Open Journal of Applied Biosensor, 2013, 2, 97-103
Published Online November 2013 (
Open Access OJAB
QCM Aptasensor for Rapid and Specific Detection
of Avian Influenza Virus
Luke Brockman1, Ronghui Wang1, Jacob Lum2,3, Yanbin Li1,2,3*
1Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, USA
2Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, USA
3Cell and Molecular Biology Program, University of Arkansas, Fayetteville, USA
Email: *
Received February 1, 2013; revised March 18, 2013; accepted April 6, 2013
Copyright © 2013 Luke Brockman 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.
There has been a need for rapid detection of Avian Influenza virus (AIV) H5N1 due to it being a potential pandemic
threat. Most of the current methods, including culture isolation and PCR, are very sensitive and specific but require spe-
cialized laboratories and trained personnel in order to complete the tests and are time-consuming. The goal of this study
was to design a biosensor that would be able to rapidly detect AIV H5N1 using aptamers as biosensing material and a
quartz crystal microbalance (QCM) for transducing method. Specific DNA aptamers against AIV H5N1 were immobi-
lized, through biotin and streptavidin conjugation, onto the gold surface of QCM sensor to capture the target virus.
Magnetic nanobeads (150 nm in diameter) were then added as amplifiers considering its large surface/volume ratio
which allows for faster movement and a higher target molecule binding rate. The result showed that the captured AIV
caused frequency change, and more change was observed when the AIV concentration increased. The nanobead ampli-
fication was effective at the lower concentrations of AIV, however, it was not significant when the AIV concentration
was 1 HA or higher. The detection limit of the aptasensor was 1 HAU with a detection time of 1 h. The capture of the
target virus on to the surface of QCM sensor and the binding of magnetic nanobeads with the virus was confirmed with
electron microscopy. Aptamers have unlimited shelf life and are temperature stable which allows this aptasensor to give
much more consistent results specifically for in field applications.
Keywords: Aptasensor; Avian Influenza; QCM; Aptamer; Nanobeads
1. Introduction
H5N1 is a highly pathogenic subtype of avian influenza
virus (AIV), influenza A virus. Since 2003, there have
been reported 615 human cases of avian influenza H5N1
resulting in 364 deaths [1]. It is estimated that H5N1 has
already cost the poultry industry over $10 billion and the
World Bank has estimated that a severe human outbreak
would cost upwards of $3 trillion to the global economy
[2]. These threats are why a sensitive, rapid detection
method is needed.
The current gold standards for avian influenza detec-
tion are viral isolation cultures and real-time RT-PCR.
They both provide high sensitivity but are time consum-
ing, expensive, and require special training and facilities
[3,4]. Rapid techniques for the detection of avian influ-
enza, such as ELISA and immunochromatographic strips,
lack sensitivity, specificity, and can have high false posi-
tive rates [5].
As an alternative to these methods, biosensors have
been studied for the detection of avian influenza virus.
Biosensors, which combine a biological sensing element,
a transducer, and a signal processing unit, have shown a
lot of promise for rapid detection of virus [5]. Surface
plasmon resonance (SPR) [6,7], optical interferometry
[8], and impedance [9,10] are a few of the more popular
biosensors being researched today. However, many of
them still lack the sensitivity and specificity required and
are not ready for in-field use.
Quartz crystal microbalance (QCM) has been gaining
popularity due to its simplicity and cost effectiveness
[11]. The QCM biosensor is based on the piezoelectric
properties of a quartz crystal wafer. When an electric
field is applied across the electrode an inverse piezoelec-
tric effect occurs, causing deformation of the crystal. The
*Corresponding author.
change of the resonant frequency of the crystal is attrib-
uted to a change in mass on the electrode surface [12].
For gas-phase measurement, the relationship between the
frequency change (Δ
) and mass change (Δ) of the
crystal is expressed by the Sauerbrey equation [13]:
where A is the electrode area, 0 is the resonant fre-
quency and
C is the sensitivity factor for the crystal.
For liquid-phase measurement, a most commonly used
model was by Kananzawa and Gordon [14] as follows:
rol lm
where h is the thickness of the quartz crystal, q
is the
density of the quartz, ro
is the resonance frequency
due to the added mass, and l
, lm
, and l
are density,
magnitude of the complex viscosity, and relative phase
angle of the liquid medium, respectively. While rapid
and easy to use, the QCM biosensor still lacks the sensi-
tivity needed to be considered an effective detection
method for avian influenza virus. There have been a few
previous studies using the QCM to rapidly detect AIV
and that use nanoparticle amplification. Liu et al. [15]
used the QCM to detect E. coli O157:H7 while also com-
paring different nanobead sizes for amplification. Hewa
et al. [16] was able to use the QCM to detect influenza
virus. Li et al. [17] was able to successfully detect H5N1
using nanobead amplification. Owen et al. [18] used aero-
solized influenza virus with the QCM and successfully
detected down to 4 virus particles/ml in a gas media.
Most recently, Wang and Li [19] developed a hydrogel
based QCM aptasensor to greatly reduce their detection
limit down to 0.0128 HAU. Improvements can still be
made not only in the sensitivity and specificity of these
tests but in the detection time as well.
Aptamers are artificially created single-stranded oli-
gonucleotides that have the ability to bind to targets such
as amino acids, drugs, proteins, cells, and viruses with
high affinity and specificity [20]. They are selected
through an in vitro process from random oligonucleotide
pools called Systematic Evolution of Ligands by Expo-
nential enrichment (SELEX) [21,22]. Aptamers show a
very high affinity to their targets, comparable to those of
monoclonal antibodies [23]. Aptamers can provide a
number of advantages over antibodies, namely the ease at
which they are designed and modified, higher thermal
stability, and a much longer shelf life [24]. In order to
improve the sensitivity and specificity of the QCM bio-
sensors, aptamers were considered by some researchers.
Bai et al. [7] used an aptamer based SPR biosensor for
the detection of AIV H5N1 and successfully detected
down to 0.128 HAU. As previously mentioned, Wang
and Li [19] developed a hydrogel based QCM aptasensor
for the detection of AIV H5N1. Cui et al. [25] was able
to use aptamers specifically as a labeling technique for
quantum dots against Influenza A particles.
In this study, we describe an improved QCM aptasen-
sor for the detection of AIV H5N1. Aptamers were used
with the advantage of being much more consistent and
stable compared to antibodies while still maintaining a
strong binding affinity to the target virus. Aptamer-la-
beled 150 nm nanobeads were used as biolabels and mass
amplifiers to increase the sensitivity of the system.
2. Materials and Methods
2.1. Biological and Chemical Reagents
Phosphate buffered saline (PBS, 10X) was purchased
from Sigma-Aldrich (St. Louis, MO) and diluted with
Milli-Q (Mill-Q, Bedford, MA) water to 10 mM (pH 7.4)
for use in all tests. Poly (ethylene glycol) methyl ether
thiol (PEG) was purchased from Sigma-Aldrich (St.
Louis, MO). PEG was dissolved in 10 mM PBS to a con-
centration of 0.1 mg/ml and prepared fresh for each test.
Streptavidin was purchased from Rockland Inc. (Gilbert-
sville, PA). It was reconstituted in 10 mM PBS and
stored in 1 mg/ml aliquots at 20˚C. The streptavidin
was diluted to 0.25 mg/ml for use in tests. All water used
in tests was obtained from a Millipore water purification
system (Mill-Q, Bedford, MA).
2.2. Virus and Aptamers
Inactivated avian influenza A/H5N1 virus was obtained
from the USDA/National Veterinary Services Laboratory
in Ames, Iowa. The virus was inactivated by the USDA
lab using β-propiolactone. The H5N1 virus used in the
tests was isolated from chickens in Scotland in 1959. The
stock concentration of the virus was 128 HAU. All dilu-
tions were done using PBS. Killed AIV H7N2, H9N2,
H5N9, H5N2, and H5N3 were obtained from Animal
Diagnostic Laboratory at Penn State University (Univer-
sity Park, PA).
The H5N1 aptamer (73 nucleotides; 5’-GTGTGCAT-
TG-3’) was previously developed in our laboratory
through the SELEX method [19]. The aptamer was syn-
thesized and biotin labeled by Integrated DNA Tech-
nologies (Coralville, IA). The aptamer was aliquoted and
stored at 20˚C. The aliquots were diluted using PBS to a
working concentration of 0.023 mg/ml (1 µM).
2.3. Instruments and Electrodes
Figure 1 shows the general aptasensor set up. All meas-
urements were taken with the QCA 922 Quartz Crystal
Open Access OJAB
Electrode: Top View
Flow Cell La ptop
QCA 922
Inlet Ourlet
Figure 1. Schematic of the aptasensor. The biological sens-
ing element is immobilized on the electrode surface. The
quartz in the electrode acts as a transducer, converting the
mass change to a frequency signal. The QCM processes and
records all of the data.
Analyzer from Princeton Applied Research (Oak Ridge,
TN) with the WinEchem software used to collect and
plot the data. The leads of the QCM were connected to
the electrode and frequency and resistance measurements
were takenat 1 s intervals. All measurements were done
in PBS at room temperature.
AT-cut quartz crystals (13.7 mm diameter) were ob-
tained from International Crystal Manufacturing (Okla-
homa City, OK). The crystals had a resonant frequency
of 7.995 MHz, and its surface was polished with gold
(5.1 mm diameter).
A flow cell from International Crystal Manufacturing
(Oklahoma City, OK) was used for mounting the crystal
electrode and holding the testing sample. The two flow
cell pieces were screwed together to hold the electrode in
place and was sealed with two O-rings. A 70 µl chamber
was located above the polished gold surface for the in-
jection of the testing samples.
2.4. Pretreatment and Aptamer Immobilization
of Electrodes
The crystal’s electrodes were first cleaned by immersing
them in 1 M NaOH for 20 min. Then a freshly prepared
piranha solution (1:3 H2O2:H2SO4) was dropped on the
gold surface for 1 min. Special care was taken to keep
this solution away from the electrode leads. The elec-
trodes were washed with deionized water and dried in a
stream of nitrogen after each pretreatment. The crystal
was then ready for installation into the flow cell.
A schematic of the electrode pretreatment and aptamer
immobilization is shown in Figures 2(a)-(c). PBS solu-
tion was first injected into the flow cell to provide a
baseline measurement. Streptavidin (0.25 mg/ml) was
then added to the flow cell for 30 min and allowed to
bind to the electrode surface through protein adsorption.
Biotin labeled H5N1 aptamer (1 µM) was injected into
the flow cell and allowed to incubate for 15 min. Next,
poly (ethylene glycol) methyl ether thiol (0.1 mg/ml) was
added for 1 h to prevent any nonspecific binding to the
St reptavidin
Biotin labeled
(a) (b)
Poly (ethylene
H5N1 virus
(c) (d)
Aptamer labeled
Figure 2. The electrode modification, virus detection and
signal amplification of the aptasensor. (a) Streptavidin ad-
sorption; (b) Aptamer immobilization by streptavidin bind-
ing; (c) PEG blocking of unbound sites; (d) Capturing of
target H5N1 virus; (e) Amplification by nanobead labeling.
electrode surface.
2.5. Preparation of 150 nm Magnetic Nanobeads
MagCellect Streptavidin Ferrofluid nanobeads with a
diameter of 150 nm were obtained from R&D systems
(Minneapolis, MN) and used at stock concentration. The
MagCellect Streptavidin Ferrofluid was a colloid of mag-
netic nanoparticles conjugated to streptavidin in solution
containing Bovine Serum Albumin (BSA) and preserva-
A 20 µl streptavidin coated nanobead solution was
mixed with 200 µl of PBS and then a magnetic field at
0.8 T was applied for 2 min using a magnetic separator
(AIBIT LLC., Jiangyin, China). The nanobeads were
then resuspended in 100 µl of aptamers and 100 µl of
PBS. The solution was rotated at 15 rpm for 30 min.
Next, the nanobeads were suspended in 100 µl of PBS
and 100 µl of biotin solution and rotated again for 10 min.
After each step the magnetic separation and washing was
repeated, resuspending the nanobeads in 200 µl of PBS.
2.6. Detection of AIV H5N1
Inactivated AIV H5N1 with titers in the range of 0.01 - 4
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HAU/50 µl in PBS were added to the flow cell for cap-
turing by the aptamer immobilized on the surface of
QCM sensor and allowed to incubate for 30 min (Figure
2(d)). All uses of HAU refer to HAU/50 µl. Next, the
aptamer coated nanobeads were injected into the flow
cell for 30 min to allow binding to the captured virus
(Figure 2(e)). Specificity tests were conducted using
inactivatedAIV H7N2, H9N2, H5N9, H5N2, and H5N3
at 2 HAU. Triplicate tests were run for each of the titers
of 0.01, 0.1, 1, 2 and 4 HAU and were used for deter-
mining the relationship between frequency change and
virus concentration. The mean and standard deviation of
frequency changes were calculated plotted using Micro-
soft Excel. The threshold for positive detection or lower
detection limit was set as signal/noise ratio of 3 where
the noise is defined as the standard deviation of the con-
trol sample.
2.7. ESEM Images
ESEM imaging was done using the Philips XL30 ESEM
(Environmental Scanning Electron Microscope, FEI, Hil-
lsboro, OR) to confirm the binding of nanobeads to the
AIV H5N1. The ESEM samples were prepared by fol-
lowing the electrode modification protocol at 4 HAU
followed by fixation with Karnovsky’s fixative and de-
hydration with ethanol.
3. Results and Discussion
3.1. Fabrication and Characterization of the
QCM Aptasensor
Figure 2 shows the stepwise modification of the fabrica-
tion of the QCM biosensor, including target binding and
signal amplification. Initial tests were run with no block-
ing agent prior to the addition of H5N1 virus and mag-
netic nanobeads. Negative control tests showed a sig-
nificant amount of non-specific binding of the virus and
nanobeads to the gold surface in the absence of aptamers.
The first solution was to use Bovine Serum Albumin
(BSA) to block the remaining free gold surface from any
non-specific binding. However, this was found to also in-
hibit the capturing of the target virus by the aptamer,
most likely due to the large size of BSA ( 12 nm) rela-
tive to the aptamer size ( 3 nm). Poly (ethylene glycol)
methyl ether thiol (PEG) was then used due to its small
size and strong blocking capabilities. PEG proved to be
very effective for not only preventing non-specific bind-
ing to the gold surface but also not interfering with the
aptamer’s ability to capture the target virus. Figure 3
shows a comparison of the two blocking agents. BSA
blocking actually caused a decrease in mass after the
addition of AIV H5N1virus at 4 HAU, most likely due to
the BSA being washed away easily from the shear stress
of the fluid flow. PEG clearly allows the aptamer to still
capture the target virus. The target H5N1 virus was then
added to the flow cell and would bind to the immobilized
aptamer. Lastly, 150 nm magnetic nanobeads modified
with the H5N1 aptamer were used as biolabels for further
amplifying the mass of the virus particles. Each of these
steps caused a decrease in the resonant frequency of the
electrode due to an increase in mass. The net response
from each step is determined by the difference in the
corresponding PBS baselines.
Figure 4 shows a typical sensorgram of virus detec-
tion with the aptasensor. The adsorption of streptavidin
to the gold surface along with the immobilization of the
aptamer were both verified in real time by the decreases
in frequency, 77 Hz and 46 Hz respectively. PEG re-
quired a long binding time of 1 h in order to completely
block the remaining electrode surface and further de-
creased the frequency by 2 Hz. Following the blocking
step, the target virus was captured by the aptamer and
caused a 44 Hz decrease in frequency. Lastly, nanobead
amplification further increased the frequency change by
10 Hz by binding to the H5N1 virus. Each step was fol-
lowed by a PBS washing step to wash away any unbound
materials and to create a stable baseline prior to the next
3.2. Detection of AI H5N1 Virus
The total detection time for the detection of virus and
Blocking Agent
Block + Virus
−ΔF (Hz)
Figure 3. Frequency shifts of the QCM aptasensor compar-
ing BSA and PEG blocking agents for AIV H5N1.
Frequency (Hz)
0 2000 4000 6000 8000 10,000 12,000
Time (sec)
Figure 4. Typical sensorgram of the QCM aptasensor for
surface modification, aptamer immobilization, target AIV
detection and signal amplification with nanobeads. The
concentration of AIV H5N1 was 1 HA in this test.
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amplification with nanobeads is 1 h for this sensor. The
detection signal was the change in frequency of the PBS
baselines due to the addition of the virus and nanobeads
and the results can be seen in Figure 5. The detection
limit of this sensor was determined to be 1 HAU. Using
the same calculation method, the detection limit of the
biosensor was also 1 HAU for H5N1 detection without
nanobead amplification.
Compared to the QCM immunosensor reported by Li
et al. [17], the aptamer seemed to be much more effec-
tive in the capturing virus than the more commonly used
antibody. Not only was the magnitude of the frequency
change higher for each respective titer, but the aptamer
also cut the detection time in half compared to antibody
tests. Also, the aptamers did not require chemical immo-
bilization which is time consuming and material inten-
sive. However, the aptamer coated nanobeads were not
as effective as a mass amplifier as hoped. While they did
slightly increase the sensitivity of the biosensor, this was
not enough to lower the detection limit from the pure
virus detection limit. It should be noted that as the titer
concentration lowered, the beadamplification became
more and more significant. At 0.1 and 0.01 HAU the
nanobeads more than doubled the frequency change due
to the capture of AIV H5N1. Unfortunately, this was still
below the lower detection limit of the aptasensor. If the
noise level could be lowered by further reducing nonspe-
cific binding of nanobeads the aptasensor would prove to
be even more sensitive. A possible explanation for the
failure of the nanobead amplification could be the non-
rigid structure of the AIV/nanobead complex. The QCM
is much more sensitive to rigid structures compared to
elastic ones. Antibodies provide a more rigid structure
and can bind to multiple virus particles as well while
aptamers are more flexible and only have one binding
site. While aptamers are still great for very specific virus
detection, they are not as strong when it comes to nano-
bead amplification because of the elasticity of their
Figure 6 shows an SEM image of the top of the QCM
electrode surface. The image confirms the capturing of
the target H5N1 virus by the aptamer and also the bind-
ing of the 150 nm magnetic nanobead to the virus.
3.3. Specificity of the Aptasensor
The immunosensor was evaluated for specicity with
ve different subtypes of avian inuenza viruses using
the same procedure described previously. These non-tar-
get viruses were chosen due to the similar properties of
their HA and NA proteins. Figure 7 shows the frequency
change due to the addition of each non-target virus at a
concentration of 2 HAU. While AIV H5N1 made 65 Hz
of frequency change, none of the five non-target subtypes
caused more than 8 Hz of frequency change, which is
well below the lower detection limit of 18 Hz frequency
change. The results proved a high specificity of the apta-
sensor to the target AIV H5N1.
4. Conclusion
The results of this study showed that the aptamer not
only increased the sensitivity of the aptasensor to the tar-
get virus but also reduced the detection time. A detection
limit of 1 HAU was determined. Unfortunately, the nano-
bead amplification proved to be insignificant by not am-
plifying the frequency change enough to lower the detec-
78 75
control0.010.11 2 4
-F (Hz)
Virus Concentration (HAU)
Figure 5. Frequency shifts of the QCM biosensor as a function of the titer (HAU) of AI H5N1 virus in PBS solution. The de-
ection limit is determined as 1 HAU. Error bars indicate the standard deviation (n = 3).
Figure 6. SEM image of the top view of the QCM surface.
Confirmation of the binding of a magnetic nanobead with a
150 nm diameter to a target H5N1 virus (80 nm diameter).
H5N1 H7N2 H9N2 H5N9 H5N2 H5N3
-F (Hz)
Virus Subtype
Figure 7. Frequency shifts of the QCM aptasensor for the
target AIV H5N1 virus along with the five non-target AIV
subtypes at 2 HAU. Error bars indicate standard deviation
(n = 3).
tion limit. If the noise level could be reduced, the sensor’s
sensitivity could be greatly increased. While the detec-
tion limit of this aptasensor was very similar to previous
studies (1 HAU), it has the advantage of being specific to
the H5N1 virus rather than just the H5 protein. The apta-
sensor was proven to have no non-specific binding to
similar non-target AIV subtypes. The detection time of
the aptasensor was also greatly reduced down to 1 h.
5. Acknowledgements
This research was supported in part by ABI. The authors
thank Dr. Huaguang Lu, Animal Diagnostic Laboratory
at Penn State University, for providing inactivated vi-
ruses and Lisa Kelso, Department of Poultry Science at
University of Arkansas, for her help in SEM imaging.
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