ISSN 0030400X, Optics and Spectroscopy, 2013, Vol. 114, No. 6, pp. 894–898. © Pleiades Publishing, Ltd., 2013. Original Russian Text © M.V. Tsurkan, N.S. Balbekin, E.A. Sobakinskaya, A.N. Panin, V.L. Vaks, 2013, published in Optika i Spektroskopiya, 2013, Vol. 114, No. 6, pp. 981–986.

LASER OPTICS

Terahertz Spectroscopy of DNA M. V. Tsurkana, N. S. Balbekina, E. A. Sobakinskayab, A. N. Paninb, and V. L. Vaksb a

St. Petersburg National Research University of Information Technologies, Mechanics, and Optics, Kronverkskii pr. 49, St. Petersburg, 197101 Russia b Institute for Physics of Microstructures, Russian Academy of Sciences, GSP105, Nizhni Novgorod, 603950 Russia email: [email protected] Received November 21, 2012

Abstract—The spectra of the degraded DNA of herring are studied using two methods of THz spectroscopy that employ a femtosecond laser and a frequency synthesizer based on a BWO and a highQ cavity. An inter pretation of the experimental results is presented. DOI: 10.1134/S0030400X13060222

1. INTRODUCTION The recent interest in the development of methods for application of THz radiation in medicine and biol ogy [1–3] has been driven mainly by the fact that the rotational spectra of light gases and lowfrequency vibrations of biologically important large molecules belong to the THz spectral range. The spectroscopic data that make it possible to characterize molecules can be used for interpretation and control of biochem ical processes and medical diagnostics at the cellular and molecular levels. The dielectric function of macromolecules in the THz spectral range is formed by lowfrequency vibra tions that represent cooperative motions of large atomic groups in a molecule. Such vibrations are sen sitive to the intramolecular structure owing to the cooperative nature and allow analysis of conforma tional states. The corresponding results are important for the study of the conformational flexibility of bio molecules (DNA, RNA, proteins, vitamins, etc.), which plays an important role in biochemical reac tions. The specificity of the THz spectra with respect to various conformers and isomers of the substances under study has been demonstrated in several works. In addition, the THz absorption bands can be sensitive to mutations and the environment [4]. The application of the THz spectroscopy in the study of nucleotides and nucleosides that represent the DNA structural units was demonstrated in [1]. The analysis of the dielectric function of the solid samples in the frequency interval 0.5–3.5 THz yields the spe cific spectral features of the four bases in the absorp tion and reflection coefficients. The THz spectra of the synthesized single and doublestranded DNA molecules were presented in [3]. In spite of a relatively low resolution, the spectral differences of the DNA samples were demonstrated. Spectrometers with

higher resolution make it possible to more accurately measure the DNA spectrum [5]. In most works, the THz spectra are measured using spectrometers based on femtosecond lasers. In such setups that employ the optical approach, optical radi ation is converted into the THz radiation [6, 7]. The advantage of the method lies in its shortterm mea surements in a wide spectral interval. However, the spectral resolution is several gigahertz and the mea sured spectral envelope allows only qualitative charac terization of the sample under study. In the framework of the optical approach, the best results are obtained using a spectrometer based on the opticalbeat oscilla tor [8]. THz radiation is generated with the aid of pho tomixers in which interaction of the signals of two optical lasers takes place. The difference frequency that results from the mixing belongs to the THz spec tral range [9]. Such a method allows significant fre quency tuning and a resolution of several megahertz. However, a relatively low THz power (about 1 µW) and the problems of the control system and frequency sta bilization impede the application of spectrometers in highprecision spectroscopy. The spectral resolution of the conventional FTIR spectrometers (several giga hertz) is also insufficient for analysis of complicated spectra. The best spectral resolution in the THz spectrome ters is reached using the microwave methods. A com monly accepted approach in the development of stable microwave oscillators involves the multiplication of the frequency of a stable reference synthesizer. Fre quency multipliers, mixers, and amplifiers are the key elements of the setups. In particular, mixers and mul tipliers based on quantum semiconductor superlat tices [10] have been used in a few highprecision methods and devices for various applications [11]. The disadvantage of the approach lies in the absence of broadband radiation sources. In particular, no less than seven BWOs are needed for the measurements in

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895

DL П L1

LA

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PM2

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Fig. 1. Block diagram of the TS5 spectrograph: (Fl1) femtosecond Yb:KGW laser, (M1–M3) plane mirrors, (BS) beamsplitter, (DL) delay line, (OMM) optomechanical modulator, (InAs) InAs crystal, (M) permanent magnet, (G) Glan prism, (PM1) and (PM2) parabolic mirrors, (F) filter, (L1) and (L2) lenses, (O) object, (CdTe) CdTe crystal, (λ/4) quarterwave retardation plate, (W) Wollaston prism, (BD) balance detector, (LA) lockin amplifier, and (PC) computer.

the frequency interval 100 GHz–1 THz. Therefore, it is effective to perform detailed analysis with the aid of the highprecision sources after general spectral mea surements of the substance under study. Thus, a combination of the broadband spectrome ters based on femtosecond lasers and spectrometers with highprecision sources allows the most efficient spectral study of complicated molecules and applica tions of the THz spectroscopy in real problems. The purpose of this work is the development and imple mentation of an experimental setup for highprecision spectral measurements and the analysis of the THz spectrum of DNA using the above methods. 2. EXPERIMENTAL SETUP AND MEASUREMENT PROCEDURE 2.1. A THz Spectrometer Based on a Femtosecond Laser At the first stage, we measure the spectrum of DNA using a spectrometer based on the femtosecond laser, characterize the spectrum in general, and determine the intervals of relatively high absorption. In this work, we employ an experimental setup that generates broadband pulsed THz radiation in a photo conducting antenna that is irradiated with femtosec ond pulses (Fig. 1) (see [12] for the details of the experimental setup). The radiation of a Solar FL1 ytterbium–KGW laser at a wavelength of 1040 nm represents a train of pulses with a duration of 160 fs and a repetition rate of 75 MHz. The mean power amounts to 1.2 W. The radiation is divided into two beams at a ratio of 1 : 100 using beamsplitter BS. The highpower beam that passes through the delay line is incident on the InAs semiconductor surface that is placed at the center of permanent magnet M where the magnetic induction is 1.8 T. The generated THz radi ation is collimated by parabolic mirror PM1, separated from the pump radiation by filter F, and focused by OPTICS AND SPECTROSCOPY

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parabolic mirror PM2 to the CdTe crystal to induce additional birefringence. The sample under study is placed into the THz beam between two confocal lenses, which allows the measurement of small sam ples. The second beam (λ = 1.04 µm) is reflected by BS, passes through polarizer P and the aperture in PM2, and is incident on the CdTe crystal. In the absence of induced birefringence in the CdTe crystal, linearly polarized radiation with wavelength λ = 1.04 µm passes through the quarterwave retardation plate (λ/4) and the Wollaston prism and is incident on two photodiodes that work as a balance detector set to zero. The nonzero output signal of the balance detec tor results from the induced birefringence. An increase in the signaltonoise ratio is reached using a FEMTO LIAMV150 lockin amplifier with the optomechan ical modulator. The data processing in the spectrometer is provided by a virtual device based on the LabView software that controls the delay and measurement procedure and allows the recording of the THzpulse time shape. The second software package makes it possible to calculate the spectrum of radiation using the Fourier transform of the measured time dependence. The transmission spectrum of the sample is calculated as the ratio of the absolute difference between the spectra of the sample and substrate to the spectrum of the substrate. The pump beam of the femtosecond laser generates the THz radiation in the InAs semiconductor crystal in the presence of the static magnetic field. The parameters of the radiation that is generated in the spectral interval 0.02–1.5 THz are as follows: mean power, about 50 µW; pulse repetition rate, 75 MHz; pulse power, no less than 200 mW; and pulse duration, 3 ps. The power is predominantly distributed over nine peaks at frequencies from 0.12 to 1 THz. The fre quency error of the detection system is less than 5.5 GHz.

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Mixer

Cavity with the sample

BWO

Power supply

Mixer

Rohde & Schwarz synthesizer

Amplifier

Agilent spectrum analyzer

Fig. 2. Block diagram of the highprecision spectrometer with QC.

In the experiments, we use solid powder of degraded DNA of herring. The DNA samples repre sent films on polyethylene or quartz substrates, which exhibit relatively high transparency in the THz spec tral range. Rectified ethanol or distilled water serve as solvents. The DNA samples are mixed with solvents at various concentrations and dried on substrates under atmospheric conditions to obtain solid films. 2.2. A HighPrecision Spectrometer Based on the BWO Frequency Synthesizer and HighQ Cavity At the second stage, we study the spectral interval 300–375 GHz in detail using a highprecision spec trometer based on the frequency synthesizer with BWO and a highQ cavity (Fig. 2), which allows a rel atively high stability of the signal and a spectral resolu tion of less than 10 kHz. The frequency synthesizer contains BWO; a BWO power supply and control unit; phaselocked system that includes a harmonic mixer, a Hewlett–Packard reference synthesizer, and phase detector PD; and quasioptical units. The detection system contains a harmonic mixer and a Rohde & Schwarz synthesizer that serves as a heterodyne. Table 1. Absorption bands of the DNA samples in spectra that are measured using the femtosecondlaser spectrometer

Substrate Polyethylene Quartz

Frequencies of ab Frequencies of ab sorption bands sorption bands of DNA samples with of DNA samples with water, GHz ethanol, GHz 159, 180, 360, 401, 525, 549, 610, 700 304, 321, 372, 467,630, 660, 689

159, 181, 331, 350, 625, 710, 749 360, 380, 655, 687

The signal of the synthesizer that passes through the sample several times is transmitted through a rect angular aperture in the mirror of the cavity. Then, the signal is fed via a waveguide to the mixer that is con nected to the Rohde & Schwarz synthesizer. The sig nal at intermediate frequency IF is fed to an Agilent spectrum analyzer via an amplifier. One method to increase the sensitivity of spectro scopic analysis involves application of highQ cavities (QCs). Spectral study of various substances with the aid of QCs can be performed using measurements of (i) variations in the resonance frequency and Q factor of the cavity or (ii) intensity and decay characteristics of the cw or pulsed radiation that passes through the QC in which the sample is placed. Even minor varia tions in the parameters (e.g., shifts of the resonance frequency and variations in the Q factor) related to the presence of the sample under study can be experimen tally measured owing to relatively high Q factors of the cavities. QC spectroscopy is well developed in the IR spectral range and employed in various applications [13–15]—in particular, analysis of biomolecules [16]. The key element of the setup is a QC made of Invar rods with a length of 25 cm and circular copper mirrors with a diameter of 11 cm. The Invar rods provide sta bility of the cavity to thermal heating. The Q factor of the cavity is about 60000. For measurements, we use the DNA solutions in distilled water that are stored in polyethylene vessels. In the experiments, we employ two DNA samples (0.5 and 1.7 g) in a solution with 20 mL of water. To establish the relationship of the measured quan tities and the absorption coefficient of the sample, we use a simple model of the cavity characteristic:

I 1(ω) = (I 0 /α) ⎡(ω − ω0 )2 + 12 ⎤ , ⎢⎣ α ⎥⎦ where I0 is the intensity of the incident radiation at fre quency ω, ω0 is the resonance frequency of the cavity, and parameter α takes into account all of the losses in the cavity and is related to the Q factor. In the presence of the sample in the cavity, the fol lowing expression is valid at relatively low absorbance: 2 I 2(ω) = (I 0 / ε) ⎡( ω − ω1 ) + 12 ⎤ . ⎣⎢ ε ⎦⎥ Here, ε = α + γ is the total loss in the cavity; γ is the quantity that is proportional to the absorption coeffi cient of the sample (γ = dδ, where d is the length and δ is the absorption coefficient of the sample); and ω1 is the modified resonance frequency of the cavity. Thus, the measurements of the transmitted intensity make it possible to determine parameter γ:

γ=

I 2 ( ω1 ) − I 1 ( ω0 ) α. I 1 ( ω0 )

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TERAHERTZ SPECTROSCOPY OF DNA T, arb. units 1.2

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358

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362 F, GHz

Fig. 3. DNA transmission spectra in water and ethanol that are measured on (a) polyethylene and (b) quartz substrates and averaged over three measurements: (1) high, (2) medium, and (3) lowconcentration solutions in water and (4) high and (5) lowconcentration solutions in ethanol.

Fig. 4. Absorption spectra of (1) water and (2) low and (3) highconcentration solutions of DNA that are measured (a) with a step of 3 GHz in the spectral interval 300– 375 GHz and (b) with a step of 200 MHz in the spectral interval 358–362 GHz.

The measurements of absorption γ of the empty polyethylene cell, the cell with distilled water, and the cell with the DNA solution make it possible to obtain the DNA absorption spectrum.

333, 348, and 360 GHz. It is seen that the results are in agreement with the results from the spectrometer based on a femtosecond laser.

3. RESULTS Figure 3 shows the transmission spectra of the DNA films on the polyethylene and quartz substrates that are measured with the aid of the spectrometer based on the femtosecond laser. Table 1 presents the most intense absorption bands of the DNA films. Six absorption bands at frequencies of 304, 321, 331, 350, 360, and 372 THz correspond to the working range of the highprecision QC spectrometer. Figure 4a shows the DNA absorption spectrum in the working range of the BWO synthesizer (300– 375 GHz) with a step of 3 GHz. The greatest absorp tion is observed at frequencies of about 306, 315, 324, OPTICS AND SPECTROSCOPY

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Then, scanning with a step of 200 MHz is per formed. Figure 4b shows an example of such scanning of the absorption band at a frequency of about 360 GHz that yields a fine structure. Table 2 presents the bands with the greatest absor bances of no less than 4 units that are measured using the highprecision spectrometer. 4. DISCUSSION AND COMPARISON WITH LITERATURE DATA We analyze the data for the spectral interval 300– 375 GHz of both experiments and compare them with the spectra of DNA of herring from [3] and solvent (water) from [17].

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Table 2. Strongest absorption bands of DNA in the spectral interval 300–375 GHz (measurements using the highpreci sion spectrometer) 304.6 325.4

305.0 347.2

310.6 360.0

311.0 360.8

313.8 361.4

Table 3. DNA absorption bands from works [3, 17] DNA of herring [3]

Liquid water [17]

306, 339, 375

321, 342

The DNA absorption spectra are measured in [3] with a spectral resolution of about 6 GHz using an IFS66 FTIR spectrometer. Note that, in [3], the spectra of dry films in the absence of solvents were measured. The measurements of water films were per formed on an FT66v IR spectrometer with a resolu tion of 7.5 GHz. Table 3 presents the frequencies of the absorption bands in the interval 300–375 GHz from [3, 17]. Figure 4a clearly shows that, with allowance for the spectral resolution of the FT66v spectrometer, the absorption bands of water peaked at frequencies of 321 ± 7.5 and 342 ± 7.5 GHz from [17] coincide with the water bands that are measured in this work using the highprecision spectrometer. A band peaked at 321 GHz is also observed in the spectra of the DNA films with water as solvent. The DNA absorption bands from [3] are also in agreement with the results of this work if the spectral resolution of the IFS66 spectrometer is taken into account. Note that the absorption band peaked at 306 ± 6 GHz in [3] is split in our measurements into two bands with relatively high absorbances peaked at 304.6 and 305.0 GHz. Note also relatively low intensities of the bands peaked at 339 ± 6 and 375 ± 6 GHz in the experiments in [3] and the measurements on the highprecision spectrometer (Fig. 4a). However, the intensities of the bands peaked at 372 ± 5.5 and 380 ± 5.5 GHz are higher in the spectra that are measured in the first part of our measurements and are reproduced on the quartz sub strate in the presence of ethanol and water as solvents. Relatively strong bands at frequencies of about 360 GHz are not observed in [3]. However, the groups of such bands are observed in our measurements using both spectrometers with different solvents and sub strates (Figs. 3 and 4b). Thus, the DNA spectra that are measured with the aid of the two spectrometers are in good agreement with each other and identification of the results is pos sible. We assume that the bands peaked at 304.6, 305.0, 339, and 372 GHz are assigned to the DNA intramo lecular vibrations.

5. CONCLUSIONS In this work, we show the advantages of a combina tion of the quasioptical and highprecision measure ments in the THz spectroscopy. A spectrometer based on a femtosecond laser allows fast measurements in a relatively wide spectral range that can be used to select the intervals for the highprecision experiments. We demonstrate a THz spectroscopic method based on the application of the frequencystabilized radiation sources and QCs that makes it possible to analyze the fine structure in the spectra of biomolecules. Such an approach can be used to solve biological problems. ACKNOWLEDGMENTS This work was supported by the federal program “Human Capital for Science and Education in Inno vative Russia (2009–2013)” (State contract no. 16.513.11.3070), Dynasty Foundation (for E.A. So bakinskaya), Russian Foundation for Basic Research (projects nos. 110297051r_povolzh’e_a and 1102 12203ofim2011), Ministry of Education and Sci ence of the Russian Federation (project no. 20121.4 120001010001), and Government of the Russian Federation (project no. 11.G34.31.0066). REFERENCES 1. B. M. Fischer, Phys. Med. Biol. 47, 3807 (2002). 2. M. Semenov, T. Bolbukh, and V. Maleev, J. Mol. Struct. 408/409, 213 (1997). 3. A. Rahman, B. Stanley, and A. K. Rahman, Proc. SPIE—Int. Soc. Opt. Eng. 7568, 756810 (2010). 4. A. Markelz, Phys. Med. Biol. 47, 3797 (2003). 5. R. Parthasarathy et al., Appl. Phys. Lett. 87, 113901 (2005). 6. G. Matthaus et al., Appl. Phys. Lett. 93, 091110 (2008). 7. D. Talbayev et al., Appl. Phys. Lett. 93, 212906 (2008). 8. F. Hindle, A. Cuisset, R. Bocquet, and G. Mouret, Comp. rend. l’Acad. Sci. 9, 262 (2008). 9. E. R. Brown, Proc. SPIE—Int. Soc. Opt. Eng. 7938, 793802 (2011). 10. D. G. Paveliev, Yu. I. Koshurinov, V. P. Koshelets, A. N. Panin, et al., in Book of Abstracts of the 16th Inter national Symposium on Space Terahertz Technology (ISSTT 2005) (Chalmers, Sweden, 2005), pp. 1–16. 11. V. Vaks, J. Infrared Milli Terahz Waves 33, 43 (2012). 12. V. G. Bespalov et al., Opt. Zh. 75 (10), 636 (2008). 13. WenBin Yan, Proc. SPIE 4648, 156 (2002). 14. J. M. Langridge et al., Opt. Express 16, 10178 (2008). 15. E. R. Crosson et al., Anal. Chem. 74 (9), 2003 (2002). 16. T. McGarvey, Opt. Express 14, 10441 (2006). 17. T. Globus et al., Proc. SPIE—Int. Soc. Opt. Eng. 6772, 67720 (2007).

Translated by A. Chikishev OPTICS AND SPECTROSCOPY

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