Biosensors and Bioelectronics 42 (2013) 148–155

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Biosensors and Bioelectronics

journal homepage: www.elsevier.com/locate/bios

Hydrogel based QCM aptasensor for detection of avian influenza virus

Ronghui Wang a,b, Yanbin Li a,b,c,n
a
Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR 72701, USA
b
College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310068, China
c
Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA

a r t i c l e i n f o a b s t r a c t

Article history: The objective of this study was to develop a quartz crystal microbalance (QCM) aptasensor based on
Received 7 August 2012 ssDNA crosslinked polymeric hydrogel for rapid, sensitive and specific detection of avian influenza virus
Received in revised form (AIV) H5N1. A selected aptamer with high affinity and specificity against AIV H5N1 surface protein was
28 September 2012
used, and hybridization between the aptamer and ssDNA formed the crosslinker in the polymer
Accepted 10 October 2012
hydrogel. The aptamer hydrogel was immobilized on the gold surface of QCM sensor using a self-
Available online 22 October 2012
assembled monolayer method. The hydrogel remained in the state of shrink if no H5N1 virus was
Keywords: present in the sample because of the crosslinking between the aptamer and ssDNA in the polymer
Aptamer network. When it exposed to target virus, the binding reaction between the aptamer and H5N1 virus
Hydrogel
caused the dissolution of the linkage between the aptamer and ssDNA, resulting in the abrupt swelling
QCM aptasensor
of the hydrogel. The swollen hydrogel was monitored by the QCM sensor in terms of decreased
Avian influenza
Virus detection frequency. Three polymeric hydrogels with different ratio (100:1 hydrogel I, 10:1 hydrogel II, 1:1
hydrogel III) of acrylamide and the aptamer monomer were synthesized, respectively, and then were
used as the QCM sensor coating material. The results showed that the developed hydrogel QCM
aptasensor was capable of detecting target H5N1 virus, and among the three developed aptamer
hydrogels, hydrogel III coated QCM aptasensor achieved the highest sensitivity with the detection limit
of 0.0128 HAU (HA unit). The total detection time from sampling to detection was only 30 min. In
comparison with the anti-H5 antibody coated QCM immunosensor, the hydrogel QCM aptasensor
lowered the detection limit and reduced the detection time.
& 2012 Elsevier B.V. All rights reserved.

1. Introduction requiring expensive equipments and high level biosafety facilities.

Avian influenza viruses (AIV) have caused global concerns due
to the potential pandemic threat for public health and enormous
economic losses. Highly pathogenic AIV H5N1 has caused devas-
tating outbreaks in poultry worldwide, and the virus has a high
mortality rate in both poultry and humans, often having a 100%
mortality rate in poultry and a 60% mortality rate in humans.
The H5N1 virus has killed 358 of 607 people infected since 2003
(WHO, 2012). Rapid and sensitive laboratory and field tests for
the diagnosis of H5N1 infection are essential for disease control
(MacKay et al., 2008). Currently, the laboratory methods for
influenza virus detection include conventional virus culture
followed by serological differentiation (Woo et al., 1997; Chen
et al., 2007), and the molecular methods such as RT-PCR and real-
time RT-PCR (Xie et al., 2006; Spackman et al., 2003). However,
these methods are time consuming, technical demanding, or
n
Corresponding author at: Department of Biological and Agricultural Engineer-
ing, University of Arkansas, 203 Engineering Hall, Fayetteville, AR 72701, USA.
Tel.: þ1 4795752881; fax: þ1 4795752846.
E-mail address: yanbinli@uark.edu (Y. Li).
Other methods include rapid influenza antigen test which lacks
the required sensitivity (Drexler et al., 2009) and ELISA which
takes relatively long analysis time due to multistage procedure
(He et al., 2007).
As an alternative, considerable effort has been directed
towards the development of simple biosensors for the detection
of influenza virus (Critchley and Dimmock, 2004; Xu et al., 2007;
Oh et al., 2008; Wang et al., 2009; Owen et al., 2007; Hewa et al.,
2009; Li et al., 2011a, 2011b; Wang et al., 2011; Lum et al., 2012).
Due to the advantages of cost-effectiveness, simplicity, direct
detection and real-time output, QCM biosensors have attracted
interest in applications for influenza virus detection (Owen et al.,
2007; Hewa et al., 2009; Li et al., 2011a). So far, most studies
focused on the development of QCM immunosensor based on
antibody–antigen interaction for influenza virus detection
(Owen et al., 2007; Hewa et al., 2009; Li et al., 2011a, 2011b).
For example, a QCM immunosensor with anti-M1 monoclonal
antibody immobilized on the quartz crystal was developed by
Hewa et al., and both influenza A and B viruses were detected
(Hewa et al., 2009). Li et al. (2011a) reported a nanobeads
amplified QCM immunosensor with polyclonal antibody as the

0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bios.2012.10.038
 R. Wang, Y. Li / Biosensors and Bioelectronics 42 (2013) 148–155
recognition ligand for detection of AIV H5N1. Since aptamers have
a number of advantages in comparison with antibodies, such as
overcoming the use of animals for their production, can be
chemically modified and labeled easily, and good thermal stabi-
lity, making them an alternative to antibodies as recognition
agents in analytical and diagnostic applications (Tennico et al.,
2010; Pan et al., 2010; Tuleuova et al., 2010; Duan et al., 2012).
Aptamers are artificial nucleic acid ligands, specifically generated
against certain targets, such as amino acids, drugs, proteins or
other organic or inorganic molecules (Jayasena, 1999). They are
generated by an in vitro selection process called SELEX (systema-
tic evolution of ligands by exponential enrichment). Aptamers
usually show a very high affinity for their targets, with dissocia-
tion constants typically from the micromolar to low picomolar
range, comparable to those of some monoclonal antibodies,
sometimes even better (Jenison et al., 1994).
Hydrogel is a three-dimensional insoluble polymeric network,
which can be homogeneously dispersed in water. The gel has
high-density capacity due to its flexible and open matrix structure
as well as high hydrophilicity, allowing a better accommodation
of biomolecules and consequently enhancing biocompatibility,
bioactivity, and molecular stability of the gel-fabricated products.
Extensive studies have been reported for hydrogel applications in
biomedical, biotechnology and biosensing fields (Dion and Burns,
2011; Chen et al., 2009; Yang et al., 2008; He et al., 2010; Tierney
and Stokke, 2009; Miyata et al., 1999; Murakami and Maeda,
2005). DNA is very suitable for in-vivo applications because of its
inherent biocompatibility. DNA can fold into unique secondary or
tertiary structures, which result in high binding affinities for
targets, leading to structural changes. Moreover, DNA is easy to
synthesize, suitable for in-vitro design, and predictable in its
molecular behavior, which is based upon the base-pairing prin-
ciple of cDNA. For these reasons, DNA has recently been regarded
as an excellent material for engineering hydrogel system and the
aptamer based hydrogel can be tailored to applications in bioa-
nalysis, biomedicine and biosensing (Lin et al., 2004; Um et al.,
2006; Liu et al., 2012). Very recently, Liu et al. (2012) reviewed
aptamer-incorporated hydrogels for visual detection, controlled
drug release, and targeted cancer therapy. These developments
have positioned aptamer-incorporated hydrogels to make a sig-
nificant impact in many areas.
Upon exclusively review of literatures, there is no publication
on hydrogel based QCM aptasensor for AIV detection. In this
research, an aptamer-ssDNA crosslinked polymeric hydrogel was
developed, and the feasibility of a QCM aptasensor was demon-
strated for rapid and sensitive detection of AIV H5N1 using the
developed responsive hydrogel as the sensor coating material.
2. Materials and methods
2.1. Chemicals and biochemicals
16-Mercaptohexadecanoic acid (MHDA), N-(3-dimethylaminopro-
pyl)-N0 -ethylcarbodiimide hydrochloride (EDC  HCL), N-hydroxysuc-
cinimide (NHS), phosphate buffered saline (PBS, 0.01 M, pH 7.4),
Acrylamide (99%), ammonium persulfate (98%), Tetramethylethyle-
nediamine (TEMED, 99%) and Tris borate–EDTA buffer (TBE) solution
were purchased from Sigma-Aldrich (St. Louis, MO). Nitrocellulose
membrane was received from Schleicher & Schuell Bioscience, Inc.
(Keene, NH). Anti-mouse IgG-alkaline phosphatase conjugate, BCIP/
NBT substrate, blocking solution and wash buffer were obtained from
KPL, Inc. (Gaithersburg, MD). Streptavidin-alkaline phosphatase con-
jugate was purchased from PerkinElmer Life And Analytical Sciences,
Inc. (Waltham, MA). Ultrapure water (18 MO cm) produced by a
Millipore-Milli-Q system (Bedford, MA, USA) was used throughout.
149
Inactivated AIV H5N1 was supplied from USDA APHIS Research
Laboratory (Ames, IA). AIV subtypes H5N2, H5N3, H5N9, H7N2,
H9N2, and anti-H5 monoclonal antibody were precious gifts from
Animal Diagnostic Laboratory, Pennsylvania State University, Univer-
sity Park, PA. The selected aptamer with high affinity and specificity
against AIV H5N1 surface protein was developed in our group and the
detailed information was described in our previous study (Li et al.,
2011b; Wang et al., 2012). Biotin labeled aptamer and aptamer with
the oligonucleotide containing 50 -terminal acrylamide group designed
for hydrogel synthesis was obtained from Integrated DNA Technolo-
gies, Inc. (Coralville, IA). The sequences are 50 -Biotin-GTG TGC ATG
GAT AGC ACG TAA CGG TGT AGT AGT AAC GTG CGG GTA GGA AGA
AAG GGA AAT AGT TGT CGT GTT G-30 and 50 -Acrydite-TCC TAA TAC
GAC TCG TGT GCA TGG ATA GCA CGT AAC GGT GTA GTA GTA ACG
TGC GGG TAG GAA GAA AGG GAA ATA GTT GTC GTG TTG TAT TGA
AAA CGC G-30 , respectively. Single-stranded DNA (ssDNA) containing
50 -terminal acrylamide group was purchased from Integrated DNA
Technologies, Inc. (Coralville, IA). The sequence of ssDNA is 50 -
Acrydite-CGC GTT TTC AAT AGA GTC GTA TTA GGA-30 .
2.2. Instruments and quartz crystals
The circular AT-cut 7.995 MHz quartz crystals with Au elec-
trodes evaporated on both sides were purchased from Interna-
tional Crystal Manufacturing Co. (Oklahoma City, OK, USA). The
quartz crystals were 14 mm in diameter and 0.2-mm thick and
the gold electrodes were 5.1 mm in diameter and 100-nm thick.
The quartz resonator was housed inside a methacrylate cell
(International Crystal Manufacturing Co. OK, USA) so that only
one side of the crystal was in contact with the solution in the cell
well. The frequency variations were continuously recorded using
a QCA922 quartz crystal analyzer (Princeton Applied Research,
Oak Ridge, TN).
2.3. Synthesis of aptamer hydrogels
The polymer hydrogels were synthesized based on a reported
procedure in synthesis of DNA-crosslinked polyacrylamide hydro-
gel (Lin et al., 2004) with some modifications. The polymeration
parameters such as reaction time, monomer concentration, and
initiator concentration were changed to obtain virus responsive
hydrogels in this research. The detailed procedures are described
as following: Stock solution of acrylamide was prepared at 2%
with deionized water, and stock solutions of aptamer and ssDNA
were prepared at 1 mmol/ml with TBE buffer, respectively. All
synthesis reactions were carried out in a 1.7 ml microcentrifuge
tubes with two holes on the tube cap for gas bubbling. Dry
nitrogen was bubbled for ten minutes through a solution 1 and
solution 2 in the tubes, respectively. For hydrogel I (acrylami-
de:aptamer:ssDNA, 100:1:1), solution 1 is a mixture of 714 ml, 2%
acrylamide and 200 ml, 0.25 mM aptamer, and solution 2 is a
mixture of 714 ml, 2% acrylamide and 200 ml, 0.25 mM ssDNA.
A 100 ml of a freshly prepared initiator–catalyst mixture consist-
ing of 0.5 ml DI water, 0.05 g ammonium persulfate, and 25 ml
TEMED was added to the solutions 1 and 2, respectively. Nitrogen
was bubbled through the mixtures for additional 10 min while
polymerization proceeded. Then, add the mixture of solution 1 to
the mixture of solution 2, and the hybridization reaction between
ssDNA and aptamer was performed for another 10 min. To
increase the viscosity of the hydrogel, the final mixture was
evaporated in half in volume at 37 1C incubator for overnight.
For hydrogel II (acrylamide:aptamer:ssDNA, 10:1:1), solution
1 is a mixture of 71.4 ml, 2% acrylamide and 200 ml, 0.25 mM
aptamer, and solution 2 is a mixture of 71.4 ml, 2% acrylamide and
200 ml, 0.25 mM ssDNA. The amount of initiator–catalyst mixture
is the same as that used in hydrogel I, and the procedures and
150 R. Wang, Y. Li / Biosensors and Bioelectronics 42 (2013) 148–155
reaction time are the same as that used in preparation of the
hydrogel I except for that the final reaction mixture was not
evaporated due to high crosslinking in the hydrogel II. For
hydrogel III (acrylamide:aptamer:ssDNA, 1:1:1), solution 1 is a
mixture of 7.14 ml, 2% acrylamide and 200 ml, 0.25 mM aptamer,
solution 2 is a mixture of 7.14 ml, 2% acrylamide and 200 ml,
0.25 mM ssDNA. Other procedures are the same as that of
hydrogel II.
2.4. Immobilization of aptamer hydrogels on the gold surface
The quartz resonators were cleaned by dipping in 1 M NaOH
for 5 min and 1 M HCl for 2 min in sequence. They were then
thoroughly washed with deionized water and dried in a steam of
nitrogen. The cleaned resonators were immersed in 10 mM
MHDA ethanol solution for overnight at room temperature to
form SAM, and then were rinsed with ethanol and water, and
dried in a steam of nitrogen. The pretreated resonator was fixed
into the detection cell and connected to the quartz crystal
analyzer. First, 200 ml of deionized water was added in the cell
well to get a baseline. Second, 200 ml EDC/NHS (75 mM/30 mM,
v/v, 1:1) solution was dropped into the cell well (10 min) for
activation. After deionized water washing, 200 ml of aptamer
hydrogel was added and incubated for 40 min. After washing
with deionized water and PBS buffer (0.01 M, pH 7.4), the
aptamer hydrogel immobilized resonator was ready for AIV
detection.
2.5. AIV detection
Target AIV H5N1 (200 ml) with different titers were added into
the detection cell and incubated for 30 min at room temperature.
After incubation, the quartz resonator was washed three times
with PBS (300 ml per time). The frequency change (DF) caused by
AIV binding was calculated by measuring the difference before
and after adding the target virus.
2.6. Dot blot analysis
Dot blot assay (Vivekananda and Kiel, 2006) was used for the
comparison study of the affinity and specificity against target AIV
H5N1 between the selected aptamer and monoclonal anti-H5
antibody.
Target AIV H5N1 (5 ml) with different titers were spotted onto
nitrocellulose membrane (BA85 Protran, 0.45 mm, Whatman,
USA) and allowed to air dry. Then these samples were followed
by blocking with blocking buffer (12.5 g casein; 4.5 g NaCl;
605 mg Tris; 100 mg Thimerosal) for 30 min and incubated with
biotinylated aptamers (2.2 mg ml  1) for 45 min at room tempera-
ture. After washing three times with 1  KPL (KPL, Gaithersburg,
MD, USA) washing solution (0.002 M imidazole, 0.02% Tween 20,
0.5 mM EDTA, 160 mM NaCl), the membrane was reacted with
streptavidin-conjugated alkaline phosphotase (1:500 dilution) for
30 min. Excess enzyme was removed by three subsequent washes
with 1  KPL washing solution. Finally, the membrane was coated
in BCIP/NBT (5-bromo-4-chloro-3-indoxyl-phosphate and nitro-
bluetetrazolium) (KPL, MD, USA) substrate in the dark for color
development. PBS buffer was served as a negative control and
monoclonal antibody (Mab) specifically against AIV H5 subtypes
(provided by Animal Diagnostic Laboratory at Pennsylvania State
University) with a concentration of 4.4 mg ml  1 was employed in
the dot blot analysis to replace the aptamer as a comparison.
3. Results and discussion
3.1. Comparison study between aptamers and monoclonal antibody
for their affinity and specificity against AIV H5N1
We have successfully obtained a DNA aptamer specifically
against AIV H5N1 using SELEX technology with a dissociation
constant of 4.65 nM as determined by surface plasmon resonance
(Li et al., 2011b; Wang et al., 2012). A comparison study between
the selected aptamer and anti-H5 monoclonal antibody
(antiH5 Mab) was carried out to check the affinity and specificity
against the target AIV H5N1 using the Dot blot assay. Target AIV
H5N1 with titer of 10-fold dilutions in the range of 0.0128 to
128 HAU was detected using the aptamer and anti-H5 Mab,
respectively, to compare the affinity. As shown in Table 1(a), the
result indicated that both antiH5 Mab and aptamer were able to
detect H5N1 with Dot-ELISA, and the observed detection limit
was 1.28 HAU for both ligands. Different H5 subtypes (H5N1,
H5N2, H5N3, H5N9) and other AIV subtypes (H7N2) were also
tested to compare the specificity, and the result is presented in
Table 1(b). All tested H5 subtypes (H5N1, H5N2, H5N3, H5N9)
appeared visible dots on the strip when using antiH5 Mab as the
recognition ligand, showing the antiH5 Mab was specific to H5
subtypes of AIV. It was found from Table 1(b) that the obtained
aptamer had great specificity against target H5N1, and other AIV
subtypes (H5N2, H5N3, H5N9, and H7N2) could not bind with the
aptamer.
3.2. Synthesis of poly(acrylamide-co-aptamer) hydrogels
In the study, we firstly designed the hydrogel structure from
the chemical structures of the monomers and the crosslinker.
To form polymer chain, we used a method developed by Mosaic
Technologies (Waltham, MA) to introduce a phosphoramidite
molecule to the 50 end of the aptamer, and based on the same
way, the phosphoramidite molecule was also grafted to the
single-stranded DNA (ssDNA). To gain the crosslinking gels, we
designed a specific ssDNA with 27 bases with the sequence
exactly matching the DNA grafted on the ends of the aptamer.
Hybridization between ssDNA and DNA-grafted aptamers enables
the crosslinking in the polymer hydrogel. Fig. 1(a) schematically
illustrates the structure of the polymer hydrogel. Part of polymer
chains consists of acrylamide and aptamer units; another part of
chains consists of acrylamide and ssDNA units in the hydrogel.
The density of aptamers on the polymer chains can be controlled
by the ratio of aptamer to acrylamide, and in the same way, the
Table 1
Dot blot analysis for the comparison study between the aptamer and H5N1
monoclonal antibody (antiH5 Mab): (a) Detection of target H5N1 in the titer range
from 0.0128 to 128 HAU; and (b) Specificity test using non-target AIV subtypes.
(a)
AIV H5N1 titer (HAU)
128 12.8 1.28 0.128 0.0128
antiH5 Mab þþ þþ þ  
Aptamer þþ þþ þ  
(b)
AI subtypes
H5N1 H5N2 H5N3 H5N9 H7N2
antiH5 Mab þþ þþ þ þþ 
Aptamer þþ    
 R. Wang, Y. Li / Biosensors and Bioelectronics 42 (2013) 148–155 151

Fig. 1. (a) Scheme of poly(acrylamide-co-aptamer) hydrogel. Part of polymer chain is comprise of acrylamide and aptamer; another part formed by acrylamide and ssDNA.
Hydrogel formed through crosslinking between aptamer and ssDNA. (b) Schematically illustrates chemical reaction mechanism and reactions used in the experiment for
polymer hydrogel synthesis. Co-polymerization of acrylamide, acrydite modified ssDNA and acrydite modified aptamer. Polymer chains crosslinked by the ssDNA and
aptamer. The polymerization reaction was initiated and catalyzed by a mixture of ammonium persulfate and TEMED. (c) Picture of poly (acrylamide-co-aptamer) hydrogels
with the ratio of acrylamide, aptamer, and ssDNA is 100:1:1 (hydrogel I), 10:1:1 (hydrogel II), and 1:1:1 (hydrogel III). It is shown that all of three hydrogel materials are
viscous transparent liquid.

ssDNA density on the polymer chains can be adjusted by changing
the ratio of the ssDNA to acrylamide. The polymerization reaction
mechanism and step-by-step synthesis reaction of crosslinking
hydrogel are shown in Fig. 1(b). Fig. 1(b) showed the two
separated polymerization reactions, reactions (1) and (2) were
firstly conducted in the presence of initiator and catalyst in a
buffer solution at room temperature. In the last reaction step, the
crosslink reaction in polymer hydrogel occurred through hybri-
dization between ssDNA and DNA-grafted aptamer.
The obtained DNA crosslinked poly(acrylamide-co-aptamer)
hydrogel was a network of polymer chains that are water-
insoluble (Fig. 1c). It was a soft jelly-like liquid material with
some degree of viscosity determined by the amount of aptamers
and ssDNA in the hydrogel. Fig. 1(c) showed the appearance of
three hydrogels with the ratios of acrylamide to aptamer and
ssDNA of 100:1:1 (hydrogel I), 10:1:1 (hydrogel II), and 1:1:1
(hydrogel III), respectively. The density of the hydrogel I, hydrogel
II and hydrogel III were measured to be 1.032, 1.064, and 1.143,
respectively. It is close to the density of water, indicating that the
hydrogel consists mainly of water. It was also found that there
was a small increase in density when the crosslink increased for
the hydrogels. Increasing viscosity of hydrogel was observed with
the increment of crosslinker of ssDNA and aptamer. All hydrogels
obtained are transformed from gel to sol at the temperature
above 90 1C, showing the dissolution of the hydrogel at high
temperature.
152 R. Wang, Y. Li / Biosensors and Bioelectronics 42 (2013) 148–155
3.3. Fabrication of QCM biosensor with the synthesized
aptamer hydrogels
The poly(acrylamide-co-aptamer) hydrogel itself contains
amino group on its polymer chains. SAM (self-assembled mono-
layer) method was used to immobilize poly(acrylamide-co-apta-
mer) polymer hydrogels on QCM sensor gold surface due to its
advantages in stability. The mechanism of the SAM method for
poly(acrylamide-co-aptamer) hydrogels immobilization is illu-
strated in Fig. 2(a). It involved the following steps. First, an
oriented monolayer of 16-mercaptohexadecanoic acid (MHDA),
a long-chain carboxylic acid-terminating alkanethiol, was formed
via the strong Au-thiolate bond with the tail carboxylic group
exposed at the monolayer-liquid interface. The monolayer of
MHDA appears to be stable indefinitely in aqueous solution at
room temperature, and it is thermally more stable than the SAMs
from short-chain disulfides or thiols. In the second step, the
MHDA-SAM was treated with 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide (EDC) and N-hydroxysuccinimide ester (NHS) to
form an NHS ester. In the third step, the active NHS ester was
replaced by the primary amines presented in the poly(acryla-
mide-co-aptamer) polymer hydrogels, and the poly(acrylamide-
co-aptamer) hydrogels were thus immobilized through the amide
bond. Affinity immobilization of biomolecules onto alkane-thiol-
100
50
1 The H5N1 responsive aptamer hydrogels were optimized
0
79 Hz
-50 4 increased sensitivity of the hydrogel II-coated QCM biosensor was
Δf (Hz)
2
-100
EDC/NHS wash
-150
3  172 Hz, respectively. PBS without target AIV was also detected
-200
Aptamer Hydrogel
-250
-300
0 500 1000 1500 2000
Time(s)
Fig. 2. (a) Schematic diagram for QCM sensor fabrication using the synthesized
poly(acrylamide-co-aptamer) polymer hydrogel; (b) The typical response of QCM
sensor for fabrication using the synthesized aptamer hydrogel: (1) SAM formed
on sensor in equilibrium with deionized water; (2) NHS/EDC activation;
(3) poly(acrylamide-co-aptamer) polymer hydrogel immobilization; (4) wash to
get the baseline.
fabricated gold-coated quartz crystals via ligand-linked hydrogels
has been demonstrated as an effective scheme for chip prepara-
tion (Chen et al., 2009; Tombelli et al., 2002) because the multi-
layer co-coating with long-chain alkanethiols, hydrogels, and
affinity ligands can provide combined effects for eliminating
undesirable surface denaturation and nonspecific binding, and
at the same time, improving chip activity, sensitivity, and stability
(Jenison et al., 1994).
The immobilization of aptamer hydrogel I, II, III on gold-coated
quartz crystals resulted in frequency shifts (DF) of  78714,
 81717, and  83719, respectively. Fig. 2(b) shows the typical
temporal responses of frequency shifts for the stepwise assembly
of the fabrication of the QCM sensor using the synthesized
aptamer hydrogel III. Based on the SAM technique, the synthe-
sized poly(acrylamide-co-aptamer) polymer hydrogel III resulted
in 79 Hz decrease of resonant frequency, indicating the successful
assembly of the aptamer hydrogel on QCM sensor surface.
3.4. Demonstration of the feasibility for detection of AIV H5N1 using
the apatmer Hydrogel based QCM biosensor
The QCM sensors coated with the poly(acrylamide-co-apta-
mer) polymer hydrogels were used to prove and demonstrate the
feasibility for detection of AIV H5N1. The original titer of AIV
H5N1 virus was 128 HA unit (HAU), and diluted to the desired
titers with PBS for further use. Three polymer aptamer hydrogels
with different ratios of acrylamide and aptamer monomer, hydro-
gel I (100:1), hydrogel II (10:1), and hydrogel III (1:1) were
investigated. 10, 15, 20, 25, and 30 mM NaOH was optimized
for regeneration, and the result showed that 25 mM NaOH with
incubation time of 20 s was the best, which was used as the
regeneration solution throughout the tests. Since real samples
from H5N1 infected poultry and human are required to be
handled under biosafety level 3 conditions in WHO H5 Reference
Laboratories, no real samples from infected objects were tested in
this study due to the limitation of our laboratory.
With the hydrogel I as the coating material, H5N1 with the
titers of 64, 6.4, 1.28 and 0.128 HAU was detected using the QCM
biosensor. A decrease in resonance frequency shift was observed
for detection of target H5N1 with the titers of 64, 6.4 and
1.28 HAU, and the corresponding response in resonance fre-
quency was  200,  52 and  22 Hz, respectively. No detectable
signal was obtained for H5N1 with the titer of 0.128 HAU. The
typical detection curve is shown in Fig. 3. The QCM biosensor
results clearly showed that the synthesized poly(acrylamide-co-
aptamer) polymer hydrogel coated sensor was capable of detec-
tion of target AIV H5N1. The decrease in resonance frequency
indicated the swelling of the hydrogel on the sensor surface.
based on the ratio of the monomers. For hydrogel II (10:1), an
obtained. Fig. 4 shows the frequency response upon exposing
H5N1 with different titers (0.64, 0.32, 0.128 and 0.0128 HAU). The
observed frequency shifts were  2676,  1773,  873,
as a negative control (blank), and a signal of 071 Hz was
obtained. The detection limit was determined as background
(blank)þ3  noise (standard deviation) (Li et al., 2011a), and a
positive result was considered when the detectable signal
was equal to or greater than the detection limit. The detection
limit of the hydrogel II coated QCM biosensor was obtained as
0.128 HAU. The specificity of the fabricated QCM aptasensor was
evaluated using non-target AIV subtypes H5N2, H9N2 and H7N2
with a titer of 6.4 HAU, and no detectable signal was observed for
these non-target viruses ( 171, 071 and  171 Hz, respectively),
indicating that the developed poly(acrylamide-co-aptamer) polymer
 R. Wang, Y. Li / Biosensors and Bioelectronics 42 (2013) 148–155
Fig. 3. Frequency shifts (DF) corresponding to different titers of AIV H5N1 (64, 6.4 and 1.28 HAU, respectively) for aptamer hydrogel I coated QCM sensor.
Fig. 4. Results of AIV H5N1 detection in the range from 0.00128 to 0.64 HAU using
QCM sensors coated with aptamer hydrogel II and aptamer hydrogel III. All tests
were repeated three times. Error bars ¼ S.D. (n¼3).
hydrogels had good specificity for the target H5N1 when they were
used in the QCM aptasensor.
For hydrogel III (1:1) coated QCM biosensor, the highest
sensitivity for detection of H5N1 was achieved (Fig. 4). H5N1
with the titers of 0.64, 0.128, 0.0128 and 0.00128 HAU induced a
signal change in resonance frequency of  5177, 25 75,
9 72 and  271 Hz, respectively. The detection limit of
H5N1 can be reached to 0.0128 HAU. The working range was
obtained up to 64 HAU of the target AIV H5N1 for the hydrogel III
(1:1) coated QCM biosensor. The repeatability of the measure-
ments was evaluated using the same titer of target AIV H5N1
(0.64 HAU) for five different cycles of detection/regeneration on
the same day. The average signal obtained was 5177 Hz with a
CV of 11%. In clinical diagnostics of poultry, real time RT-PCR has
been broadly accepted. The Dot-ELISA has also been used as a
rapid screening and diagnostic test to identify AIV in various
sources of surveillance samples and clinical diagnostic specimens
153
(Lu, 2003). The titer of avian influenza virus to cause early symp-
toms on poultry is about 103 EID50/ml (EID50: 50% Egg Infection
Dose), which equals to  0.0128 HAU.
To prove the enhanced sensitivity of hydrogel coated QCM
biosensors, we conducted a comparison test with anti-H5 antibodies
for label-free detection of AIV H5N1. Anti-H5 antibodies were
immobilized on the QCM sensors using the same SAM method as
above for immobilization of hydrogels on the QCM sensors because
there are rich amino groups in antibody molecules. The immobiliza-
tion of anti-H5 antibodies resulted in 204722 Hz decrease in
frequency. The QCM sensors coated with anti-H5 antibodies were
applied for AIV H5N1 detection. The comparison result is listed in
Table 2. Table 2(a) indicates the amount of coating on both types of
biosensors. They were very comparable to each other, and the
obtained coating amount was 5.7  10  3 nmol for the immunosen-
sor and 5.8  10  3 nmol for the aptasensor, respectively. It can be
clearly seen from Table 2(b) that the hydrogel III coated QCM sensor
induced 2575 Hz change in frequency and the anti-H5 antibody
coated QCM sensor can only generate 674 Hz change in fre-
quency for the same titer of H5N1 of 0.128 HAU. The detection limit
of H5N1 with anti-H5 antibody coated QCM sensor was 0.128 HAU
and the detection limit of aptamer hydrogel based QCM biosensor
was 0.0128 HAU. The reasons for the improved sensitivity obtained
by the aptasensor were possibly due to (1) the artificial ligand-
aptamer and (2) the hydrogel. Aptamers are self-refolding, single-
chain, and redox-insensitive. They also lack the large hydrophobic
cores of proteins and thus do not aggregate. They can tolerate harsh
physical and chemical conditions that proteins usually cannot.
Therefore, even the coating amount of antibodies was similar to that
of aptamers in the test, the antibodies might partially lose their
activities during the chemical covalent bonding procedure, and
resulted in a decrease of sensitivity. Moreover, aptamers are capable
of greater specificity and affinity than antibodies (Jayasena, 1999).
QCM measures both mass and viscosity in processes occurring on the
surfaces. The change of hydrogels from shrink to swelling may result
in the change of viscosity and consequently the increased sensitivity.
When both the aptamer and anti-H5 antibody were employed as the
recognition ligands for detection of AIV H5N1 using the conventional
Dot blot assay, the same detection limit of 1.28 HAU was observed
for both ligands (Table 1a). After synthesis of the poly(acrylamide-co-
aptamer) polymer hydrogel and employed it for QCM biosensor
154 R. Wang, Y. Li / Biosensors and Bioelectronics 42 (2013) 148–155
Table 2
Comparison between the anti-H5 antibody immobilized QCM biosensor (immunosensor) and the aptamer hydrogel based QCM biosensor (aptasensor): (a) amount of
coating used for biosensing; and (b) detection of AIV H5N1.
(a)
QCM Biosensor Frequency change (Hz)
in coating
Immunosensor  204
Aptasensor  83
n
The measured values of frequency change are the means of three replicates.
n
According to Sauerbrey equation DF ¼  2.3  106F20DM/A [F0: Resonant frequency (Hz); DF: Frequency change (Hz); DM: Mass change (g); A: Piezoelectrically active
crystal area (cm2)], for the used QCM sensor (F0 ¼ 7.995 Hz and A ¼0.2 cm2), 1 Hz frequency change corresponds to 1.4 ng mass change.
(b)
QCM Biosensor Frequency Change (Hz), Mean7 S.D.
H5N1 titer (HAU)
1.28
Immunosensor  377 16
Aptasensor  907 17
n
three replicates.
development and applied for AIV detection, the detection sensitivity
was improved by one order of magnitude by lowering the detection
limit from 0.128 to 0.0128 HAU. In addition, the detection time for
the anti-H5 antibody based biosensor was 1 h and the aptamer
hydrogel based QCM biosensor was only 30 min. Therefore, in
comparison with the anti-H5 antibody coated QCM biosensor, the
developed aptamer hydrogel coated QCM biosensor lowered the
detection limit and reduced the detection time.
4. Conclusions
In this study, we have developed an aptamer-ssDNA crosslinked
polymeric hydrogel material, and demonstrated the feasibility of
constructing an aptamer hydrogel based QCM biosensor for rapid
and sensitive detection of AIV H5N1. The main innovation of the
study was to introduce a designed ssDNA and the specific aptamer
against AIV H5N1 surface protein to the polymer backbones to
form a ‘smart’ AIV-responsive hydrogel, and then employed it for
QCM biosensor development with applications for AIV detection.
Three polymeric hydrogels with different ratios (100:1, 10:1, 1:1)
of acrylamide and the aptamer monomer were synthesized. The
fabrication of QCM biosensor with the obtained three aptamer
hydrogels was carried out through a self-assembled monolayer
method. The QCM biosensor results showed that the synthesized
aptamer hydrogel coated sensor was capable of detection of target
AIV H5N1. Among the three polymer hydrogels, higher sensitivity
was observed with lower ratio of acrylamide and the aptamer, and
the hydrogel with the ratio of 1:1 gave the best sensitivity and the
lowest detection limit (0.0128 HAU). The total detection time was
only 30 min for the developed aptamer hydrogel based QCM
biosensor. No interference was observed from non-target AIV
subtypes. A comparison study in terms of detection time and
detection limit was carried out between the anti-H5 antibody
immobilized QCM biosensor and the aptamer hydrogel based QCM
biosensor for detection of AIV H5N1.
Acknowledgments
This research was supported in part by Arkansas Bioscience
Institute (ABI). The authors thank Dr. Huaguang Lu at Animal
Mass change (ng) Molecular Coating
in coating weight (kDa) amount (nmol)
286 50 5.7  10  3
116 20 5.8  10  3
0.64 0.128 0.0128 0.00128
 19 76 674 271 –
 51 77  25 7 5 972  27 1
Diagnostic Laboratory, Penn State University for his help in
preparing samples used in the tests.
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