A nanobeads amplified QCM immunosensor for the detection of avian influenza virus H5N1

Biosensors and Bioelectronics 26 (2011) 4146–4154

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A nanobeads amplified QCM immunosensor for the detection of avian influenza virus H5N1 Dujuan Li a,b , Jianping Wang a,∗ , Ronghui Wang b , Yanbin Li b,c,∗∗ , Daad Abi-Ghanem d , Luc Berghman d , Billy Hargis c , Huaguang Lu e a

College of Biosystems Engineering and Food Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR 72701, USA c Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA d Department of Poultry Science and Veterinary Pathobiology, Texas A&M University, College Station, TX 77843, USA e Animal Diagnostic Laboratory, Penn State University, State College, PA 16802, USA b

a r t i c l e

i n f o

Article history: Received 12 January 2011 Received in revised form 6 April 2011 Accepted 6 April 2011 Available online 13 April 2011 Keywords: Avian influenza (AI) H5N1 virus Magnetic nanobeads Quartz crystal microbalance (QCM) Immunosensor Anti-H5 antibodies

a b s t r a c t As a potential pandemic threat to human health, there has been an urgent need for rapid detection of the highly pathogenic avian influenza (AI) H5N1 virus. In this study, magnetic nanobeads amplification based quartz crystal microbalance (QCM) immunosensor was developed as a new method and application for AI H5N1 virus detection. Polyclonal antibodies against AI H5N1 virus surface antigen HA (Hemagglutinin) were immobilized on the gold surface of the QCM crystal through self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid (MHDA). Target H5N1 viruses were then captured by the immobilized antibodies, resulting in a change in the frequency. Magnetic nanobeads (diameter, 30 nm) coated with anti-H5 antibodies were used for further amplification of the binding reaction between antibody and antigen (virus). Both bindings of target H5N1 viruses and magnetic nanobeads onto the crystal surface were further confirmed by environmental scanning electron microscopy (ESEM). The QCM immunosensor could detect the H5N1 virus at a titer higher than 0.0128 HA unit within 2 h. The nanobeads amplification resulted in much better detection signal for target virus with lower titers. The response of the antibody–antigen (virus) interaction was shown to be virus titer-dependent, and a linear correlation between the logarithmic number of H5N1 virus titers and frequency shift was found from 0.128 to 12.8 HA unit. No significant interference was observed from non-target subtypes such as AI subtypes H3N2, H2N2, and H4N8. The immunosensor was evaluated using chicken tracheal swab samples. This research demonstrated that the magnetic nanobeads amplification based QCM immunosensor has a great potential to be an alternative method for rapid, sensitive, and specific detection of AI virus H5N1 in agricultural, food, environmental and clinical samples. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The highly pathogenic avian influenza (AI) H5N1 virus has been considered as a potential pandemic threat to human health. The first human disease caused by H5N1 was reported in Hong Kong in 1997, with 18 cases and six deaths (Claas et al., 1998; Subbarao et al., 1998; Yuen et al., 1998). In December 2003 and January 2004, outbreaks of AI H5N1 were reported in eight Asian countries (Sims et al., 2005). And till now, there have been 302 cases of human

∗ Corresponding author. Tel.: +86 571 88982350; fax: +86 571 88982350. ∗∗ Corresponding author at: Department of Biological and Agricultural Engineering, Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA. Tel.: +1 4795752881/2424; fax: +1 4795752846. E-mail addresses: [email protected] (J. Wang), [email protected] (Y. Li). 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.04.010

death caused by avian influenza H5N1 virus since 2003 according to the report of WHO (2010). This panzootic outbreak only can be stopped by the killing of the entire domestic poultry population within the territory. H5N1 has greatly impacted both economic and social wellbeing. This threat makes it necessary to detect AI H5N1 as early, rapid, specific, and sensitive as possible. Virus isolation is a sensitive technique, but typically requires 5–7 days for testing with very labor-intensive and time consuming procedures (Storch, 2000). Although RT-PCR is becoming more commonly available in diagnostic laboratories, it requires expensive equipment, appropriate laboratory facilities, and a trained technician (Di Trani et al., 2006; Payungporn et al., 2006). The antigen capture immunoassays can provide rapid test results, but suffer from low sensitivity and high cost (Woolcock and Cardona, 2005). Another two methods for the detection of AI virus are DNA microarrays which also require dedicated equipment (Liu

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et al., 2006; Townsend et al., 2006) and ELISA which takes relatively long analysis time due to multistage procedure (He et al., 2007). As an alternative, biosensors have been studied for the detection of AI H5N1 virus (Kukol et al., 2008; Pavlovic et al., 2008; Deng et al., 2009; Ho et al., 2009; Tam et al., 2009; Ting et al., 2009; Wang et al., 2009) for rapid and reliable testing of influenza with minimal sample handling and laboratory skill requirements (Amano and Cheng, 2005). Microgravimetric quartz crystal microbalance (QCM), owing to its simplicity and cost effectiveness, has been extensively investigated as a transducer for virus detection such as influenza A and B viruses (Owen et al., 2007; Hewa et al., 2009), dengue virus (Wu et al., 2005; Chen et al., 2009a), airborne vaccinia viruses (Lee et al., 2008), flavivirus (Tai et al., 2006), bovine ephemeral fever virus (Lee and Chang, 2005; Lee et al., 2005), hybridization of hepatitis B virus (Yao et al., 2008), human rhinovirus (HRV) and the foot-and-mouth disease virus (FMDV) (Tai et al., 2005,2006; Jenik et al., 2009), and other viruses (Abad et al., 1998; Eun et al., 2002). Based on the Web of Science database, to date there is only one report published on QCM immunosensor for AI virus detection (Liu et al., 2008), however, this study was only type-specific viral detection and could not be used for the avian influenza virus subtype identification. Due to lower sensitivity and higher detection limit of QCM immunosensor compared to the traditional methods, many methods have been studied for the improvement of QCM immunosensor for virus detection, including optimizations of antibody immobilization (Wu et al., 2005) and signal amplification strategies using nanoparticles (Hewa et al., 2009). Previous studies (Mao et al., 2006; Liu et al., 2007; Hewa et al., 2009) demonstrated the signal amplification strategy using nanoparticles to improve the sensitivity of the QCM immunosensor owing to the outstanding performances of nanoparticles. Nanoparticles are defined as particles with size in the range of 1–100 nm at least in one of the three dimensions. Due to strong magnetic properties and low toxicity, magnetic nanobeads are widely used for biochemical applications in immunoassays and cell separation (Shimazu et al., 2005; Tsai et al., 2007; Chen et al., 2009b; Di Corato et al., 2009; Soelberg et al., 2009; Li et al., 2010). Magnetic nanobeads with functional derivatives can separate the compound of biological activity from others in biological samples under magnetic fields. It is known that magnetic beads less than 30 nm will exhibit superparamagnetism. Strictly speaking, the beads are not magnetic but superparamagnetic, meaning they are only magnetic in a magnetic field which permits the beads to be redispersed without magnetic aggregate formation which prevents clumping and allows for easy dispersion of the beads (Mikhaylova et al., 2004; Chiang et al., 2005). Compared to magnetic microbeads (1–5 ␮m in diameter), magnetic nanobeads (≤30 nm in diameter) promise high-performance in biological separations because of their large surface/volume ratios, faster movement, and high target molecule binding rate (Sun et al., 2000; Josephson et al., 2002; Tripp et al., 2002; Gu et al., 2003; Yavuz et al., 2006). In this study, we demonstrated a magnetic nanobeads amplification based QCM immunosensor as a new approach for the detection of AI H5N1 virus. Magnetic nanobeads with a diameter of 30 nm were employed as “mass enhancer” to amplify target signal. The immunosensor was fabricated using 16-mercaptohexadecanoic acid (MHDA) to modify the crystal surface followed by immobilization of anti-H5 antibodies onto MHDA with N-hydroxysuccinimide (NHS) ester as a reactive intermediate. Both H5N1 virus binding onto the crystal surface and magnetic nanobeads binding onto the target virus were further confirmed by environmental scanning electron microscopy (ESEM). The specificity of the QCM immunosensor was determined using non-target subtypes such as AI subtypes H3N2, H2N2, and H4N8. Tracheal swab samples from chickens were utilized to evaluate the QCM immunosensor.

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2. Materials and methods 2.1. Chemicals and biochemicals 16-Mercaptohexadecanoic acid, N-(3-dimethylaminopropyl)N -ethylcarbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), ethanolamine, and phosphate buffered saline (PBS, 0.01 M, pH 7.4) containing 0.154 M NaCl were supplied from Sigma–Aldrich (St. Louis, MO, USA). Deionized water (18.2 M cm) produced by a Millipore-Milli-Q system (Bedford, MA, USA) was used throughout. Bovine serum albumin (BSA) from Sigma–Aldrich (St. Louis, MO, USA) was prepared in PBS (5.0%, w/v) as a blocking solution. Sulfo-NHS-Biotin was purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). D-Biotin was ordered from Molecular Probes (Eugene, OR, USA). 2.2. Instruments and electrodes A QCA922 quartz crystal analyzer (Princeton Applied Research, Oak Ridge, TN, USA) was used to monitor frequency change, F, in real time during antibody immobilization, AI virus binding, and magnetic nanobeads amplification. AT-cut quartz crystals (International Crystal Manufacturing, Oklahoma City, OK, USA), which had a diameter of 13.7 mm, a polished Au electrode (5.1 mm diameter, 1000 A˚ thickness) deposited on each side, and a resonant frequency of 7.995 MHz, was employed to develop the immunosensor. A flow cell from International Crystal Manufacturing (Oklahoma City, OK, USA) was used for fixing the QCM crystals. The cell consists of an upper and lower piece held together with two screws. The sensor was sealed between two O-rings in the upper and lower pieces. The ESEM tests were performed using a PHILIPS XL 30 SEM (FEI Company, OR, USA). 2.3. 30 nm magnetic nanobeads The magnetic nanobeads with a diameter of 30 nm were supplied from Ocean NanoTech LLC (Springdale, AR, USA), which is a group of water soluble iron oxide nanocrystals with amphiphilic polymer coating. Their surface functional group was streptavidin. Their organic layers consisted of a monolayer of oleic acid and a monolayer of amphiphilic polymer. The thickness of the total organic layers is approximately 4 nm. The hydrodynamic size of the nanocrystals is about 8–10 nm larger than their inorganic core size measured by TEM. The streptavidin molecules are linked to polymers. The density of the magnetic nanobeads is 5.2 g/cm3 . 2.4. Production of polyclonal anti-H5 antibody Two New Zealand White Rabbits were immunized subcutaneously at 3-week intervals with 50 ␮g of recombinant influenza hemagglutinin (rHA) protein of the strain A/Vietnam/1203/2004, subtype H5N1 (Protein Sciences, Meriden, CT). The first injection was administered in Complete Freund’s Adjuvant, followed by Incomplete Freund’s Adjuvant for all subsequent injections. One week after each immunization, blood samples were collected from the central ear artery and were assayed for anti-H5 response by ELISA. Briefly, an ELISA plate (Corning, NY) was coated overnight at 4 ◦ C with 2 ␮g/ml of rHA protein. The plate was then washed with PBST (0.02 M phosphate, 0.9% NaCl, and 0.05% Tween-20, pH 7.4), serum samples were added and incubated for 4 h at room temperature. The plate was then washed and incubated with a 1:3000 dilution of peroxidase-conjugated goat anti-rabbit IgG (Jackson Immunoreserach Laboratories, West Grove, PA) for 1 h at room temperature. Detection of bound antibody was completed by

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addition of BD OptEIATM TMB substrate (BD Biosciences, San Diego, CA). The reaction was stopped by the addition of 10% sulfuric acid, and absorbances were read at 450 nm in a Wallac plate reader (PerkinElmer, Boston, MA). Rabbit anti-H5 was purified from the final bleed of the best responding rabbit by ammonium sulfate precipitation. Briefly, serum lipids were removed by treatment of the serum sample with CleanasciteTM (Biotech Support Group, North Brunswick, NJ), overnight at 4 ◦ C. The preparation was then centrifuged at 1000 × g for 15 min, and immunoglobulins in the supernatant were precipitated by gradual addition of ammonium sulfate to 45% saturation. The IgG fraction was collected by centrifugation at 5000 × g for 20 min, dissolved in PBS, and finally dialyzed against PBS and sterile-filtered. Antibody concentration was calculated by absorbance measurement at 280 nm. 2.5. Pure virus preparation Killed avian influenza A/H5N1 virus (Scotland 59) was provided by USDA-APHIS National Veterinary Services Laboratory (NVSL, Ames, IA, USA). The AI H5N1 viruses were inactivated with ␤propiolactone similar to the procedure of Sever et al. (1964) where viral infectivity was eliminated while maintaining hemagglutination ability (Goldstein and Tauraso, 1970). The original titer of AI H5N1 virus was approximately 128 HA unit, and diluted to the desired titers with PBS for further use. 2.6. Beads modification with anti-H5 antibody 2.6.1. Biotin labeling of anti-H5 antibody Three microliters (3 (l) of freshly prepared Sulfo-NHS-Biotin water solution (10 mM) and 100 (l anti-H5 antibody PBS solution (1–5 mg/ml) were added into 200 (l PBS. Then, they were incubated for the reaction at room temperature for 60 min. The sample was injected into Slide-A-Lyzer Cassette (Thermo Scientific, Rockford, IL, USA) using a syringe (Thermo Scientific, Rockford, IL, USA) for dialysis after the incubation. Dialysis was conducted through immersing the cassette in 80 ml of PBS at room temperature. The PBS was changed after 2 h of dialysis and for another 2 h. The cassette was then immersed in new PBS overnight. The sample was removed by a syringe after totally dialyzing. The biotin-labeled antibody was further diluted 1:2 with PBS and stored at 4 ◦ C. 2.6.2. Immobilization of anti-H5 antibody onto the surface of magnetic nanobeads Anti-H5 antibody was immobilized onto the surface of magnetic nanobeads through streptavidin–biotin affinity binding. 75 ␮l of 30 nm streptavidin-coated magnetic nanobeads (0.5 mg/ml) and 70 ␮l of biotin conjugated antibody were added to 100 ␮l of PBS. The mixture was rotated for 50 min at 15 rpm. D-biotin (0.5 mg/ml) and BSA (5% in PBS) were then added into the mixture serially and rotated 30 min at 15 rpm to block non-specific binding sites on the surface of the magnetic nanobeads. After completely blocking, a magnetic field (1.2 T) was applied for 1 h to separate the antibody–nanobeads complex from the background. Finally, the complex was resuspended in 200 ␮l of PBS. The antibody modified magnetic nanobeads were stored at 4 ◦ C if they were not used immediately.

2.7.1. Pretreatment of crystals The crystals were immersed in 1 M NaOH for 5 min and 1 M HCl for 2 min in sequence. And then, freshly prepared piranha etch solution (1:3 (30% v/v) H2 O2 –H2 SO4 ) was dropped on the gold surface for 2 min, with special care to avoid the contamination of the electrode leads. The crystals were rinsed with deionized water successively and dried in a stream of nitrogen after each pretreatment. After above cleaning procedure, the crystal was ready for surface modification and antibody immobilization. 2.7.2. Fabrication of the immunosensor The pretreated crystals were immersed in 10 mM MHDA ethanol solution for overnight at room temperature to form an SAM via the strong Au–thiolate bond with the tail carboxylic group exposed at the monolayer–liquid interface. The crystals were then rinsed by spraying ethanol and water successively, and dried in a stream of nitrogen. The MHDA modified crystal was mounted in a 70 ␮l acrylic flow cell with only one side exposed to the solution. First, deionized water was injected into the flow cell to get a baseline. And then, EDC/NHS (75 mM/15 mM, v/v, 1:1) solution was injected into the sample chamber for 10–20 min to convert the terminal carboxylic group to an active NHS ester. After EDC/NHS reaction with the crystal surface, deionized water and PBS were injected into the flow cell in sequence. And anti-H5 antibodies (0.75 mg/ml, 200 ␮l) in PBS was introduced into the chamber immediately after the injection of PBS without waiting for baseline and incubated for 1 h. After washing with PBS, ethanolamine (pH 8.5, 1 M water solution) was injected into the flow cell to block nonspecific binding sites. After 5 min blocking, PBS was introduced to wash the immunosensor surface successively to get the baseline. Finally, the immunosensor was ready for AI virus binding. 2.7.3. Detection of AI virus AI virus binding was performed by injecting 300 ␮l of killed AI H5N1 virus into the flow cell fixed with anti-H5 antibody coated crystal, and incubating it at room temperature for 1 h. After incubation, the crystal was rinsed with PBS to obtain a baseline. The frequency change caused by AI virus binding was calculated by measuring the difference before and after adding the target virus. 2.7.4. Amplification of signals by magnetic nanobeads For further amplifying the binding reaction between the antibody and antigen (virus), 200 ␮l of anti-H5 antibody coated magnetic nanobeads was injected into the flow cell mounted with the target virus bound crystal, and then incubated for 1 h at room temperature. Finally, PBS was injected to wash the crystal successively to get the final baseline. The frequency shift was measured before and after injecting nanobeads as the amplified signal for detection of target virus in a sample. 2.8. Specificity and swab sample tests

2.7. Procedure for detection of AI virus

2.8.1. Non-specific subtypes Killed AIV subtypes H3N2, H2N2, and H4N8 provided from Animal Diagnostic Laboratory, Penn State University were used for specificity tests of the QCM immunosensor in the detection of AI H5N1 virus. The original titers AIV subtype H3N2, H2N2, and H4N8 were 64, 128, and 32 HA unit, respectively. The three subtypes were all diluted with PBS to 1.28 HA unit for further use. Each subtype was tested with 3 replicates. The change in frequency and resistance caused by three subtypes were compared with that from the H5N1 at the same titer, 1.28 HA unit.

The whole experimental procedure contains four steps: crystal pretreatment, antibody immobilization, AI virus binding, and nanobeads amplification.

2.8.2. Swab sample preparation Two tracheal swabs were taken from two healthy chickens and then placed in a tube containing 3 ml of Viral Transport

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Media (VTM). The VTM included 500 ml of Minimum Essential Media (MEM), 7.5 ml of Hepes Buffer (1 M), 10 ml of Gentamycin (10 mg/ml), 2.5 ml of Kanamycin (10 mg/ml), 5 ml of Antibiotic/Antimycotic (Pen-Strep-Amp), and 5 ml of Horse Serum (heat inactivated at 56 ◦ C for 30 min). First, two swabs were mixed with VTM solution sufficiently using a votex mixer. After mixing, each swab was pressed against the tube wall several times to squeeze out the solution and then was discarded. Four tubes of tracheal swab solutions were mixed together to prepare a uniform tracheal swab solution from eight swab samples for further tests. Finally, the solution was filtered using a syringe filter (0.45 ␮m) and spiked with AI H5N1 virus for further use. The original AI H5N1 virus titers were diluted to 12.8, 1.28, and 0.128 HA unit using the swab solution. Swab solution without spiking was used as a control. Each titer was repeated 3 times in the tests.

3. Results and discussion 3.1. Preparation and characterization of the QCM immunosensor Fig. 1A is a schematic of the stepwise fabrication of the QCM immunosensor, target virus binding, and signal amplification in our system. The QCM immunosensor was developed through an oriented monolayer of MHDA. MHDA was directly chemically adsorbed onto the gold surface of the AT-cut quartz crystals via the strong Au–thiolate bond with the tail carboxylic group exposed at the monolayer–liquid interface (Bain et al., 1989). The second step is the activation of the monolayer, which involves a stepwise formation and replacement of terminal EDC and NHS in sequence to form an NHS ester. Then, polyclonal anti-H5 antibodies were immobilized on the sensor surface through the amide bond due to replacement of the active NHS ester by the primary amines of the antibody. As a result, MHDA mediated antibodies immobilization could lead to a highly efficient immunoreaction. Then, ethanolamine was applied to block the residual reacting sites and reduce non-specific adsorptions. Finally, in detection of the target H5N1 viruses, a specific binding event occurs between the immobilized antibodies and the antigens on the virus surface, resulting in a frequency change. The change in the frequency caused by AI samples was correlated to the concentration or different titer of H5N1 AI virus present in the solution. Magnetic nanobeads modified with anti-H5 antibodies were used as biolabels for further specifically amplifying the binding reaction of the antibody–antigen (virus). A typical QCM sensorgram of antigen detection in our system is shown in Fig. 1B. To eliminate the background interference, the frequency change between every two neighboring baselines of PBS buffer was calculated as the net response. The immobilization of anti-H5 antibodies caused about 220 Hz drop of frequency in 1 h, which confirmed the successful immobilization of the probe. As shown in Fig. 1B, upon the introduction of ethanolamine solution, a drastic frequency drop was observed partially due to the significant change of properties (density, viscosity, etc.) of the liquid contacting with quartz crystal. After blocking the residual reacting sites using ethanolamine, the frequency was increased while ethanolamine solution was replaced with PBS and stabilized shortly after. The frequency shifts less than 5 Hz before and after ethanolamine blocking suggested the adsorption of ethanolamine to the gold surface. After anti-H5 antibodies immobilization and ethanolamine blocking, the target H5N1 viruses were firstly introduced into the sample room to specifically bind with the antibodies. As shown in Fig. 1B, the frequency change caused by the positive H5N1 viruses (12.8 HA unit) was huge, which indicated the affinity immunoreaction between anti-H5 antibodies and H5N1 virus. Furthermore, the signal was significantly amplified by the following nanobeads amplification step. The frequency decreased over time

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while the nanobeads were conjugated to AI H5N1 captured target virus. 3.2. Preparation protocols for modification of magnetic nanobeads with anti-H5 antibodies In order to maximize the nanobeads amplification signal and minimize the non-specific noise, five different protocols were studied for the preparation of antibody coated nanobeads. Pure bare nanobeads without any modification were also tested as a reference. Fig. 2 shows the nonspecific response of the antibody-coated crystal to the magnetic nanobeads prepared with different protocols. The tests of pure bare nanobeads indicated that the bare nanobeads without streptavidin coating could result in a huge nonspecific response of the antibody-coated crystal. It was found that surface charges and electrostatic interactions play a dominant role in determining the magnetic nanoparticles binding to a substrate (Palma et al., 2007). The bare magnetic nanobeads tested in this study is a group of water soluble iron oxide nanocrystals with positively charged surface (Catalog Number: SMG30, Ocean NanoTech LLC, Springdale, AR, USA). When the magnetic nanobeads are positively charged, it is electrostatically attracted to the negatively charged substrates. Even after PBS washing steps, signals remain constant, indicating that the magnetic nanobeads were strongly and nonspecifically adsorbed to the substrate. Antibody coated nanobeads without blocking (Protocol 2) resulted in 50 Hz decrease in frequency, showing non-specific signal on the QCM immunosensor. Therefore, it is necessary to apply block reagents on the nanobeads. According to the test’s results of the pure bare nanobeads, BSA was utilized to block the bare surface of the streptavidin coated nanobeads. Due to streptavidin-coated nanoparticles could be strongly adsorbed onto the gold surface of the crystal after probe immobilization to cause false-positive signal (Mao et al., 2006), D-biotin was also employed to block the free streptavidin on the nanobeads surface. As shown in protocols 5 and 6, both D-biotin and BSA were used to block the nanobeads surface. Experimental results indicated that the nonspecific noise was negligible after using both D-biotin and BSA to block the nanobeads surface. Compromising the amplification signal and non-specific noise, protocol 6 was selected as the preparation method in the following tests. 3.3. Environmental scanning electron microscopy (ESEM) imaging Environmental scanning electron microscopy (ESEM) was used to further confirm the binding of target AI H5N1 virus with the antibody immobilized on the crystal surface and the magnetic nanobeads. Fig. 3A and B demonstrates both the binding of AI virus on the crystal surface and the binding of the magnetic nanobeads to the virus. Fig. 3C shows the nonspecific attachment of the magnetic nanobeads onto the crystal surface resulting in the noise as we discussed in Section 3.2. Comparing Fig. 3B with Fig. 3C, the size of bare nanobeads were smaller than the biomaterial modified beads, which is consistent with the information provided by the supplier. The results also demonstrated that the QCM immunosensor based on anti-H5 antibodies coated magnetic nanobeads amplification was capable of detecting AI H5N1 virus. 3.4. Detection of pure AI H5N1 virus Effective loading of active antibodies onto the crystal surface is critical for the sensitivity of the QCM immunosensor. The antibodies at different concentrations (0.5, 0.75, and 1.6 mg/ml) were investigated to optimize the performance of the immunosen-

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Fig. 1. (A) Schematic illustration of the QCM immunosensor fabrication, detection, and amplification procedure. (B) Typical sensorgram of the QCM immunosensor for target detection and signal amplification.

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Fig. 2. Frequency shifts caused by magnetic nanobeads modified with anti-H5 antibody with different preparation protocols. S.D., n = 3. Protocols 1–6 were defined as inset table, respectively.

sor. The corresponding changes of frequency were observed to be 166 ± 18, 228 ± 16, and 235 ± 21 Hz, respectively, showing the increase of frequency shift along with the increase of antibody’s concentration (the data is shown in Fig. S1 in the supplementary information). The relatively steady state was obtained when the concentration of antibodies was ≥0.75 mg/ml. Thus, 0.75 mg/ml was selected as the antibody’s concentration in the immunosensor considering the detection sensitivity and material cost. Killed AI H5N1 viruses with titers in the range of 0.00128–23.27 HA unit in PBS were tested using the magnetic nanobeads amplification based QCM immunosensor. The total detection time of this immunosensor was 2 h, including virus binding and nanobeads amplification. Frequency shift caused by both virus and nanobeads was used as the detection signal of this immunosensor. The results are shown in Fig. 4. Triplicate tests were done for each measured titer, and the standard deviations (S.D.) are shown as error bars in the figure. A linear relationship was found between the frequency shift and the log number of the H5N1 titers ranging from 0.128 HA unit to 12.8 HA unit, and a corresponding equation was described as: f = −37.67 log N − 44.295 (R2 = 0.99). Table 1 shows the detail frequency shifts of the QCM immunosensor caused by different titers of AI H5N1 virus and corresponding amplification signal of 30 nm magnetic nanobeads. The

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Fig. 4. Frequency shifts of the QCM immunosensor as a function of the titer (HA unit) of AI H5N1 virus in PBS suspension. The detection limit is determined as 0.0128 HA unit. A linear correlation between the frequency change and logarithmic number of virus titers was found from 0.128 to 12.8 HA unit. Error bars indicate the standard deviation (n = 3).

threshold for the positive detection was set as background (blank) signal + 3 × noise (standard deviation) (Mao et al., 2006) and the detection limit of the magnetic nanobeads based QCM immunosensor was determined as 0.0128 HA unit. Using the same calculation method, the detection limit of the QCM immunosensor was 1.28 HA unit for H5N1 detection without amplification. The data indicated that the magnetic nanobeads amplification resulted in an improved detection limit of 0.0128 HA for the QCM immunosensor, which is two orders lower than the QCM immunosensor without nanobeads amplification. The results indicate that the magnetic nanobeads amplification based QCM immunosensor was more sensitive than ELISA for AI H5N1 detection (Ho et al., 2009). The estimated detection limit of the antigen capture enzyme-linked immunosorbent assay (AC-ELISA) using H5- and N1-specific monoclonal antibodies was 1–2 HA titers, which showed that the QCM immunosensor developed with polyclonal anti-H5 antibodies was comparable with the ELISA technology in terms of sensitivity for H5N1 detection even without nanobeads for signal amplification. The result demonstrated that the magnetic nanobeads amplification is a good method to improve the sensitivity of the QCM immunosensor for H5N1 virus detection.

Fig. 3. SEM images of the top view of QCM gold surface. (A) The binding of a H5N1 virus; (B) the binding of a 30 nm magnetic nanobead to a H5N1 virus; (C) the non-specific binding of three 30 nm magnetic nanobeads.

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Table 1 Frequency shifts of the QCM immunosensor caused by different titers of AI H5N1 virus and corresponding amplification signal of 30 nm magnetic nanobeads. S.D., n = 3. Titers of AIV H5N1 (HA unit)

F (Hz) ± S.D. (virus)

F (Hz) ± S.D. (nanobeads)

F(nanobeads) F(virus)

rt

Total frequency shifts (Hz)

23.27 12.8 1.28 0.128 0.0128 0.00128 PBS

−104 ± 17 −58 ± 21 −37 ± 16 −6 ± 5 −2 ± 1 −2 ± 5 0±1

−37 ± 13 −28 ± 5 −10 ± 2 −5 ± 3 −9 ± 2 −7 ± 8 −4 ± 2

35.6% 48.6% 27.0% 88.8% 386.3% 419.2% /

2.8 2.06 3.7 1.13 0.26 0.24 /

−141 ± 28 −87 ± 26 −47 ± 14 −11 ± 5 −11 ± 2 −9 ± 3 −3 ± 2

3.5. Amplification of target signal through magnetic nanobeads It can be seen from Table 1 that the magnetic nanobeads amplification signal was lower than the virus binding signal at titers equal to or higher than 0.128 HA unit. When the titers were lower than 0.128 HA unit, the nanobeads amplification signal was much larger than the virus binding signal. To explain this phenomenon, a theoretical calculation was done as shown below:

H5N1 virus Nanobeads

Diameter (nm)

Density (g/cm3 )

Numbers of particles resulted in 10 Hz shift

80 30

1.185 (Reimer et al., 1966) 5.2

4.41 × 107 1.91 × 108

Note: 1.4 ng was considered as the mass resulting in 1 Hz of frequency shift.

The result indicated that the virus and magnetic nanobeads could cause the same frequency change when there were average 4.32 particles of magnetic nanobeads bound onto each virus. Assumed that there was only one magnetic nanobead bound onto each virus on average, the same amount (4.41 × 107 particles) of nanobeads just could result in 2.31 Hz of frequency shift. Therefore, an equation for calculating the ratio between the frequency shifts caused by virus and nanobeads was obtained: r=

10 2.31 × a

where a is the average number of the magnetic nanobeads particles bound onto each virus. Using this equation, the r was calculated with corresponding a: a

1

2

3

4

...

rc

4.33

2.16

1.44

1.08

...

16 0.27

17 0.25

18

showed that the magnetic nanobeads (diameter, 30 nm) amplified signal much better at lower titers, which made the immunosensor very suitable for applications to early screening of H5N1 virus. 3.6. Specificity, stability, and reusability of the sensor Specificity. Different subtypes of AI virus were assayed and the assay was specific for H5N (Figs. S2 and S3 in supplementary information). As shown in Fig. S4 (the data is shown in supplementary information), the signal resulting from those non-target subtypes (H3N2, H2N2 and H4N8) were −6, −8, −2 Hz, respectively. The results indicated that the detection of AI H5N1 virus at 1.28 HA unit titer could be distinguished from the blank buffers and other subtypes solutions (Fig. S4 in supplementary information). The specificity of the QCM immunosensor is mainly dependent upon the antibodies immobilized on the crystal surface. The result also demonstrated that the polyclonal anti-H5 antibody developed in this study worked well in the specific detection of AI H5N1 virus. Stability and reusability. Stability between different crystals were studied through comparing frequency shifts caused by anti-H5 antibody with the same concentration (0.75 mg/ml). The stability between different antibody modified crystals showed a RSD of 0.05%, which indicates satisfied stability between different crystals. For the same crystal, regeneration solution of NaOH (10 mM) was applied for 10 min, however, about 20% loss of activity was observed. Therefore, different titers of H5N1 virus were tested using new crystal respectively in this study. 3.7. Detection of tracheal swab samples spiked with AI H5N1 virus

0.24

Comparing the ratio rt of tests (Table 1) with the calculation results for rc , it can be concluded that there were one or two magnetic nanobeads bound to each target virus on average while the titer was 23.27 HA unit. The conclusion is consistent with the result of ESEM tests (Fig. 3B) and the sensorgram (Fig. 1B). The binding number of magnetic nanobeads increased gradually along with the decreasing target titers from 23.27 to 0.128 HA unit. The ratio of 0.26 and 0.24 meant that there were more than 16 magnetic nanobeads particles bound onto each virus at the titer of 0.0128 and 0.00128 HA unit on average. The comparison between rt and rc indicated that the magnetic nanobeads amplified the target virus signal much better at low titers than high titers. Based on the calculation of the percentage of bound magnetic nanobeads, there was less than 1% of nanobeads bound onto the virus and more than 99% nanobeads were still free in the system, indicating that the total number of magnetic nanobeads applied to the biosensor system was sufficient to bind the H5N1 virus. There is no need to add more nanobeads for signal amplification. However, the observed phenomenon indicated that the signal was more amplified at lower virus titer but not at high virus titer, it was possibly due to the steric hindrance for the virus at high titer limited the binding sites available for nanobeads, and resulting in less number of nanobeads bind to each target virus. The analytical results

The experimental spiked tracheal swab samples were used to test the performance of the immunosensor for the detection of viral

Fig. 5. Frequency shifts caused by negative controls and tracheal swab samples spiked with AI H5N1 virus ranging from 0.128 to 12.8 HA unit. A linear correlation between logarithmic value of AI H5N1 virus titers and the frequency shifts was found from 0.128 to 12.8 HA unit. Error bars = S.D. (n = 3).

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antigens in complex solution (Fig. 5). As shown in Fig. 5, the titer of 0.128 HA was a suspense sample (between positive and negative), so the QCM immunosensor could detect tracheal swab samples higher than 0.128 HA unit when the frequency shift of pure tracheal swab was taken as the control background. A linear relationship between the frequency shift and logarithmic value of AI H5N1 virus concentration was found in a range of 0.128–12.8 HA unit, and the correlation coefficient is 0.87. The results showed that the magnetic nanobeads based QCM immunosensor was able to detect AI H5N1 virus in tracheal swab samples. However, compared with the experimental results of the pure H5N1 virus detection (Fig. 3), the detection limit of the QCM immunosensor for tracheal swab samples increased from 0.0128 to 0.128 HA unit. This is probably due to the interference/noise of larger components presented in the tracheal swab samples interfered with the binding of target virus to the sensor surface. 4. Conclusions In this study, a magnetic nanobeads amplification based QCM immunosensor was developed as a new approach for rapid, sensitive, and specific detection of AI H5N1 virus in swab samples. The use of nanobeads effectively amplified the signals in frequency shifts, and improved the detection limit by 2 orders for pure virus (0.0128 HA unit), in comparison with the QCM immunosensors without nanobeads amplification. The magnetic nanobeads amplified the target signal much better at low titers than high titers, which made the immunosensor very promising for more sensitive detection of H5N1 virus. A linear quantitative relationship was found between the measured signal and the titers of AI H5N1 virus in the range of 0.128–12.8 HA unit, which enabled the sensor to enumerate the target virus. Both the binding of target AI H5N1 virus onto the antibody-modified crystal surface and magnetic nanobeads onto the H5N1 virus were further confirmed by environmental scanning electron microscopy. Magnetic nanobeads further amplified the binding reaction of the antibody–antigen (virus) due to its specific and strong binding receptors for AI H5N1 virus. The specificity of the nanobeads amplification based QCM immunosensor was tested at AI virus subtype H5 for the first time. The immunosensor could distinguish AI H5N1 from AI subtypes H3N2, H2N2, and H4N8. The detection limit of the QCM immunosensor for tracheal swab samples was 0.128 HA unit. Although preparation of antibody immobilized crystals requires many steps and takes too much time, chemicals and labors, the detection could be completed within 2 h, which was shorter or comparable with other methods (Storch, 2000; He et al., 2007). The results indicated that the QCM immunosensor has a great potential to be an alternative method for the detection of AI virus H5N1 in agricultural, environmental, and clinical samples. However, the optimization of amplification conditions needs to be conducted in the future research. Reproducibility and regeneration of the immunosensor also need to be further studied for the sensor’s improvement performance in real practical applications. Acknowledgements This research was supported in part by China Scholarship Council (CSC) and the Center of Excellence for Poultry Science at University of Arkansas. The authors thank Dr. Andrew Wang of Ocean Nanotech. LLC for providing magnetic nanobeads. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2011.04.010.

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