Abstract:
A method and apparatus of detecting biological molecules, the method including the steps of: performing Terahertz (THz) absorption spectroscopy, performed in a first frequency range of 0.2 to 2.2 THz (10-79.2 cm−1), on at least one sample including a substance comprising the biological molecules, the substance being selected from at least one of tryptophan, albumin bovine, DNA, nucleotides, bacillus subtilis, spore, and DPA; calculating a frequency-dependent absorption value of biological molecules; performing THz absorption spectroscopy on at least one reference substance; detecting the substance through the frequency-dependent absorption value by comparison of absorption peaks; and outputting information proving existence of the substance in the sample. The method further creates a library of known THz frequency modes on spectra to identify the presence of unknown substance in biological and chemical composite media.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is related to U.S. Provisional Patent Application Ser. No. 60/463,354 filed Apr. 17, 2004, the contents of which are incorporated herein by reference. 
     
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The present invention relates generally to Terahertz (THz) absorption spectroscopy and more specifically to a simple, cost effective, high-performance method of detection of biological molecules using THz absorption spectroscopy. 
         [0004]    2. Description of Related Art 
         [0005]    Low frequency collective vibration modes of biological molecules in proteins, DNA, virus, and bacteria can provide information about type of bio-molecules present in these substances and their conformational state. Low frequency collective vibration modes are associated with collective motion of the subunits of molecules moving with respect to one another or coherent movement of a portion of a structural subunit. Similar motions are associated with conformational movements that occur during ligand binding and are critical to protein function, folding, isomerization and riboswitches. 
         [0006]    Terahertz (THz) spectroscopy offers a new tool to probe for the presence of biological molecules in an area. As described in A. G. Markelz, A. Roitherg and E. J. Heilweil, “Pulsed Terahertz Spectroscopy Of DNA, Bovine Serum Albumin And Collagen Between 0.1 And 2.0 THz.” Chem. Phys. Lett., (2000) 320, 42-48, which is incorporated herein by reference, (hereinafter “Markelz”), E. W. Prohofsky and collaborators have predicated the helix, base twisting, and librational modes of DNA in the 20-100 cm −1  range. 
         [0007]    As described in R. Nossal and H. Lecar, “Molecular And Cell Biophysics”, 1 st  edition (1991) Addison-Wesley, Redwood City, Calif.; W. Zhuang, Y. Feng and E. W. Prohofsky, “Predication Of Modes With Dominant Base Roll And Propeller Twist In B-DNA Poly (dA)-Poly (dT)” (1990) Phys. Rev. A41 7020-7023; and L. Young, V. V. Prabhu, E. W. Prohofsky, “Calculation Of Temperature Dependence Of Interbase Breathing Motion Of A Guanine-Cytosine DNA Double Helix With Adenima Thymine Insert” (1991) Phys. Rev. A41, 1049-1053, all of which are incorporated herein by reference, proteins are close-packed structures where changes in the arrangement of subunits in the protein take place due to photo-isomerization and enzyme actions on a sample. A conformational transition from one structure to another involves these collective modes. 
         [0008]    As discussed in Austin, R. H., Hong, M. K., Moser, C., and J. Plombon. “Far-Infrared Perturbation Of Electron Tunneling In Reaction Centers.” (1991) Chem. Phys. 158: 473-486, which is incorporated herein by reference, molecules excited up the vibrational ladder can cross the transitional energy barrier. The dynamics of the collective modes generally occur via anharmonic interactions with other normal molecular modes leading to energy exchange. According to Austin, R., Roberson, M., and P. Mansky, “Far-Infrared Perturbation Of Reaction Rates In Myoglobin At Low Temperature.” (1989) Phys. Rev. Lett., 62: 1912-1915 and Xie, A. Meer, Alexander F. G Van Der, and Robert H. Austin. “Excited-State Lifetimes Of Far-Infrared Collective Modes In Proteins.” (2002) Phys. Rev. Lett. 88: 018102-4, which are incorporated herein by reference, it is believed that the low-frequency collective modes are responsible for the directed flow of conformational energy for a variety of biological processes ranging from primary photoisomerization events of vision to enzyme action. The motions of molecular sub-units within proteins are associated with different functions. These processes involve well-defined torsional modes along one of the C═C bonds of the polyene chain. The THz region offers a way to detect these biological molecules like in the visible, UV, and infrared region. 
         [0009]    Far-infrared (FIR), in the range from 10μ to 1000μ, studies of materials have been limited due to weak sources and low signal to noise ratios, especially below 100 cm −1 . Pulsed THz time-domain spectroscopy (TDS) can be used to overcome these difficulties and have become a versatile tool for spectroscopy in FIR. Both picosecond (ps) and femtosecond (fs) time probes as well as THz frequency can be used. The THz technique has been applied to examine motion of DNA and other bio-molecules (see Markelz) for studies, not detection of the DNA. 
         [0010]    What is needed is a method for separating molecules along the main THz absorption lines of biological molecules, where THz can be used as fingerprints to distinguish different bio-molecules and detect the presence of these bio-molecules, such as bacteria and virus, in a given area using infrared radiation. 
     
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0011]    The foregoing and other objects, aspects, and advantages of the present invention will be better understood from the following detailed description of preferred embodiments of the invention with reference to the accompanying drawings that include the following: 
           [0012]      FIG. 1  is a schematic diagram of a Terahertz time-domain spectrometer; 
           [0013]      FIG. 2  is a graph of measured Terahertz temporal profiles for a polyethylene substrate alone and a graph of measured Terahertz temporal profiles for a tryptophan film on a polyethylene substrate; 
           [0014]      FIG. 3  is a graph of power spectra of the polyethylene substrate alone and the power spectra of the tryptophan film on the polyethylene substrate illustrated in  FIG. 2 , in which the insert is a graph showing logarithmic dependence of the power spectra on frequency (v); 
           [0015]      FIG. 4  is a graph of absorbance of the tryptophan film, in which the dash line indicates a fit to the absorption data according to a sum of six Lorentzian oscillators; 
           [0016]      FIG. 5  is a graph of the Terahertz absorption spectrum of bacillus subtilis; 
           [0017]      FIG. 6  is a graph of the Terahertz absorption spectrum of spore; and 
           [0018]      FIG. 7  is a graph of the Terahertz absorption spectrum of albumin bovine. 
         SUMMARY OF THE INVENTION 
         [0019]    The invention describes a method of detecting biological molecules, comprising the steps of: performing Terahertz (THz) absorption spectroscopy, performed in a first frequency range of 0.2 to 30 THz (10-79.2 cm −1 ), on at least one sample including a substance comprising the biological molecules, the substance being selected from at least one of tryptophan, albumin bovine, DNA, RNA, nucleotides, bases, bacillus subtilis, spore, and dipicolinic acid (DPA), viruses, proteins, amino acids; calculating a frequency-dependent absorption value of biological molecules; performing THz absorption spectroscopy on at least one reference substance; detecting the substance through the frequency-dependent absorption value by comparison of absorption peaks; and outputting information proving existence of the substance in the sample. According to the present invention, a library of modes of biological and chemical substances and molecules in THz range frequency is developed by collecting absorption specters of all substances to be tested. 
           [0020]    The presence of the biological molecules in the substance of the at least one sample is detected by comparison of electrical signal specters of the sample and the reference, where there is the presence of pre-determined absorption lines in the electrical signal specter of the sample. The electrical signals are created by conversion from optical signals in a balance detector. The converted electrical signals are intense and have a signal to noise ratio of 5000 to 1 over a large THz bandwidth. 
           [0021]    The described absorption spectroscopy is frequency-dependent and is obtained from a mode-locked Ti:Sapphire amplifier system providing pulses greater than 90-fs, for example 200-fs pulses at a wavelength of 800 nm, with a repetition rate of 250 kHz. The amplifier system produces a strong THz pulse radiation by using optical rectification in a nonlinear medium, e.g., a ZnTe Crystal via χ (2) . Other lasers can be used, for example a Cr 4+  Forsterite laser operating in 1150 nm-1300 nm and a Cr 4+  YAG laser operating in 1300 nm-1600 nm range to produce THz radiation for developing a spectrometer unit using optical rectification and/or optical switching. 
           [0022]    The first frequency range covers collective vibrational and torsional modes occurring in the sample substance to measure absorption peaks. The low frequency of the first frequency range is responsible for directed flow of conformational energy for a variety of biological motions. A second frequency range covers torsional modes along one of the C═C bands of the chain and other groups including C—H, C—N, H—O, D-O, CH 3 , C—S, CH 2 , CO, OO, and HO. 
           [0023]    The invention describes measuring the absorption spectrum of biological-molecules, such as tryptophan, albumin bovine, bacillus subtilis, spore, and DPA, in the range from 0.2 to 2.2 THz (10-79.2 cm −1 ). The THz absorption lines are used as characteristics to distinguish different biological-molecules, such as bacteria and viruses, and also to detect the existence of the biological-molecules, visual pigments, photosynthesis molecules, bacteriochlorophyll and bacteriorhodopsin. The visual pigments may include rhodopsin, photosysthesis molecules, and chromophores such as bacteriorhodopsin and bacteriochlorophyl. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0024]    The invention describes a method of using Terahertz (THz) spectroscopy for detecting biological molecules in a substance, such as bacteria. The THz absorption spectra of biological molecules, such as tryptophan, albumin bovine and bacteria, e.g., bacillus subtilis, spore, and dipicolinic acid (DPA) have been found in the range from 0.2 to 3 THz (10-99 cm −1 ). Different absorption lines were found for different biological molecules. These THz absorption lines caused by the torsional and rotational motion of molecules, can be used to distinguish biological molecules. 
         [0025]    A THz time-domain spectroscopy (TDS) system  10  for measurement collection is shown in  FIG. 1 . A mode-locked Titanium-sapphire or Ti:sapphire laser (“Light Amplification by Stimulated Emission of Radiation”)  102  emits near-infrared light (light is a form of electromagnetic radiation). Infrared radiation is electromagnetic radiation of a wavelength longer than visible light, but shorter than microwave radiation. The Ti:sapphire laser  102  is tunable in the range from 750 nm to 1,100 nm. Titanium-sapphire refers to a lasing medium, a crystal of sapphire (Al 2 O 3 ) that is doped with titanium ions. Ti:sapphire lasers operate most effectively at a wavelength of 800 nm. Ti:sapphire amplifier system  102  provides 200-fs pulses at a wavelength of 800 nm with a repetition rate of 250 kHz. Femtosecond (fs) is a very small unit of time equal to one million billionth of a second, e.g., 1 fs=10 −15  s. 
         [0026]    A biological and chemical sample  132  is positioned between an emitter crystal  128  and a detector crystal  136 . The THz-TDS system  10 , makes and collects the measurements of the THz absorption spectra of biological and chemical sample  132 ; these measurements are made by THz time-domain spectroscopy at room temperature. The THz-TDS system  10  is enclosed in dry nitrogen purged boxes (not shown) to diminish the Tera absorption due to ambient humidity. Because the THz possesses superior penetration over other materials, the sample  132  is deposited on a cell made of polyethylene substrate (not shown). The thickness of the polyethylene substrate is selected to be at least 4 mm to avoid interference from multiple reflections from the two layers of the cell substrate. 
         [0027]    The Ti:sapphire laser  102  sends a beam of light  104  to a wedge beam splitter  106 , which splits the light beam  104  in to a main beam  108 , including up to 90% of the original light beam, and a control beam  110 . Using pulses  108  and  110  of different optical duration ranging from a picosecond (ps), which is a very small unit of time equal to one trillionth i.e., one million millionth, of a second, e.g., 1 ps=10 −12  s, to fs described above, can produce Far-Infrared (FIR) radiation in χ (2)  material. For all of the components of the original light beam  104  to reach a zinc tellurium (ZnTe) detector crystal  136  at the same time, the control beam  110  is time delayed by being directed to a time delay prism  112 . Using a mirror  114 , the control beam  110  is directed through a lens  116  and a polarizer  118  to meet up with the main beam  108  reaching a parabolic mirror  134 . 
         [0028]    The path of the main beam  108  is redirected by mirrors  120  and  122 , which are positioned in a manner as to allow the direction of the main beam  108  to be parallel to the direction of the original light beam  104 . It is understood by those skilled in the art that the path of the beams described with reference to  FIG. 1  is for illustrative purposes only. Any path of the beams  104 ,  108 , and  110 , leading to results described herein below is acceptable. 
         [0029]    After path correction performed by the mirrors  120  and  122 , the main beam  108  passes through a beam chopper  124 , where the beam is modulated, a lens  126 , and a ZnTe emitter crystal  128 . Transition through the ZnTe crystal  128  produces THz radiation by optical rectification in a nonlinear medium, namely ZnTe via χ (2)  material. The electric field of the THz pulses  131  is reflected by a parabolic mirror  130  and passes through a sample of material  132  for which a specter is being graphed. After passing through the sample material  132 , the electric field of the THz pulses  133  is collected by a parabolic mirror  134  and is united with the control beam  110  to result in a collected beam  135 . 
         [0030]    The collected beam  135  is detected in a second ZnTe crystal  136  via electro-optic sampling described below. The collected beam  135  passes through a cross polarizer  138 , a quarter wave plate  140 , and finally a Wollaston prism  142 . The Wollaston prism consists of two orthogonal prisms, whose optical axes lie perpendicularly to each other and perpendicular to the direction of propagation of the incident light, in the present example collected beam  135 . Light striking the surface of incidence at right angles is refracted in the first prism into an ordinary (O) ray and an extraordinary (A) ray. 
         [0031]    A balanced detector  144  detects both rays and performs the optical rectification in a nonlinear medium and the electro-optic sampling, which is discussed in Wu, Q., Litz, M., and X. C. Zhang, “Broadband Detection Capability of ZnTe Electro-Optic Field Detectors.” (1996), Appl. Phys. Lett., 68: 2924-2926, incorporated herein by reference, (hereinafter referred to as “Wu”) and Yu, B. L., and R. Alfano. “Probing Dielectric Relaxation Properties of Liquid CS2 With Terahertz Spectroscopy. (2003) Appl. Phys. Lett. (to be published), incorporated herein by reference, (hereinafter referred to as “Yu”). 
         [0032]    The electrical signal measurements, converted from the optical by the balanced detector  144 , are measured by a lock-in device  146  and are stored and displayed on a computing device  148  having a video and audio display, a printer, and networking capabilities (not shown). The computing device may make audio announcements, e.g., via a speaker, and transmit the analyzed findings to other computing devices via a network, for example the Internet. 
         [0033]      FIG. 2  illustrates the electrical signal measurement specters in a graph (a), a reference graph of the THz temporal profiles after transmission through an empty polyethylene cell, and in a graph (b), the graph of THz profiles after transmission through a tryptophan film.  FIG. 3  shows power curves marked with letters (a) and (b) respectively resulting from performance of a Fourier Transform of the temporal profiles of graphs of (a) and (b) of  FIG. 2  for both the substrate and the deposition of tryptophan on the substrate. The frequency-dependent absorption of the sample  132  can be determined by performing the following calculation: ln(P sample /P reference ). The absorption peaks of tryptophan in the THz region from 0.2 to 2.2 THz is shown in  FIG. 4 . 
         [0034]    In another example, shown in  FIG. 5 , the THz frequency-dependent absorption of  bacillus subtilis  (used as sample  132 ) in the THz frequency region is shown. In the shown spectrum, some water vapor absorption lines such as: 1.09, 1.41, 1.60, 1.71 THz are found. Other lines, such as 1.38, 1.49 1.53, 1.88 THz are found characteristic of bacteria. 
         [0035]      FIG. 6  illustrates a frequency-dependent absorption of bacteria spore (used as sample  132 ) in the THz frequency region. In the spectrum, some lines are the same as those of bacillus subtilis of  FIG. 5  while others are different, indicating distinct characteristics. 
         [0036]      FIG. 7  illustrates a frequency-dependent absorption of protein albumin bovine (used as sample  132 ) in the THz frequency region. As can be seen, distinct characteristic lines, except for vapor lines, are seen in the spectrum. 
         [0037]    The frequency-dependent main THz absorption peaks of specific bio-molecules: L-tryptophan, protein, albumin bovine, DNA, e.g., salmon tests, nucleotide, bacteria, e.g., bacillus subtilis, spore, and dipicolinic acid (DPA) in the range of 0.2 to 2.2 THz are summarized in Table 1 below. As can be seen from the Table, the absorption peaks for different bio-molecules are different. These differences can be used as fingerprints to distinguish bio-molecules. These exact frequencies can change depending on the environment that these substances are located in and surrounded by due to polar and nonpolar environments and pH. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                 Bacteria 
                   
                 DPA 
               
               
                 L-Tryptophan 
                 ( bacillus   
                 DNA (salmon tests) 
                 (Dipicolinic acid) 
               
               
                 (THz) 
                   subtilis ) (THz) 
                 (THz) 
                 (THz) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                   
                 0.853 
                   
               
               
                   
                 1.051 
               
               
                   
                   
                 1.134 
               
               
                 1.200 
                 1.238 
               
               
                 1.435 
                   
                 1.472 
               
               
                   
                 1.538 
               
               
                   
                   
                   
                 1.622 
               
               
                 1.711 
                 1.725 
                 1.702 
               
               
                 1.842 
                   
                   
                 1.81 
               
               
                   
                 1.924 
                 1.908 
                 1.997 
               
               
                   
                 2.04 
               
               
                 2.114 
                 2.119 
                 2.178 
                 2.142 
               
               
                 2.264 
                 2.231 
                 2.231 
               
               
                   
               
             
          
         
       
     
         [0038]    While the invention has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.