Open Journal of Applied Biosensor, 2013, 2, 112-119
Published Online November 2013 (http://www.scirp.org/journal/ojab)
http://dx.doi.org/10.4236/ojab.2013.24015
Open Access OJAB
Development of a QCM (Quartz Crystal Microbalance)
Biosensor to the Detection of Aflatoxin B1
Katia Spinella1, Lucia Mosiello1, Giuseppe Palleschi2, Fabio Vitali1
1ENEA, Italian National Agency for New Technologies, Energy and the Environment, Rome, Italy
2Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma Tor Vergata, Rome, Italy
Received January 30, 2013; revised March 14, 2013; accepted April 1, 2013
Copyright © 2013 Katia Spinella et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
In this study, we have used a direct immunoassay where the simple binding between antigen and an antibody is detected.
Immunoassays were performed in a drop system, monitoring the frequency decrease of the quartz-crystal microbalance
device because of mass increasing during immunoreaction. The QCM sensor was coated on both sides by gold elec-
trodes, only one side of the crystal (liquid side) was in contact with the solution; the other side (contact side) was al-
ways dry. We tested a piezoelectric immunosensor for aflatoxin B1 (AFLA-B1) mycotoxin detection through the immo-
bilization of DSP-anti-AFLAB1 antibody (AFLA-B1-Ab anti AFLAB1) on gold-coated quartz crystals (AT-cut/5 MHz).
The DSP (3,3’-Dithiodipropionic-acid-di-N-hydroxysuccinimide ester) was used for the covalent attachment of the pro-
teins. The piezoelectric crystal electrodes were pretreated by DSP for 15 min, rinsed with water and dried in a gentle
flow of nitrogen gas. Then the DSP-coated crystals were installed in a sample holder and exposed to the anti-AFLAB1
antibody and to the AFLA-BI. Frequency and resistance shifts (Δf and ΔR) were measured simultaneously. Δf versus
AFLA-BI concentrations in the range of 0.5 - 10 ppb exhibited a perfect linear correlation with a coefficient of above
0.998.
Keywords: Immunosensor; Quartz Crystal Microbalance; Aflatoxin B1; Antibody; Gold Electrodes
1. Introduction
The term mycotoxins includes numerous secondary me-
tabolites with high toxicity, products in suitable micro-
climatic conditions by microscopic fungi and filamentous,
better known by the term “mold”, that colonize the plants
and/or the foodstuffs during their growth [1]. Aflatoxin
B1 (AFLA-B1) is an example of a group of highly toxic
difurancoumarin derivatives that are produced by many
strains of Aspergillusflavus and A. parasiticus which
often contaminate a variety of food and animal feed
stored. The four major aflatoxins have been designated as
B1, B2, G1 and G2 based on their fluorescence under
UV light and their relative chromatographic mobility
during thin layer chromatography. AFLA-B1 has been
classified as a group 1 human carcinogen and aflatoxins
G1, G2 and B2 belong to a group human carcinogens [2].
These toxins exhibit carcinogenic, teratogenic and muta-
genic properties and have now been isolated from a wide
variety of agricultural products [3,4]. AFLA-B1 can enter
the food chain mainly through the ingestion of contami-
nated human or animal food. The intake of AFLA-B1
over a long period of time, even in low concentrations,
may be very deleterious to health [5]. The Food and Ag-
ricultural Organization 2004 report on mycotoxins [6]
revealed that as of December 2003, at least 99 countries
worldwide had regulations in place for permitted my-
cotoxin levels in food/or feed, and have set limits for
AFLA-B1 alone or for the sum of aflatoxins B1, B2, G1
and G2. The maximum permissible level for AFLA-B1
in food was set at 2 μg/kg (2 ppb). Analytical methods
for the determination of mycotoxins in food products
generally provide a first stage of extraction of the toxin
from the matrix (with a solution of methanol-water or
ethanol-water, depending on the type of analysis later), a
second purification step, and finally the revelation by an
appropriate instrumental technique. Typically, the meth-
ods used are of type chromatographic or immunochemi-
cal but in recent years are being developed analytical
techniques that use instead of antibodies, such as bio-
logical elements, aptamers. In particular for the determi-

K. SPINELLA ET AL. 113
nation of OchratoxinA (OTA) have been developed on
solid phase extraction columns containing aptamers spe-
cific for the OTA [6]. Generally tests are employed qua-
litative or semi-quantitative rapid screening for as many
samples (tendentially with immunochemical methods)
and subsequent phases of confirmation with a precise
quantification for positive samples (tendentially with
chromatographic methods), these include thin-layer chro-
matography (TLC) [7] and high-performance liquid chro-
matography (HPLC) [8]. Though these techniques have
excellent sensitivities they typically require skilled op-
erators, extensive sample pre-treatment and expensive
equipment [9]. The goal of more recent studies has been
to simplify and expedite the method of detection while
attempting to maintain or improve the sensitivity. Re-
sponding to the need to achieve high sensitivity and
move to the use of disposable probes, several electro-
chemical immunosensors have been reported in literature
for the detection of AFB1 in corn and barley [10-12] and
AFM1 in milk [13]. Among the immunochemical ap-
proaches, the enzyme-linked immunosorbent assay (ELI-
SA) method is the most widely applied. Spectrophoto-
meric ELISAs specific for AFLAB1 [14,15], total afla-
toxins [16,17] and AFLAM1 [18,19] have been devel-
oped and their simplicity, adaptability and sensitivity
have been demonstrated. In order to achieve higher sen-
sitivity and move to the use of disposable probes, elec-
trochemical immunosensors for aflatoxins based on indi-
rect competitive ELISA format have been proposed [20].
These immunosensors require the use of labeled secon-
dary antibodies for detection. To achieve label-free im-
munosensors, direct electrochemical immunosensors for
AFB1 based on electrochemical impedance spectroscopy
[21], optical waveguide lightmode spectroscopy [22] and
room temperature ionic liquids [23] have been reported.
However, results on ELISAs need several incubation,
washing, and separation steps. Because of the labor of
ELISAs, a great interest in developing label-free and less
time-consuming on-line detection methods, such as the
use of biosensors, is undiminished. With this purpose,
piezoelectric quartz crystals are convenient for affinity-
based sensors, particularly for immunosensors. The com-
bination of low cost with increased sensitivity, selectivity,
simplicity, and possible reusability make piezoelectric
immunosensors a valuable alternative to other existing
like optical (e.g., plasmon resonance or molecular fluo-
rescence) and electrochemical immunosensors. The
search for a simple and label-free piezoelectric immuno-
sensor is of considerable interest. The mass sensing of
the quartz-crystal microbalance (QCM) removes the need
of any labelling step for the signal transduction and dis-
play detection sensitivities up to a 1- to 100-fold linear
ΔF vs. mass range with limit detections as few as nano-
grams per milliliter levels. The QCM immunosensor is
usually comprised of a quartz crystal with an antigen or
antibody immobilised on its surface, which allows the
label-free detection with a direct quantification of the im-
munocomplex (Ab-Ag). A QCM that responds to changes
in mass on the electrode surface and has a sensitivity in
the ngand is used for a wide range of applications in the
medical field [24,25]. Currently, the piezoelectric immu-
nosensors are used to determine tumor markers in clini-
cal diagnostics, since they allow performing the analysis
in real time. Chou et al. [26] and Zhang et al. [27] have
proposed respectively a piezoelectric immunosensor for
the human ferritin and one for hCG (human chorionic
gonadotropin). In this investigation, a piezoelectric im-
munosensor for the detection of AFLA-B1 was devel-
oped through the drop-coating of DSP-anti-AFLAB1
antibody (AFLA-B1-Ab anti AFLAB1) on gold-coated
quartz crystals (AT-cut/5 MHz). Details of the prepara-
tion, characterization and application of the immunosen-
sor are described.
2. Experimental Section
2.1. Instrumentation
Research Quartz Crystal Microbalance (RQCM) and the
piezoelectric quartz crystals (AT-cut, 5 MHz, 1-inch dia-
meter) were from Inficon (USA).The quartz crystals
were obtained by cutting the quartz with an angle of 35˚
25′ relative to the axis crystallographic z. The main ad-
vantage of this cut is the obtainment of quartzes little
sensitive to temperature, at least in a range that goes from
10˚C to 50˚C. The crystals are coated on both sides by
gold electrodes, the crystals were installed in a sample
holder and exposed to the anti-mycotoxin antibody and
to the analyte (mycotoxin). Frequency and resistance shi-
fts (Δf and ΔR) were measured simultaneously. Only one
side of the crystal was in contact with the solution; the
other side was always dry. All date is recorded and dis-
played graphically using an integrated software based on
Windows system. The heart of this system is an oscillator,
a phase locked loop (PLO, Phase Lock Oscillator) which
allows creating a signal whose phase has a fixed rela-
tionship with that of a reference signal. The circuit also
includes the ability to adjust the electrical capacity of the
crystal, and this is essential for accurate measurements of
thin films and for applications that provide a measure of
the analyte in the flow. The PLO uses an internal oscil-
lator indicated as a voltage controlled oscillator (VCO,
Voltage Controlled Oscillator) for driving the crystal.
2.2. Immunochemicals and Chemicals
Polyclonal rabbit antibody for Aflatoxin B1 (Rabbit anti-
Aflatoxin B1) was from Sigma Aldrich (St. Louis, USA),
monoclonal rabbit antibody immobilized on beads which
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K. SPINELLA ET AL.
Open Access OJAB
114
were recorded as a function of time at an interval of 300
s for a longtime monitoring (about 5 h).
were extracted from immunoaffinity columns were from R-
Biopharms.r.l, (Italy). Aflatoxin B1 from A. flavus (As-
pergillusflavus), 3,3’-Dithiodipropionic-acid-di-N-hydro-
xysuccinimide ester (DSP),acetone (C3H6O) and sepha-
roase beads, similar to those usually used for the coating
of Protein A in the production of affinity columns for the
Fc region (Fragment crystallizable) immunoglobulin (IgG),
were purchased from Sigma-Aldrich. Working buffer
was the phosphate buffer saline (PBS) of pH = 7.4 (1.4
mM KH2PO4; 8.0 mM Na2PO4; 136 mM NaCl; 2.7 mM
KCl). Caution AFLA-B1 is a potent carcinogen molecule,
and extreme caution is therefore necessary to avoid con-
tact with this. Contaminated materials must be appropri-
ately discarded.
2.4. Immobilization of AFLA-B1 Antibody
The coating of quartz was carried out under a hood to
avoid contamination. We have developed two procedures
for immobilization of Ab-specific antigen (Ab anti-afla-
toxin) on the surface of the quartz crystals: absorption of
specific antibodies directed and use of antibodies immo-
bilized on the beads (Figure 1). As regards the direct
absorption, antigen specific antibodies were directly
added in drops on the surface of the quartz crystals pre-
viously coated with DSP.
The antibodies immobilized on the solid support were
taken from immunoaffinity columns and added in a drop
on quartz pre-treated with DSP.
2.3. Materials and Methods Used for the Coating
of Quartz Crystals
The piezoelectric crystal electrodes were pretreated with
20 ul DSP (3,3’-Dithiodipropionic-acid-di-N-hydroxy-
succinimide ester) for 15 min rinsing with water, dried in
a gentle flow of nitrogen gas and then were installed in a
sample holder. The DSP was used for the covalent at-
tachment of the proteins. The DSP binds covalently to
proteins, including antibodies, which generally have dif-
ferent primary amines in the amino acid side chains (ad.
Example amines present in the chain of Lysine R), these
residues and the N-terminal of each polypeptide are avai-
lable as targets for the DSP. This phase functionalization
of quartz crystals is extremely critical, you must obtain a
homogeneous coating of the surface to be treated. Clean
gold surfaces were immersed in a 3 mL solution of hy-
drogen peroxide (30%), ammonia (25%) and deionized
water heated to a temperature of about 75˚C for 5 min-
utes. Immediately rince liberally with deionized water
and dry in a gentle flow of nitrogen gas. Then the DSP-
coated crystals were installed in a sample holder and ex-
posed to the anti-mycotoxin antibody and to the analyte
(mycotoxin). Frequency and resistance shifts (Δf and ΔR)
were measured simultaneously. The frequency shifts
2.5. Method of Extraction of Aflatoxin from
Contaminated Matrices
For the extraction of aflatoxin B1 from real samples
(peanuts) considered in this work applies a standard pro-
tocol: the homogenized sample with a water-alcohol so-
lution, then you make a dilution of the extract by the ad-
dition of a phosphate buffer solution (PBS). The purifi-
cation is carried out by passing it through the immunoaf-
finity column (IAC) containing antibodies specific for
AFB1; the mycotoxin is then eluted with methanol and
quantified via RQCM. Initially were weighed directly
homogenizer 5 g of sample (accuracy ± 0.1 g), and added
0.5 g of sodium chloride (NaCl) and 2 mL of extraction
solvent (methanol: water in the proportions 80:20 v/v).
The sample was agitated at high speed for 3 minutes. The
extract was filtered on a paper filter and 2 mL of filtrate
is taken and diluted with 2 mL of PBS (Phosphate Buff-
ered Saline). The diluted sample was applied to the im-
munoaffinity column as described in the next section.
The aflatoxin B1 was eluted directly into 5 mL volumet-
ric flask, following a two-step procedure:
QCM sensore
DSP
Analita beads
Ab-antigene specifico
(a) (b)
Figure 1. Diagram of immobilization of Ab on the surface of the electrode of gold of the quartz crystal. (a) Coating of the gold
surface with DSP and Ab-specific antigen; (b) Coating of the gold surface with DSP and beads with monoclonal Ab specific
mycotoxin.

K. SPINELLA ET AL. 115
 By applying 1 mL of methanol and leaving the IAC
flowing by gravity.
 Waiting for 1 minute, and applying a second portion
of 1 mL of methanol.
Subsequently we collected the eluting solvent residue
passing, with a 10 mL syringe, through the IAC, a volu-
me of air equal to two /three times the volume of the
same. It was brought to volume with water, the sample
was then stored at 4˚C until analysis with RQCM.
3. Results and Discussion
3.1. Characterization of the Quartz Crystal
Surface
For accurate results of the QCM the characterization of
the gold surface of the quartz crystals are highly recom-
mended. The surface of the quartz crystal functionalized
with the DSP was characterized AFM (Atomic Force
Microscope) is shown in Figure 2. The phase imaging is
able to bring out details hidden in the normal image re-
construction, highlighting edges and grooves which can
be correlated with the different physic-chemical charac-
teristics of the sample.
From the phase imaging shows that the treated surface
is homogeneous, the gradation of gray indicates the
thickness of the coating, the lightest areas are those that
present a greater layer DSP, 90 nm thick coating.
3.2. Standard Curve for Aflatoxin B1
For the determination of mycotoxins through an immu-
nosensor-QCM was initially developed a direct immu-
noassay which used a polyclonal antibody, by which it
was possible to identify the mycotoxin AflatoxinB1 in
solutions at different concentrations (standard solutions)
prepared in the laboratory. In these experiments, con-
ducted essentially in order to define and characterize our
system, we used the standard concentrations of Aflatox-
inB1 of 0.5, 1, 5, 10 ppb (ng/mL). Applying the method-
ology described were conducted immunoassays and di-
rect from the data obtained we were able to construct the
standard curve shown in Figure 3. We report the follow-
3500 nm
−90 nm
−40 nm
−0 nm X: 3.5 umY: 3.5 um
20 nm
0 nm
(a) (b)
Figure 2. AFM image of surface of the quartz crystal functionalized with the DSP: (a) Phase-imaging; (b) Topography 3D
image.
[AflaB1] ng/mL
0 2 4 6 8 10 12
Frequenza (MHz)
y = −0.0002x + 5
R
2
= 0.9846
Time
00:53:56.35
Deta
Frequency#1
−1376.29
○Deta Frequency#1 (Hertz)
100.00
−2200.00
5001
50,005
5
49,995
4999
49,985
4998
49,975
Mass#1
24.354
Resistance#1
779.2
ΔMass#1 (ugm/cm
2
)
100.000
□Resistance#1 (Ohms)
1100.0
−10.000 0.00
00:00:00.00 Time (Minutes) 03:00:00.00
Figure 3. Standard curve by immunoassay using polyclonal antibodies directed to different concentrations of AflaB1 (on left)
and relationship between Δf (red line) a function of mass (violet line) and resistance (green line).
Open Access OJAB

K. SPINELLA ET AL.
116
ing values:
 LOD = 0.3 ng/ml
 Working range: 0.5 - 10 ng/ml
The detection limit (LOD) is estimated from the mean
of the blank, the standard deviation of the blank and
some confidence factor. The value of is given by the
equation:
11
L
bb
x
xks (1)
where 1b
x
is the mean of the blank measures, 1b
s
is the
standard deviation of the blank measures, and k is a nu-
merical factor chosen according to the confidence level
desired.
The RQCM allows to measure simultaneously the Δf
and ΔR, the frequency has a decreasing trend, as de-
scribed by Sauerbrey equation [28] with increasing mass
deposited on the surface of the quartz crystal is a reduc-
tion of the resonance frequency of the crystal.
3.3. Standard Curve for Aflatoxin B1
(Immunoassay Performed with a
Monoclonal Antibody mAb Anti-Aflatoxin
B1 Immobilised on Beads)
Using the protocol described before and through the use
of another type of antibody specific for the Aflatoxin B1
(a monoclonal antibody), we conducted a series of ex-
periments, especially in order to improve the sensitivity
of our system and could construct a second standard
curve. In this case, we used monoclonal antibodies to the
Aflatoxin B1 immobilized on beads. The results obtained
are shown in Figure 4. We have built a standard curve
for the Aflatoxin B1, obtaining very encouraging results
about the possibility to propose our system as a screening
test for this mycotoxin. As shown by the graphs, the two
different systems have similar results especially in terms
of measurement sensitivity. The system using polyclonal
antibody would seem to be more sensitive and certainly
better meets the analytical needs required for the deter-
mination of Aflatoxin B1.
Monoclonal antibodies, in fact, generally show a grea-
ter selectivity and affinity for the antigens against which
they are produced, compared to polyclonal immunoglo-
bulins. For the extraction of aflatoxin B1 from real sam-
ples (peanuts) considered in this work, we used a stan-
dard protocol described previously. In Figure 5 we have
a graph that relates the frequency as a function of con-
centration, each point on the curve corresponds to the ad-
dition of Ab specific monoclonal AflaB1 and to the sam-
ple of Aflatoxin B1 diluted 1:100, 1:50, 1:10 and 1:5, the
curve has a decreasing trend which corresponds to the
decrease of the resonance frequency of the quartz crystal.
For the development immunoassay direct we used the
monoclonal antibodies immobilized on beads as gener-
ally show greater selectivity for the antigens against
which they are produced, compared to the polyclonal
immunoglobulins.
4. Conclusion
These results, since they are related to a direct immuno-
assay using monoclonal antibodies specific for Aflatoxin
B1 (mAb anti-AflaB1) immobilized on beads, bound on
the surface of quartz pre-treated, by means of which in-
creases the mass on the surface of the quartz crystals is a
reduction of the resonance frequency of the crystal, are
of particular importance, since they show the applicabil-
ity and feasibility of direct immunoassay format with im-
mobilized antibodies. Unfortunately, we have found that
the step of functionalization of the quartz crystals affect
the success of the experiment, however, since the greater
advantage of the QCM results in a label-free system that
allows the identification of different analytes, in our sys-
tem that characteristic not only is preserved, but, being
○Deta Frequency#1 (Hertz)
4800.00
Deta
Frequency#1
2722.49
−220.00
ΔMass#1 (ugm/cm
2
)
100.000
□Resistance#1 (Ohms)
2400.0
Time
01:58:03.80
Mass#1
−48.022
Resistance#1
2066.3 −100.000 0.00
00:00:00.00 Time (Minutes) 05:50:00. 0 0
[AflaB1] ng/mL
0 2 4 6 8 10 12
Frequenza (Mhz)
y = −0.0001x + 49,948
R
2
= 0.9864
49,952
4995
49,948
49,946
49,944
49,942
4994
49,938
49,936
49,934
Figure 4. Standard curve by direct immunoassay using monoclonal antibodies immobilized on beads (on left) and relation-
ship between Δf (red line) a function of mass (violet line) and resistance (green line).
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K. SPINELLA ET AL. 117
Dilutions AflaB1 (ng/mL)
0.00 0.05 0.10 0.15 0.20 0.25 0.30
Frequenza (MHz)
y = −0.0091x + 5
R
2
= 0.9804
Time
01:14:12.70
Deta
Frequency#1
−292.02
○Deta Frequency#1 (Hertz)
100.00
−1000.00
5001
50,005
5
49,995
4999
49,985
4998
49,975
Resistance#1
785.6
Mass#1
5.143
ΔResistance#1 (Ohms)
970.0
□Mass#1 (ugm/cm
2
)
100.000
0.00 −10.000
00:00:00.00 Time (Minutes) 03:40:00.00
0
1:100
1:50
1:10
1:5
Figure 5. Standard curve by direct immunoassay using monoclonal antibodies for AFLAB1 applied in the sample (on left)
and relationship between Δf (red line) a function of mass (green line) and resistance (violet line).
applied to the identification of mycotoxins, can assume
new application scenarios of the QCM, based on the use
of antibodies immobilized on a solid support in the field
of food security and, therefore, human health. Frequency
and resistance shifts (Δf and ΔR) were measured simul-
taneously. Δf versus mycotoxins concentrations in the
range of 0.5 - 10 ppb exhibited a perfect linear correla-
tion with a coefficient of above 0.998. Monoclonal anti-
bodies, in fact, generally show a higher and better speci-
ficity for antigens against which they are produced, as
compared to polyclonal immunoglobulin. The QCM bas-
ed sensing label free procedure for mycotoxin detection,
developed in our laboratory, can be considered a simple,
cost effective, real time and no time and labor consuming
technique in comparison with conventional assay proce-
dures, as GC chromatography. It is important to remem-
ber the QCM measures frequency changes and not mass
changes, then a strictly keeping constant of the experi-
mental conditions while a typical QCM immunosensor
experiment is necessary.
5. Acknowledgements
This work was supported by ENEA, Italian National
Agency for New Technologies, Energy and Sustainable
Economic Development. The contributions of Dr. Bar-
bara De Santis (IstitutoSuperioredi Sanità, ISS, Rome,
Italy), Ing. NicolaDonato and Dr. Giovanni Neri (Uni-
versity of Messina, Italy) are also strongly acknowl-
edged.
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K. SPINELLA ET AL. 119
Biographies
Katia Spinella received her MSc degree in industrial
biotechnology from University of Rome, TorVergata, in
2012. She is pursuing her PhD in materials for health,
environment and energy in the University of RomeTor-
Vergata. She is currently working onthe development
aptamers based biosensors for food contaminants.
Lucia Mosiello received her MSc degree in biology
from University of Rome, Sapienza, in 1986. Since 1990
she is a senior researcher at ENEA, Italian National
Agency for New Technologies, Energy and Sustainable
Economic Development, Department of Biotecnology,
Laboratory of Agroindustry Innovation. Her research in-
terests include biosensors, chemosensors, nanotechno-
logy and study of biomolecular interactions using quartz
crystal microbalance.
Giuseppe Palleschi is the head of Chemistry Depart-
ment of the University of Rome “Tor Vergata”. Since
1989 member of the Scientific Committee for the “Emer-
gency Plan for National Defense Pollution of hydrocar-
bons or other harmful substances caused by marine acci-
dents”, Department of Civil Protection of the Presidency
of the Council of Ministers. The activity of prof. Pal-
leschi, Professor of Analytical Chemistry, is mainly fo-
cused on the development and application of chemical
sensors, biosensors and immunosensors for food, envi-
ronmental and clinical analysis.
Fabio Vitali is a senior researcher at ENEA, Italian
National Agency for New Technologies, Energy and Su-
stainable Economic Development, Laboratory of Agro-
industry Innovation. He is participating to most of those
National and European projects on biosensors in which
ENEA is involved. The activities include basic research
for the advancement of the frontiers of knowledge, the
development and transfer of technology “enabling” a
multi-sectoral and boosting industrial research, method-
ologies, services and systems approaches to support the
processes of innovation and competitiveness of defined
territorial contexts.
Open Access OJAB