di Bari A. Moro - Via Orabona, 4 70126 Bari, Italy
CNR-IPCF, Istituto per i Processi Chimico-Fisici - Via Orabona, 4 70126 Bari, Italy

CNR-IMIP, Istituto di Metodologie Inorganiche e dei Plasmi - Via Orabona, 4 70126 Bari, Italy

Dipartimento di Chimica Universita degli Studi di Firenze via della Lastruccia, 3 50019 Sesto Fiorentino, Italy

CSGI Universita degli Studi di Firenze via della Lastruccia, 3 50019 Sesto Fiorentino, Italy

S Supporting Information
*

ABSTRACT: A Functional Bio-Interlayer Organic Field-Eect Transistor (FBI-OFET)

sensor, embedding a streptavidin protein capturing layer, capable of performing label-free
selective electronic detection of biotin at 3 part per trillion (mass fraction) or 15 pM, is
proposed here. The response shows a logarithmic dependence spanning over 5 orders of
magnitude of analyte concentration. The optimization of the FBI analytical performances is
achieved by depositing the capturing layer through a controllable Layer-by-Layer (LbL)
assembly, while an easy processable spin-coating deposition is proposed for potential lowcost production of equally highly performing sensors. Furthermore, a Langmuirian
adsorption based model allows rationalizing the analyte binding process to the capturing
layer. The FBI-OFET device is shown to operate also with an antibody interlayer as well as
with an ad hoc designed microuidic system. These occurrences, along with the proven extremely high sensitivity and selectivity,
open to FBI-OFETs consideration as disposable electronic strip-tests for assays in biological uids requiring very low detection
limits.
bilayer is ipped so that the Functional Biological Interlayer
(FBI) is placed underneath the OS,1517 in direct contact with
the dielectric and right at the interface where the OFET twodimensional transport occurs.18,19 As already reported,15 the
direct interfacing between the biological capturing layer and the
two-dimensional organic channel allows for electronically
probing subtle changes occurring in the capturing layer while
exposed to an external agent, eventually permitting the
measurement of an extremely sensitive response. A detailed
comparison between this technology and the state-of-the-art in
OFET biosensors is extensively provided elsewhere.15,20,21
Indeed, the FBI-OFETs show good electronic properties, while
the biological activity of the interlayer is retained.
The so far proposed FBI-OFETs included staking bilayer
structures realized by spin-coating both the biological and the
organic layers.15,20 This paper proposes the study of FBIOFETs embedding a streptavidin (SA) layer deposited either
by electrostatic layer-by-layer (LbL) assembly22 or by spincoating, providing an assessment of the analytical performances
for both structures. The LbL assembly approach allows for a
more controlled deposition, and here it is used to optimize the
analytical performance of the capturing layer. This deposition
involves, on the other hand, procedural steps that are not easily

lectronic bioanalytical detection performed by means of

disposable plastic or paper devices has the potential to
revolutionize the current approach to strip-testing. Presently,
low-cost tests largely rely on lateral ow strips1 that can deliver
an analogic output, inherently not processable. An organic
electronic sensing device would be capable, in principle, of
producing a digital output allowing data processing for
quantication purposes. Such devices can be produced by
printing procedures likely at costs comparable to those of
presently commercially available analogic strips. Eventually, the
idea of a paper strip printed with an electronic biosensor circuit
to be used as a disposable cartridge can be conceived as being
capable of directly feeding a miniaturized reading system with a
digital output. Such systems would be ideal to perform reliable
analytical quantication at the point-of-need and at low-costs.
Such a perspective is triggering a great amount of research
activities in the eld of organic electronic biodetection
worldwide.24 These endeavors involve the use of transistor
sensing devices and see contributions proposing properly
biofunctionalized silicon or carbon nanostructured electronic
materials5 as well as organic semiconductor (OS) thin-lms.68
The latter involved so far either the so-called electrolyte gated
Organic Field-Eect Transistors (OFETs)9,10 or bottom-gate,
top-contact OFETs bearing a bilayer architecture as active
layer.1113 The elicited bilayer is formed by a bioactive coating
grafted on top of the OS, oering the advantage of the low-cost
manufacturing procedures typically employed in organic
electronics.14 In a novel OFET sensor approach, the elicited
2013 American Chemical Society

Received: September 21, 2012

Accepted: January 16, 2013
Published: January 16, 2013
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The lms morphology was inspected by means of Atomic

Force Microscopy (AFM) on a XE-100E (Park System Corp.,
Korea) in Non-Contact Mode using silicon tips (curvature
radius <10 nm, force constant 40 N/m).
Fabrication of FBI-OFETs. The devices were fabricated on
a highly n-doped silicon substrate acting as the gate contact,
covered by a 300 nm thick thermally grown SiO2 gate dielectric.
The SiO2 surface was rinsed with solvents of increasing polarity
before depositing the dierent capturing layers. The P3HT lm
was then deposited, from a chloroform solution (2.6 mg/mL),
directly over the capturing layer by spin-coating at 2000 rpm
for 30 s. The P3HT lm thickness was about 20 nm, and its
uniformity was inspected by optical microscopy. Source (S),
drain (D), and gate (G) contacts were deposited by thermal
evaporation (8 107 Torr) of gold through a shadow mask
resulting in a sequence of 38 rectangular pads (4 mm wide, this
being the channel width, W) spaced by 200 m (this being the
channel length, L). A device top view micrograph picture is
provided in the TOC entry.
Electronic Response Measurements. The FBI-OFETs
were measured in the common-source conguration, and
electronic parameters (FET, on/of f ratio, and VT) were
extracted from the ID-VG transfer-characteristics, keeping VD
xed at 80 V. The measurements were performed in the dark
and under a continuous nitrogen purging; the curve sweeping
time was 45 s. The baseline (reference) ID-VG transfer curve
measurement was taken by depositing a droplet (2 L) of water
HPLC grade, directly on the P3HT surface in the OFET
channel, i.e. between two contiguous source and drain pads.
The device was subsequently incubated for 15 min and dried
under a nitrogen ow. The ID-VG transfer characteristic was
then measured, and the I0 value was taken as ID at VG = 100
V. The response to biotin was measured afterward, by placing a
2 L droplet, at the lowest biotin concentration, on the same
OFET channel and incubating the device for 15 min.
Subsequently, the excess solution was rinsed with water and
after drying the OFET in nitrogen, the ID current was
measured; the ID (analyte) value was taken also at VG =
100 V. The biosensor response was nally evaluated as the ID
fractional change, i.e. as (ID (analyte) I0)/I0 = I/I0. Five
increasingly higher biotin concentrations were evaluated too on
the very same OFET channel, following the aforementioned
procedure. Error bars assessing the responses reproducibility
are evaluated as the relative standard deviations (RSDs) on the
same set of responses taken from three dierent channels. This
approach allows collecting a set of uniform response data
throughout all the calibration curve, but it does not allow
evaluating reproducibility of a single response over the whole
chip of 37 OFETs. To this aim, responses at one randomly
chosen concentration were taken on three dierent OFET
channels laying on the same chip. The measure of the whole
calibration curve by this procedure involved the evaluation of
up to 21 dierent sensors overall. The response was evaluated
also in this case as the ID fractional change (I/I0) at VG =
100 V.
The measurements with the microuidic system were
performed in continuous f low with all the previously described
steps carried out by subsequently pumping the liquid (water or
biotin solution) with a micro syringe in the microuidic system
running into the transistor channel (see TOC and video entry
for details). The ID-VG transfer characteristics were measured,
this time, while the liquid resided on the channel.

scaled-up; besides it can be implemented only with charged

species. This latter limitation does not hold for spin-coating
deposition procedures that are also more compatible with
organic electronics based low-cost production processes. A
Langmuirian adsorption based model is also proposed to
rationalize the analyte binding to the streptavidin molecules
loaded in the FBI, and the analytical performances of the LbL
and spin-coated capturing layers are discussed accordingly.
From a more applicative point of view, the device is shown to
operate also through an ad hoc designed microuidic system.
This, along with the proven extremely high sensitivity and
selectivity, opens to FBI-OFETs appraisal as disposable
electronic strip-tests for assays in biological uids requiring
highly sensitive detection, such as saliva or even eye-drops.

EXPERIMENTAL SECTION
Materials. Streptavidin (SA), biotin, and poly(dimethyldiallylammonium chloride) (PDDA) were purchased
from Sigma-Aldrich. Alexa Fluor488 streptavidin and antibiotin
monoclonal antibody (Biotin, Mouse IgG1-cod. 033700) were
purchased from Invitrogen. Poly(3-hexylthiophene-2,5-diyl) P3HT - (Sepiolid P200, regioregularity >98% from BASF) was
puried as reported elsewhere.23
Capturing Layer Deposition Procedures. The streptavidin FBI layers have been deposited, directly on the Si/SiO2
substrate, either by layer-by-layer electrostatic deposition (LbL)
or by spin-coating. The former deposition procedure started
from a negatively charged SiO2 surface. This charging was
achieved through an oxidative cleaning the SiO2 substrate in a
piranha solution (oleum H2SO4 and H2O2, 7:3 v/v) for 15
min at 0 C with subsequent rinsing in water for 5 min. To
adsorb the rst polycation layer, the substrate was immersed in
a 2 mg/mL PDDA water solution for 30 min. Then, the sample
was washed in water for 2 min, and a layer of streptavidin was
adsorbed by dipping it into a 100 g/mL protein solution for
30 min. The sample was rinsed with water, and the above steps
were repeated a number of times to obtain the required number
of PDDA/streptavidin layers. The thickness of a two-layer LbL
deposit was evaluated with a prolometer to be 30 2 nm. To
spectroscopically quantify the amount of SA deposited by LbL,
SA molecules conjugated with Alexa Fluor488 dyes were
deposited on a quartz optical window (1 cm 3 cm from
Hellma); after the deposition of each SA layer, the absorbance
of the peak at 494 nm was measured. The extinction coecient
of the Alexa Fluor dye at 494 nm is 494 = 75 106 cm2 mol1.
On average, ve molecules of dye are conjugated to a protein
molecule (information from the manufacturer), and the
spectrophotometric measurement probes the streptavidin
adsorbed on both slide sides. Taking into account the above
pieces of information the amount of SA moles per square
centimeter was evaluated.
For the spin-coated lm, 50 L of a 10 g/mL aqueous
solution of SA was spin deposited at 200 rpm for 40 min. The
resulting SA lm was 35 5 nm thick and the estimated SA
surface density was 10 pmol/cm2, this being comparable to a
fully packed SA monolayer (6.2 pmol/cm2).24 Analogous
procedures were used to spin-deposit the SAbiotin complexes
preformed in solution, the antibiotin antibody as well as the
bovine serum albumin (BSA) proteins. Specically, 50 L of an
aqueous solution of antibiotin (100 g/mL), BSA (100 g/
mL), and an SAbiotin complex (SA 10 g/mL and biotin 0.2
g/mL) were spin deposited at 200 rpm for 40 min.
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RESULTS AND DISCUSSION

FBI-OFETs exploiting the streptavidinbiotin receptor-analyte

system are studied here to provide a proof-of-principle of the
ultimate performance limits that can be reached in electronic
bioanalytical assay operating in pure water. In fact, the SA
biotin complex dissociation constant is extremely low (KD 1014
M)25 allowing, in principle, detection down to the fM
concentration level. Indeed, the integration of most commonly
available lower anity biosystems would result in higher
detection limits. Due to the high anity, SAbiotin chemistry
has been widely exploited in previous works either to detect
biotin (by using SA as receptor) or to quantify SA (by using
biotin as receptor). However, the detection capability of the
two approaches is quite dierent as the binding of a very large
protein (SA weights 60.000 Da) to a small molecule (just 244
Da for biotin) is likely to perturb the transducing system much
more than in the opposite way around. Accordingly, label-free
electronic quantication has been limited to SA detection, so
far. Specically, biotinylated lipid bilayers supported on carbon
nanotube FETs have been proven to provide SA detections in
the 5 M or 312 part per million (mass fraction, ppm) range,26
while silicon nanowires transistor delivered 2.5 M (156
ppm).27 Better performances were achieved with a biotinylated
OS blend, capable of detecting 0.8 nM (0.05 ppm) of SA.28 The
best performance level reached was 10 pM (0.6 part per billion,
ppb) SA detection obtained with an extremely sensitive system
such as the biotinylated single silicon nanowire transistor is.29
None of these studies involved the assessment of analytical
gures of merit such as limit of detection, limit of
quantication, analytical sensitivity, repeatability, or reproducibility.
As anticipated, the quantication of small molecules is a
rather demanding task, and, in the case of biotin, nM
concentration levels have been detected at most, even with
extremely sensitive nonelectronic determinations such as an
electrochemical30 and a label needing uorescent31 assay, both
carried out in solution. In the label-free assay proposed here
biotin is detected at the pM level, by using an SA FBI as
receptor layer. This approach involves the so far avoided
integration of a protein capturing layer into an electronic device
(vide inf ra), obtaining direct interfacing between the capturing
layer and the OS transistor electronic channel. Such a device
conguration allows, for the rst time to the best of our
knowledge, the electronic (not electrochemical) probing of
biotin. Besides, the protein integration approach proposed is
quite versatile; indeed preliminary results on the integration of
an antibody and an enzyme are presented too.
Streptavidin Functional Biointerlayer OFET. A schematic representation of a FBI-OFET structure comprising a
streptavidin (SA) layer interfaced to a poly(3-hexylthiophene)
(P3HT) layer is reported in Figure 1a. The currentvoltage
characteristics obtained for a FBI-OFET integrating an SA layer
deposited by spin-coating are reported in Figure 1b. Similar
curves were recorded also for FBI-OFETs including a LbL
deposited SA assembly. The IV characteristics of the FBIOFET bioelectronic device exhibit current modulation with
well-shaped linear and saturated regions as well as fairly low
leakage current at low source-drain voltage. The device gures
of merit have been acquired by extrapolation from the sqrt |ID|
vs VG curve, following an assessed procedure.32 To this aim, the
capacitance per unit area of the FBI-OFET was measured to be
in the nF/cm2 range. This capacitance is comparable to that of

Figure 1. (a) FBI-OFET structure comprising a streptavidin (SA) FBI

layer interfaced to a P3HT organic semiconductor (OS) layer. (b) IDVD output characteristics for a spin-coated SA FBI-OFET; VG ranges
from +20 V to 100 V. Inset: sqrt |ID| vs VG at VD = 80 V.

the sole 300 nm SiO2 dielectric as the capacitance of the SA

layer is much higher. Due to the relatively thick dielectric layer
used in this case, quite high biases have been applied to operate
the OFETs. It has been extensively demonstrated, however,
that OFET sensors can be operated even in the subvolt regime
if a thinner dielectric is used.3336
The bioelectronic device gures of merit, averaged over 10
samples, are reported in Table S-1; a comparison with the bare
P3HT device is also provided. While a reduction in
performance is observed for the bioelectronic FBI-OFET
device, the operational level is however high enough to allow
their use for sensing purposes.
FBI-OFET Integrating a LbL Assembled Capturing
Layer. The optimization of the SA capturing layer was
performed by using the LbL deposition procedure as it allows,
compared to spin-coating, a better control of the uniformity of
the lm coverage and thickness, hence, of the available SA
binding sites. In Figure 2a a spectroscopic quantication for the
LbL SA deposits is proposed by using the dye conjugated SA
molecule. As shown in the inset of Figure 2a, the amount of
bound streptavidin per unit area (S) increases by ca. 5 pmol/
cm2 for each SA layer deposited. In Figures 2b and 2c the
uorescent microscopy images of a single and double SA LbL
deposits are shown, respectively. The dierent degree of
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Figure 2. (a) Absorption spectra of PDDA/Alexa Fluor488 SA

multilayers; inset: S values vs the number of deposited layers.
Fluorescence microscopy images of a single and double LbL deposit of
PDDA/Alexa Fluor488 labeled SA are reported in panels (b) and (c)
respectively.

coverage achieved, the double LbL deposit being more uniform

and compact, is readily apparent.
In Figure 3a the LbL FBI-OFET transfer characteristics (ID
vs VG at VD = 80 V) measured upon exposure to dierent
concentrations of biotin are reported; the response to pure
water is used as reference, and all the curves are taken from the
very same device. A current decrease, scalable with the biotin
concentration, occurs even at a concentration as low as 10 part
per trillion. Figure 3b reports the biosensor response for FBIOFETs embedding single and double SA LbL assemblies,
showing that a saturation occurs at lower concentration on the
single deposit, that also exhibits a lower maximum response.
This proves that a two LbL deposit is needed to ensure that a
sucient surface density of SA capturing sites is available to
perform the sensing over ve dierent biotin concentrations on
the same OFET channel. Accordingly, the surface density

Figure 3. (a) LbL SA FBI-OFET transfer characteristics (ID vs VG at

VD = 80 V) measured on the same transistor channel after exposure
to pure water and to solutions at dierent biotin concentrations. Each
curve takes 45 s to be acquired. (b) FBI-OFETs responses as a
function of biotin concentration for devices based on a single and
double SA LbL deposits. Measurements were performed by adding
increasingly more concentrated biotin solutions, to the same channel.
Lines represent the ts according to eqs 1 and 3; best ts parameters
are as follows: single LbL deposits a = 1.1 0.3, c = 0.042 0.001
decade1, S 2 2 pmolescm2; double LbL deposit a = 1.9 0.1, c =
0.070 0.006 decade1, S 6 3 pmolescm2. (c) Left ordinate
(diamonds): biotin calibration plot for a FBI-OFET based on two LbL
SA deposits. Measurements were performed acquiring the response to
a single concentration on three dierent OFETs and the data points
are the average over the three replicates, while error bars are the
relevant standard deviations. The limit of detection and limit of
quantication are also marked on the graph. Line represents the t
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response reproducibility at a single concentration. For these

data the limit of detection (LOD), estimated3739 as three
times the standard deviation of the reference solution is 3 ppt
(or 15 pM), while the estimation of the limit of quantication
(LOQ) as ten times the reference standard deviation results in
10 ppt. Further on, the biosensor response at the lowest
concentration assayed is found to be 8.8 times higher than that
of the reference solution (average over 8 replicates). The
analytical sensitivity (dened as the slope of the response vs the
logarithm of the concentration) is 0.07 decade1 as measured in
the 0.01 ppb10 ppb dynamic range, while the error bars,
evaluated as the relative standard deviations (RDS) over three
replicates, are within 14%.
Interestingly, the SA FBI-OFET electronic response varies
logarithmically with the delivered biotin concentration. This is a
unique feature as in Figure 3c the response of the assay based
on the quenching of the SA intrinsic uorescence in solution is
shown to be proportional to the number of bound biotin.39
Here, F/F [(FF)/F with F being the uorescence in the

Figure 3. continued
according to eqs 1 and 3; best ts parameters are as follows: a = 1.8
0.1, c = 0.070 0.007 decade1, S 13 7 pmolescm2. Right
ordinate: fractional decrease (F/F0) of the intrinsic SA uorescence
after biotin binding in solution (squares and triangles represent two
dierent sets of measurements); the line is the best-t assuming that
F/F0 is proportional to the fraction of SA binding (S/4S) as
described in eq 3.

parameter (S of ca. 10 pmol/cm2) and the thickness (30 2

nm) of the two LbL SA assembly were chosen as optimal ones
for further investigations.
To assess the analytical performance level of this type of SA
OFET to a further extent, a calibration curve is produced on a
chip embedding a double LbL SA assembly. This time the
electronic responses are measured at a randomly chosen
concentration, from three dierent devices (Figure 3c). As
anticipated, this procedure allows a better evaluation of the

Figure 4. (a) Spin-coated SA FBI-OFET transfer characteristics (ID vs VG at VD = 80 V) measured on the same transistor channel after exposure to
pure water and to solutions at dierent biotin concentrations. Each curve takes 45 s to be acquired; (b) transfer ID VG characteristics for a FBIOFET embedding an antibiotin monoclonal antibody exposed to pure water and to 0.01 ppb and 1 ppb biotin solutions; (c) response of a spincoated SA FBI-OFET to dierent biotin concentrations. Each data point is the I/I0 mean value over three replicates measured on dierent OFET
devices. Error bars, barely visible are the standard deviations. Line represents the t according to eqs 1 and 3; best ts parameters are as follows: a =
1.8 0.1, c = 0.070 0.007 decade1. (d) Response to dierent biotin concentrations for FBI-OFETs embedding several capturing layers. Triangles:
saturated streptavidinbiotin complexes; squares: P3HT-OFET; diamonds: BSA FBI-OFET.
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characteristics (ID vs VG at VD = 80 V) measured in the

presence of dierent concentrations of biotin; the response to
pure water is reported as a reference. Also in this case a current
decrease, scalable with the biotin concentration, occurs even at
concentration as low as 10 part per trillion. The data reported
in Figure 4a demonstrate that the detection performed with the
FBI-OFET is also fast as the potential swept takes 45 s. In
Figure 4b similar curves are reported for a FBI-OFET
embedding an antibiotin monoclonal antibody; this is a positive
control experiment exhibiting responses comparable to those of
the SA FBI-OFET, even at the lowest biotin concentration. In
Figure 4c the ID fractional changes (sensor responses) are given
in a semilogarithmic plot resulting in slightly higher estimated
limit of detection (10 ppt, 50 pM) and limit of quantication
(20 ppt, 100 pM) both being nevertheless, extremely low. The
analytical sensitivity is 0.09 decade1 as measured in the 0.01
ppb10 ppb dynamic range, while the error bars, evaluated as
the RSD over three replicates on dierent OFETs, are within
15%. In Figure 4d, negative control experiments show that
biotin induces negligible changes in the bare P3HT-OFET
(maximum I/I0 response below 4%). Similarly, exposure to
biotin is ineective on FBI-OFETs whose biological interlayer
is formed by presaturated streptavidinbiotin complexes. The
dierences observed between FBI-OFETs based on unsaturated
and saturated SA molecules demonstrate that the electronic
response really probes specic binding events at an extremely
low concentration level. The third negative control experiment
in Figure 4d is relevant to the device embedding a spin-coated
BSA protein not capable indeed of any specic binding.
Sensor measurements can also be performed when the
analyte is delivered through a properly designed microuidic
system. In Figure 5a a photograph of the FBI-OFET chip is
reported, while Figure 5b shows an optical microscope image of
six source and drain pads with the microuidic system running
into the OFET channel and dispersing the analyte far from the
source and drain contact regions. The output data, for a spincoated SA FBI-OFET at 10 ppt of biotin concentration, are
reported in Figure 5c. Also in this case a clear current decrease
can be seen at very low concentration, and the response is
provided in few minutes. It is also interesting to note that the
electronic measurements could be performed, while the liquid
resided on the channel, despite the high biases applied, because
the microuidic system was designed to run in the middle of
the transistor channel, avoiding contacts between the S and D
pads and the liquid (see Figure 5b).
A video showing the measurement of a 10 ppt biotin solution
performed with an SA FBI-OFET endowed with the ad hoc
designed microuidic system is also provided as the Supporting
Information.
Modeling of Biotin Binding Process in FBI-OFETs. Due
to the extremely low SAbiotin complex dissociation, the
delivered biotin molecules are readily bound to any active SA
binding site and saturation is achieved, at least in solution, in
the presence of one to four protein-to-ligand mole ratio.
According to the experimental evidence previously presented,
the responses of SA FBI-OFETs have been modeled through a
logarithmic dependence on the number of occupied sites. This
assumption is based here on a mere phenomenological
observation, whose rationalization being presently still under
scrutiny. Indeed, such dependence could be in principle
ascribed to an electrochemical potential variation occurring at
the gate dielectric interface, in analogy to what was already
proposed for ISFET pH sensors.43 However, preliminary

presence of biotin and F the baseline signal] is reported on the

right side ordinate axis. The comparison unambiguously shows
how the FBI-OFET logarithmic response allows analyte
quantication over an exceptionally large dynamic concentration range.
Characterization of a Spin-Coated Streptavidin
Capturing Layer. The analytical performances of the LbL
capturing layer are of very high level; however, this deposition
procedure is not easily scalable in a printing fabrication process.
In this respect, a spin-coating or a drop casting procedure
would be preferable; besides the LbL deposition is applicable
only for charged biorecognition elements, while spin-coating
does not have this limitation. By using the same capturing
surface sites density and lm thickness parameters already
optimized on the LbL capturing layer, a spin-coated SA lm
was deposited in the FBI-OFET conguration. In Figure S-1a a
uorescent microscope image of a spin-coated SA layer, holding
a nominal S of 10 pmol/cm2 and a thickness of 35 5 nm, is
reported. The image shows indeed a uniform deposit
resembling that reported in Figure 2c.
Atomic Force Micrographs of a spin-coated P3HT lm, a SA
layer, and a SA-P3HT bilayer are reported as Supporting
Information in Figure S-1b, Figure S-1c, and Figure S-1d,
respectively. The AFM investigation performed on the P3HT
deposited directly on the atomically at SiO2 (Figure S-1b)
clearly shows that the morphology is composed of granular
domains ca. 1020 nm wide, along with voids of comparable
size. This is in full agreement with data already published on
RR-P3HT morphology.40 A spin-coated SA deposit is pictured
in Figure S-1c exhibiting larger features with an average size up
to 200 nm that can be associated with aggregates of SA
molecules. The SA lm coated with P3HT is featured in Figure
S-1d; here a very structured surface is seen resembling that of
the underneath biodeposit. Also in this case the granular
features of the P3HT are seen on the very top. An image
analysis, in fact, clearly shows a features size bimodal
distribution in this case. Voids are also present that are likely
to allow analyte molecules to percolate through the OS down
to the SA layer.
One of the major potential pitfalls in the FBI-OFET
conguration is in fact the analyte eective capability to ow
through the P3HT layer reaching the underneath biological
recognition elements. To demonstrate that this is not a limiting
step, a lm of the horseradish peroxidase (HRP) enzyme was
embedded into a FBI-OFET, and it was allowed to interact with
a chemiluminescent (CL) system including luminol molecules
as described in the S3.2 paragraph and in Figures S-2a and S-2b.
This experiment proves that luminol molecules can percolate
through the P3HT lm and also that the HRP enzymes
bioactivity is retained when subjected to the FBI-OFET device
fabrication processing, in particular the chloroform spreading.
This latter occurrence is in line with what was stated by the
wide literature concerning the use of enzymes and proteins in
dry organic solvents.41,42 As a further step it was also proven
that not only small molecules such as biotin (or luminol) can
percolate but also a biological molecule such as insulin
(molecular weight 5808 Da) and even streptavidin (60.000
Da) can in fact pass through the P3HT as proven by the
experiments reported in Figures S-2c and S-2d respectively.
Spin-Coated SA FBI-OFET Biosensor Analytical Performances. The biosensing performances of a FBI-OFET
including a spin-coated SA layer are assessed in the experiments
reported in Figure 4. Panel 4a shows the elicited device transfer
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delivered biotin (b) and on the surface density of SA

molecules (S).
The interaction between biotin and SA obeys the following
binding isotherm
S
[b]
=
4S
KD + [b]

(2)

where KD is the dissociation constant, [b] is the equilibrium

concentration of the ligand b (biotin in the present case), and
the term 4S accounts for the four binding sites of SA. Equation
2, written in terms of KD1, gives the Langmuirs isotherm of
biotin to the SA layer. The amount of unbound biotin is the
dierence between the number of delivered biotin molecules
and the number of occupied binding sites. Taking into account
that the sensing is performed using a droplet of volume Vd = 2
L that covers an area ad = 0.03 cm2, and denoting the
concentration of delivered biotin as b, the concentration of
unbound biotin molecules [b] can be written as [b] = (b/Vd
4Sad/Vd). As a result eq 2 becomes

S 2
V b
V b
S VdKD

0 + d
+ 1 + d
=0
0
4S
4Sad
4Sad
4S 4S ad

(3)

To test the consistency of this model, the calibration curves

have been tted simultaneously to eqs 1 and 3, xing KD at
1014 M. Typical tting curves are shown in Figures 3b, 3c, and
4c, and the best-t parameters are reported in the relevant
gures captions. It turned out that all the inspected devices
share mutually close intercept a values, while their sensitivity (c
parameter in eq 1) depends markedly on the number of
immobilized SA molecules, being 0.042 decade1 for a single
layer LbL and 0.07 decade1 for two layers LbL deposition. The
sensitivity in the case of spin-coated FBI-OFET is higher (0.09
decade1) suggesting a slightly higher surface density of binding
sites in this case. The ts of the data to eqs 1 and 3 allow also
an independent estimation of the SA surface density (S).
Although the extracted value for this last parameter is aected
by a fairly large uncertainty, the resulting gures order of
magnitude well compare with the spectroscopic determination.
In the case of LbL deposition the S values increase with the
number of deposited layers, going from S = 2 2 pmol/cm2
for the single layer to S = 6 3 pmol/cm2 for the double SA
layers. These values are in reasonable agreement with the
spectroscopic quantication resulting in S = 5 pmol/cm2 for a
single and S = 10 pmol/cm2 for a double layer.
Comparison of LbL and Spin-Coated SA FBI-OFETs
Analytical Performances. Following a standard approach44,45
the comparison is carried out through the assessment of
sensors calibration curves gures of merit. As it is clear from
the data presented before, the analytical gures of merit are not
signicantly dierent for the two layer LbL assembly and the
spin-coated one. Therefore these two biosensors should be
considered as equivalently highly performing. On the other
hand, a comparison with a state-of-the-art SA electronic assay
performed on a silicon nanowire FET,29 evidences that 3 orders
of magnitude in mass fractions have been gained with the FBIOFET biotin assay although, due to the dierent masses of the
SA and biotin analytes, comparable absolute concentrations
were detected. Besides, the FBI-OFET platform can oer
undoubted advantages over a nanotechnological based
approach as far as production costs and scalability are
concerned. Much worse performance levels were achieved by

Figure 5. (a) Picture of the microuidic system implemented on the

SA FBI-OFET chip. (b) Microscope image of the microuidic system
composed of one single channel that runs through the electronic
channels of three transistors of the chip. (c) ID-VG transfer
characteristics at VD = 80 V for the SA FBI-OFET with the analyte
at a concentration of 10 ppt dispensed through the microuidic
system. The response to pure water, also dispensed through the
microuidic system, is provided as reference.

results show that the transduction mechanism appears as more

complex for FBI-OFETs compared to ISFETs, the former
involving not only a variation of the threshold voltage but also
of the eld-eect mobility. The variation of both these two
parameters is likely to be responsible for the systematically
observed current and fractional current decreases upon SA
biotin complex formation, and a detailed study will be
published elsewhere. According to the elicited assumption,
the FBI-OFET responses have been tted by the following
equation

I
= a + cln S
I0

(1)

where a and c are constant parameters, and S is the number of

occupied binding sites per unit area that depend on the
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dx.doi.org/10.1021/ac302702n | Anal. Chem. 2013, 85, 38493857

Analytical Chemistry

Article

FlexSMELL - Gas Sensors on Flexible Substrates for Wireless

Applications - FP7-PITN-GA-2009-238454 partially supported the research study.

all the other studies proposing SA electronic detection.

Furthermore FBI-OFET assay works at concentration levels
outperforming even state-of-the-art biotin assays, such as
uorescent (LOD = 0.2 nM)30 and electrochemical (LOD =
1 M)31 ones performed in solution.
From a more applicative point of view it is acquired that
biotin sensing is not interesting per se. However, in this study
also antibiotin as well as the HRP enzyme are successfully used,
proving that the FBI-OFET platform is capable of working even
when an antibody or an enzyme is used as capturing layer. This
signicantly widens its potential applications spectrum.
Relevant to note is also that preliminary results (not reported)
carried out exposing the FBI-OFET to a 10 mM phosphate
buer solution to which the biotin analyte is added show
calibration curves that allow specic and repeatable detection
also at the ppt concentration level. This set of evidence
combined with the recent progress in the eld of organic
electronics,46 along with the need for versatile, compact,
inexpensive, high-throughput, and eld-deployable chemical
and biological sensors, makes the perspective of realizing plastic
or even paper FBI-OFET electronic sensors very appealing.

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CONCLUSIONS
In conclusion, SA FBI-OFETs are shown to probe liquid
analytes at the low part per trillion concentration level in a
highly selective and repeatable fashion. The FBI-OFET
fabrication procedure, including a spin-coated capturing layer,
allows for perspective applications in plastic or even paper
electronic strips fabricated by printing processes. The study
includes also a session where the critical issue concerning the
analyte percolation through the FBI-OFET semiconductor
layer is discussed providing evidence that even bioanalytes as
large as insulin or streptavidin eciently ow through this
electronic layer. Although the study is focused on the
streptavidinbiotin interaction, also antibodies and enzymes
are used, thus signicantly widening the potential applications
spectrum of this platform.
The excellent functioning also when the analyte is dispensed
through a microuidics system along with the extremely high
sensitivity allows conceiving FBI-OFET employment in
applications such as digital and possible disposable strip-tests
for determinations in biological uids such as saliva or even eye
drops, where ultralow detection limits are needed.

ASSOCIATED CONTENT

S Supporting Information
*

Table S1, Figures S-1 and S-2, and a video. This material is
available free of charge via the Internet at http://pubs.acs.org.

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: maria.magliulo@uniba.it.
Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
We thank Dr. Paolo Greco of Scriba Technologies for support
in the microuidics implementation. Dr. Serana Cotrone and
Dr. Maria Daniela Angione are acknowledged for useful
discussions. The PRIN 09 project Electronic and electrochemical biosensors 2009AZKNJ7 founded by Italian Ministry
of Education, University and Research (MIUR) and the
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