Tatyana Khromova

THz-Spectroscopy of Biological Molecules.

The terahertz frequency absorption spectraof DNA molecules reflect low-frequencyinternal helical vibrations involvingrigidly bound subgroups that are connectedby the weakest bonds, including thehydrogen bonds of the DNA base pairs,and/or non-bonded interactions. Althoughnumerous difficulties make the directidentification of terahertz phonon modes inbiological materials very challenging, ourresearch has shown that such measurementsare both possible and fruitful. Spectra ofdifferent DNA samples reveal a large numberof modes and a reasonable level ofsequence-specific uniqueness. In an attemptto show that the long wavelength absorptionfeatures are intrinsic properties ofbiological materials determined by phononmodes, a normal mode analysis has been usedto predict the absorption spectra ofpolynucleotide RNA Poly[G]-Poly[C]. Directcomparison demonstrated a correlationbetween calculated and experimentallyobserved spectra of the RNA polymers, thusconfirming that the fundamental physicalnature of the observed resonance structureis caused by the internal vibration modesin the macromolecules.In this work we demonstrate results fromFourier-Transform Infrared (FTIR)spectroscopy of DNA macromolecules andrelated biological materials in theterahertz frequency range. Carefulattention was paid to the possibility ofinterference or etalon effects in thesamples, and phenomena were clearlydifferentiated from the actual phononmodes. In addition, we studied thedependence of transmission spectra ofaligned DNA and polynucleotide film sampleson molecule orientation relative to theelectromagnetic field, showing the expectedchange in mode strength as a function ofsample orientation. Further, the absorptioncharacteristics were extracted from thetransmission data using the interferencespectroscopy technique, and a stronganisotropy of terahertz characteristics wasdemonstrated.

Terahertz Fourier transform characterization of biological materials in a liquid phase

Significant progress has been achieved during the last several years relating to experimental and theoretical aspects of terahertz (or submillimetre wave) Fourier transform spectroscopy of biological macromolecules. However, previous research in this spectral range has been focused on bio-materials in solid state since it was common opinion that high water absorption will obscure the spectral signatures of the bio-molecules in solutions. At the same time, the biological functions of DNA and proteins take place in water solutions. In this work, the spectra of DNA samples have been measured in liquid phase (gel) over the spectral range 10–25 cm−1 and compared with spectra obtained from solid films. The results demonstrate that there is very little interference between the spectral features of the material under test and the water background except for the band around 18.6 cm−1. Multiple resonances due to low frequency vibrational modes within biological macromolecules in solutions are unambiguously demonstrated. Higher level of sensitivity and higher sharpness of vibrational modes are observed in the liquid environment in comparison with the solid phase, with the width of spectral lines 0.3–0.5 cm−1. Gel sample spectra are found to be polarization-dependent. The ability of THz spectroscopy to characterize samples in liquid phase could be very important since it permits examination of DNA interactions in real (wet) samples. One demonstrated example of practical importance is the ability to discriminate between spectral patterns for native and denaturated DNA.

Dielectric properties of biological molecules in the Terahertz gap

In this work, results from parallel measurements of reflection and transmission spectra of biological molecules were utilized to enable detailed and direct calculation of the refractive index and absorption coefficient spectra in the Terahertz gap. The DNA samples from herring and salmon, as well as the protein Ovalbumin sample, have been characterized. The modeling technique is described. The reflection spectra have resonance features similar to those demonstrated earlier for transmission, thereby reaffirming molecular vibrational modes in biological materials. The dispersion of refractive index and absorption coefficient is demonstrated within the Terahertz gap of 10cm−1to25cm−1. The data yielded higher refractive index and absorption coefficient for the single stranded salmon DNA than for the double stranded counterpart, with several different vibrational modes.

Highly Resolved Sub-Terahertz Vibrational Spectroscopy of Biological Macromolecules and Cells

Sub-terahertz (sub-THz) vibrational spectroscopy for biosensing is based on specific resonance features, vibrational modes or group of modes at close frequencies, in the absorption (transmission) spectra of large biological molecules and entire bacterial cells/spores. Further improvements in sensitivity, especially in the discriminative capability of sub-THz vibrational spectroscopy for detection, characterization, and identification of bacterial organisms, require spectral resolution adequate to the width of spectral features. Evidences exist for long-lasting relaxation processes for atomic dynamics (displacements) resulting in narrow spectral lines and justifying the development and application of highly resolved vibrational spectroscopy. Here we describe a new continuous-wave frequency-domain spectroscopic sensor with imaging capability operating at room temperature in the sub-THz spectral region between 315 and 480 GHz. We present experimental spectra from biological macromolecules and species obtained using this spectrometer and compare some spectra with simulation results using molecular dynamics. Observed multiple intense and specific resonances in transmission/absorption spectra from nano-gram samples with spectral line widths as small as 0.1 cm-1 provide conditions for reliable discriminative capability, potentially to the level of the strains of the same bacteria, and for monitoring interactions between biomaterials and reagents in near real-time.

Terahertz-Regime Attenuation Signatures in Bacillus subtilis and a Model Based on Surface Polariton Effects

A summary is provided for terahertz attenuation signatures measured in spore-laden samples of Bacillus subtilis in three different forms: 1) concentrated powder; 2) dilute powder; and 3) aerosol. In addition to a surprising spectral narrowness, some signatures also display an increase in peak signature strength (per spore) with dilution of the sample. A model is constructed to explain this phenomenology based on the presence of optical phonons and electromagnetic interaction with the spore wall. Specifically, the spheroidal Bacillus spores admit surface modes that interact with radiation via polaritonic coupling and are underdamped if isolated from each other through a dilution or aerosol levitation. Hence, the results defy longstanding assumptions that the biomolecular-related terahertz vibrations are necessarily overdamped and have immeasurably weak attenuation

An analysis of the THz frequency signatures in the cellular components of biological agents

The development of an effective biological (bio) agent detection capability based upon terahertz (THz) frequency absorption spectra will require insight into how the constituent cellular components contribute to the overall THz signature. In this work, the specific contribution of ribonucleic acid (RNA) to THz spectra is analyzed in detail. Previously, it has only been possible to simulate partial fragments of the RNA (or DNA) structures due to the excessive computational demands. For the first time, the molecular structure of the entire transfer RNA (tRNA) molecule of E. coli was simulated and the associated THz signature was derived theoretically. The tRNA that binds amino acid tyrosine (tRNAtyr) was studied. Here, the molecular structure was optimized using the potential energy minimization and molecular dynamical (MD) simulations. Solvation effects (water molecules) were also included explicitly in the MD simulations. To verify that realistic molecular signatures were simulated, a parallel experimental study of tRNAs of E. coli was also conducted. Two very similar molecules, valine and tyrosine tRNA were investigated experimentally. Samples were prepared in the form of water solutions with the concentrations in the range 0.01-1 mg/ml. A strong correlation of the measured THz signatures associated with valine tRNA and tyrosine tRNA was observed. These findings are consistent with the structural similarity of the two tRNAs. The calculated THz signature of the tyrosine tRNA of E. coli reproduces many features of our measured spectra, and, therefore, provides valuable new insights into bio-agent detection.

Optical characteristics of biological molecules in the terahertz gap

Terahertz Spectroscopy has been recently introduced as a promising technique for the collection of signature data in transmission spectra of biological materials including warfare agent simulants. To characterize material rather than sample, it is always desirable to obtain the materials optical properties as functions of frequency. In this work, we present results from parallel measurements of reflection and transmission spectra of biological molecules to enable detailed and direct calculation of refractive index and absorption coefficient spectra in the terahertz gap. DNA samples from herring and salmon as well as samples of Ovalbumin and Bacillus Subtillus spores have been characterized. The technique for simulation is described. Reflection spectra reveal resonance features similar to those demonstrated earlier for transmission, thereby affirming molecular vibrational modes in biological materials. The dispersion of refractive index and absorption coefficient is demonstrated within the Terahertz gap of 10 cm-1 to 25 cm-1.

Reliability Analysis of THz Characterization of Modified and Unmodified Vector Sequences

As part of a broad study to survey and parse the fundamental spectroscopic features of double stranded DNA in the THz domain, we have performed a comparative study of a 2900 bp basic sequence, 3200 bp basic sequence with inserts, and segments of the basic sequence of 1000 and 500 bp over the range 10-25 cm-1. Samples were generated by amplifying regions of the firefly luciferase gene modified and unmodified pGEM vector sequences using the polymerase chain reaction. Rich spectra have been obtained from each type of sample at varying concentrations. Major features appear to be relatively unaffected by concentration, over the range of concentrations studied. The effect of inserts, filtering, and length of DNA was shown to be distinguishable. One of the spectra of each length was deconvoluted into about 25 separated bands.

Low-terahertz spectroscopy of liquid water

In this work we present the results on combined experimental and computational study of sub-THz spectra of liquid water. The important new result is the detection of hydrogen bonds in liquid water. The experimental study was performed by employing Fourier Transform terahertz (THz) spectroscopy with spectral resolution of 0.25 cm-1. Resonance features in transmission spectra of water layers between thin film substrates are demonstrated in the sub-THz range. The theoretical approach for computer simulation of THz absorption spectra from liquid water is also discussed. The molecular dynamical (MD) simulations of water were performed using Amber 8 and the TIP3P, SPCE (Extended Single Point Charge) and TIP4P water models. Several examples of modeling results are presented. The experimental spectra are compared with the theoretical predictions. The SPCE model better correlates with experimental spectra compare to two other models.

THz absorption signature detection of genetic material of E. coli and B. subtilis

The development of efficient biological agent detection techniques requires in-depth understanding of THz absorption spectral features of different cell components. Chromosomal DNA, RNAs, proteins, bacterial cell wall, proteinaceous coat might be essential for bacterial cells and spores THz signature. As a first step, the DNAs contribution into entire cell THz spectra was analyzed. The experimental study of cells and DNAs of E. coli and cells/spores and DNA of Bacillus subtilis was conducted. Samples were prepared in the form of water solutions (suspension) with the concentrations in the range 0.01-1 mg/ml. The measurable difference in the THz transmission spectra of E. coli and Bacillus subtilis DNAs was observed. The correlation between chromosomal DNA signature and a corresponding entire spore/cell signature was observed. This correlation was especially pronounced for spores of Bacillus subtilis and their DNA. These experimental results justify our approach to develop a model for THz signatures of biological simulants and agents. In parallel with the experimental study, for the first time, the computer modeling and simulation of chromosome DNAs of E. coli and Bacillus subtilis was performed and their THz signatures were calculated. The DNA structures were optimized using the Amber software package. Also, we developed the initial model of the DNA fragment poly(dAT)-poly(dTA) solvated in water to be used in the simulations of genetic material (DNA and RNA) of spores and cells. Molecular dynamical simulations were conducted using explicit solvent (3-point TIP3P water) and implicit solvent (generalized Born) models. The calculated THz signatures of E. coli and Bacillus subtilis DNAs and poly(dAT)-poly(dTA) reproduce many features of our measured spectra. The results of this study demonstrate that THz Fourier transform infrared spectroscopy is a promising tool in generating spectral data for complex biological objects such as bacterial cells and spores.