Sub-Terahertz (THz) vibrational spectroscopy is an emerging technique for fingerprinting biological molecules and species. Very recently the frequency and wavelength domains shown below were named the “Terahertz Gap” – so little was known about sub-THz.


Sub-THz radiation has the unique ability to excite low-frequency internal molecular vibrations involving atomic subgroups that are connected by the weakest bonds, including hydrogen bonds and/or non-bonded interactions. These internal motions are sensitive to molecular composition and topology, have an impact on the main processes related to the transfer of genetic information and eventually can give information regarding the flexibility of biological macromolecules and their biological functions. Experimental results from sub-THz and THz spectroscopy demonstrated numerical resonances that are associated with low frequency vibrational modes in qualitative agreement with the theoretical predictions.
Sub-Terahertz (THz) Vibrational Spectroscopy of Biological Molecules and Cells
Bacteria and spores are complex biological objects. Since the absorption of bio materials in the THz range is not very strong (absorption coefficient ~ 10-100 cm-1), the radiation propagates through the entire object, allowing genetic material (DNA and RNA), proteins, and other molecular components to contribute to the THz signature of bacterial cell/spore. THz vibrational spectroscopy technique for identifying and characterizing biological objects is based on the specificity of their absorption spectra that is manifested as resonances at characteristic frequencies.

The spectral range below 1 THz (sub-THz) is especially attractive for practical applications because there is low disturbance from liquid water and water vapor. Sensors in the sub-THz range do not require evacuation or purging with dry nitrogen; material in solutions can be characterized as well as solid samples. In addition, THz signatures of large macromolecules and organisms are better resolved and are more specific in the sub-THz range, and due to the uniqueness of low energy vibrations, the information provided is quite different compared to other frequencies, including IR and far-IR.
Progress in THz spectroscopy in the last several years (sample preparation techniques, repeatability and reliability of spectroscopic features, intensity of features) significantly enhanced experimental spectroscopic features, thus resulting in more reliable signatures from macromolecules and bacterial cells/spores. A big advantage is that this is an optical, label and reagent free method that does not require special sample preparation procedures.
Bacillus subtilis (BG) cells (Bruker FT66v). The spectral resolution is  0.25 cm–1 and the standard  deviation for a set of similar samples is better than 0.5%.
   Vibratess produces the major breakthrough in subTHz Spectroscopic
Sensing of Bio-materials
To be able to resolve vibrational modes from biological molecules and organisms, the spectra have to be measured with a relatively good spectral resolution that is adequate to the widths of spectral features. To date, most THz spectroscopy has been done at frequencies above 1 THz, for relatively small biomolecules. Full exploitation of THz vibrational spectroscopy is still impeded because of the absence of spectroscopic systems that simultaneously satisfy all the requirements of good spectral and spatial resolution, along with high sensitivity and room temperature operation. In addition, a simple sample preparation procedure, good reproducibility, and user-friendly operation are also important for widespread adoption of this technique.

Vibratess, LLC is a start up, women-owned small business company, orginazed in 2007 as a spin-off from the University of Virginia to develop and commercialize a novel low-THz resonance spectroscopy technology for detecting, identifying and characterizing biological macromolecules and microorganisms. Since then, a Sub-THz spectroscopic sensor with high sensitivity, good spectral resolution, and spatial resolution below diffraction limit has been developed.
Frequency domain sub-THz spectroscopic bio-sensor system (Vibratess, LLC)
This novel constant wave, frequency-domain instrument is based on a patented strong local enhancement of the electro-magnetic field, thus allowing increased coupling of the THz radiation with the sample biomaterials. The invention uses the discontinuity edge effect and the extraordinary transmission of a sub-wavelength-slit conductive structure [B. Gelmont, T. Globus, et al “Method of Local Electro-Magnetic Field Enhancement of Terahertz (THz) Radiation in Sub Wavelength Regions and Improved Coupling of Radiation to Materials through the Use of the Discontinuity Edge Effect”, Patent No. 8,309,930 issued November 26. 2012].

Absorption spectrum of protein thioredoxin  from E. coli: MD simulation and experimental results as measured using Vibratess’ spectroscopic sensor.
For scientific applications, our new instrument opens opportunities for better understanding the physics of interaction between THz radiation and bio material and time scales of intramolecular dynamics relaxation processes. Sub-Thz spectroscopy coupled with theoretical prediction can become a powerful tool for the investigation of the molecular structure and possible biological function.
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Vibratess has a strong theoretical group. It focuses on two aspects: 1. Using molecular dynamics (MD) and other computational tools to predict, to verify and confirm experimental signatures from known and unknown biological objects. We have significantly improved the predictive capability of MD simulation for proteins and developed a novel approach for a computational modeling of THz vibrational spectra from biological macromolecules. In parallel with experimental characterization, simulated absorption spectra have been received for DNA molecules from different strains of E.coli, from protein Thioredoxin, and from artificial engineered molecular structures - DNA monocrystals. Vibratess has clearly demonstrated that the frequencies of resonances and absorption spectra patterns for different biomaterials are unique. Comparison between simulated and experimental spectra enhances understanding of the physics of intramolecular atomic motions and dynamics relaxation processes in biological macromolecules. 2. Optimizing experimental setups to improve interaction between THz radiation and biological materials, to more effectively collect and transfer the information.
Validation: Computational Modeling
 Continuing Development
Vibratess is constantly working on refinements and improvements, including customized biosensors, designed for specific wavelengths and bio-targets. Additionally, we are developing a novel, inexpensive microfluidic chip with multiple ports to allow precise control of the sample, as well as the introduction of reagents. The fluid can be blood, to detect cancer cells and signaling molecules, for example, or an environmental water sample. Ultimately, our goal is to reduce the system to a portable, field-ready version. Vibratess also continues experiments with new biomaterials. Most recently, Vibratess successfully identifies the unique spectroscopic signature or “fingerprint” of ovarian cancer cells in cell culture. The system can detect and identify a single cancer cell by interrogation of specific resonances caused by intra-molecular motions within the cell. With the addition of the novel microfluidic sample device that is currently in development, the system can identify individual cells in liquids, detect signaling molecules circulating in blood, and assess therapeutic responses in cancer or other targeted cells. The multi-port device is also expected to expedite testing and development of treatment modalities. Current work is aimed at identifying signatures for normal vs. malignant cells.
Advantages
The Vibratress system achieves good spectral resolution, spatial resolution below diffraction limit, and is highly sensitive. It requires only nanograms of material for fast (approximately 10 minute) sample characterization at room temperature with reproducible results. Sample preparation is simple, with no reagents or labels. Since there is low disturbance from water or other solvents, detection in solution is possible. Vibratess offers a sturdy system that is relatively small and inexpensive and operates with user-friendly software. The system, unlike others, has actual experimental results to demonstrate its capabilities. The first patent has issued on this technology, which has been further validated by continuing government support; two more patents are pending. Vibratess carefully manages its intellectual property portfolio, which includes a high level of expertise.
Potential Applications
The Vibratess system has broad applications:
1) Creating reference libraries of pathogens, biohazards, diseased cells, contaminants and so on for rapid field and lab testing. Faster identification means a faster response time.
2) Providing an in-house tool for hospitals to rapidly analyze bio-cell and tissue to screen, diagnose, and evaluate disease states, such as cancer.
3) Enhancing drug discovery, biomaterial characterization and pharmokinetics studies; the multi-port device allows evaluation of a target’s response to various compounds; process control, product inspection.
4) Expediting research into cellular and biological processes, including mechanisms producing disease; real-time monitoring of biological processes – folding-unfolding, and conformational changes, for example.
5) Improving national security by the rapid detection of chemical and biological agents, and environmental contaminants.
6) Promoting research and environmental science in universities, national laboratories, and the commercial sector

 Markets
Vibratess is entering a young market. The first commercial THz spectrometers were introduced less than a decade ago. Since then, the market has grown to more than $10 million in annual sales, with more than half a dozen vendors. These systems however require relatively large amounts of material, and in most cases fail to produce sufficient spatial and spectral resolution to detect and characterize the unique fingerprints of biomaterials.

The sub-THz vibrational spectroscope is an advanced, unique diagnostic, screening and research tool. The overall goal for Vibratess is to have its spectroscopic sensor as ubiquitous and taken for granted as a microscope. This system will provide unique possibilities in detection, separation, classification and identification applications. The system is a stand-alone product, but Vibratess also is developing and refining its component parts, such as biosensors and sample holders-chips, to sell as customized devices that interface with existing THz spectroscopy systems.
      Oncology:  Vibratess brings a vital new tool for diagnosis, prognosis, treatment, and early detection. The timing is just right, since cancer is being studied at the molecular level to find diagnostic and therapeutic targets. The annual global market for next-generation cancer diagnostics was $776 million in 2010 and growing at a compound annual growth rate of 47%.
      Antibiotics: Antibiotics generate sales of over $42 billion and grow at about 4% a year. A single drug family, cephalosporin, makes up $11.9 billion of that, with 28% of the market. However, as patents expire and antibiotic resistance becomes an increasing problem, there is a trend to first identify pathogens with lab tests instead of routinely prescribing antibiotics based on symptoms. Antibiotic-resistant organisms have led to a demand for new types of antibiotics that more precisely target pathogens and reduce side effects. Presently, this is laborious and uncertain work. Vibratess offers more rapid diagnosis and a new technique to study targets and evaluate the activity of candidate therapeutic compounds.


      National Security: Detection of biothreats is a high priority. Vibratess has the potential to identify pathogens in minutes, rather than days, as is standard now. Recently ARO and ECBC have launched a multi-disciplinary research program under the support of the U.S. Defense Threat Reduction Agency (DTRA) that seeks to develop new devices that can obtain THz signatures from target bio-molecules. These government agencies and national laboratories will be priority customers for Vibratess.
      Water-borne and other contaminants: The Centers for Disease Control (CDC) has created a special agency to address the global problem of prevention and control of diseases caused by zoonotic, vector-borne, food-borne, water-borne, mycotic, and related infections. Testing household water and bodies of water in the environment must be rapid and reliable. Appropriate detection methods are also needed to obtain clinical and environmental samples from the air and ground to detect parasitic and other contaminants.
The room temperature test results, requiring only nanograms of material with no labels or reagents, demonstrate spectral resolution of at least 1 GHz or 0.03 cm-1, and increased sensitivity by an order of magnitude compared to a commercial Bruker FTIR spectrometer operating with the liquid helium cooled bolometer (1.7 K). Highly resolved signatures from biological materials have been measured. The results demonstrate multiple, very intense and narrow spectral features from biological molecules and bacteria with a width between 0.05 and 0.2 cm-1.

Improved spectral resolution enables higher discriminative capability of sub-THz vibrational spectroscopy. The results confirmed that different strains of the same bacteria can be detected and discriminated. The highly sensitive system that utilizes label and reagent free vibrational resonance technology has also demonstrated sufficient resolution to detect and identify a single cancer cell.

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