Abstract:
Disclosed is a method and device for detection of  H. Pylori  in breath emissions utilizing an unlabelled urea, in which a patient ingests a safe quantity of unlabelled urea. After ingestion, expired breath of the patient is analyzed for ammonia, with a detection based on levels of ammonia lower than 50 parts per billion to 500 ppm to detect  helicobacter pylori.

Description:
PRIORITY 
       [0001]    This application claims priority to U.S. Patent application Ser. No. 60/973,066, filed Sep. 17, 2007, the contents of which are incorporated herein by reference. 
     
    
     BACKGROUND 
       [0002]    1. Field of the Invention 
         [0003]    The present invention relates generally to a medical device and protocols to facilitate diagnosis of  Helicobacter pylori  ( H. pylori ) based on administration of unlabelled urea. 
         [0004]    2. Background of the Invention 
         [0005]    Exhaled breath has long been known to enable non-invasive disease detection. Exhaled gases, such as ammonia, nitric oxide, aldehydes and ketones have been associated with kidney and liver malfunction, asthma, diabetes, cancer, and ulcers. Other exhaled compounds like ethane, butane, pentane, and carbon disulfide have been connected to abnormal neurological conditions. However, though analysis of body fluids (blood, sputum, urine) for disease diagnoses and monitoring is routine clinical practice, human breath analysis methodologies that exploit the non-invasive nature of such diagnoses are still under-developed and conventional technologies lack specificity, are excessively expensive or lack portability. 
         [0006]    Sensors have been produced to measure gases in a variety of settings, including automotive and biological applications. See U.S. Pat. No. 7,017,389 to Gouma, the contents of which are incorporated by reference, regarding detection of No x  emissions in the automotive field. Technologies for monitoring exhaled breath require complex and expensive apparatuses that are difficult to calibrate and are often not sufficiently sensitive to provide a high degree of certainty in regard to medical condition diagnosis. Such biological systems pose challenges that include sensitivity to extremely low levels of gases, presence of reducing and oxidizing gases, organic vapors (VOCs), etc. See U.S. Pat. No. 7,220,387 to Flaherty et al., the contents of which are incorporated by reference, regarding disposable sensors to measure gaseous sample analytes. 
         [0007]    A conventional apparatus disclosed by Kearney, D, et al.,  Breath Ammonia Measurement in Helicobacter pylori Infection,  Digestive Diseases and Sciences, Vol. 47, No. 11, pp. 2523-2530 (2002), provides a fiber optic device placed in the stream of expelled breath that is connected to an optical sensor for detecting whether a patient has  H. pylori  by measuring for ammonia excreted by the lungs, utilizing a hydrophobic TFE-based membrane to avoid affect of dissolved ions such as ammonia. Also see, WO 03/041565 A2 of Hubbard et al., the contents of which are incorporated by reference,  H. pylori  detection. 
         [0008]    Diagnostic tests for  H. pylori  include a) serologic testing to detect anti- H. pylori  antibodies in blood, b) upper gastrointestinal endoscopy with mucosal biopsies, c)  H. pylori  culture, including antimicrobial susceptibility testing, and d) detection of  H. pylori  antigens in stool. Serologic testing, however, cannot distinguish current from old infection. Upper gastrointestinal endoscopic biopsies are submitted for rapid urease testing or histological examination, and this approach has the drawbacks of the invasive nature of endoscopy and the suboptimal performance of histopathology.  H. Pylori  culture is invasive and cumbersome and detection of  H. pylori  antigens in stool is limited by the low acceptance of stool testing and suboptimal specificity/sensitivity. 
         [0009]    Several diagnostic tests are based on the ability of  H. pylori  to convert urea to CO2 and NH3 using its enzyme urease.  H. Pylori  produces large amounts of urease which often comprises about five percent of its total protein. Urease activity is assessed in two general ways: Biopsy-based rapid urease testing and various urea breath tests. Biopsy-based rapid urease tests require endoscopy for sample acquisition. Biopsy samples are placed in an agar gel or paper strip containing a pH indicator. In addition to requiring an invasive endoscopy, biopsy-based rapid urease tests provide a less-than-optimal test due to the time required for the diagnosis, which is 3-24 hours, a less than 90% specificity, and reduced sensitivity in children. Moreover, conventional devices, particularly point-of-care devices, are expensive, particularly to assess  H. pylori,  which discontinuously colonizes the gastroduodenal mucosa. 
         [0010]    Conventional testing is performed utilizing instrumentation that ranges from variations of mass spectrometers to IR detectors that are costly and require a trained operator. Breath sample transportation is also an issue with most conventional devices. The limited availability of instruments operable by patients and available at the point of care requires samples to be shipped to central testing facilities, adding cost and inconvenience. In regard to Urea Breath Testing (UBT) for the diagnosis of  H. pylori  infection, there are two versions of the UBT, based upon the type of urea being used as a substrate:  13 C labeled urea and  14 C labeled urea.  14 C is a radioactive isotope of carbon.  13 C is a stable, non-radioactive isotope, encountered in nature. The FDA has approved both  13 C- and  14 C-based UBTs for the diagnosis of  H. pylori,  though the  14 C-based assay is rarely used. The  14 C-based UBT is associated with exposure to radioactivity, which albeit small for an otherwise healthy adult, is nevertheless present and patients should not be exposed to it in the absence of safe alternatives. It is because of the risk associated with the radioactivity of the  14 C-urea that the  14 C-based UBT is contraindicated in pregnant women and children. See,  Using Breath Tests Wisely in a Gastroenterology Practice: An Evidence - Based Review of Indications and Pitfalls in Interpretation,  Romagnuolo, et al., Am J Gastroenterology 97:1113-1126 (2002). 
         [0011]    A further difficulty arising with the UBT is the high cost of  13 C-urea, as well as the cost and operational expenses of instruments to detect exhaled  13 CO 2 . 
         [0012]    To solve this shortcoming, the present invention departs from detection of  13 CO 2 by using unlabeled urea as a substrate, detecting ammonia in breath instead of CO 2 . The present invention provides an ammonia-specific nanosensor and provides a simple, inexpensive hand-held device for the detection of breath NH 3 . 
         [0013]    Accordingly, the present invention provides a highly accurate, economical, easy to operate, portable and sufficiently sensitive medical device for diagnosis of  H. Pylori.  The present invention departs from detection of  13 CO 2  and provides a simplified assay that uses lower cost unlabeled urea as a substrate. 
       SUMMARY OF THE INVENTION 
       [0014]    The present invention substantially solves the above shortcomings and provides at least the following advantages. 
         [0015]    The present invention obviates the need for serologic testing, for upper gastrointestinal endoscopy with mucosal biopsies for the detection of  H. pylori,  which is invasive and cumbersome, and for detection of  H. pylori  antigens in stool. 
         [0016]    In a preferred embodiment, a medical device is provided to sample breath emitted from a patient&#39;s mouth, to analyze ammonia content and the composition of a gaseous sample via contact with sensing electrodes, particularly gold substrates arranged on a TO8 substrate. 
         [0017]    Another embodiment of the present invention provides a method for using the medical device of the present invention to analyze a patient&#39;s breath sample to diagnose the presence of a medical condition, by obtaining a breath sample from a patient; analyzing volatile components of the patient sample to provide a breath profile that includes both qualitative and quantitative data; comparing the patient&#39;s breath profile to a database of breath profiles, with each database profile being characteristic of at least one medical condition, to provide information pertinent to diagnosis of the presence or absence of a medical condition. 
         [0018]    In a preferred embodiment, a single sample is used for an independent or multiple tests, which may be combined to produce a template or pattern representative of a patient&#39;s condition or representative of the presence of a particular compound or set of compounds. In a preferred embodiment high sensitivity nanomorphs of metal oxides prepared by sol-gel practices are used for a more selective and quantitatively precise analysis. 
         [0019]    In a preferred embodiment, the invention utilizes arrays of biocomposite and bio-doped films to provide a low cost, portable analyzer for detection of chemical products of biochemical reactions, such as ammonia and NO, in a real-time manner. 
     
    
     
       DETAILED DESCRIPTION OF THE FIGURES 
         [0020]    The above and other objects, features and advantages of certain exemplary embodiments of the present invention will be more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which: 
           [0021]      FIG. 1  is a schematic representation of an embodiment of a test device for the present invention; 
           [0022]      FIGS. 2   a  and  2   b  show heater and sensing electrodes utilized in  FIG. 1 ; 
           [0023]      FIGS. 3   a  and  3   b  show sensor response of the test device of  FIG. 1 ; 
           [0024]      FIGS. 4   a  and  4   b  show NH 3  sensing and sensor response when exposed only to CO 2 ; 
           [0025]      FIG. 5  shows NH 3  sensing with a CO 2  filter; and 
           [0026]      FIG. 6  provides a block diagram of an apparatus of an embodiment of the present invention. 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0027]    The below description of detailed construction of preferred embodiments provides a comprehensive understanding of exemplary embodiments of the invention. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the invention. Descriptions of well-known functions and constructions are omitted for clarity and conciseness. 
         [0028]    Analysis of breath and skin emission samples for diagnostic purposes has the advantage that the sample to be analyzed is collected from the patient in a non-invasive manner with a minimum of discomfort or inconvenience. Basic components of the medical device used for analysis in accordance with a preferred embodiment of the present invention are shown in  FIG. 1 . In preferred embodiments of the invention, breath samples are quantitatively and qualitatively processed. Notably, the sensor is tuned to detect NH 3  levels lower than 50 parts per billion (&lt;50 ppb) and as high as 500 ppm, thereby covering all NH 3  levels encountered in humans, and in particular in patients undergoing UBT. Quantitative analyzers preferably include a sensing substrate surrounded by a gold substrate surrounded by a TO8 substrate. The medical device of the present invention is preferably qualitatively used to test exhaled gas. Qualitative tests performed by the test device usable with the present invention may test carbon dioxide content, alcohol content, lipid degradation products, aromatic compounds, thio compounds, ammonia and amines or halogenated compounds. 
         [0029]    In a preferred embodiment, multiple different tests performed on a single sample may be independent, or may be the result of several tests combined to produce a template or pattern representative of a patient&#39;s condition or representative of the presence of a particular compound or set of compounds. The high sensitivity of the nanomorphs of metal oxides prepared by sol-gel practices used in the medical device of the present invention are both more selective and more quantitatively precise than similar information obtained by currently available electronic nose technology. As a result, correlating the data pattern or changes in the data pattern over time identifies a wider range of conditions or compounds. 
         [0030]    The present invention departs from detection of  13 CO 2  and provides a simplified assay that uses unlabeled urea as a substrate and detects ammonia in breath instead of CO 2  utilizing Equation (1): 
         [0000]      CO(NH 2 ) 2 +HOH-urease→CO 2 +2NH 3    (1)
 
         [0000]    In an embodiment of the present invention, a nanosensor is provided to detect breath ammonia and a simple, portable, inexpensive hand-held device is thereby provided to detect breath NH 3 . The nanosensor is tuned according to the method described below for other breath gases, and the nanosensor is in a preferred embodiment provided as a plug-in component. The sensor is constructed of a metal oxide that is not crystalline, raising sensitivity to ammonia and other gases. 
         [0031]    In  FIG. 1 , a gas sample, i.e. breath or skin emission, accesses analyzer  110  via entry and exit orifices  102  and  104 . A stainless steel chamber preferably connects the orifices to avoid absorption/distortion. Sensing electrode  122  and heater electrode  124  are positioned within the analyzer  110 . The sensing electrode  122  includes a sensor  130  having gold substrate  132 , sensing substrate  134  and TO8 substrate  136 . Heater and sensing electrodes  122  and  124  of an embodiment of the present invention are shown in  FIGS. 2   a  and  2   b.  Those of skill in the art recognize use of the TO8 substrate. Hirata et al. in U.S. Pat. No. 5,252,292, the contents of which are incorporated by reference herein, disclose a type of ammonia sensor. 
         [0032]    In the present invention, the sensing electrode  124  is selectively tuned by spin or drop coating of sensing substrates generating a film of MoO 3 . In a preferred embodiment, a gel-sol synthesis was employed to produce three-dimensional (3-D) networks of nanoparticles, with the sol-gel processing preparing a sol, gelating the sol and removing the solvent. Molybdenum trioxide (MoO3) was prepared by an alkoxide reaction with alcohol according to Equation (2): 
         [0000]      Molydenum (VI) Isopropoxide+1-Butanol→Precursor (0.1 M)   (2)
 
         [0033]    The prepared sol was spin coated and drop coated onto sensing substrates generating thin films of MoO 3 . The sensing substrates (3 mm×3 mm) were made of Al 2 O 3  and were patterned with interdigitated Pt electrodes. Pt heater electrodes were embedded on the rear of the sensor. The amorphous films were then calcined at higher temperatures generating polymorphic form. Differential scanning calorimetry confirmed the phase transformation. 
         [0034]      FIG. 3   a  shows sensor response to NH 3 , with the sensor generating a clear and measurable response to two NH 3  concentrations, 50 and 100 ppb. The measured amounts of ppb, i.e. parts per billion, are much lower than amounts typically expected in human breath, allowing for more accurate and expedited measurement and results.  FIG. 3   b  shows sensor response to various breath gases, and the specificity regarding same. Shown in  FIG. 3   b  are NH 3 , NO 2 , NO, C 3 H 6  and H 2 , gases that potentially interfere with NH 3  determination. 
         [0035]      FIG. 4   a  shows NH 3  sensing without a CO 2  filter, as gas-sensing properties of the nanosensor. As shown in  FIGS. 4   a - b,  when the sensor was exposed to various concentrations of NH 3  gas in a background mixture of N 2  and O 2  simulating ambient air, NH 3  was detected easily, down to 50 ppb, and even lower concentrations. 
         [0036]    In  FIG. 4   a,  CO 2  and NH 3 , each at 1 ppm, produce similar responses to gas pulses, shown as vertical lines in  FIG. 4   a.  Sensor response when exposed only to CO 2  gas, in the presence of the CO 2  filter, is shown in  FIG. 4   b.  The CO 2  filter completely eliminates CO 2  from the gas stream, abrogating the sensor response to it. 
         [0037]    Sensor specificity, in regard to sensing of NH 3 , was evaluated by exposing the sensor to various gases typically encountered in human breath, including NO 2 , NO, C 3 H 6 , and H 2 , each up to 490 ppm. Conductivity changes were measured in dry N 2  with 10% O 2 . At 440° C. the film was very sensitive to NH 3 , with 490 ppm increasing the conductivity by approximately a factor of 70, approximately 17 times greater than the response to the other gases. The NH 3  response, however, was relatively unaffected by 100 ppm of NO 2 , NO, C 3 H 6 , and H 2 . X-ray photoelectron spectroscopy (XPS) showed that the increased conductivity in the presence of NH 3  was accompanied by a partial reduction of the surface MoO 3 . The resistance of the films increased after extended time at elevated temperatures. 
         [0038]    CO 2  is an important component of human breath, with its concentration in expired breath reaching up to 5%. Under test conditions, CO 2  interfered with NH 3  sensing. To overcome this limitation, a commercially available CO 2  filter (NaOH premixed with Vermiculite (V-lite) used in a 10:1 ratio; Decarbite absorption tube, PW Perkins and Co) was used. Decarbite reacts only with highly acidic gases such as CO 2 , H 2 S, thus excluding the possibility of cross adsorption; and the latter was verified. Exposing the sensor to various concentrations of NH 3  and CO 2 , in the presence of N 2  and O 2 , indicated that the presence of CO 2  did not affect NH 3  sensing. This was found to be true even when the two gases were at equal concentrations ranging between 0.5 and 10 ppm. 
         [0039]    The data shown in  FIGS. 4   a - b  are from experiments with a low CO 2  concentration (1 ppm). In the present invention, the NaOH Decarbite traps CO 2  more efficiently at high CO 2  concentrations. 
         [0040]      FIG. 5  shows NH 3  sensing with a CO 2  filter. In  FIG. 5 , the sensor is exposed to NH 3  in the presence of the filter, with no interference of the measurement.  FIG. 6  shows a prototype for sensing breath, having a sensor, acquisition module, memory/computation module and displays. 
         [0041]    Combining NH 3  and CO 2  generated similar results, with the filter eliminating the experimental 1 ppm of CO 2  in the gas stream. Even at low concentrations, interference by CO 2  is eliminated. Operation of the apparatus of the present invention is preferably based on sensor response modifying electrical resistance. That is, the MoO 3  sensor is prepared with properties required for its intended use, with lower limits of detection for NH 3  well below the NH 3  concentrations typically found in human breath and, of course, below the increased NH 3  levels of a positive UBT. 
         [0042]    Another embodiment includes colloidal synthesis of hexagonal WO 3  nanowire and sheets. Lithium ion batteries are vital for advancing the field of portable electronics. They operate by reversibly inserting Li+ ions from the electrolyte into the electrodes and for generating electricity. Reversible intercalation of Li+ ions into the host matrix is crucial for battery operation and can be accomplished by having electrode materials that have relatively open crystal structures. Thermodynamically stable crystal structures are typically close-packed, to whereas metastable oxide phases have open lattices that promote very high diffusion rates for intercalating ions. 
         [0043]    Building smaller and more efficient batteries is imperative for advancing nanoelectronics. The synthesis of novel materials with reduced dimensionalities for battery electrodes is the key factor in improving battery performance. One-dimensional nanomaterials with high aspect ratio such as nanowires are used to construct miniaturized power units and to increase the surface area of the electrodes in order to increase energy density. Building 3-dimensional architectures of micro-/nano electrodes allows for a reduced footprint area for the battery, while at the same time the high aspect ratio of the nanowires serves to increase the energy density tremendously. 
         [0044]    In the area of resistive gas sensing, the attraction of hexagonal WO 3  lies in the structural similarity it shares with the orthorhombic form of Mo0 3 . Both these crystal structures have layered oxygen octahedra, in other words an open lattice structure, that provides long paths for small, diffusing gas molecules and facilitates easy removal of oxygen ions from the lattice. The present invention utilizes the key role played by the crystal structure in determining selectivity of the sensing matrix. Orthorhombic MoO 3  has been shown to be selective to ammonia in the presence of other gases. MoO 3  with its low sublimation temperature is not a suitable candidate for prolonged use at elevated temperatures. WO 3  on the other hand has higher structural integrity than MoO 3  and hence ideal for high temperature sensor applications. Also the high aspect ratio of the nanowires will serve to improve the energy density of the batteries without increasing the effective volume of the battery. 
         [0045]    Also, metal oxides with lower dimensionalities have been the focus of intense research activity for applications requiring high surface-area to volume ratio such as gas sensing. 
         [0046]    Nanowires of h-WO 3  are fabricated by hydrolysis and subsequent annealing of a sub-stoichiometric metal alkoxide precursor (tungsten (V) isopropoxide-W (i Pr)5) in air at a maximum temperature limit of 515° C. The sol-gel reaction occurring with the metal alkoxide precursor is outlined in Equation (3): 
         [0000]      W ( i Pr)5+H—OH→W (OH)5+R—OH (1)   (3)
 
         [0047]    The hydrolysis and subsequent condensation occur by alcoxolation, i.e. by removal of water. The isopropoxide functional group is removed as isopropanol, which then dries out. The precursor for the hexagonal lattice requires an additive for stabilizing the framework. The substoichiometric isopropoxide precursor on reaction with atmospheric moisture results in the formation of H0.24WO 3  that is known to transform to h-WO 3  on oxidation in air. The metal alkoxide is sub-stoichiometric and removal of two molecules of water from the W(OH)5in Equation (3) results in a lone hydrogen atom that can be accommodated in the interstitial spaces of WO 3  framework. 
         [0048]    Advantages of this embodiment include single step synthesis of a novel metastable phase of WO 3 ; unique crystal structure of the material enables reversible intercalation of Li ions for rechargeable batteries; higher thermal stability; high aspect ratio nanostructures such as nanowires, nanocubes and nanosheets for high surface area to volume ratio applications. 
         [0049]    While this invention has been particularly shown and described with reference to 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 scope of the invention encompassed by the appended claims.