QCM Aptasensor for Rapid and Specific Detection of Avian Influenza Virus

doi:10.4236/ojab.2013.24013

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Open Journal of Applied Biosensor, 2013, 2, 97-103

Published Online November 2013 (http://www.scirp.org/journal/ojab)

http://dx.doi.org/10.4236/ojab.2013.24013

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: *yanbinli@uark.edu

Received February 1, 2013; revised March 18, 2013; accepted April 6, 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

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.

L. BROCKMAN ET AL.

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]:



Δf

fCf



 







(1)

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:

Δ42

2π

rol lm

fsin





















(2)

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-

GGATAGCACGTAACGGTGTAGTAG-ATACGT-GC-

GGGTAGGAAGAAAGGGAAATAGTTGTCCTGT-

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

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L. BROCKMAN ET AL. 99

Electrode: Top View

Flow Cell La ptop

QCA 922

Inlet Ourlet

Waste

Electrode

Syringe

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

aptame

(a) (b)

Poly (ethylene

glycol)

H5N1 virus

Aptamer labeled

nanobeads

(e)

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-

tives.

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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L. BROCKMAN ET AL.

100

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

injection.

3.2. Detection of AI H5N1 Virus

The total detection time for the detection of virus and

120

100

0BSA PE

Blocking Agent

Block

Block + Virus

−ΔF (Hz)

Figure 3. Frequency shifts of the QCM aptasensor compar-

ing BSA and PEG blocking agents for AIV H5N1.

7,979,900

7,979,850

7,979,800

7,979,750

7,979,700

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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L. BROCKMAN ET AL.

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101

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

structure.

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 speciﬁcity with

ﬁve different subtypes of avian inﬂuenza 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-

017

913

78 75

115

-20

100

120

140

160

control0.010.11 2 4

-∆F (Hz)

Virus Concentration (HAU)

Virus

Virus+Beads

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).

L. BROCKMAN ET AL.

102

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).

-10

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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