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Protein functionalized ZnO thin film bulk acoustic resonator as an odorant biosensor
Sensors and Actuators B 163 (2012) 242–246
Contents lists available at SciVerse ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Protein functionalized ZnO thin film bulk acoustic resonator as an odorant biosensor Xiubo Zhao a,b,∗ , Gregory M. Ashley c , Luis Garcia-Gancedo d , Hao Jin e , Jikui Luo c,e , Andrew J. Flewitt d , Jian R. Lu a,∗∗ a
Biological Physics Group, School of Physics & Astronomy, University of Manchester, Oxford Road, Manchester M13 9PL, UK Department of Chemical and Biological Engineering, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield S1 3JD, UK c IMRI, University of Bolton, Deane Road, Bolton BL3 5AB, UK d Electrical Engineering Division, University of Cambridge, JJ Thomson Avenue, Cambridge CB3 0FA, UK e Department of Information Science & Electronic Engineering, Zhejiang University, 38 Zheda Road, Hangzhou 310027, PR China b
a r t i c l e
i n f o
Article history: Received 8 November 2011 Received in revised form 12 January 2012 Accepted 13 January 2012 Available online 25 January 2012 Keywords: FBAR Biosensor Odorant binding protein DEET Gas sensor
a b s t r a c t ZnO thin film bulk acoustic resonators (FBARs) with resonant frequency of ∼1.5 GHz have been fabricated to function as an odorant biosensor. Physical adsorption of an odorant binding protein (AaegOBP22 from Aedes aegypti) resulted in frequency down shift. N,N-diethyl-meta-toluamide (DEET) has been selected as a ligand to the odorant binding protein (OBP). Alternate exposure of the bare FBARs to nitrogen flow with and without DEET vapor did not cause any noticeable frequency change. However, frequency drop was detected when exposing the OBP loaded FBAR sensors to the nitrogen flow containing DEET vapor against nitrogen flow alone (control) and the extent of frequency shift was proportional to the amount of the protein immobilized on the FBAR surface, indicating a linear response to DEET binding. These findings demonstrate the potential of binding protein functionalized FBARs as odorant biosensors. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Biosensors are important tools for medical diagnostics and studying biomolecular interactions in biology and biotechnology [1]. Sensitive, label-free and disposable biosensors are particularly promising [2]. Surface acoustic wave (SAW) and film bulk acoustic resonator (FBAR) sensors have been developed because of the much higher resonant frequency (hence greater sensitivity), lower cost, and smaller size than quartz crystal microbalance (QCM) sensors [3]. Over the past few years, FBAR sensors have been used as mass sensors for many biological applications. For example, Lee et al. [3] reported the use of AlN FBAR sensors for the detection of carcinoembryonic antigens which have been widely used as tumor markers. Common to all these applications is the shift of resonant frequency with the deposition of protein and subsequent
∗ Corresponding author at: Department of Chemical and Biological Engineering, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield S1 3JD, UK. ∗∗ Corresponding author at: Biological Physics Group, School of Physics & Astronomy, University of Manchester, Oxford Road, Manchester M13 9PL, UK. Tel.: +44 161 306 3926. E-mail addresses: [email protected] (X. Zhao), [email protected] (J.R. Lu). 0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2012.01.046
antigen binding onto the biosensor surface. FBAR sensors have also been used for the label-free detection of DNA [2,4]. Zhang et al. [5] demonstrated that FBAR can detect single base pair mismatch of the DNA hybridization. Auer et al. [6] recently reported that FBAR have the capability to detect 1 nM oligonucleotide dissolved in 1% serum. Xu et al. [7,8] have used FBAR sensors for the detection of aptamer–thrombin binding pair and for the real-time in situ monitoring of the competitive adsorption/exchange of proteins. In combination with a competitive antibody–antigen association process, it has been demonstrated that FBARs have the capability of detecting small drug molecules such as cocaine (molecular weight 303 g/mol) and heroin (molecular weight 369 g/mol) [9]. Meanwhile, FBAR has also been explored as gas sensors. Penza et al. [10] reported that AlN/Si3 N4 thin film FBARs functionalized with a layer of single-walled carbon nanotubes have the capability of detecting organic vapors of acetone, ethylacetate, and toluene with high sensitivity, fast response and good repeatability. In this paper, we describe the use of FBAR as an odorant sensor for the detection of mosquitoes repellent by coating the odorant binding protein onto the top electrode of the sensors. DEET (N,N-diethyl-meta-toluamide) is the most common active ingredient in insect repellents and is highly effective against mosquitoes [11–13]. Therefore, it has been selected as a ligand to the odorant binding protein (OBP, AaegOBP22) originated from Aedes aegypti
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mosquitoes. It was found that bare FBARs do not respond to DEET while a resonant frequency shift was observed when exposing the OBP coated FBARs to DEET. This observation indicates that FBAR sensors have the sensitivity in detecting molecular interaction between proteins and small odorant molecules (the molecular weight of DEET is 191 g/mol) and has the potential to be explored as odorant sensors. 2. Materials and methods 2.1. Materials The odorant binding protein (AaegOBP22 from A. aegypti) was provided by Prof. Shuguang Zhang from Centre for Biomedical Engineering, MIT. The expression, purification and functional analysis have been described previously [14]. In brief, AaegOBP22 was expressed by heterologous Escherichia coli extracellular secretion. Protein purification was achieved by Strep-Tactin affinity binding and size-exclusion chromatography. Analysis by SDS–PAGE and mass spectrum confirmed protein purity and molecular weight. CD spectra showed that AaegOBP22 underwent a pH dependent conformational change of the secondary structure. After purification, the protein functions of AaegOBP22 were tested by fluorescent probe 1-NPN binding assays. The results showed AaegOBP22 proteins have characteristics of selective binding with various ligands. N,N-diethyl-meta-toluamide (DEET) was purchased from Sigma–Aldrich. Ultra high quality (UHQ) water with purity to 18.2 M cm was produced from PURELAB® ultra laboratory water purification system.
Fig. 1. (A) Schematic cross-sectional view of the FBAR device used in the experiment (not to scale). The active sensing area is where the top and bottom electrodes overlap. (B) Top view of one of the devices fabricated.
2.2. FBAR sensor fabrication and characterization FBAR devices were fabricated on double-side-polished 4 in. silicon wafers covered with 2 m of thermally grown SiO2 (PI-KEM Ltd., Staffordshire, UK). 5 nm Cr adhesion layer and 60 nm of Au were evaporated on top of the SiO2 surface as the bottom electrode using a thermal evaporator and a standard lift-off photolithography process. The ZnO thin film was then deposited with a thickness between 1.5 and 2.5 m. The film was wet etched in a 2% glacial acetic acid and phosphoric acid solution to form the Via etch holes ZnO for electrical connection. The top electrode was then patterned with a second lift-off photolithography process. Finally the SiO2 on the back of the wafer was patterned and removed with CF4 plasma to expose the Si, which was subsequently removed with a deep reactive ion etching (DRIE) process to release the membrane. The SiO2 suspension layer serves as a barrier, preventing the ZnO from being etched away with the DRIE process. Fig. 1A shows the schematic cross sectional view (not to scale) of FBAR devices and Fig. 1B shows the image of one of the FBAR devices fabricated. The yield of the devices fabricated is higher than 95%. The devices were then wired onto 50 transmission line PCBs using Al wires. Before characterization the devices were ozone cleaned for at least 30 min followed by UHQ water rinsing and N2 drying. Electrical characterization of the FBAR devices was then carried out using an Agilent E5062A network analyzer controlled by software written with LabVIEW to continuously monitor and record the resonance spectrum of the FBAR. A typical response at around 1.56 GHz is shown in Fig. 2 as an example. The sharp resonance of the FBAR indicates a very high quality factor (Q). The quality factor (Q) of the FBAR was determined by measuring the transmission signal (S21 ) and 3 dB bandwidth [15–18] using Eq. (1) Q−3 dB =
f0 f−3 dB
Fig. 2. A typical frequency response (S21 parameter) of the fabricated device. The sharp resonance of the ZnO indicates a very high quality factor.
3 dB Q of the FBARs was found typically around 800. The reduction of the Q factor was found after protein loading. For example, the Q factor of one device reduced from 883 (bare FBAR) to 798 after protein loading. 2.3. Protein loading onto FBAR sensor OBP was loaded onto clean FBAR sensor surface by physical adsorption. A drop of 20 l protein solution (0.35 mg/ml) in 1× PBS (phosphate buffered saline) was placed on top of the active area of the FBAR sensor. Adsorption was allowed from 5 to 15 min followed by UHQ water rinse and N2 drying to achieve different amount of protein adsorbed on the surface. Longer adsorption time led to greater amount of adsorption. 2.4. Measurement setup
(1)
where f0 is the resonant frequency and f−3 dB is the bandwidth at 3 dB, both extracted from the transmission signal spectrum. The
The schematic diagram of the experimental setup of the FBAR sensor for odorant measurement is shown in Fig. 3. The dry N2 supply passed through a flow controller to a three-way tap that
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Fig. 3. Schematic diagram of the FBAR sensor experimental setup.
leads to two identical 100 ml bottles. The N2 from either of the bottles then comes to another three-way tap and goes into and out of the gold coated brass isolation box which hosts the FBAR sensor on the PCB board linked to the network analyzer through two transmission cables. The signal from the network analyzer is then collected by the computer using the GPIB channel. The isolation box is placed on an anti-vibration table and the temperature of the isolation box is controlled by a Haake (K20) bath and kept constant with a variation of no more than ±0.5 ◦ C. Control experiment is carried out to monitor the frequency change of the bare FBAR when switching between the pure N2 flow and N2 flow containing DEET vapor to see the response of the bare FBAR sensor to the DEET vapor. Control experiment is also carried out to monitor the frequency change of the OBP loaded FBAR when switching the N2 flow between two identical empty bottles to see the frequency changes caused by switching the three-way taps. Finally, the frequency changes of the OBP loaded FBAR sensor are recorded when switching between the pure N2 flow and N2 flow containing DEET vapor to see the responses of the OBP to the ligand DEET.
Fig. 4. Response of a bare FBAR sensor to N2 gas with and without DEET vapor.
3. Results and discussion Before coating the FBAR sensor for odorant detection, a control experiment was carried out to investigate the frequency response of the bare FBAR to the DEET. Fig. 4 records the frequency change of a clean bare FBAR against the time when switching between the N2 flow without and with DEET vapor. Apart from the resonant frequency drifting down at around 5 kHz/2000 s (it was found that some FBAR devices might have resonant frequency drifts either up or down a bit), switching resulted in sparks due to the pressure changes during the switching process. No noticeable frequency shift was detected with and without the presence of DEET in N2 . This control experiment confirms that the bare FBAR is not sensitive to DEET. To investigate the response of the OBP coated FBAR to N2 flow, experiment was carried out by switching the N2 gas between two identical empty bottles (Fig. 5). Similarly, no frequency shift was detected apart from the sparks during the switch and a natural resonant frequency drifting down at around 5 kHz/2000 s. This experiment demonstrates that OBP coated FBAR did not respond to N2 gas and that the two N2 pathways did not have any contribution to the frequency change.
Fig. 5. Responses of an OBP loaded FBAR sensor to the two N2 pathways, showing that the spikes were caused by pressure jumps associated with switching.
The OBP coated FBAR was then exposed alternatively to N2 flow containing DEET vapor. Fig. 6 compares the responses of the OBP loaded FBAR sensors to N2 gas with and without DEET vapor. Protein immobilization on the FBAR used for Fig. 6A and B studies
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0.1% DEET in pure water for 4 cycles. The adsorption of the protein resulted in an adsorbed amount of 0.6 mg/m2 . Water washed off some loosely bound proteins and reduced the amount to 0.5 mg/m2 . Replacing water with the 0.1% DEET solution brought the adsorbed amount to the starting value, confirming the binding of DEET to the OBP. In contrast, replacing with water again reduced the adsorbed amount by some 0.1 mg/m2 due to the reversible binding. This process was repeated for 4 times and the data indicated that the process was reproducible and consistent with the findings from the FBAR results. 4. Conclusions In this study, we have fabricated ZnO film bulk acoustic resonators with sensitive resonant frequency shifts upon surface mass loading and successfully used them as odorant biosensors. These FBARs, based on thickness longitudinal mode (TLM) using thin ZnO layer, have their working resonant frequencies around 1.5 GHz and Q factors around 800. The results showed that the bare FBARs do not respond to DEET vapor while the odorant binding protein coated FBAR showed frequency shifts down when exposed to DEET vapor. In addition, the binding of DEET to the odorant binding protein was largely reversible: the resonant frequency shifts back to its initial value when switching to pure N2 flow. The frequency response is also related to the amount of protein on the FBAR surface: higher amount protein resulted in greater frequency shift due to a larger mass load, consistent with the loading of more protein on to the surface providing more binding sites for DEET molecules. This work has thus demonstrated the huge potential of FBARs as odorant biosensors. Acknowledgments
Fig. 6. Responses of OBP loaded FBAR sensors to N2 gas with and without DEET vapor. (A) With a higher OBP loading, exposing the FBARs to DEET resulted in 8–10 kHz frequency drop and (B) with a lower OBP loading, exposing the FBARs to DEET resulted in 2–3 kHz frequency drop.
The authors wish to thank EPSRC for financial support under grants EP/F062966/1, EP/F063865/1 and EP/F06294X/1. The authors would also like to thank Professor Shuguang Zhang at MIT for providing the odorant binding protein. References
resulted in frequency drops of 9.7 MHz and 1.3 MHz, indicating different amounts of protein loaded. Distinct frequency drop was detected when exposed to the N2 gas containing DEET vapor. In the first three cycles as shown, exposure to the DEET vapor results in the shift down of 8–10 kHz in Fig. 6A while the frequency rises to the normal resonant frequency when switching back to N2 flow (a bump that came out at the second exposure was due to the gas leaking of the tubular connection). When a smaller amount of protein is loaded onto the FBAR surface, its response to DEET is much smaller as well (Fig. 6B), indicating that the FBAR response to DEET is related to the amount of the protein immobilized onto the FBAR surface. More protein on the surface provides more binding sites and can therefore accommodate more DEET molecules. Thus, the main observation from Fig. 6 is the almost reproducible cycling of DEET adsorption and desorption in each cycle, suggesting the robustness of the protein molecules immobilized and measurement setup. It was found however that if the adsorption/desorption is recycled 8–10 times, the gap between DEET adsorption and desorption in each cycle became narrower, showing possible structural damage of the protein in a dry gas condition. To support the FBAR results, ellipsometry measurements have been carried out (data not shown). Dilute OBP solution was preadsorbed onto hydrophobic modified silica surface followed by a general wash using pure water. Measurements were carried out by mentoring the mass changes when replacing the pure water with
[1] G. Wingqvist, AlN-based sputter-deposited shear mode thin film bulk acoustic resonator (FBAR) for biosensor applications—a review, Surface & Coatings Technology 205 (2010) 1279–1286. [2] M. Nirschl, A. Blüherc, C. Erler, B. Katzschnerc, I. Vikholm-Lundind, S. Auerd, J. Vörösb, W. Pompec, M. Schreitera, M. Mertig, Film bulk acoustic resonators for DNA and protein detection and investigation of in vitro bacterial S-layer formation, Sensors and Actuators A: Physical 156 (2009) 180–184. [3] T.Y. Lee, J.T. Song, Detection of carcinoembryonic antigen using AlN FBAR, Thin Solid Films 518 (2010) 6630–6633. [4] R. Gabl, H.-D. Feucht, H. Zeininger, G. Eckstein, M. Schreiter, R. Primig, D. Pitzer, W. Wersing, First results on label-free detection of DNA and protein molecules using a novel integrated sensor technology based on gravimetric detection principles, Biosensors and Bioelectronics 19 (2004) 615–620. [5] H. Zhang, M.S. Marma, S.K. Bahl, E.S. Kim, C.E. McKenna, Sequence specific labelfree DNA sensing using film-bulk-acoustic-resonators, IEEE Sensors Journal 7 (2007) 1587–1588. [6] S. Auer, M. Nirschl, M. Schreiter, I. Vikholm-Lundin, Detection of DNA hybridisation in a diluted serum matrix by surface plasmon resonance and film bulk acoustic resonators, Analytical and Bioanalytical Chemistry 400 (2011) 1387–1396. [7] W. Xu, S. Choi, J. Chae, A contour-mode film bulk acoustic resonator of high quality factor in a liquid environment for biosensing applications, Applied Physics Letters 96 (2010) 053703. [8] W. Xu, X. Zhang, S. Choi, J. Chae, A high-quality-factor film bulk acoustic resonator in liquid for biosensing applications, Journal of Microelectromechanical Systems 20 (2011) 213–220. [9] G. Wingqvist, J. Bjurströma, A.-C. Hellgren, I. Katardjiev, Immunosensor utilizing a shear mode thin film bulk acoustic sensor, Sensors and Actuators B: Chemical 127 (2007) 248–252. [10] M. Penza, P. Aversa, G. Cassano, D. Suriano, W. Wlodarski, M. Benetti, D. Cannatà, F.D. Pietrantonio, E. Verona, Thin-film bulk-acoustic-resonator gas sensor functionalized with a nanocomposite Langmuir–Blodgett layer of carbon nanotubes, IEEE Transactions on Electron Devices 55 (2008) 1237–1243.
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[11] Z. Syed, W.S. Leal, Mosquitoes smell and avoid the insect repellent DEET, Proceedings of the National Academy of Sciences of the United States of America 105 (2008) 13598–13603. [12] B.W. Bissinger, C.S. Apperson, D.E. Sonenshine, D.W. Watson, R.M. Roe, Efficacy of the new repellent BioUD® against three species of ixodid ticks, Experimental & Applied Acarology 48 (2009) 239–250. [13] G. Paluch, L. Bartholomay, J. Coats, Mosquito repellents: a review of chemical structure diversity and olfaction, Pest Management Science 66 (2010) 925–935. [14] G. Yang, G. Winberg, H. Ren, S. Zhang, Expression, purification and functional analysis of an odorant binding protein AaegOBP22 from Aedes aegypti, Protein Expression and Purification 75 (2011) 165–171. [15] K.M. Lakin, G.R. Kline, K.T. McCarron, High-Q microwave acoustic resonators and filters, IEEE Transactions on Microwave Theory and Techniques 41 (1993) 2139–2146. [16] A.D. Wathen, F. Munir, W.D. Hunt, A high-Q hybrid acoustic mode in thin film ZnO solidly mounted resonators, Applied Physics Letters 95 (2009) 123509. [17] J.K. Coakley, J.D. Splett, M.D. Janezic, R.F. Kaiser, Estimation of Q-factors and resonant frequencies, IEEE Transactions on Microwave Theory and Techniques 51 (2003) 862–868. [18] J.D. Larson III, S.M. PDB, S.M. SW, R.C. Ruby, Modified Butterworth–Van Dyke circuit for FBAR resonators and Automated measurement system, Proceedings of IEEE Ultrasonics Symposium 861 (2000) 863–868.
Biographies Xiubo Zhao received his Ph.D. degree in biological physics from the University of Manchester in 2006. He is currently a Lecturer in the Department of Chemical and Biological Engineering at the University of Sheffield, before which he was an EPSRC funded Postdoctoral Research Fellow at the University of Manchester. His research interests include biomarker immobilization and detection for biosensoring, peptide self-assembly, non-viral gene and drug delivery, interfacial adsorption of biomolecules, biomaterials and surface biocompatibility. Dr Zhao is a member of the Institute of Physics, UK. Gregory M. Ashley received his B.Sc. degree in microbiology from the University of Liverpool in 1996, then M.Sc. degree in Biosensors from the University of Manchester in 2000. He went on to obtain his Ph.D. in Biosensors in the University of Cranfield on RF MEMS based Biosensors. He is at present a Postdoctoral Research Fellow at the University of Bolton, working on FBAR based biosensors. Luis Garcia-Gancedo received a B.Sc. in physics from the University of Oviedo, Spain in 2003. He then joined the University of Brighton (UK) where he received a Ph.D. for
his work on magnetostrictive ultrasonic transducers for SONAR applications. After completing his Ph.D., Luis worked as a Research Fellow at the University of Birmingham (UK), in a multidisciplinary project fabricating ultrasonic transducers and arrays for ultrahigh resolution real time biomedical imaging. He joined Cambridge University (UK) in January 2009 and is at present a Research Associate at the Electrical Engineering Division, working on the development of thin film bulk acoustic resonators (FBARs) based ultrahigh sensitive biosensor arrays for diagnostic applications. Since October 2010 Luis is also a Lecturer in Engineering at Newnham College, University of Cambridge. Hao Jin received his Ph.D. degree in electronic science and technology from Zhejiang University, China in 2006. He then worked as a Postdoctoral Research Fellow between 2007 and 2009 in the Department of Information Science & Electronic Engineering at Zhejiang University. He was appointed to an associate professor in the same Department in 2009. His research interests include vacuum science and technology, thin film electronics and microwave-radio technology. Jack Luo received his Ph.D. from the University of Hokkaido, Japan in 1989. He worked in Cardiff University as a research fellow, in Newport Wafer Fab. Ltd., Philips Semiconductor Co. and Cavendish Kinetics Ltd. as an engineer, senior engineer and manager, and then in Cambridge University as a senior researcher from 2004. From January 2007, he became a Professor in MEMS at the Centre for Material Research and Innovation (CMRI), University of Bolton. His current research interests focus on microsystems and sensors for biotechnology and healthcare applications, and third generation thin film solar cells using novel low cost materials. Andrew J. Flewitt received the B.Sc. degree in physics from the University of Birmingham, U.K. in 1994 and the Ph.D. degree in scanning tunneling microscopy of amorphous silicon from the University of Cambridge in 1998. Following this, he was a Research Associate studying the low-temperature growth of silicon-based materials in the Engineering Department, University of Cambridge. He was appointed to a Lectureship in the same Department in 2002. Since 2009, he has held the position of University Reader in Electronic Engineering. His research interests span a broad range of large area electronics and related fields, including thin film transistors and MEMS devices. Dr. Flewitt is a Chartered Physicist and a Member of the Institute of Physics and the Institution of Engineering and Technology. Jian R. Lu is professor of biological physics and head of the Biological Physics Group at the University of Manchester. He obtained his Ph.D. degree from Hull University in 1991 in surface chemistry and went on a post-doctoral fellowship in Physical and Theoretical Chemistry Laboratory at Oxford University. From 1995 to 2000 he worked as Lecturer and Reader at Surrey University before taking the chair at Manchester in 2001. He has developed an international reputation in the use of neutron reflection and related techniques for studying biointerfaces.
Contents lists available at SciVerse ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Protein functionalized ZnO thin film bulk acoustic resonator as an odorant biosensor Xiubo Zhao a,b,∗ , Gregory M. Ashley c , Luis Garcia-Gancedo d , Hao Jin e , Jikui Luo c,e , Andrew J. Flewitt d , Jian R. Lu a,∗∗ a
Biological Physics Group, School of Physics & Astronomy, University of Manchester, Oxford Road, Manchester M13 9PL, UK Department of Chemical and Biological Engineering, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield S1 3JD, UK c IMRI, University of Bolton, Deane Road, Bolton BL3 5AB, UK d Electrical Engineering Division, University of Cambridge, JJ Thomson Avenue, Cambridge CB3 0FA, UK e Department of Information Science & Electronic Engineering, Zhejiang University, 38 Zheda Road, Hangzhou 310027, PR China b
a r t i c l e
i n f o
Article history: Received 8 November 2011 Received in revised form 12 January 2012 Accepted 13 January 2012 Available online 25 January 2012 Keywords: FBAR Biosensor Odorant binding protein DEET Gas sensor
a b s t r a c t ZnO thin film bulk acoustic resonators (FBARs) with resonant frequency of ∼1.5 GHz have been fabricated to function as an odorant biosensor. Physical adsorption of an odorant binding protein (AaegOBP22 from Aedes aegypti) resulted in frequency down shift. N,N-diethyl-meta-toluamide (DEET) has been selected as a ligand to the odorant binding protein (OBP). Alternate exposure of the bare FBARs to nitrogen flow with and without DEET vapor did not cause any noticeable frequency change. However, frequency drop was detected when exposing the OBP loaded FBAR sensors to the nitrogen flow containing DEET vapor against nitrogen flow alone (control) and the extent of frequency shift was proportional to the amount of the protein immobilized on the FBAR surface, indicating a linear response to DEET binding. These findings demonstrate the potential of binding protein functionalized FBARs as odorant biosensors. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Biosensors are important tools for medical diagnostics and studying biomolecular interactions in biology and biotechnology [1]. Sensitive, label-free and disposable biosensors are particularly promising [2]. Surface acoustic wave (SAW) and film bulk acoustic resonator (FBAR) sensors have been developed because of the much higher resonant frequency (hence greater sensitivity), lower cost, and smaller size than quartz crystal microbalance (QCM) sensors [3]. Over the past few years, FBAR sensors have been used as mass sensors for many biological applications. For example, Lee et al. [3] reported the use of AlN FBAR sensors for the detection of carcinoembryonic antigens which have been widely used as tumor markers. Common to all these applications is the shift of resonant frequency with the deposition of protein and subsequent
∗ Corresponding author at: Department of Chemical and Biological Engineering, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield S1 3JD, UK. ∗∗ Corresponding author at: Biological Physics Group, School of Physics & Astronomy, University of Manchester, Oxford Road, Manchester M13 9PL, UK. Tel.: +44 161 306 3926. E-mail addresses: [email protected] (X. Zhao), [email protected] (J.R. Lu). 0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2012.01.046
antigen binding onto the biosensor surface. FBAR sensors have also been used for the label-free detection of DNA [2,4]. Zhang et al. [5] demonstrated that FBAR can detect single base pair mismatch of the DNA hybridization. Auer et al. [6] recently reported that FBAR have the capability to detect 1 nM oligonucleotide dissolved in 1% serum. Xu et al. [7,8] have used FBAR sensors for the detection of aptamer–thrombin binding pair and for the real-time in situ monitoring of the competitive adsorption/exchange of proteins. In combination with a competitive antibody–antigen association process, it has been demonstrated that FBARs have the capability of detecting small drug molecules such as cocaine (molecular weight 303 g/mol) and heroin (molecular weight 369 g/mol) [9]. Meanwhile, FBAR has also been explored as gas sensors. Penza et al. [10] reported that AlN/Si3 N4 thin film FBARs functionalized with a layer of single-walled carbon nanotubes have the capability of detecting organic vapors of acetone, ethylacetate, and toluene with high sensitivity, fast response and good repeatability. In this paper, we describe the use of FBAR as an odorant sensor for the detection of mosquitoes repellent by coating the odorant binding protein onto the top electrode of the sensors. DEET (N,N-diethyl-meta-toluamide) is the most common active ingredient in insect repellents and is highly effective against mosquitoes [11–13]. Therefore, it has been selected as a ligand to the odorant binding protein (OBP, AaegOBP22) originated from Aedes aegypti
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mosquitoes. It was found that bare FBARs do not respond to DEET while a resonant frequency shift was observed when exposing the OBP coated FBARs to DEET. This observation indicates that FBAR sensors have the sensitivity in detecting molecular interaction between proteins and small odorant molecules (the molecular weight of DEET is 191 g/mol) and has the potential to be explored as odorant sensors. 2. Materials and methods 2.1. Materials The odorant binding protein (AaegOBP22 from A. aegypti) was provided by Prof. Shuguang Zhang from Centre for Biomedical Engineering, MIT. The expression, purification and functional analysis have been described previously [14]. In brief, AaegOBP22 was expressed by heterologous Escherichia coli extracellular secretion. Protein purification was achieved by Strep-Tactin affinity binding and size-exclusion chromatography. Analysis by SDS–PAGE and mass spectrum confirmed protein purity and molecular weight. CD spectra showed that AaegOBP22 underwent a pH dependent conformational change of the secondary structure. After purification, the protein functions of AaegOBP22 were tested by fluorescent probe 1-NPN binding assays. The results showed AaegOBP22 proteins have characteristics of selective binding with various ligands. N,N-diethyl-meta-toluamide (DEET) was purchased from Sigma–Aldrich. Ultra high quality (UHQ) water with purity to 18.2 M cm was produced from PURELAB® ultra laboratory water purification system.
Fig. 1. (A) Schematic cross-sectional view of the FBAR device used in the experiment (not to scale). The active sensing area is where the top and bottom electrodes overlap. (B) Top view of one of the devices fabricated.
2.2. FBAR sensor fabrication and characterization FBAR devices were fabricated on double-side-polished 4 in. silicon wafers covered with 2 m of thermally grown SiO2 (PI-KEM Ltd., Staffordshire, UK). 5 nm Cr adhesion layer and 60 nm of Au were evaporated on top of the SiO2 surface as the bottom electrode using a thermal evaporator and a standard lift-off photolithography process. The ZnO thin film was then deposited with a thickness between 1.5 and 2.5 m. The film was wet etched in a 2% glacial acetic acid and phosphoric acid solution to form the Via etch holes ZnO for electrical connection. The top electrode was then patterned with a second lift-off photolithography process. Finally the SiO2 on the back of the wafer was patterned and removed with CF4 plasma to expose the Si, which was subsequently removed with a deep reactive ion etching (DRIE) process to release the membrane. The SiO2 suspension layer serves as a barrier, preventing the ZnO from being etched away with the DRIE process. Fig. 1A shows the schematic cross sectional view (not to scale) of FBAR devices and Fig. 1B shows the image of one of the FBAR devices fabricated. The yield of the devices fabricated is higher than 95%. The devices were then wired onto 50 transmission line PCBs using Al wires. Before characterization the devices were ozone cleaned for at least 30 min followed by UHQ water rinsing and N2 drying. Electrical characterization of the FBAR devices was then carried out using an Agilent E5062A network analyzer controlled by software written with LabVIEW to continuously monitor and record the resonance spectrum of the FBAR. A typical response at around 1.56 GHz is shown in Fig. 2 as an example. The sharp resonance of the FBAR indicates a very high quality factor (Q). The quality factor (Q) of the FBAR was determined by measuring the transmission signal (S21 ) and 3 dB bandwidth [15–18] using Eq. (1) Q−3 dB =
f0 f−3 dB
Fig. 2. A typical frequency response (S21 parameter) of the fabricated device. The sharp resonance of the ZnO indicates a very high quality factor.
3 dB Q of the FBARs was found typically around 800. The reduction of the Q factor was found after protein loading. For example, the Q factor of one device reduced from 883 (bare FBAR) to 798 after protein loading. 2.3. Protein loading onto FBAR sensor OBP was loaded onto clean FBAR sensor surface by physical adsorption. A drop of 20 l protein solution (0.35 mg/ml) in 1× PBS (phosphate buffered saline) was placed on top of the active area of the FBAR sensor. Adsorption was allowed from 5 to 15 min followed by UHQ water rinse and N2 drying to achieve different amount of protein adsorbed on the surface. Longer adsorption time led to greater amount of adsorption. 2.4. Measurement setup
(1)
where f0 is the resonant frequency and f−3 dB is the bandwidth at 3 dB, both extracted from the transmission signal spectrum. The
The schematic diagram of the experimental setup of the FBAR sensor for odorant measurement is shown in Fig. 3. The dry N2 supply passed through a flow controller to a three-way tap that
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Fig. 3. Schematic diagram of the FBAR sensor experimental setup.
leads to two identical 100 ml bottles. The N2 from either of the bottles then comes to another three-way tap and goes into and out of the gold coated brass isolation box which hosts the FBAR sensor on the PCB board linked to the network analyzer through two transmission cables. The signal from the network analyzer is then collected by the computer using the GPIB channel. The isolation box is placed on an anti-vibration table and the temperature of the isolation box is controlled by a Haake (K20) bath and kept constant with a variation of no more than ±0.5 ◦ C. Control experiment is carried out to monitor the frequency change of the bare FBAR when switching between the pure N2 flow and N2 flow containing DEET vapor to see the response of the bare FBAR sensor to the DEET vapor. Control experiment is also carried out to monitor the frequency change of the OBP loaded FBAR when switching the N2 flow between two identical empty bottles to see the frequency changes caused by switching the three-way taps. Finally, the frequency changes of the OBP loaded FBAR sensor are recorded when switching between the pure N2 flow and N2 flow containing DEET vapor to see the responses of the OBP to the ligand DEET.
Fig. 4. Response of a bare FBAR sensor to N2 gas with and without DEET vapor.
3. Results and discussion Before coating the FBAR sensor for odorant detection, a control experiment was carried out to investigate the frequency response of the bare FBAR to the DEET. Fig. 4 records the frequency change of a clean bare FBAR against the time when switching between the N2 flow without and with DEET vapor. Apart from the resonant frequency drifting down at around 5 kHz/2000 s (it was found that some FBAR devices might have resonant frequency drifts either up or down a bit), switching resulted in sparks due to the pressure changes during the switching process. No noticeable frequency shift was detected with and without the presence of DEET in N2 . This control experiment confirms that the bare FBAR is not sensitive to DEET. To investigate the response of the OBP coated FBAR to N2 flow, experiment was carried out by switching the N2 gas between two identical empty bottles (Fig. 5). Similarly, no frequency shift was detected apart from the sparks during the switch and a natural resonant frequency drifting down at around 5 kHz/2000 s. This experiment demonstrates that OBP coated FBAR did not respond to N2 gas and that the two N2 pathways did not have any contribution to the frequency change.
Fig. 5. Responses of an OBP loaded FBAR sensor to the two N2 pathways, showing that the spikes were caused by pressure jumps associated with switching.
The OBP coated FBAR was then exposed alternatively to N2 flow containing DEET vapor. Fig. 6 compares the responses of the OBP loaded FBAR sensors to N2 gas with and without DEET vapor. Protein immobilization on the FBAR used for Fig. 6A and B studies
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0.1% DEET in pure water for 4 cycles. The adsorption of the protein resulted in an adsorbed amount of 0.6 mg/m2 . Water washed off some loosely bound proteins and reduced the amount to 0.5 mg/m2 . Replacing water with the 0.1% DEET solution brought the adsorbed amount to the starting value, confirming the binding of DEET to the OBP. In contrast, replacing with water again reduced the adsorbed amount by some 0.1 mg/m2 due to the reversible binding. This process was repeated for 4 times and the data indicated that the process was reproducible and consistent with the findings from the FBAR results. 4. Conclusions In this study, we have fabricated ZnO film bulk acoustic resonators with sensitive resonant frequency shifts upon surface mass loading and successfully used them as odorant biosensors. These FBARs, based on thickness longitudinal mode (TLM) using thin ZnO layer, have their working resonant frequencies around 1.5 GHz and Q factors around 800. The results showed that the bare FBARs do not respond to DEET vapor while the odorant binding protein coated FBAR showed frequency shifts down when exposed to DEET vapor. In addition, the binding of DEET to the odorant binding protein was largely reversible: the resonant frequency shifts back to its initial value when switching to pure N2 flow. The frequency response is also related to the amount of protein on the FBAR surface: higher amount protein resulted in greater frequency shift due to a larger mass load, consistent with the loading of more protein on to the surface providing more binding sites for DEET molecules. This work has thus demonstrated the huge potential of FBARs as odorant biosensors. Acknowledgments
Fig. 6. Responses of OBP loaded FBAR sensors to N2 gas with and without DEET vapor. (A) With a higher OBP loading, exposing the FBARs to DEET resulted in 8–10 kHz frequency drop and (B) with a lower OBP loading, exposing the FBARs to DEET resulted in 2–3 kHz frequency drop.
The authors wish to thank EPSRC for financial support under grants EP/F062966/1, EP/F063865/1 and EP/F06294X/1. The authors would also like to thank Professor Shuguang Zhang at MIT for providing the odorant binding protein. References
resulted in frequency drops of 9.7 MHz and 1.3 MHz, indicating different amounts of protein loaded. Distinct frequency drop was detected when exposed to the N2 gas containing DEET vapor. In the first three cycles as shown, exposure to the DEET vapor results in the shift down of 8–10 kHz in Fig. 6A while the frequency rises to the normal resonant frequency when switching back to N2 flow (a bump that came out at the second exposure was due to the gas leaking of the tubular connection). When a smaller amount of protein is loaded onto the FBAR surface, its response to DEET is much smaller as well (Fig. 6B), indicating that the FBAR response to DEET is related to the amount of the protein immobilized onto the FBAR surface. More protein on the surface provides more binding sites and can therefore accommodate more DEET molecules. Thus, the main observation from Fig. 6 is the almost reproducible cycling of DEET adsorption and desorption in each cycle, suggesting the robustness of the protein molecules immobilized and measurement setup. It was found however that if the adsorption/desorption is recycled 8–10 times, the gap between DEET adsorption and desorption in each cycle became narrower, showing possible structural damage of the protein in a dry gas condition. To support the FBAR results, ellipsometry measurements have been carried out (data not shown). Dilute OBP solution was preadsorbed onto hydrophobic modified silica surface followed by a general wash using pure water. Measurements were carried out by mentoring the mass changes when replacing the pure water with
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Biographies Xiubo Zhao received his Ph.D. degree in biological physics from the University of Manchester in 2006. He is currently a Lecturer in the Department of Chemical and Biological Engineering at the University of Sheffield, before which he was an EPSRC funded Postdoctoral Research Fellow at the University of Manchester. His research interests include biomarker immobilization and detection for biosensoring, peptide self-assembly, non-viral gene and drug delivery, interfacial adsorption of biomolecules, biomaterials and surface biocompatibility. Dr Zhao is a member of the Institute of Physics, UK. Gregory M. Ashley received his B.Sc. degree in microbiology from the University of Liverpool in 1996, then M.Sc. degree in Biosensors from the University of Manchester in 2000. He went on to obtain his Ph.D. in Biosensors in the University of Cranfield on RF MEMS based Biosensors. He is at present a Postdoctoral Research Fellow at the University of Bolton, working on FBAR based biosensors. Luis Garcia-Gancedo received a B.Sc. in physics from the University of Oviedo, Spain in 2003. He then joined the University of Brighton (UK) where he received a Ph.D. for
his work on magnetostrictive ultrasonic transducers for SONAR applications. After completing his Ph.D., Luis worked as a Research Fellow at the University of Birmingham (UK), in a multidisciplinary project fabricating ultrasonic transducers and arrays for ultrahigh resolution real time biomedical imaging. He joined Cambridge University (UK) in January 2009 and is at present a Research Associate at the Electrical Engineering Division, working on the development of thin film bulk acoustic resonators (FBARs) based ultrahigh sensitive biosensor arrays for diagnostic applications. Since October 2010 Luis is also a Lecturer in Engineering at Newnham College, University of Cambridge. Hao Jin received his Ph.D. degree in electronic science and technology from Zhejiang University, China in 2006. He then worked as a Postdoctoral Research Fellow between 2007 and 2009 in the Department of Information Science & Electronic Engineering at Zhejiang University. He was appointed to an associate professor in the same Department in 2009. His research interests include vacuum science and technology, thin film electronics and microwave-radio technology. Jack Luo received his Ph.D. from the University of Hokkaido, Japan in 1989. He worked in Cardiff University as a research fellow, in Newport Wafer Fab. Ltd., Philips Semiconductor Co. and Cavendish Kinetics Ltd. as an engineer, senior engineer and manager, and then in Cambridge University as a senior researcher from 2004. From January 2007, he became a Professor in MEMS at the Centre for Material Research and Innovation (CMRI), University of Bolton. His current research interests focus on microsystems and sensors for biotechnology and healthcare applications, and third generation thin film solar cells using novel low cost materials. Andrew J. Flewitt received the B.Sc. degree in physics from the University of Birmingham, U.K. in 1994 and the Ph.D. degree in scanning tunneling microscopy of amorphous silicon from the University of Cambridge in 1998. Following this, he was a Research Associate studying the low-temperature growth of silicon-based materials in the Engineering Department, University of Cambridge. He was appointed to a Lectureship in the same Department in 2002. Since 2009, he has held the position of University Reader in Electronic Engineering. His research interests span a broad range of large area electronics and related fields, including thin film transistors and MEMS devices. Dr. Flewitt is a Chartered Physicist and a Member of the Institute of Physics and the Institution of Engineering and Technology. Jian R. Lu is professor of biological physics and head of the Biological Physics Group at the University of Manchester. He obtained his Ph.D. degree from Hull University in 1991 in surface chemistry and went on a post-doctoral fellowship in Physical and Theoretical Chemistry Laboratory at Oxford University. From 1995 to 2000 he worked as Lecturer and Reader at Surrey University before taking the chair at Manchester in 2001. He has developed an international reputation in the use of neutron reflection and related techniques for studying biointerfaces.