WO2016045750A1

WO2016045750A1 - A technique for detecting helicobacter pylori

Info

Publication number: WO2016045750A1
Application number: PCT/EP2014/070679
Authority: WO
Inventors: Patrick Fröse; Erhard Magori; Roland Pohle; Angelika Tawil; Oliver von Sicard
Original assignee: Siemens Aktiengesellschaft
Priority date: 2014-09-26
Filing date: 2014-09-26
Publication date: 2016-03-31

Abstract

A technique for analyzing a test sample for presence of Helicobacter pylori by determining an extent of ammonia present in the test sample is presented. The test sample includes chloride ions. A first electrode and a second electrode of a Helicobacter pylori sensor are contacted with the test sample. At least the first electrode includes silver. Subsequently, a voltage is applied, for a first period of time, across the first and the second electrodes such that at least a part of the silver in the first electrode is converted to silver chloride. A current flowing across the first and the second electrodes is measured, for a second period of time. The second period of time at least partially overlaps with the first period of time. Finally, the extent of ammonia present in the test sample is calculated from the current flowing across the first and the second electrodes.

Description

A technique for detecting Helicobacter pylori This invention relates generally to a method and a sensor for analyzing a test sample for presence of Helicobacter pylori and more particularly to a method and a sensor for Helicobac^¬ ter pylori based on electrochemical interactions between the sensor and the test sample.

Helicobacter pylori (hereinafter referred to as, HBP) are rod-shaped bacteria, which can colonize the human stomach and are responsible for a number of gastrointestinal disorders. Besides other pathological conditions caused by HBP, the gas- trointestinal disorders include peptic ulcers such as stomach ulcers and duodenal ulcers. In chronic conditions, HBP can also cause stomach cancer. The prevalence of HBP is about 50% worldwide. Therefore, an investigation of infection with HBP represents an integral part of the diagnosis of gastrointes- tinal diseases.

In modern medicine, a HBP infection may, for example, be treated with eradication therapy, that involves simultaneous^¬ ly using a combination of different antibiotics. However, be- fore such eradication therapy can be started, an exact diag^¬ nosis is necessary.

For HBP detection, various direct and indirect detection methods are known, for example, non invasive testing can be performed with a blood antibody test, stool antigen test, urine ELISA test or with the carbon urea breath test (in which the patient drinks 14C—labeled urea or 13C-labeled urea, which the HBP metabolizes, producing labeled carbon di^¬ oxide that can be detected in the breath of the patient) .

Another method for detecting H. pylori infection is the so called endoscopy or gastroscopy method. In this method, the investigator i.e. the gastroenterologist performs a biopsy on a tissue sample collected from the gastrointestinal tract of the test subject. The biopsy involves a rapid urease test, histological examinations, and microbial culture from the tissue sample. In rapid urease test, the biopsy sample is placed in a test medium. The test medium contains a nutrient solution for HBP, urea and an indicator such a phenol red. If HBP is present in the biopsy sample, the HBP produces urease that hydrolyzes urea to ammonia and carbon dioxide. In pres^¬ ence of ammonia the pH of the medium is raised and thus the color of the specimen changes from yellow (urease from HBP not present) to red (urease from HBP present) . However, all of these detection methods as well as other known methods have their drawbacks such as delay in getting test results, being unpleasant to the test subject i.e. the patient, and being expensive.

Another technique for examination of the stomach to detect a settlement of HBP is disclosed in WO2010108759 Al which at^¬ tempts to provide an alternate to the above disclosed test methods. WO2010108759 Al presents a Helicobacter pylori sen^¬ sor. The Helicobacter pylori sensor comprises a slide with a measuring area, a first electrode made of a precious metal which cannot be attacked by hydrochloric acid, and a second electrode which is made of silver and has a silver chloride layer, wherein the first electrode and the second electrode extend at least partially into the measuring area, and a change in an electrical variable can be measured when the measuring area and the two electrodes are at least partially wetted with a measurement solution and when ammonia is pre- sent in the measurement solution between the first electrode and the second electrode. The Helicobacter pylori sensor ac^¬ cording to the disclosure in WO2010108759 Al is compact and of simple design and makes it possible to reliably detect Helicobacter pylori in a very short time.

However, the Helicobacter pylori sensor of WO2010108759 Al has its drawbacks. First, there is a requirement of having a silver chloride coating or layer on the silver electrode i.e. the second electrode of WO2010108759 Al . Thus, either the se^¬ cond electrode is pre-functionalized (addition of silver chloride on the silver electrode) , thus requiring an addi^¬ tional manufacturing step or is required to be functionalized (addition of silver chloride on the silver electrode) before being used to analyze the sample solution, thus requiring an additional step before analyzing the sample solution. Since measurement of ammonia in WO2010108759 Al is dependent on the silver chloride layer in the second electrode of WO2010108759 Al, for proper results from the Helicobacter pylori sensor of WO2010108759 Al it is required that the functionalization step of the second electrode of WO2010108759 Al be carried out accurately either during manufacturing of the second electrode for WO2010108759 Al or before use of the Helicobac- ter pylori sensor of WO2010108759 Al, if the second electrode is not pre-functionalized. Thus, the technique of

WO2010108759 Al is cumbersome and time consuming.

It is therefore an object of the present invention to provide a technique for analyzing a test sample for presence of Heli^¬ cobacter pylori which is not dependent on achieving complete and accurate functionalization of the electrodes of the Heli^¬ cobacter pylori sensor. This object is achieved by a method described in claim 1 and by the Helicobacter pylori sensor described in claim 8. The dependent claims describe advantageous embodiments of the method and the Helicobacter pylori sensor. According to an aspect of the present technique, a method for analyzing a test sample for presence of Helicobacter pylori (hereinafter, HBP) by determining an extent of ammonia present in the test sample, is presented. The test sample in^¬ cludes chloride ions. In the method, a first electrode and a second electrode of a Helicobacter pylori sensor (hereinaf^¬ ter, HBP sensor) are contacted with the test sample to be an^¬ alyzed. At least the first electrode of the HBP sensor in^¬ cludes silver. Subsequently, a voltage is applied, for a first period of time, across the first electrode and the se^¬ cond electrode of the HBP sensor such that at least a part of the silver in the first electrode is converted to silver chloride. In the method, a current flowing across the first and the second electrodes of the HBP sensor is measured, for a second period of time. The second period of time at least partially overlaps with the first period of time. Finally, the extent of ammonia present in the test sample is calculat^¬ ed from the current flowing across the first and the second electrode of the HBP sensor.

In an embodiment of the method the voltage applied is a con^¬ stant voltage. Application of constant voltage presents a simple way of working the method of the present technique.

In another embodiment of the method the voltage applied is a time varying voltage. Application of time varying voltage presents a simple way of working the method of the present technique in which regeneration of the electrodes of the Hel- icobacter pylori sensor is achieved by the application of the time varying voltage.

In another embodiment of the method the first and the second periods of time are entirely concurrent. Thus, the applica- tion of the voltage across the first and the second elec^¬ trodes and the measurement of the current flowing across the first and the second electrodes is done simultaneously. Thus buildup of silver chloride on the silver electrode with the application of the voltage is monitored throughout the entire first period of time i.e. throughout the entire period of ap^¬ plication of the voltage across the first and the second electrodes. This provides comprehensive data in form of the measured current flowing across the first and the second electrodes which in turn ensures accuracy of the method.

In another embodiment of the method the current flowing across the first and the second electrodes of the Helicobac^¬ ter pylori sensor is measured continuously. Thus buildup of silver chloride on the silver electrode during the second pe^¬ riod of time is monitored continuously which in turn provides comprehensive data in form of the measured current flowing across the first and the second electrodes ensuring accuracy of the method.

In another embodiment of the method the current flowing across the first and the second electrodes of the Helicobac^¬ ter pylori sensor is measured intermittently. Thus buildup of silver chloride on the silver electrode during the second pe^¬ riod of time is monitored intermittently and so lesser number of measurements is needed for the application of the method which in turn makes the method computationally less inten^¬ sive .

In another embodiment of the method the first period of time and/or the second period of time are predetermined. This pro^¬ vides a simple to implement embodiment of the present method. Moreover comparison of results obtained from multiple appli- cation of the method is facilitated.

According to another aspect of the present technique, a Heli^¬ cobacter pylori sensor (hereinafter, HBP sensor) for analyzing a test sample for presence of Helicobacter pylori (here- inafter, HBP) by determining an extent of ammonia present in the test sample, is presented. The HBP sensor includes a first electrode, a second electrode, a voltage source adapted to apply a voltage across the first and the second elec^¬ trodes, a measuring unit to determine a current flowing across the first and the second electrodes, and a processing and control unit. The first electrode includes silver. The processing and control unit is adapted to initiate the volt^¬ age source to apply, for a first period of time, a voltage across the first electrode and the second electrode such that at least a part of the silver in the first electrode is con^¬ verted to silver chloride. The processing and control unit is also adapted to initiate the measuring unit to measure, for a second period of time, the current flowing across the first and the second electrodes. The second period of time at least partially overlaps with the first period of time. Further^¬ more, the processing and control unit is adapted to calculate the extent of ammonia present in the test sample from the current flowing across the first and the second electrodes as determined by the measuring unit.

In an embodiment of the HBP sensor, the processing and control unit is adapted to control the measuring unit and the voltage source such that the first and the second periods of time are entirely concurrent. Thus, the HBP sensor is

equipped to apply the voltage across the first and the second electrodes and to measure the current flowing across the first and the second electrodes, simultaneously. Therefore, the HBP sensor is capable of monitoring buildup of silver chloride on the silver electrode with the application of the voltage throughout the entire first period of time i.e.

throughout the entire period of application of the voltage across the first and the second electrodes. This provides comprehensive data in form of the measured current flowing across the first and the second electrodes which in turn en^¬ sures accuracy of the HBP sensor.

In another embodiment of the HBP sensor, the processing and control unit is further adapted to control the measuring unit to determine the current flowing across the first and the se^¬ cond electrodes, continuously. Thus buildup of silver chlo^¬ ride on the silver electrode during the second period of time is monitored by the HBP sensor continuously which in turn provides comprehensive data in form of the measured current flowing across the first and the second electrodes ensuring accuracy of the HBP sensor.

In another embodiment of the HBP sensor, the processing and control unit is further adapted to control the measuring unit to determine the current flowing across the first and the se^¬ cond electrodes, intermittently. Thus buildup of silver chlo^¬ ride on the silver electrode during the second period of time is monitored by the HBP sensor intermittently and so lesser number of measurements is needed for use of the HBP sensor making the use of HBP sensor simple. Moreover, the HBP sensor is computationally less intensive and thus economical to fab- ricate.

In another embodiment of the HBP sensor, the voltage source is adapted to apply a constant voltage across the first and the second electrodes. Application of constant voltage pre- sents a simple way of working the HBP sensor of the present technique .

In another embodiment of the HBP sensor, the voltage source is adapted to apply a time varying voltage across the first and the second electrodes. Application of time varying volt^¬ age presents a simple way of working the HBP sensor of the present technique in which regeneration of the electrodes is made easily during the working of the HBP sensor. In another embodiment of the HBP sensor, the second electrode includes silver. This eliminates the requirement of selecting different material for the second electrode. Moreover, the regeneration of the electrodes to their default state, i.e. before the HBP sensor was used to analyze the sample, is made easily possible by reversing the polarities of the voltage applied .

It may be noted that by using the present technique a pres^¬ ence or absence of the ammonia in the test sample, and ac- cordingly the presence or absence of HBP in the test sample, may be detected. Furthermore, in test samples where the ammo^¬ nia is found or detected to be present, an extent of ammonia present i.e. an amount of ammonia present in the test sample may be determined which leads to determination of an amount of the HBP present in the test sample. The present technique is further described hereinafter with reference to illustrated embodiments shown in the accompany^¬ ing drawings, in which: is a flow chart illustrating a method for analyzing a test sample of a test subject for presence of Helicobacter pylori; is a schematic representation of an exemplary embodiment of a Helicobacter pylori sensor in accordance with aspects of the present technique; is a schematic representation of another exemplary embodiment of the Helicobacter pylori sensor at an intermediate stage of the method; is a schematic representation of a graph depicting exemplary curves, a first curve for a test sample with ammonia and a second curve for a test sample without ammonia, in accordance with aspects of the present technique.

Hereinafter, above-mentioned and other features of the pre^¬ sent technique are described in details. Various embodiments are described with reference to the drawings, wherein like reference numerals are used to refer to like elements throughout. In the following description, for purpose of ex^¬ planation, numerous specific details are set forth in order to provide a thorough understanding of one or more embodi- ments. It may be noted that the illustrated embodiments are intended to explain, and not to limit the invention. It may be evident that such embodiments may be practiced without these specific details.

The basic principle of the detection of Helicobacter pylori (hereinafter HBP) is based on detecting the presence or absence of ammonia (NH₃) in the test sample. HBP characteristi^¬ cally produces bacterial urease, an enzyme that catalyzes the hydrolysis of urea [( H₂)₂CO] into carbon dioxide (C0₂) and ammonia as shown in the following chemical equation:

(NH₂)₂CO + H₂0 → C0₂ + 2NH₃

Ammonia is not present under normal circumstances in a hollow organ of the gastrointestinal tract (hereinafter, GI tract) such as the stomach. Even if present, ammonia is present only in insignificantly small amounts. However, in test samples or in test subjects i.e. patients suffering from HBP infection the amount of ammonia present in the GI tract or in the test culture to which the test sample is added is significantly increased due to the bacterial urease produced by HBP. Thus, determining an extent of ammonia present in the test sample is a definitive conclusion of the presence of HBP.

Detection of ammonia is performed by observing a net effect of buildup and dissolution of silver chloride (AgCl) . Silver in presence of chloride ions results in buildup of silver chloride on the silver as per the following chemical equa^¬ tion :

Ag(s) + Cl^"(aq) → AgCl (s) Ammonia in aqueous state reacts with AgCl in solid state to form a readily water soluble silver diamine complex as per the following chemical equation:

AgCl(s) + 2NH₃(aq) → [Ag (NH₃) ₂] ⁺ (aq) + Cl^"(aq)

This results in a loss of silver chloride by dissolution into the test sample.

Thus when a silver electrode is introduced in a test sample which includes chloride ions, a buildup of silver chloride will take place on the silver electrode. However, if ammonia is also present in the test sample then at least some of the silver chloride will also dissolve into the test sample, sim- ultaneously with the buildup of silver chloride. In test sam^¬ ples without any ammonia this simultaneous dissolution of silver chloride will not take place. Thus, with other parame^¬ ters, such as time, kept constant, a test sample with ammonia will have a thinner layer of silver chloride buildup on sil^¬ ver electrode compared to the silver chloride buildup in a test sample without ammonia. This difference created in the silver chloride buildup in presence or absence of ammonia in the test sample is determined by the current flowing across the electrodes and concludes the presence or absence of ammo^¬ nia which in turn concludes the presence or absence of Heli^¬ cobacter pylori.

The above described principle of determining presence of HBP is used in the present technique.

FIG 2 is a schematic representation of an exemplary embodi^¬ ment of a Helicobacter pylori sensor 1 (hereinafter HBP sensor 1) in accordance with aspects of the present technique and FIG 1 is a flow chart illustrating a method 1000 for ana^¬ lyzing a test sample 2 (shown in FIG 2) of a test subject for presence of Helicobacter pylori (hereinafter, HBP) bacteria.

In the present technique, the presence of HBP is determined by determining an extent of ammonia present in the test sam^¬ ple 2. The phrase "extent of ammonia", as used herein means, absence or presence of ammonia i.e. zero amount of ammonia or non-zero amount of ammonia in the test sample 2. Furthermore, the phrase "extent of ammonia", when in non-zero amount i.e. when ammonia is present in the test sample 2, includes the quantitative assessment of the ammonia present in the test sample 2.

For the purposes of the present technique, the term "analyz- ing" or like terms, as used herein, means probing, checking, evaluating, testing, scrutinizing or examining the test sample. The phrase "analyzing the test sample for presence of Helicobacter pylori" means analyzing the test sample to de- termine or detect a presence of HBP and may optionally in^¬ clude quantifying HBP in the test sample.

The "test sample", as used herein, means and includes an in vivo sample or in vitro sample. For probing the test sample in vivo, the HBP sensor 1 is required to be introduced inside the body of the test subject i.e. the patient. This can be achieved by integrating the HBP sensor 1 with a suitable invasive device such as a gastroscope, an endoscope, an endos- copy capsule, a biopsy catheter, so on and so forth. An exam^¬ ple of the test sample, in vivo, may be, but not limited to, gastric juice within the stomach of the test subject or con^¬ tents or mediums within other parts of the GI tract. For probing the test sample in vitro, the test sample may be a biological specimen collected from the test subject for exam^¬ ple a specimen of the gastric juice of the test subject. The test sample, in vitro, may also include test sample prepared with additives such as a suitable test buffer or water for dilution .

The HBP sensor 1 includes a first electrode 4 including sil^¬ ver, a second electrode 6, a voltage source 8, a measuring unit 10 and a processing and control unit 12. The terms

"first" and "second" are used in this disclosure in their relative sense only. It will be understood that, unless oth^¬ erwise noted, those terms are used merely as a matter of con^¬ venience in the description of one or more of the embodi^¬ ments. The first and the second electrodes 4, 6 may have var^¬ ious shapes and configurations for example the first and the second electrodes 4, 6 may be simple wire electrodes or stick electrodes or may be miniaturized on a substrate, for example printed on a chip, to form a compact or miniature HBP sensor 1. The voltage source 8 is adapted to apply a voltage across the first 4 and the second electrodes 6. The voltage source 8 is capable of providing a constant voltage across the first 4 and the second electrodes 6 and/or a voltage that varies with time across the first 4 and the second electrodes 6. The voltage that varies with time across the first 4 and the se^¬ cond electrodes 6 may be in form of sinusoidally varying voltage. The measuring unit 10 is adapted to determine a cur- rent flowing across the first 4 and the second electrodes 6. The measuring unit 10 may be, but not limited to, an ammeter, a microammeter, or any other device that is designed to meas^¬ ure current flowing in a circuit. The processing and control unit 12 (hereinafter PCU 12) is adapted to control the voltage source 8 such that the voltage source 8 applies, for a first period of time, the voltage across the first electrode 4 and the second electrode 6. The voltage applied by the voltage source for the first period of time is such that at least a part of the silver in the first electrode 4 starts converting to silver chloride 5 (as shown in FIG 3) . The conversion of silver of the first electrode 4 to the silver chloride 5 happens due to the chloride ions present in the test sample 2. The chloride ions may be pre- sent in the test sample 2 in form of hydrochloric acid, which is present in gastric juice of animals, especially human be^¬ ings. The conversion of silver of the first electrode 4 to the silver chloride 5 begins with application of the voltage i.e. with the start of the first period of time and occurs continuously during the first period of time. The PCU 12 also controls the measuring unit 10 to measure, for a second peri^¬ od of time, the current flowing across the first 4 and the second electrodes 6. It may be noted as essential to the present technique, that the second period of time at least partially overlaps with the first period of time, which means that the measuring of the current flowing across the first 4 and the second elec^¬ trode 6 is performed by the measuring unit 10 during the con- tinuous buildup of the silver chloride 5 on the first elec^¬ trode 4. In one embodiment of the HBP sensor 1, the PCU 12 controls the voltage source 8 and the measuring unit 10 such that the first and the second periods of time are entirely concurrent, meaning thereby that the first and the second pe^¬ riods of time begin at the same instance, continue for the same length of time, and stop at the same instance. This en^¬ sures that the measurement unit 10 measures the current flow- ing across the electrode 4,6 during the buildup of the silver chloride 5 in the first electrode 4 from the beginning of the first period of time to the end of the first period of time. The measurement of the current flowing across the electrodes 4, 6 is performed by the measuring unit 10 either continuously or intermittently with respect to second period of time.

The PCU 12 is also adapted to calculate the extent of ammonia present in the test sample 2 from the current flowing across the first 4 and the second electrodes 6 as determined by the measuring unit 10. As mentioned hereinabove, the rate of net buildup of the silver chloride 5 is dependant upon: (a) buildup of the silver chloride 5 due to presence of chloride ion in the test sample 2 and application of the voltage across the first 4 and the second electrode 6 and (b) the dissolution of the silver chloride 5 due to presence of ammo^¬ nia, if any, in the test sample 2. This rate of net buildup of the silver chloride 5 has a manifestation in the current flowing across the first 4 and the second electrode 6. As may be now clear to one skilled in the art, the fact that whether ammonia is present or not and if present then concen^¬ tration of ammonia present in the test sample has an effect on the rate of net buildup of the silver chloride 5. The rate of net buildup of the silver chloride 5 in turn effects the current flowing across the electrodes 4,6. Thus, by measuring the current flowing across the electrodes 4,6, the presence or absence of ammonia in the test sample 2 is determined which in turn informs about presence or absence of HBP in the test sample 2. Moreover, the current measured by the measure- ment unit 10 also informs about the concentration of ammonia present in the test sample 2 which in turn informs about a concentration of HBP in the test sample 2. The method 1000 of the present technique, the measurements of currents obtained by the measuring unit 10 and their inter^¬ pretation to determine the extent of ammonia present in the test sample 2 and thus the presence or absence or amount of HBP present in the test sample 2 is explained hereinafter with FIG 1 and FIG 4 in combination with FIG 2 and FIG 3.

In the method 1000, in step 100, the first electrode 4 and the second electrode 6 of the HBP sensor 1 are contacted with the test sample 2 to be analyzed. As mentioned above, at least the first electrode 4 includes silver. This stage of the method 1000 is depicted in FIG 2.

In one embodiment of the HBP sensor 1, the second electrode 6 is constituted of a material that is inert to ammonia and/or hydrochloric acid and/or other contents of the test sample 2. In this embodiment, even when the test sample 2 comes in con^¬ tact with the second electrode 6 there is no chemical reac^¬ tion between the second electrode 6 with ammonia and/or hy- drochloric acid i.e. the chloride ions and/or other contents of the test sample 2. In an exemplary embodiment of the HBP sensor 1, the second electrode 6 is made of inert elements or inert metallic compounds such as Gold (Au) , Platinum (Pt) , and so on and so forth. In this embodiment of the HBP sensor 1, buildup of the silver chloride 5 happens only at the first electrode 4. Thus, the current flowing across the electrodes 4, 6 is affected only by the first electrode 4.

In another embodiment of the HBP sensor 1, the second elec- trode 6 includes silver. In this embodiment, depending on the polarity of the voltage applied across the first 4 and the second electrode 6, the silver chloride 5 buildup may occur at either of the electrodes 4, 6 at a given instance of time. For example, on application of the time varying voltage, say for example, an alternating voltage, the buildup of silver chloride 5 occurs once at the first electrode 4 and then with reversal of polarity of the applied voltage the silver chlo^¬ ride 5 is dissolved from the first electrode 4 and buildup of the silver chloride 5 now occurs simultaneously at the second electrode 6, and so on and so forth.

Subsequently, in the method 1000, in a step 200, the voltage is applied, for a first period of time, across the first electrode 4 and the second electrode 6 of the HBP sensor 1. As a result of the application of the voltage, the silver chloride 5 starts to buildup at the first electrode 4, as de^¬ picted in FIG 3. Furthermore, in the method 1000, in a step 300, the current flowing across the first 4 and the second electrodes 6 of the HBP sensor 1 is measured, for a second period of time. It may be noted that the second period of time at least partially overlaps with the first period of time, and in one embodiment of the method 1000, the second period of time entirely or completely overlaps with the first period of time i.e. the first and the second periods of time are concurrent. Thus the step 300 may initiate instantaneous^¬ ly with the initiation of step 200 or may initiate after the initiation of step 200 but before the end of step 200. Fur- thermore, the first period of time and/or the second period of time may be predetermined.

Finally in the method 1000, in a step 400, the extent of am^¬ monia present in the test sample 2 is calculated from the current flowing across the first 4 and the second electrodes 6 of the HBP sensor 1. FIG 4 schematically represents an ex^¬ emplary graph 50 depicting exemplary curves - a first curve 56 for a test sample 2 without ammonia and a second curve 58 for a test sample 2 with ammonia. The ^λΧ' axis represented by reference numeral 52 represents time and the ^λΥ' axis repre^¬ sented by reference numeral 54 represents current flowing across the electrodes 4, 6 observed by the measurement unit 10 during the second period of time. It may be noted that graph 50 and the curves 56 and 58 are for exemplary purposes only, and are presented as examples of standard curves and not intended to be limitations of the present technique.

Thus, by observing the current flowing across the electrodes 4, 6 the presence or absence of ammonia in the test sample 2 is determined. In a similar way, i.e. by use of standard curves, the extent of ammonia i.e. the amount or concentra^¬ tion of ammonia present in the test sample is determined. In the present technique, the rate of change of the current is used to calculate the extent of ammonia present in the test sample 2 by using reference curves representing the net rate of buildup of the silver chloride 5 on the first elec^¬ trode 4 and the correlation between the rate of change of current with the amount or concentration or extent of ammonia present in the test sample. Similarly, the amount or concen^¬ tration or extent of ammonia present in the test sample may be used to calculate the quantity of HBP present in the test sample by using reference curves representing the extent of ammonia and its correlation to the quantity of HBP present for standard samples. The technique of using such reference curves is well known and pervasively used in the art of ana^¬ lytical chemistry and physics and thus the same has not been described herein for sake of brevity.

In the present disclosure FIG 4 has been illustrated for the method 1000 when the voltage applied across the electrodes 4,6 is constant with time. However, as may be appreciated by one skilled in the art, when the voltage applied across the electrodes 4,6 is varying with time, a different graph or curves will be obtained as opposed to the graph 50 and the curves 56, 58 shown in FIG 4. Moreover, when both electrodes 4, 6 are silver electrodes the application of time varying voltage will essentially result in buildup of the silver chloride 5 initially on one of the electrodes 4,6 for example say the first electrode 4, then with reversal of polarity of the applied voltage the silver chloride 5 will dissolve, in presence of ammonia, from the first electrode 4 but simulta^¬ neously there will be a buildup of the silver chloride 5 at the second electrode 6. Again, with time as the polarity of the applied voltage is reversed once again, the silver chlo^¬ ride 5 that is buildup on the second electrode 6 will dis^¬ solve but simultaneously there will be buildup of the silver chloride 5 again at the first electrode 4. This will ensure regeneration of the first electrode 4 either completely or partially with the help of second electrode 6 and thus the HBP sensor 1 will be more stable for use for repeated analyz- ing of the test sample 2 and/or for longer periods of analyzing of one or more test samples 2.

It may be noted that, for the purposes of the present disclo^¬ sure only the term "current" and its measurements have been used, however, as may be appreciated by a person skilled in the art of electrical sciences, from knowledge of other elec^¬ trical properties, the current can be easily calculated, and the phrase "measurement of current" and like phrases as used in the present technique include such other electrical prop- erties.

While the present technique has been described in detail with reference to certain embodiments, it should be appreciated that the present technique is not limited to those precise embodiments. Rather, in view of the present disclosure which describes exemplary modes for practicing the invention, many modifications and variations would present themselves, to those skilled in the art without departing from the scope and spirit of this invention. The scope of the invention is, therefore, indicated by the following claims rather than by the foregoing description. All changes, modifications, and variations coming within the meaning and range of equivalency of the claims are to be considered within their scope.

Claims

Patent claims

1. A method (1000) for analyzing a test sample (2) for pres^¬ ence of Helicobacter pylori by determining an extent of ammo- nia present in the test sample (2), the test sample compris^¬ ing chloride ions, the method (1000) comprising:

- contacting (100) a first electrode (4) and a second elec^¬ trode (6) of a Helicobacter pylori sensor (1) with the test sample (2) to be analyzed, wherein at least the first elec- trode (4) comprises silver;

- applying (200), for a first period of time, a voltage across the first electrode (4) and the second electrode (6) of the Helicobacter pylori sensor (1) such that at least a part of the silver in the first electrode (4) is converted to silver chloride (5) ,

- measuring (300), for a second period of time, a current flowing across the first (4) and the second electrodes (6) of the Helicobacter pylori sensor (1), wherein the second period of time at least partially overlaps with the first period of time, and

- calculating (400) the extent of ammonia present in the test sample (2) from the current flowing across the first (4) and the second electrodes (6) of the Helicobacter pylori sensor (1) .

2. The method (1000) according to claim 1 wherein the voltage applied is a constant voltage.

3. The method (1000) according to claim 1 wherein the voltage applied is a time varying voltage.

4. The method (1000) according to any of claims 1 to 3, wherein the first and the second periods of time are entirely concurrent .

5. The method (1000) according to any of claims 1 to 4, wherein the current flowing across the first (4) and the se- cond electrodes (6) of the Helicobacter pylori sensor (1) is measured continuously.

6. The method (1000) according to any of claims 1 to 4, wherein the current flowing across the first (4) and the se^¬ cond electrodes (6) of the Helicobacter pylori sensor (1) is measured intermittently.

7. The method (1000) according to any of claims 1 to 6 where- in the first period of time and/or the second period of time are predetermined.

8. A Helicobacter pylori sensor (1) for analyzing a test sample (2) for presence of Helicobacter pylori by determining an extent of ammonia present in the test sample (2), the Helico^¬ bacter pylori sensor (1) comprising:

- a first electrode (4) comprising silver,

- a second electrode (6),

- a voltage source (8) adapted to apply a voltage across the first (4) and the second electrodes (6),

- a measuring unit (10) to determine a current flowing across the first (4) and the second electrodes (6),

- a processing and control unit (12) adapted to initiate the voltage source (8) to apply, for a first period of time, a voltage across the first electrode (4) and the second elec^¬ trode (6) such that at least a part of the silver in the first electrode (4) is converted to silver chloride (5), to initiate the measuring unit (10) to measure, for a second pe^¬ riod of time, the current flowing across the first (4) and the second electrodes (6), wherein the second period of time at least partially overlaps with the first period of time, and to calculate the extent of ammonia present in the test sample (2) from the current flowing across the first (4) and the second electrodes (6) as determined by the measuring unit (10) .

9. The Helicobacter pylori sensor (1) according to claim 8, wherein the processing and control unit (12) is adapted to control the voltage source (8) and the measuring unit (10) such that the first and the second periods of time are en^¬ tirely concurrent.

10. The Helicobacter pylori sensor (1) according to claim 8 or 9, wherein the processing and control unit (12) is further adapted to control the measuring unit (10) to determine the current flowing across the first (4) and the second elec^¬ trodes (6), continuously.

11. The Helicobacter pylori sensor (1) according to claim 8 or 9, wherein the processing and control unit (12) is further adapted to control the measuring unit (10) to determine the current flowing across the first (4) and the second elec- trodes (6), intermittently.

12. The Helicobacter pylori sensor (1) according to any of claims 8 to 11, wherein the voltage source (8) is adapted to apply a constant voltage across the first (4) and the second electrodes (6) .

13. The Helicobacter pylori sensor (1) according to any of claims 8 to 11, wherein the voltage source (8) is adapted to apply a voltage that varies with time across the first (4) and the second electrodes (6) .

14. The Helicobacter pylori sensor (1) according to any of claims 8 to 13, wherein the second electrode (6) comprises silver .