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Dielectric Properties of Monoclonal Antibodies to Influenza Using Terahertz Spectroscopy

Yiwen Sun1, Emma MacPherson2, Zexuan Zhu3 1.Shenzhen Key Lab of Biomedical Engineering, School of Medicine, Shenzhen University, Shenzhen, 518060, China, ywsun@szu.edu.cn 2.Department of Electronic & Computer Engineering, The Hong Kong University of Science and Technology, Kowloon, Hong Kong, eeemma@ece.ust.hk 3.College of Computer Science and Software Engineering, Shenzhen University, Shenzhen, 518060, China, zhuzx@szu.edu.cn
Abstract. In this paper we use terahertz spectroscopy to study the effects of the monoclonal antibody to influenza A and the monoclonal antibody to influenza B. The complex permittivity was measured over the frequency range 0.1-1.7THz. The investigated antibodies can be distinguished by the frequency dependence of the dielectric properties. Therefore terahertz spectroscopy may be useful for medical diagnoses where the presence of antibodies is indicative of disease. Keywords: protein; dielectric properties; Terahertz; spectroscopy 1. Introduction Recently, there has been renewed interest in the far-infrared (FIR) region of the spectrum, especially in the terahertz (THz = 1012Hz) regime, which lies between the microwave and infrared regions of the EM spectrum, shown as figure 1. The terahertz regime is typically defined as ranging from 0.1 to 10 THz in frequency, or 3.33 cm-1 to 33.3 cm-1 in wavenumbers [1]. This region has only recently been explored due to a previous lack of appropriate sources and detectors. In fact, it was commonly referred to as the terahertz gap before advances in semiconductor physics enabled the so-called gap to be bridged [2-3]. THz radiation is non-destructive, non-invasive and non-ionising [4-6]. Towards the far-infrared end of the terahertz range there are vibrational modes corresponding to protein tertiary structural motion: such inter-molecular interactions are present in many biomolecules. Other molecular properties that can be probed in the Terahertz range include bulk dielectric relaxation modes and phonon modes. Terahertz spectroscopy is therefore emerging as an important and highly sensitive tool to determine biomolecular structure and dynamics [7-8]. For example Fischer et al. demonstrated that even when the molecular structure differs only in the direction of a single hydroxyl group with respect to the ring plane, a pronounced difference in the terahertz spectra is observed [9].

Fig. 1 The THz region and molecular transitions in the electromagnetic spectrum Proteins influence both the spatial and dynamic arrangement of neighboring liquid layers through weak inter-molecular interactions [10]. The collective vibrational modes are associated with the proteins tertiary structure and lie in the far infrared or terahertz frequency range. With the advent of pulsed terahertz techniques, low frequency dielectric characterization of proteins can be accomplished. In this paper we conduct terahertz spectroscopy to study the frequency dependence of the dielectric properties of the antibodies in liquid solutions. Influenza is an infectious disease caused by Orthomyxoviridae RNA viruses, including Influenza A, Influenza B, and Influenza C. Monoclonal antibodies that are broadly reactive with influenza A or influenza B viruses were produced as stable reagents for typing influenza viruses. Briefly, monoclonal antibodies to influenza A were specific for either matrix (M) protein or nucleoprotein (NP). The antibodies to influenza B were specific for nucleoprotein or hemagglutinin protein. In an enzyme immunoassay procedure, influenza A antibodies detected HlN1, H2N2, and H3N2, and recently H5N1, are responsible for the majority of serious human illness and death. Influenza B has a limited host range (primarily human) [11]. Thus, we investigate the terahertz properties of antibodies to influenza to determine if terahertz spectroscopy could potentially be used for antibody detection in the future. 2. Experimental Methods A modified version of the TPI Imaga 1000 (TeraView Limited, Cambridge, UK) was used to determine the terahertz

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dielectric response. The similar optical set up has been described in figure 2. Briefly, this system uses photoconductive antennas for generation and detection of terahertz radiation, an ultrafast laser (Vitesse, Coherent Inc.) emits 90 fs pulses centered at a wavelength of 800 nm, with a 80 MHz repetition rate and an average power of about 200 mW. The laser beam is separated into the pump and probe beams by a beam splitter, but in contrast to the TPI Imaga 1000TM, the laser in this system used for optical excitation reaches the antennas via a fiber optic, rather than through free space optics. The probe is used to contact the liquid sample directly with an angle of incidence of 3.4 and the reflected terahertz pulse from the quartz/sample interface is detected coherently. The THz beam is scanned up and down across the center line of the window to form a line scanning. A more detailed description of the system can be found in reference [12].

Fig. 2 Schematic diagram of a terahertz pulsed imaging system TPITM in reflection geometry. Two types of antibodies were selected for this investigation. The Monoclonal Antibody to Influenza A and the Monoclonal Antibody to Influenza B (Medix Biochemica, Kauniainen, Finland) at the same protein concentration of 5.4mg/ml in citrate-NaCl buffer with 0.1% NAN3 as preservative. 3. Results and Discussion Figure 3 shows the absorption coefcients () of the monoclonal antibody to influenza A (blue line) and the monoclonal antibody to influenza B (red line) in the frequency range from 0.1 to 1.7 THz at the room temperature. Two dominated absorption peaks at around 1.18 and 1.49 THz were observed for anti-influenza B. Only one peak at about 1.18THz was still found for anti-influenza A. Influenza viruses are identified though the expression of hemagglutinin protein (HA) and neuraminidase (NA) protein antigens on the viral envelope. Monoclonal antibodies to influenza A were specific for either matrix protein or nucleoprotein, while the antibodies to influenza B were specific for nucleoprotein or hemagglutinin protein. These peaks, which are either due to the difference of the binding site on the monoclonal antibodies or the chemical structure, ensure the virus is detected sensitively.

Figure 3 Absorption coefcient vs. frequency of the monoclonal antibody to influenza A (blue line) and the monoclonal antibody to influenza B (red line). The complex permittivity * is expressed by the real part and the imaginary part (*= - i ) in which the real part corresponds to the dielectric constant and the imaginary part, known as the dielectric loss factor, is a measure of the energy absorption per cycle [13]. The real and imaginary parts of the complex permittivity were calculated from the frequency-dependent optical constants n() and (), and are plotted in figure 4 for the monoclonal antibody to influenza A and the monoclonal antibody to influenza B respectively.
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Figure 4 Cole-Cole plots of the complex permittivity for the monoclonal antibody to influenza A (blue line) and the monoclonal antibody to influenza B (red line). In figure 4 we compare measurements of the complex permittivity of antibodies. At the room temperature, the dielectric constant of anti-influenza A is lower than that of anti-influenza B for real part, but has a greater dielectric loss. The dielectric loss factor is related by the general fluctuation-dissipation theorem to the spectrum of polarization fluctuations of a dielectric sample that originate from thermally induced translational stochastic motions of charge carriers and the rotational tumbling motion of dipolar entities. Due to the relative broadness of the dielectric spectra the different relaxation processes will only be resolved adequately if the polar molecules under investigation differ significantly in size. Furthermore, since the dielectric response is associated with the collective vibrational modes of protein tertiary structure and sensitive to the conformational changes of the protein molecules due to the structure, which we have reported in our previous work [14], these preliminary results suggest that terahertz pulsed spectroscopy could potentially be used for detecting the presence of antibodies in an aqueous phase. 4. Conclusions THz time-domain spectroscopy has been applied to examine dielectric relaxation properties of monoclonal antibody to influenza A and the monoclonal antibody to influenza B. This work reports the frequency-dependent absorption coefcient and index of refraction in the antibodies over a wide range of THz frequencies and measurements of the real and imaginary parts of the complex dielectric response function. Therefore terahertz spectroscopy may be useful for medical diagnoses where the presence of antibodies is indicative of disease. In further work we will also investigate different dielectric relaxation models of distribution function for more complex protein-liquid systems, so as to understand and relate the measurements to the molecular changes occurring. 5. Acknowledgment The authors gratefully acknowledge partial financial support for this work from Natural Science Foundation of SZU (Grant No.: 201115), Research Grants Council of the Hong Kong Government, Hong Kong Shun Hing Institute of Advanced Engineering, National Natural Science Foundation of China (Grant No.: 61001185) and Foundation for Distinguished Young Talents in Higher Education of Guangdong, China, (Grant No.: LYM10113). References [1] S. W. Smye, J. M. Chamberlain, A. J. Fitzgerald, and E.Berry, The interaction between Terahertz radiation and biological tissue, Phys. Med. Biol.,vol. 46, pp. R101R112, 2001. [2] D. H. Auston, Picosecond Optoelectronic Switching and Gating in Silicon, Applied Physics Letters 26, pp.101-103, 1975. [3] Ian S. Gregory, Colin Baker, William R. Tribe, Ian V. Bradley, Michael J. Evans, Edmund H. Lineld, A. Giles Davies and M. Missous, Optimization of Photomixers and Antennas for Continuous-Wave Terahertz Emission, IEEE Journal or Quantum Electronics 41, pp. 717-728, 2005. [4] V. P. Wallace, P. F. Taday, A. J. Fitzgerald, R. M. Woodward, J. Cluff, R. J. Pyeand D. D. Arnone, Terahertz pulsed imaging and spectroscopy for biomedical and pharmaceutical applications, Faraday Discussions 126, pp. 255-263, 2004. [5] P. H. Siegel, Terahertz technology in biology and medicine, IEEE Transactions on Microwave Theory and Techniques 52, pp. 24382447, 2004. [6] R.M.Woodward, V.P.Wallace, R.J.Pye, B.E.Cole, D.D.Arnone, E.H.Lineld and M. Pepper, Terahertz pulse imaging of ex vivo basal cell carcinoma, Journal of Investigative Dermatology 120, pp. 72-78, 2003.

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[7] B. M. Fischer, M. Hoffmann, H. Helm, R. Wilk, F. Rutz, T. Kleine-Ostmann, M. Koch and P. U. Jepsen, Terahertz time-domain spectroscopy and imaging of articial RNA, Optics Express 13, pp. 5205-5215, 2005. [8] Walther M, Fischer B M, Schall M, Helm H and Jepsen P U, Far-infrared vibrational spectra of all-trans 9-cis and 13-cis retinal measured by THz time-domain spectroscopy, Chem. Phys. Lett. 332, 389-395, 2000. [9] Bernd M. Fischer, Hanspeter Helm, and Peter Uhd Jepsen, Chemical recognition with broad and THz spectroscopy, Proceedings of the IEEE 95(8), pp. S246-S253, 2007. [10] R. Pethig, "Protein-water interactions determined by dielectric methods," Annu. Rev. Phys. Chem., vol. 43, pp. 177, 1992. [11] H. H. Walls, M. W. Harmon, J. J. Slagle, C. Stocksdale, and A. P. Kendal, "Characterization and evaluation of monoclonal antibodies developed for typing influenza A and influenza B viruses" J. Clin. Microbiol., vol.23, pp. 240, 1986. [12] Y.W. Sun, Investigating Biomolecular Interactions using Terahertz Pulsed Spectroscopy, This thesis is submitted for the degree of Doctor of Philosophy, The Chinese University of Hong Kong, 2010. [13] S.N. Vinogradov and R.H. Linnell, "Hydrogen Bonding," 1971. [14] Y.W. Sun, E. Pickwell-MacPherson, "Probing Antibody Interactions with Polar Liquids using Terahertz Pulsed Spectroscopy,"The 34th International Conference on Infrared, Millimeter, and Terahertz Waves, Busan, September 2009.

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