Recently we introduced a Sub-THz spectroscopic system for characterizing vibrational resonance features from
biological materials. This new, continuous-wave, frequency-domain spectroscopic sensor operates at room temperature
between 315 and 480 GHz with spectral resolution of at least 1 GHz and utilizes the source and detector components
from Virginia Diode, Inc. In this work we present experimental results and interpretation of spectroscopic signatures
from bacterial cells and their biological macromolecule structural components. Transmission and absorption spectra of
the bacterial protein thioredoxin, DNA and lyophilized cells of <i>Escherichia coli (E. coli)</i>, as well as spores of <i>Bacillus
subtillis</i> and <i>B. atrophaeus</i> have been characterized. Experimental results for biomolecules are compared with absorption
spectra calculated using molecular dynamics simulation, and confirm the underlying physics for resonance spectroscopy
based on interactions between THz radiation and vibrational modes or groups of modes of atomic motions. Such
interactions result in multiple intense and narrow specific resonances in transmission/absorption spectra from nano-gram
samples with spectral line widths as small as 3 GHz. The results of this study indicate diverse relaxation dynamic
mechanisms relevant to sub-THz vibrational spectroscopy, including long-lasting processes. We demonstrate that high
sensitivity in resolved specific absorption fingerprints provides conditions for reliable detection, identification and
discrimination capability, to the level of strains of the same bacteria, and for monitoring interactions between
biomaterials and reagents in near real-time. Additionally, it creates the basis for the development of new types of
advanced biological sensors through integrating the developed system with a microfluidic platform for biomaterial
Radiation in the Terahertz frequency range interacts with vibrations in the weakest molecular couplings such as hydrogen bonding, van der Waals forces, and hydrophobic interactions. The work presented demonstrates our efforts towards the development of a microfluidic device as the sample cell for presenting liquid samples within the detection region of a novel sub-THz spectrometer. The continuous-wave, frequency-domain spectrometer, operating at room temperature between 315 and 480 GHz with spectral resolution of 0.3 GHz, already demonstrated highly intense and specific signatures from nanogram samples of dry biological molecules and whole bacterial cells. The very low absorption by water in this sample cell will allow for the use of liquid samples to present cells and molecules in their natural environment. The microfluidic device design utilizes a set of channels formed with metal sidewalls to enhance the interaction between the THz radiation and the sample, increasing the sensitivity of the system. Combined with near field effects, through use of a detection probe close to the surface of the sample cell, spatial resolution less than the diffraction limit can be achieved, further reducing the amount of sample required for analysis. This work focuses on the design, and fabrication methods, which will allow implementation of the microfluidic sample cell device within the THz spectrometer. The device will be utilized for characterization of different cell types, showing that THz interrogation of liquid samples is possible.
We investigated resonance spectroscopic features from several widely used explosives materials including RDX and
PETN in the low THz range with the goal of understanding the mechanism of interaction between radiation and material
in the form of solid films, gels and dilute solutions (suspensions). FTIR spectroscopy was used to measure spectra in
transmission and reflection modes. We demonstrated that very small amount of material with a simple sample
preparation technique can be used still providing very accurate results. Spectral features are specific not only for main
ingredients but for modifications with different plasticizers. The consistency of results for different amount of material
was observed. Computational modeling confirmed the lowest frequency modes.
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<sup>-1</sup>. 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
This paper reports on the THz frequency characterization of highly diluted mixtures of Bacillus Subtilis (BG) cells and spores pressed into pellets with high densities of polyethylene (PE) micro-powders. This technique of forming matrices of sparsely distributed biological (bio) materials by mixing them into larger concentrations of PE is one that has been previously applied to study spectral signature phenomenology. In particular, previously results have suggested that isolating the microscopic bio-particles leads to an enhancement of the THz signatures - i.e., yielding sharper and stronger absorption resonances. However, it is important to minimize the influence of etalon effects because they can introduce sharp artificial fringes in the transmission spectra that obscure the underlying THz signatures. This paper will present results from optimally prepared PE matrices containing reduced concentrations of BG cells and spores. Here, the BG-PE matrices are of thickness of approximately 30 micrometers and contain BG concentrations from 0.1 to 0.3 %. As will be shown, these thinner layers allow for the more accurate characterization of smaller amounts of bio samples and reveal new information on the THz signatures of BG cells and spores. In particular these results show very close correlations between the spectra of BG cells and spores and suggest both contain common genetic components. These studies provide new information that will be useful in the future development of THz-based sensors.
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 <i>E. coli</i> 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 <i>E. coli </i>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 <i>E. coli</i> reproduces many features of our measured spectra, and, therefore, provides valuable new insights into
Only few studies have attempted to characterize biological materials by THz spectroscopy. Most of these used either solid samples or biological tissues. In this work, we present results of THz spectroscopic characterization of dilute solutions of DNA samples. Water and heavy water (D<sub>2</sub>O) have strong absorbance that overlap significantly with important absorption bands of biomolecules in conventional FTIR spectroscopy. Cumbersome spectral subtractions and highly concentrated samples are therefore required to partially overcome problems of water interference in FTIR spectra of biomolecules. Although liquid water absorbs and contributes to
background in the THz spectral range of interest, the level of water absorption in the low THz range is at least 2.5 orders of magnitude less than in the far IR. Here, we demonstrate that reproducible spectra of dilute solutions of DNA in the frequency range 10-24 cm<sup>-1</sup> can be obtained. We show that dilute aqueous samples of DNA produce THz spectra with signals that do not overlap with those of water. This is a significant achievement towards the goal of developing THz resonance spectroscopy as a useful tool for the biological sciences because all biological functions of DNA and proteins take place in aqueous environments. A simple technique for sample preparation and characterization is described. Samples containing as little as 100 ng of DNA in 10 μl of water (0.01 mg/ml or 0.001%) have been prepared and measured. The signal/noise ratios of THz spectra of these samples are sufficient to detect reproducible resonances at several characteristic frequencies. The effect of orientation on different substrates and the mechanism of sensitivity enhancement in these samples is discussed. It should be possible to extend these methods to also study proteins in dilute solutions. Advantages of using dilute samples include small quantities of biological material required, the absence of interference from interactions between neighboring molecules, and the absence of problems with light-scattering that are often encountered with short wave-length optical techniques.
A new concept is presented for realizing midwave-infrared vertical- cavity lasers based on an interband-resonant-tunneling-diode (I-RTD). Model equations are derived in terms of material and structure parameters for predicting the output power in an I-RTD laser device constructed of InAs/AlGaSb layers. Simulation made for midwave- infrared lasers suggest that the radiation output density power in this I-RTD laser can be achieved of the order of 40 W/cm<sup>2</sup>.
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 DNA's contribution into entire cell THz spectra was analyzed.
The experimental study of cells and DNAs of <i>E. coli</i> and cells/spores and DNA of <i>Bacillus subtilis</i> 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 <i>E. coli</i> and <i>Bacillus subtilis</i> 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 <i>Bacillus subtilis</i> 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 <i>E. coli</i> and <i>Bacillus subtilis</i> 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 <i>E. coli</i> and <i>Bacillus subtilis</i> 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.
This work demonstrates application of Fourier Transform Infrared Spectroscopy (FTIR) technique in the low terahertz frequency range of 10-25 cm<sup>-1</sup> to discriminate between different protein conformations and evaluate possible application of THz spectroscopy for monitoring of protein folding-unfolding process. A specific procedure developed earlier for unfolding lysozyme by salt (KSCN) precipitation and refolding the lysozyme molecules by removing of KSCN and dissolving in sodium acetate was used to prepare three different forms of lysozyme. In addition, two standard procedures were used to prepare samples in unfolded conformation: denaturation at high temperature ~95° C followed by fast freezing, and dissolution in 6 M guanidine. Thin, air dried protein films were characterized as well as material in the form of gel. Spectra reveal resonance features in transmission which represent vibrational modes in the protein samples. A great variability of spectral features for the different conformational states showed the sensitivity of vibrational frequencies to the three dimensional structure of proteins. The results obtained on liquid (gel) samples indicate that THz transmission spectroscopy can be used for monitoring folding-unfolding process in a realistic, aqueous environment.
Collaboration with the University of Virginia (UVa) and the University of California, Santa Barbara (UCSB) has resulted in the collection of signature data in the THz region of the spectrum for ovalbumin, Bacillus Subtilis (BG) and RNA from MS2 phage. Two independent experimental measurement systems were used to characterize the bio-simulants. Prior to our efforts, only a limited signature database existed. The goal was to evaluate a larger ensemble of biological agent simulants (BG, MS2 and ovalbumin) by measuring their THz absorption spectra. UCSB used a photomixer spectrometer and UVa a Fourier Transform spectrometer to measure absorption spectra. Each group used different sample preparation techniques and made multiple measurements to provide reliable statistics. Data processing culminated in applying proprietary algorithms to develop detection filters for each simulant. Through a covariance matrix approach, the detection filters extract signatures over regions with strong absorption and ignore regions with large signature variation (noise). The discrimination capability of these filters was also tested. The probability of detection and false alarm for each simulant was analyzed by each simulant specific filter. We analyzed a limited set of Bacillus thuringiensis (BT) data (a near neighbor to BG) and were capable of discriminating between BT and BG. The signal processing and filter construction demonstrates signature specificity and filter discrimination capabilities.
A biological(bio)-molecular inspired electronic architecture is presented that offers the potential for defining nanoscale sensor platforms with enhanced capabilities for sensing terahertz (THz) frequency bio-signatures. This architecture makes strategic use of integrated biological elements to enable communication and high-level function within densely-packed nanoelectronic systems. In particular, this architecture introduces a new paradigm for establishing hybrid Electro-THz-Optical (ETO) communication channels where the THz-frequency spectral characteristics that are uniquely associated with the embedded bio-molecules are utilized directly. Since the functionality of this architecture is built upon the spectral characteristics of bio-molecules, this immediately allows for defining new methods for enhanced sensing of THz bio-signatures. First, this integrated sensor concept greatly facilitates the collection of THz bio-signatures associated with embedded bio-molecules via interactions with the time-dependent signals propagating through the nanoelectronic circuit. Second, it leads to a new Multi-State Spectral Sensing (MS3) approach where bio-signature information can be collected from multiple metastable state conformations. This paper will also introduce a new class of prototype devices that utilize THz-sensitive bio-molecules to achieve molecular-level sensing and functionality. Here, new simulation results are presented for a class of bio-molecular components that exhibit the prescribed type of ETO characteristics required for realizing integrated sensor platforms. Most noteworthy, this research derives THz spectral bio-signatures for organic molecules that are amenable to photo-induced metastable-state conformations and establishes an initial scientific foundation and design blueprint for an enhanced THz bio-signature sensing capability.
This work presents the first demonstration of the characterization of cancer cells using Fourier Transform Infrared Spectroscopy (FTIR) technique in the low terahertz frequency range of 10-25 cm<sup>-1</sup> and shows how this technique can be used to discriminate between two types of cancer (prostate and bladder) in cell culture. Samples of cancer cells suspended in buffer solution with the ratio of dry material to liquid ~ 1:10 were prepared and measured. The work will serve as the foundation for future work defining specific spectra associated with different tumor histologies and tumor progression and metastasis.
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 material's 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<sup>-1</sup> to 25 cm<sup>-1</sup>.
This work presents spectroscopic characterization results for biological simulant materials measured in the terahertz gap. Signature data have been collected between 3 cm<sup>-1</sup> and 10 cm<sup>-1</sup> for toxin Ovalbumin, bacteria Erwinia herbicola, Bacillus Subtilis lyophilized cells and RNA MS2 phage, BioGene. Measurements were conducted on a modified Bruker FTIR spectrometer equipped with the noise source developed in the NRAL. The noise source provides two orders of magnitude higher power in comparison with a conventional mercury lamp. Photometric characterization of the instrument performance demonstrates that the expected error for sample characterization inside the interval from 3 to 9.5 cm<sup>-1</sup> is less then 1%.
Significant progress has been achieved during the last several years relating to experimental and theoretical aspects of Terahertz (or Sub-millimeter wave) Fourier transform spectroscopy of biological macromolecules. Multiple resonance due to low frequency vibrational modes within biological macromolecules have been unambiguously
demonstrated. However till now only solid films of bio-materials have been used for experimental characterization in this spectral range since it was common opinion that high water absorption will prevent from receiving the information on bio-molecules in a liquid phase. At the same time, all biological function of DNA and proteins take place in water solutions. In this work spectra of DNA samples and proteins have been measured in liquid phase (gel) in a spectral range
10-25 cm<sup>-1</sup> and compared with spectra obtained from solid films. The results demonstrate that there is almost no interference between spectral features of material in test and water background except for the band around 18.6 cm<sup>-1</sup>. Much higher level of sensitivity and higher sharpness of vibrational modes in liquid environment in comparison with solid phase is observed with the width of spectral lines 0.3-0.5 cm<sup>-1</sup>. Gel samples demonstrate effects of polarization. The ability of THz spectroscopy to characterize samples in liquid phase could be very important since it permits to look at DNA interactions, protein-protein interactions in real (wet) samples. One demonstrated example of practical
importance is the ability to discriminate between spectral patterns for native and denaturated DNA.
Low-frequency phonon modes of DNA and RNA molecules can serve as a signature of their structure, flexibility and, hence, their biological function. To investigate the relationship between RNA structure and far IR absorption spectra, we performed FTIR measurements on RNA molecules with known sequence in the spectral range from 10 cm<SUP>-1</SUP> to 25 cm<SUP>-1</SUP> and calculated their internal vibrations. To understand which phonon modes are determined by a double helical topology of nucleic acids, we compared the spectra of single stranded and double stranded RNA molecules. Homopolymers PolyA, polyU, polyC, and polyG, and double stranded homopolymers PolyA-polyU and polyC-polyG were investigated. Theoretical conformational analysis of the double stranded RNA molecules was performed and utilized to calculate the low-frequency vibrational modes. Conformational energy was minimized in the space of internal coordinates of a molecule using standard A-helical topology as an initial approximation. Normal modes were calculated as eigenfrequencies and eigenvectors of the matrix of energy second derivatives. Oscillator strengths were calculated for all the vibrational modes in order to evaluate their weight in the absorption spectrum of a molecule. The obtained phonon modes were convoluted to derive the far IR spectrum of a molecule. These predicted spectra were compared to those obtained by FTIR spectroscopy. Our results confirm that very far IR absorption spectra of biopolymers reflect specific dynamical properties resulting from their structure and topology and, therefore, can be used as fingerprints for specific molecules.
Sensitive and robust heterodyne mixers are needed for future atmospheric remote sensing missions. This data from satellites such as NASA's Earth Observing System (EOS) lends great insight into molecular interactions in our environment. The Microwave Limb Sounder (MLS) on EOS will detect radiation emitted from 0<SUB>3</SUB>, ClO, and OH molecules which are critical to our understanding of ozone depletion and greenhouse warming. The heterodyne mixers on MLS must exhibit sufficient spectral sensitivity, wide bandwidth, low noise, and minimal LO power requirements. Planar GaAs Schottky diodes currently are the most promising technology for space-borne radiometers where cryogenic cooling is not desirable. In this work we present progress on a novel wafer bonding technology, MASTER, used to integrate submillimeter wavelength planar GaAs Schottky mixer diodes with quartz microstrip circuitry. Problems associated with wafer expansion after bonding, open- circuited devices, and Ti/Pt/Au metallization removal have been solved and device yield is significantly improved. FTIR measurements of the bonding adhesive's properties at submillimeter wavelengths are discussed. We have fabricated 640 GHz subharmonic mixers for EOS-MLS which nearly match state-of-the-art performance at this frequency with DSB T<SUB>mix</SUB> equals 2396 K and L<SUB>mix</SUB> equals 10.98 dB using 4.67 mW of LO power. RF testing of a new higher yield batch of MASTER mixers is in progress.