GB2442748A

GB2442748A - Device and method for measuring a nitric oxide metabolite such as nitrite or nitrate

Info

Publication number: GB2442748A
Application number: GB0620342A
Authority: GB
Inventors: Paul James Davis; Sandra Hemmington; Mark Burnapp; David Eagle
Original assignee: Mologic Ltd
Current assignee: Mologic Ltd
Priority date: 2006-10-13
Filing date: 2006-10-13
Publication date: 2008-04-16
Also published as: GB0620342D0; WO2008044043A1

Abstract

A nitrate detection product (1) for detecting nitrate in a sample comprises (i) a reaction zone (5) containing a reducing agent such as zinc, cadmium iron or vanadium and adapted so as to receive the sample and contact it with the reducing agent in the presence of a source of iodide (as oxidizing agent) to produce a reaction product (iodine, I3<->); and (ii) a detection zone (10) for presenting a signal in response to the detection of a component of the reaction product. The detection may be colorimetric, detecting the complex formed with starch or polyvinyl alcohol (PVA), or electrochemical. The device may be a vertical flow chamber comprising a reaction module 5 to hold the sample and a frit 6 holding the reducing agent. A method of detecting nitrate in a sample comprises the steps of (i) contacting the sample with a reducing agent in the presence of a source of iodide in a reaction zone (5) to produce a reaction product; and (ii) presenting a signal in response to the detection of a component of the reaction product in a detection zone (10). A nitrite detection product requires only the iodide as nitrite itself acts as a reducing agent.

Description

S

NITRIC OXIDE AND RELATED METABOLIC DIAGNOSTIC

The present invention relates to a nitrate (or nitrite) detection product and also to a method for detecting the presence of nitrate (or nitrite) in a sample. The present invention also relates to a nitrite detection product, a metabolite detection product (wherein the metabolite is a metabolite of nitric oxide) and methods for detecting nitrite and metabolites in a sample.

Nitric oxide (NO) is a small gaseous molecule that plays a role in many physiological processes (see Michel T, Feron 0., 1997 Nitric Oxide synthases: Which, where, how and why?, J. GUn. Invest; 100 (9), 2146-2152). For example, nitric oxide functions as a neurotransmitter in the central and peripheral nervous system and plays a role in the homeostasis of the cardiovascular system. Nitric oxide also has a key role during * : :* infection and inflammation, particularly in relation to wound healing. It is known that * 15 patients presenting with impaired wound healing function may have low basal levels S...

of nitric oxide production (see US-6,312,663, US-6,344,181 and US-6,436,366). It, * : therefore, is useful to be able to determine the nitric oxide levels in patients, for example to allow for the detection and treatment of certain diseases and conditions, : * such as patients with impaired wound healing through low levels of nitric oxide. I...

The presence and amount of nitric oxide in a given sample (for example urine or serum) can be measured using chemiluminescence and electrochemical methods (see Bateman C., Ellis M., Sharpe M., Mehta S. and Freeman D., 2001, Effect of hemolyzed plasma on the batch measurement of nitrate by nitric oxide chemiluminescence, Clin. Chem., 47, 10, 1847-1851). Whilst such methods work acceptably well in some circumstances, they require analytical instrumentation which can be expensive and bulky. Thus, these methods can be complicated to conduct and are unsuitable for use at the point of patient care.

As an alternative, metabolites of nitric oxide can be detected and measured to provide an indication of nitric oxide levels in a sample. This is possible because nitric oxide is an unstable, short lived free radical that is subsequently metabolised to nitrate (NO3), nitrite (NO2), s-nitrothiols (RSNO, where R represents alkyl) and others.

C

Thus, levels of metabolites such as nitrate and nitrite can be used as an indication of nitric oxide levels.

Various methods are known in the art for detecting and measuring amounts of nitric oxide metabolites in samples.

Many such methods involve the detection and measurement of the nitrite metabolite.

One such method involves derivatising the sample (containing the nitrite) with Griess reagent, which interacts with nitrite to form a coloured compound (see Griess J. P., 1864, Phil. Trans. R. Soc. London). The amount of coloured compound formed can be measured with a spectrophotometer or a microtitre plate reader. In brief, this technique uses Iwo reagents under acidic conditions to yield a reaction between * nitrite and sulfanilic acid to form a diazonium cation, which is followed by the formation of a diazo chromophore with a-napthylamine; the absorbance can be measured at 540 nm. This method is limited in use because nitrite is not freely available unless some form of reducing agent is present. Additionally, this method * S....

* : requires equipment to monitor or measure the result in the absence of a standardised *SSSS* * visual method (such as a colour chart). * S * . S S...

Commercially produced assay kits based on the Griess method discussed above are *. . . . available and are intended for use within a laboratory. These kits require a pre-treatment of the sample (containing the metabolites of nitric oxide) to reduce nitrate to nitrite. This reduction usually is achieved using nitrate reductase enzyme or by contacting the sample with a reducing agent, such as a cadmium based reducing agent (for example cadmium coupled with copper or zinc, see Bones P. N., Bones C., 1995, Nitrate determination in biological fluids by an enzymatic one-step assay with nitrate reductase, Clin. Chem., 41(6) 904-907 and Cortes N., Wakid N., 1990, Determination of inorganic nitrate in serum and urine by a kinetic cadmium reduction method, Clin. Chem., 36 (8), 1440-1443). The presence and amount of nitrite is then determined by spectrophotometry. This method requires numerous sample manipulations and comparison of the reduced samples to a standard curve. Thus, the method is complicated and time consuming to conduct and again is unsuitable for use at the point of patient care. (

Other known methods require expensive and sophisticated equipment, technical expertise and long testing times. These methods include spectrophotometry, high-performance liquid chromatography (HPLC), gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), ion chromatography, capillary electrophoresis (CE) and fluorimetry (see Ellis G., Adatia L, Yazdanpanah M. and Makela S., 1998, Nitrite and Nitrate Analyses: A Clinical Biochemistry Perspective, Clinical Biochemistry, 31(4), 195-220). Many of these techniques require a sample pie-treatment and/or derivitisation step so as to render the sample volatile for measurable by the appropriate detection method. The reliability of test results is dependant upon meticulous sample handling and preparation.

JP-6331 7764 describes a method and apparatus for detecting the oxidising power of * ,* fuming nitric acid, by bringing the acid into contact with an aluminium oxide layer that S.... carries potassium iodide. Nitrogen dioxide in the fuming nitric acid reacts with the potassium iodide to form iodine and the amount of purple (iodine) colour produced * provides a measure of the oxidising power of the fuming nitric acid. * a

S

S.....

* Thus, many of the known methods for detecting nitric oxide and its metabolites are : * * complicated and time consuming to conduct and are unsuitable for the analysis of S...

urine, plasma or serum due to the complexity of the sample. This makes the methods unsuitable for use at the point of patient care The present invention seeks to alleviate one or more of the above problems.

According to the present invention, there is provided a nitrate detection product for detecting nitrate in a sample, the product comprising: (i) a reaction zone containing a reducing agent and adapted so as to receive the sample and contact it with the reducing agent in the presence of a source of iodide to produce a reaction product; and (ii) a detection zone for presenting a signal in response to the detection of a component of the reaction product. (

The nitrate detection product of the present invention is simple and easy to use, may be disposable and requires minimal sample manipulation. Additionally, the nitrate detection product is made from known materials that are readily available.

In particular, the nitrate detection product of the present invention is suitable for point of care operation. The nitrate detection product is particularly suitable for use in wound care applications because it allows the rapid assessment of nitrate levels in patients presenting with acute or chronic wounds. A particularly vulnerable patient category includes diabetics, who are prone to ulcers and who have an impaired ability to heal. The rapid delivery of results can have immediate impact on patient care, which is particularly useful for treatment of outpatients during a hospital appointment. * .. * * *

The references herein to zones" are intended to relate to a suitable area in which the specified reaction or detection may occur. In particular, the "zones" may take the form of modules, by which is meant independent units that may be constructed to * : form a more complex structure or product.

I..... * *

: * * The nitrate detection product may be used to detect nitrate in any suitable sample, **.

which sample preferably is in a liquid form. Suitable samples include wound fluid, whole blood samples, urine, plasma, serum, tissue or other specimens. As discussed above, the nitrate typically is present as a metabolite of nitric oxide and the detection of nitrate in the sample is used as an indication of nitric oxide levels in the sample.

Other suitable samples include environmental samples, for example samples taken from the environment (such as rivers, lakes, pond and suchlike) where it would be desirable to detect nitric oxide and its metabolites.

The sample may be tested in any suitable form. As the skilled person would appreciate, in some circumstances, the sample wilt be subjected to one or more pre-treatment steps prior to testing. Such pre-treatment steps may include diluting.

filtering and/or centrifuging the sample.

Typically, when the sample to be tested is a whole blood sample, the sample must be separated before it is introduced to the reaction zone for contact with the reducing agent and source of iodide. Suitable means of separation include a blood separation membrane. Thus, in one aspect the nitrate detection product of the invention may comprise a blood separation zone, such as a blood separation membrane, positioned before the reaction zone, for example at the sample application point.

Samples that contain a particulate material visible to the human eye may need to be clarified by a centrifugation step (for example at 10,000 RPM for about 5 minutes) prior to testing. Following the centrifugation step, it is the supematant that is removed for testing.

When the sample to be tested is a wound fluid, the sample may require an extraction step following the collection of the sample by use of a sterile swab. For example, the "" 15 extraction step may be by a pre-prepared extraction buffer. Thus, in one aspect the nitrate detection product of the invention may comprise an extraction zone, for I..

* example including an extraction buffer, positioned before the reaction zone, for S...,, * -example at the sample application point. Alternatively, the sample may be contacted : , * with the extraction buffer prior to its introduction into the nitrate detection product (for example in a swab extraction tube).

In the reaction zone of the nitrate detection product, the sample is contacted with the reducing agent in the presence of a source of iodide to produce a reaction product In particular, the nitrate in the sample is reduced by the reducing agent. This source of iodide may be present as the reduction occurs or may be released directly after the reduction reaction. Thus, the specified component of the reaction product is produced when nitrate is present in the sample. As the skilled person would appreciate, the reducing agent may reduce other components of the sample in addition to the nitrate, however it is the reduction of the nitrate in the sample that is fundamental to the nitrate detection product and method of the present invention.

The reaction zone can be defined as the area of the detection product in which the desired, for example aforementioned, reaction(s) occur.

S

As the skilled person would appreciate, the reference to contacting the sample with the reducing agent in the presence of a source of iodide is intended to refer both to conducing the reduction reaction in the presence of the source of iodide, as well as to conducting the reduction reaction and then directly contacting the reduction reaction product with the source of iodide (for example immediately following the reduction).

As the skilled person would appreciate, any suitable reducing agent may be contained in the reaction zone. Suitable reducing agents include (but are not limited to) any one or more of zinc, cadmium, iron and vanadium. In particular, the reducing agent is zinc. The use of zinc as the reducing agent is advantageous because it is easily and conveniently incorporated into the nitrate detection product and presents little or no toxicity to the operator of the nitrate detection product or the environment. * *.

The reducing agent may be contained in the reaction zone in any suitable form. For example, the reducing agent may be contained in the reaction zone in a solid form for example embedded in a suitable carrier material, or in a liquid or gel form for example carried on an inert reaction medium or pad.

*.*I*s * : * * As the skilled person would appreciate, any suitable source of iodide may be * ** contacted with the sample. Suitable sources of iodide include (but are not limited to) one or both of potassium iodide and sodium iodide. Other suitable sources of iodide may include calcium iodide and magnesium iodide. In particular, the source of iodide is potassium iodide. The use of potassium iodide as the source of iodide is advantageous because it is readily available and is highly water soluble.

The source of iodide may be contacted with the sample by any suitable means or method, provided that it is present in the reaction zone when the reducing agent contacts the sample or directly after the reduction has occurred. For example, in one aspect, the source of iodide may be present in the reaction zone, along with the reducing agent, prior to addition of the sample to the reaction zone and/or the source of iodide may be added to the reaction zone before, during and/or immediately following the addition of the sample. t

Thus, in one aspect, the reaction zone contains a reducing agent and a source of iodide and is adapted so as to receive the sample and contact it with the reducing agent and the source of iodide to produce the reaction product.

In one aspect, the reaction zone is further adapted so as to contact the sample with the reducing agent at a pH in the range of from I to 9, preferably of from 2 to 5, more preferably of from 2 to 3, for example of about 3. Typically, the preferred pH is achieved by contacting the sample with the reducing agent in the presence of a suitable acid. The desired pH may be achieved by adding an appropriate amount of a suitable acid to the sample before it is received in the reaction zone. Preferably, however, the desired pH is achieved by incorporating a suitable acid in the reaction zone so as to effect release of the acid upon addition of the sample to the reaction zone. Suitable acids include (but are not limited to) aqueous hydrogen chloride, citric : *** acid, phosphoric acid and acetic acid. Preferably, the desired pH in the reaction a...

zone is maintained by the use of a suitable buffer, for example a citrate containing S... . buffer. Preferably, the buffer is incorporated in the reaction zone so as to effect * : * release of the buffer upon addition of the sample to the reaction zone.

I.....

* When a sample containing nitrate is contacted with the reducing agent (such as zinc) : * :. 20 in the presence of the source of iodide (such as potassium iodide) at the appropriate . pH, then the nitrate is reduced to form nitrite. Any nitrite formed then reacts with the source of iodide to liberate iodine. Thus, when nitrate (or nitrite once the nitrate has been converted to nitrite) is present, a component of the reaction product produced in the reaction zone is iodine.

The nitrate detection product further comprises a detection zone for presenting a signal in response to the detection of a component of the reaction product, for example in response to the detection of iodine. The detection zone is the area of the detection product where detection and signal presentation occurs.

The signal presented at the detection zone or module is indicative of the outcome of the test. The presence of a signal (such as a colour or electrochemical change) at the detection zone or module indicates the presence of nitrate in the sample whereas ( the absence of a signal (such as a colour or electrochemical change) at the detection zone or module indicates the absence of nitrate in the sample. When a colour is observed at the detection zone or module, the intensity of the colour is indicative of the amount of nitrate in the sample; the darker the colour produced, the higher the amount of nitrate in the sample. Thus, references herein to detecting the presence of nitrate in a sample are intended to refer to detecting the presence or absence of nitrate in the sample.

Any suitable signal may be presented in the detection zone. Preferably the signal presented in the detection zone is a visual signal, such as the formation of a colour, i.e. a colouration in the detection zone. As a person skilled in the art would appreciate, by the term "colour" we mean an entity that absorbs light in the range of : from 375 to 740 nm, i.e. so as to appear coloured to the human eye.

15 Preferably, the detection zone comprises a reactant capable of reacting with the component of the reaction product to present the signal. In one aspect, the reactant I* S* is an organic polymer capable of forming a complex with the component of the I.....

* reaction product, for example an organic polymer capable of forming a complex with : * * iodine. By the term "organic polymer" we mean a polymer comprised of carbon, S...

oxygen, nitrogen and sulphur atoms only. *5**

As the skilled person would appreciate, when it is present in solution iodine has a brown colour and contains complex ions with iodide, i.e. lj ions. Without wishing to be bound by any theory, it is believed that the organic polymer complexes with the complex 13 ions of iodine in a helical structure to give a red to brown adduct, which amplifies the colour change produced upon the formation of iodine. The coloured adduct is effectively immobilised in the nitrate detection product and is stable for several hours. Thus, references herein to iodine" are intended to include complex ions, such as l3, typically present in iodine solutions.

Any suitable organic polymer may be used provided that it is capable of forming a complex with the component of the reaction product, for example capable of forming a complex with iodine (for example including complex l ions) which complex is coloured. Suitable organic polymers include (but are not limited to) one or more of starch and polyvinyl alcohol. In particular, the complex formed between the iodine and the organic polymer is a red/brown colour to the human eye. However, as the skilled person would appreciate, the colour of the complex formed will depend on various factors, such as the particular organic polymer used.

In one aspect, the detection zone is adapted so as to contact the reaction product with the organic polymer at a pH that is lower than the pH in the reaction zone. For example, the detection zone may be adapted so as to contact the reaction product with the organic polymer at a pH in the range of from 3 to 5, preferably of about 3.

This pH is advantageous in the detection zone because it aids in the formation of iodine from iodide in the presence of nitrite (see Vogel's Qualitative Inorganic : *., Analysis, 6th Ed., 1987, G. Svehla, p 179-180, Longman). Typically, the preferred pH S...

is achieved by contacting the sample with a suitable acid. The desired pH may be achieved by adding an appropriate amount of a suitable acid to the sample before it * .... is received in the product. Preferably, however, the desired pH is achieved by : incorporating the suitable acid in the detection zone so as to effect release of the acid upon addition of the sample to the zone. Suitable acids include (but are not limited : .: . 20 to) aqueous hydrogen chloride, citric acid, acetic acid and phosphoric acid. *..* * . *ISS

Preferably, the preferred pH of the reactants in the detection zone is maintained by the use of a suitable buffer, for example a citrate, acetate or glycine containing buffer.

Preferably, the buffer is incorporated in the detection zone so as to effect release of the buffer upon addition of the sample to that zone.

The detection zone may further contain an agent to assist the complexation of the component of the reaction product (such as iodine) to the reactant as described above. This may be achieved by any suitable agent, for example an agent that liberates borate ions into solution. Without wishing to be bound by any theory, it is believed that the presence of borate ions rigidities the helix formed as discussed above through cross-linking and improves the durability of the colour produced. For example, when the reactant is polyvinyl alcohol and the reaction product is iodine, the agent may be sodium tetraborate. Thus, it is believed that the agent may help to prevent the loss of colour from the detection zone as it is formed.

The signal presented in the detection zone may alternatively be an electrochemical signal. For example, the electrochemical signal may be a voltage andlor current measured so as to indicate the conversion of iodide ions to iodine. Thus, in this aspect, the detection zone may comprise means to measure a voltage and/or a current passing through the sample, i.e. voltametric and/or an amperometric means.

In one aspect of the invention, the product may further comprise a neutralisation zone located between the reaction and detection zones. The neutralisation zone may be used to modify the pH of the sample as it passes from the reaction zone to the detection zone. For example, the neutralisation zone may modify the pH of the sample to a pH in the range of from 7 to 10. This is advantageous because it provides an environment in which the stability of the source of iodide is increased (for a..

example compared to pH's of less than 7) so ensuring that substantially all of the *:: iodide source is available for reaction with the nitrite formed. In particular, the use of a neutralisation zone is advantageous when the reduction in the reaction zone is : * conducted at a pH of less than about 7. S...

Accordingly, the present invention provides a nitrate detection product for detecting the presence of nitrate in a sample, the product comprising: (I) a reaction zone containing zinc as a reducing agent and adapted so as to receive the sample and contact it with the zinc in the presence of potassium iodide to produce a reaction product containing iodine (when nitrate is present in the sample); and (ii) a detection zone for presenting a signal in response to the detection of the iodine.

Accordingly, the present invention provides a nitrate detection product for detecting the presence of nitrate in a sample, the product comprising: (I) a reaction zone containing zinc as a reducing agent and potassium iodide, the reaction zone being adapted so as to receive the sample and contact it with the zinc and the potassium iodide to produce a reaction product containing iodine (when nitrate is present in the sample); and (ii) a detection zone for presenting a signal in response to the detection of the iodine.

The nitrate detection product of the present invention may take any suitable form.

Preferably, the reaction zone and the detection zone are comprised in a single body, for example as reaction and detection modules. In use, a sample is received in the reaction zone and is passed directly from the reaction zone to the detection zone without removal of the sample from the product or any manipulation of the sample by the operator. This distinguishes the nitrate reaction product from previous known systems for detecting nitrate, wherein a sample containing nitrate is manipulated ::. and/or reacted in some way and then the resulting sample is transferred to a suitable . 15 machine for analysis. Thus, the nitrate detection product of the present invention is simple, convenient and quick to use, for example compared to the known systems of

the prior art. s. * .

Typically, the nitrate detection product is arranged and dimensioned so as to allow : :s 20 for easy handling by the operator at the point of patient care and to provide efficiency * * in terms of materials required for manufacture of the product.

In one aspect of the invention, the nitrate detection product comprises a vertical flow product. In this aspect, the nitrate detection product comprises a casing (preferably a cylindrical casing or column) including the reaction zone and the detection zone. In particular, the reaction and detection zones take the form of modules in the vertical flow product. Preferably, each zone or module is defined by at least one disc (otherwise described as a pad or frit) positioned at a particular location along the length of the casing. In use, the operator runs the test by simply passing a sample through the casing, such that the sample runs through the length of the casing and through the reaction zone to the detection zone. In other words, the reaction zone and the detection zone together define a flow path, along which a sample is capable of flowing. Preferably, the flow path is generally linear. r

In the vertical flow product, the reaction zone or module may comprise a reduction disc, pad or fit (hereinafter referred to as a reduction disc) in which the reducing agent is embedded. In one aspect, the source of iodide may also be embedded in the reduction disc. A suitable acid and/or buffer may additionally be embedded in the reduction zone. In use, the aforementioned reagents are released from the disc upon contact with the sample, so as to contact the reagents with the sample.

The reduction disc may be made from any suitable material, for example from a porous, fluid conducting membrane, such as a polyethylene membrane appropriately treated to render it hydrophilic. Such membranes are made by Porvair Filtration Limited under the brand name Vyon . Another suitable membrane may be a cofoam, such as a polyurethane foam.

:.:::. The reducing agent and, optionally, the source of iodide, acid and//or buffer may be *:::" applied to the disc, for example the porous, fluid conducting membrane, by any suitable method. For example, the reducing agent (for example zinc) may be applied " by contacting the membrane with a suspension of the reducing agent in a suitable ":" solvent (such as water) and then drying the membrane so as to embed the reducing : agent therein. * 20 *.*.

S..' In the vertical flow product, the detection zone or module may comprise a detection disc, pad or frit (hereinafter referred to as a detection disc) in which a reactant capable of reacting with the component of the reaction product is embedded. As discussed above, the reaction product typically is iodine and the reactant may be an organic polymer such as polyvinyl alcohol or starch. In one aspect, a suitable acid andlor buffer and/or an agent to assist complexation may also be embedded in the detection disc, Any suitable acid, buffer and/or agent may be used, as discussed above.

The detection disc may be made from any suitable material, for example from a porous, fluid conducting membrane, such as a polyethylene membrane appropriately treated to render it hydrophilic. Such membranes are made by Porvair Filtration Limited under the brand name Vyon . Another suitable membrane may be a cofoam, such as a polyurethane foam.

The reactant may be applied to a porous, fluid conducting membrane using any suitable method. For example, the reactant (such as polyvinyl alcohol or starch) may be applied by contacting the membrane with a solution or suspension of the reactant in a suitable solvent (such as water) and then drying the membrane so as to embed the reactant therein. When an acid, buffer and/or an agent to assist complexation is present, these may be included in the solution or suspension with which the membrane is contacted. When present, the agent to assist complexation typically is added to the solution or suspension after the other components to prevent the formation of a gel.

: ** The vertical flow product may include one or more further discs, pads or frits as e appropriate. For example, the vertical flow product may further include a one or more control discs positioned at appropriate ocations along the flow path. The * : control discs may be made from any suitable material, for example blocked * : nitrocellulose orpolyethersulfone. The nitrocellulose or polyethersulfone may be blocked with 5% Marvel (milk powder) to prevent protein binding. When present, a control disc preferably is positioned after the reaction zone or module (and preferably ** before the detection zone or module) in the direction of the flow path and acts advantageously to prevent the reducing agent from flowing through the casing with the sample. Thus, the control disc restricts the carnage of any loose reducing agent (such as zinc powder) into the detection zone or module, where it may affect measurement or colour determination. Any suitable number of control discs may be included, typically one or two being sufficient.

The vertical flow product may further include one or more barrier discs, pads or frits positioned at appropriate locations along the flow path. The barrier discs may be made from any suitable material, for example a water-soluble material such as cellulose, pectin or gelatine. In use, the barrier disc must be dissolved before the sample can resume its progress through the fluid flow path. When present, a barrier disc preferably is positioned after the reaction zone or module and before or after the detection zone or module in the direction of the flow path. The barrier discs act advantageously to regulate the speed of flow of the sample through the casing.

Thus, the barrier discs allow an appropriate reaction time and facilitate pH changes when required by the release of buffers. Any suitable number of barrier discs may be included, typically one or two being sufficient.

The vertical flow product may further include a neutralisation zone or module including one or more neutralisation discs, pads or frits positioned at appropriate locations along the flow path. The neutralisation disc may be made from any suitable material, for example from a porous, fluid conducting membrane, such as a polyethylene membrane made by Porvair Filtration Limited under the brand name Vyon or a cofoam, such as a polyurethane foam. Typically, the neutralisation disc comprises a suitable base (such as sodium hydroxide) embedded therein, such that the base is released from the disc upon contact with the sample so as to modify the pH of the sample. Preferably, the neutralisation zone or module modifies the pH of the sample to a pH in the range of from 7 to 10. The base may be applied to the S...., . . * * porous, fluid conducting membrane using any suitable method. Any suitable number of neutralisation discs may be included, typically one or two being sufficient. * .

Preferably, the vertical flow product further comprises a sump on the flow path, the *..* sump comprising an absorbent material in fluid communication with the detection zone or module. The sump may be made from any suitable material, for example from a porous, fluid conducting membrane, such as a polyethylene membrane made by Porvair Filtration Limited under the brand name Vyon or a cofoam, such as a polyurethane foam. Preferably, the sump is made from highly absorbent cellulose paper or fine grade glass fibre.

The vertical flow product may further comprise a blood separation zone or module, such as a blood separation membrane, positioned before the reaction zone or module, for example at the sample application point as discussed above.

Advantageously, the vertical flow product further comprises a supply of a wash fluid.

Once the colouration has developed in the detection zone or module of the vertical flow product, a semi-quantitative result may be provided by comparison to a pre-prepared, prescribed colour range. Alternatively the vertical flow product may comprise a monitor operable to measure the colour intensity so as to provide a quantitative result.

In another aspect of the present invention, the nitrate detection product comprises a booklet style product. In the booklet style product, the reaction zone and the detection zone are hingedly connected to each other, such that the zones may be brought into contact with each other by flexing about the hinge. Advantageously, at least one of the reaction zone and detection zone has an adhesive area extending around its perimeter for sealingly contacting the reaction zone with the detection zone. * ** * * * *.*

*:::* 15 The booklet style product may comprise two or more leaves (for example two or three leaves) incorporating components of the reaction and detection zones, which * " leaves are hingedly connected to each other.

a..... * .

* * In the booklet style product, the reaction zone may comprise a reaction film (or pad) that contains a gel comprising the reducing agent. The gel comprising the reducing as..

agent may, optionally, also contain the source of iodide and/or a suitable acid and/or buffer. Particularly, the gel may contain a suitable buffer. Alternatively, the source of iodide may be contained in a gel applied to a separate reaction film to the reaction film containing the reducing agent. In this embodiment, the gels containing the reducing agent and the source of iodide may optionally also contain a suitable acid and/or buffer.

The reaction film (or pad) may be made from any suitable material, such as (but not limited to) gelatine, pectin or agarose.

In the booklet style product, the detection zone may comprise a visualisation film (or pad) comprising a reactant capable of reacting with the component of the reaction product to generate the signal. Conveniently, the visualisation film is made from a material which allows uniform colour development. The visualisation film (or pad) may be made from any suitable material, preferably from the reactant capable of reacting with the component of the reaction product to generate the signal, such as (but not limited to) polyvinyl alcohol. The visualisation film may contain a gel comprising a suitable acid and/or buffer, particularly a suitable buffer.

As the skilled person would appreciate, any suitable gel may be included in the booklet style product. Suitable gels include (but are not limited to) gelatine. The use of a gel is advantageous because the gel may be formed into a transparent colourless film. By the term "gel" we mean a colloidal system comprising interconnected solid particles (for example colloidal or polymenc particles) holding liquid between them such that the liquid does not flow out of the solid matrix, i.e. syneresis. * .* * * . **.

Conveniently, the reaction zone is mounted on one or more leaves, for example on one or two leaves. * S

Conveniently, the detection zone is mounted on a leaf, there being provided an : * aperture in the leaf, for viewing of the detection zone. Preferably, the aperture is covered by a transparent window. **** * * S. ,*

Optionally, the reaction zone has a sump made from an absorbent material located adjacent to it.

Conveniently, at least one of the reaction zone and detection zone is covered by a removable protective film. Preferably, both the reaction zone and the detection zone are covered by a removable protective film.

The booklet style product may further comprise a hand held reader operable to measure a colorimetric response and interpret the data using simple software and algorithm to determine the nitrate concentration. For example, the hand held reader may comprise a colorimeter comprising an aperture of the same size as the detection zone. In use, the booklet may be placed over the aperture of the colorimeter with the visualisation film face down. The developed/developing booklet is then measured across a series of parameters. These are hue, saturation, intensity, redness, blueness and greenness.

The present invention also provides a method of detecting nitrate in a sample, the method comprising the steps of: (i) contacting the sample with a reducing agent in the presence of a source of iodide in a reaction zone to produce a reaction product; and (ii) presenting a signal in response to the detection of a component of the reaction product in a detection zone.

The method further includes the step of passing the reaction product from step (i) directly to step (ii), i.e. passing the reaction product from the reaction zone in step (i) along a flow path directly to the detection zone in step (ii). In other words, the *::* 15 method further comprises, after step (i), contacting the reaction product with the detection zone.

* ***** * S Step (i) preferably is conducted at a pH in the range of from 1 to 9, preferably of 2 to * * 5, more preferably of from 2 to 3, for example about 3. The desired pH typically is achieved by adding a suitable acid and/or a suitable buffer. Suitable acids and **** buffers are defined above.

Step (ii) preferably is conducted at a pH in the range of from 3 to 5, such as about 3.

The desired pH typically is achieved by adding a suitable acid and/or a suitable buffer. Suitable acids and buffers are defined above.

Any suitable reducing agent may be used in the method of the present invention.

Suitable reducing agents are defined above.

Any suitable source of iodide may be used in the method of the present invention.

Suitable sources of iodide are discussed above.

As discussed above, when a sample containing nitrate is contacted with the reducing agent (such as zinc) in the presence of the source of iodide (such as potassium iodide) at the appropriate pH, then the nitrate is reduced to form nitrite. Any nitrite formed then reacts with the source of iodide to liberate iodine. Thus, when nitrate is present, a component of the reaction product produced in the reaction zone is iodine.

Conveniently, in step (ii), a signal is presented in the detection zone in response to the detection of the component of the reaction product. In one aspect, the signal presented is a visual signal, for example a colouration. In this aspect, step (ii) therefore comprises observing the signal, i.e. colouration.

Particularly, step (ii) may comprise contacting the reaction product (such as iodine) with a reactant capable of reacting with the component of the reaction product to present the signal. In other words, step (ii) comprises contacting the reaction product with a reactant capable of reacting with the component of the reaction product (for example iodine) to present the signal (i.e. colouration). Suitable such * : reactants are organic polymers, as defined above.

****.. * *

* * Step (ii) may additionally comprise contacting the reaction product (such as iodine) with a reactant capable of reacting with the component of the reaction product to present the signal in the presence of an agent to assist the complexation of a component of the reaction product to the reactant. Suitable such agents are defined above.

In another aspect, in step (ii) the signal presented in the detection zone is an electrochemical signal. In this aspect, step (ii) therefore comprises measuring a voltage and/or a current to present the signal.

As discussed above, the sample may require an appropriate pre-treatment before it is tested. Thus, the method of the present invention may include an appropriate pre-treatment step prior to step (i). (

Advantageously, step (I) is carried out on a reaction substrate and step (ii) is carried out on a detection (for example visualisation) substrate. Suitable substrates are defined herein.

Accordingly, the present invention provides a method of detecting the presence of nitrate in a sample, the method comprising the steps of: (i) contacting the sample with zinc as a reducing agent in the presence of potassium iodide in a reaction zone to produce a reaction product containing iodine (when nitrate is present in the sample); and (ii) presenting a signal in response to the detection of iodine in a detection zone.

Accordingly, the present invention provides a method of detecting the presence of nitrate in a sample, the method comprising the steps of.

(i) contacting the sample with zinc as a reducing agent in the presence of potassium iodide in a reaction zone to produce a reaction product containing iodine (when nitrate is present in the sample); (ii) presenting a signal in response to the detection of iodine in a * * detection zone by contacting the reaction product with a reactant (such as polyvinyl alcohol or starch) capable of reacting with the * iodine so as to form a coloured complex.

Advantageously, the method described above uses a nitrate detection product of the present invention.

According to another aspect of the present invention, there is provided the use of a nitrate detection product as described above to detect the presence of nitrate in a sample.

The nitrate detection product and the method according to the present invention are useful in any application in which it is desirable to detect the presence (or absence) of nitrate in a sample. The product and method are particularly useful where it is intended to use the indication of nitrate levels in a sample to ascertain what levels of nitric oxide are present in a sample obtained from an animal, such as a human.

It is desirable to ascertain what levels of nitric oxide are present in a sample obtained from an animal such as a human, because this may be indicative of the onset or presence of a particular disease or condition. For example, low levels of nitric oxide in an animal are believed to be indicative of impaired wound healing in that animal (for example human). Thus, the determination of nitric oxide levels may allow for the detection of impaired wound healing and enable appropriate action to be taken to prevent and/or treat the condition.

Prevention and/or treatment of impaired wound healing may, for example, be by the * use of advanced wound dressings, which deliver nitric oxide to a wound. If impaired wound healing may be diagnosed dunng a visit to the clinic, this will reduce the ** number of visits to the clinic required by the patient and provides more appropriate and timely treatment regimes.

I..... * .

Thus, the nitrate detection product and the method of the present invention are useful : * * in the diagnosis of a disease or condition, such as impaired wound healing, in an *.

animal, such as a human. Suitable steps to prevent and/or treat the disease or condition may be taken following the diagnosis. Accordingly, the present invention provides a method of diagnosing a disease or condition, such as impaired wound healing, in an animal, such as a human, compnsing the step of detecting nitrate in a sample obtained from the animal.

According to the present invention, there is further provided a kit for detecting the presence of nitrate in a sample, the kit comprising a nitrate detection product as described above and a colour chart showing the degree of colouration provided for specified levels of nitrate in a sample. Preferably, the kit further comprises a set of written instructions for use of the nitrate detection product.

The nitrate detection product, method and kit of the present invention are suitable for home use (for example by a medical professional), as well as for use by a medical professional in a hospital or suchlike. In particular, the nitrate detection product, method and kit of the present invention are suitable for point of care operation.

According to another aspect of the present invention, there is provided a nitrite detection product for detecting nitrite in a sample, the product compnsing: (i) a reaction zone adapted so as to receive the sample and contact it with a source of iodide to produce a reaction product; and (ii) a detection zone for presenting a signal in response to the detection of a component of the reaction product, which detection zone comprises (a) a reactant capable of reacting with the component of the reaction product to present a visual signal or (b) an electrochemical device to present an electrochemical signal. * **

Another aspect of the invention provides a method of detecting nitrite in a sample, S...

the method comprising the steps of: (i) contacting the sample with a source of iodide in a reaction zone to S....

produce a reaction product; and *.*S.* * * (ii) presenting a signal in response to the detection of a component of the : * reaction product in a detection zone, wherein the signal is presented S...

(a) by contacting the reaction product with a reactant capable of reacting with the component of the reaction product to generate a visual signal or (b) by measuring a voltage and/or current to generate an electrochemical signal.

In the aforementioned product or method for detecting nitrite in a sample, the visual signal may be a colouration, for example by the formation of a coloured complex with the component of the reaction product.

The product or method for detecting nitrite in a sample may be used to detect nitrite, for example when the detection of nitrite is required instead of the detection of nitrate.

This may be useful in relation to samples that contain relatively high levels of nitrite.

This product and method could, of course, alternatively be used when a sample has been subjected to a separate pre-treatment step to reduce nitrate to nitrite prior to testing. A person skilled in the art could select an appropriate pre-treatment step.

According to another aspect of the present invention, there is provided a metabolite detection product for detecting a metabolite of nitric oxide in a sample, the product comprising: (I) a reaction zone adapted so as to receive the sample and contact it with a source of iodide to produce a reaction product; and (ii) a detection zone for presenting a signal in response to the detection of a component of the reaction product, which detection zone comprises (a) a reactant capable of reacting with the component of the reaction product to present a visual signal or (b) an electrochemical device to : present an electrochemical signal.

This metabolite detection product may be especially useful for detecting the nitrite metabolite in a sample.

* *.**. * *

In the aforementioned metabolite detection product, the reaction zone may further * * contain a reducing agent, so as to receive the sample and contact it with the reducing agent in the presence of the source of iodide to produce the reaction product. Such a product would be especially useful for detecting the nitrate metabolite in a sample.

Another aspect of the invention provides a method of detecting a metabolite of nitric oxide in a sample, the method comprising the steps of: (i) contacting the sample with a source of iodide in a reaction zone to produce a reaction product; and (ii) presenting a signal in response to the detection of a component of the reaction product in a detection zone, wherein the signal is presented (a) by contacting the reaction product with a reactant capable of reacting with the component of the reaction product to present a visual signal or (b) by measuring a voltage and/or current to present an electrochemical signal.

S

This metabolite detection method may be especially useful for detecting the nitrite metabolite in a sample.

In the aforementioned method, step (i) may further comprise contacting the sample with a reducing agent in the presence of the source of iodide to produce the reaction product. Such a method would be especially useful for detecting the nitrate metabolite in a sample.

In the aforementioned product or method for detecting a metabolite in a sample, the visual signal may be a colouration, for example by the formation of a coloured complex with the component of the reaction product.

According to another aspect of the present invention, there is provided a metabolite detection product for detecting a metabolite of nitric oxide in a sample, the product comprising: (I) an oxidation zone adapted so as to receive the sample and contact it * with an agent for oxidation to produce a reaction product; and (ii) a detection zone comprising an electrochemical device for measuring : * * the extent of oxidation of the agent for oxidation upon contact with the sample. **e.

This metabolite detection product may be especially useful for detecting the nitrite metabolite in a sample. In the detection zone, a signal will be presented in response to the measurement of the extent of oxidation of the agent for oxidation upon contact with the sample.

The metabolite detection product may further comprise a reaction zone, the reaction zone comprising a reducing agent so as to receive the sample and contact it with the reducing agent prior to addition to the oxidation zone. Such a metabolite detection product would be especially useful for detecting the nitrate metabolite in a sample.

In the metabolite detection product, the agent for oxidation may be selected from a source of iodide (for example potassium iodide) and hydroquinone. Of course, another term for an agent for oxidation would be a reducing agent.

Another aspect of the invention provides method of detecting a metabolite of nitric oxide in a sample, the method comprising the steps of: (i) contacting the sample with an agent for oxidation in an oxidation zone to produce a reaction product; and (ii) measuring the extent of oxidation of the agent for oxidation upon contact with the sample.

This metabolite detection method may be especially useful for detecting the nitrite : *". metabolite in a sample. S... S...

The aforementioned method may further comprise the step of contacting the sample with a reducing agent in a reaction zone prior to addition to the oxidation zone. Such S.....

* a method would be especially useful for detecting the nitrate metabolite in a sample.

I..... * e

: *. * In the aforementioned method, the agent for oxidation may be selected from a source of iodide (for example potassium iodide) and hydroquinone.

In the measurement of the extent of oxidation of the agent for oxidation upon contact with the sample in step (ii), a signal will be presented.

The nitrite and metabolite products and methods defined above may comprise reaction and detection zones or modules as described above in relation to the nitrate detection product and method, where appropriate. Additionally, the sample, iodide source, reaction product, reducing agent and/or signal presentation may be as described above in relation to the nitrate detection product and method, where appropriate.

References herein to detecting the presence of nitrate, nitrite or a metabolite of nitric oxide in a sample are intended to refer to detecting the presence or absence of nitrate, nitrite or metabolite in the sample.

The nitrite and metabolite products and methods are suitable for diagnosis of certain diseases and conditions as discussed above (especially impaired wound healing), as would be appreciated by the skilled person.

In the present invention, when an electrochemical signal is presented, the detection zone or module comprises an electrochemical test module. The test module comprises working and reference electrodes and, optionally, a counter electrode.

The sample is applied to the test module so as to contact the electrodes and the : **. extent of oxidation of a particular reagent (for example of an agent for oxidation) is measured. As the skilled person would appreciate, the extent of oxidation may be e..s measured in relation to the source of iodide and its conversion to iodine (for example in the products and methods described above wherein a sample containing nitrate is * contacted with a reducing agent in the presence of a source of iodide to reduce I.....

* nitrate to nitrite or where a sample containing nitrite is contacted simply with a source : * * of iodide). Alternatively, the extent of oxidation may be measured in relation to the *0*I oxidation of the agent for oxidation, such as a source of iodide (for example potassium iodide) and hydroquinone.

For example, in the aspects of the invention discussed above where nitrite reacts with a source of iodide (such as potassium iodide) to generate iodine, the iodide ion of the potassium iodide is oxidised to form iodine and a change in the electrochemical properties of the sample occurs, i.e. a change in the voltage and/or current measurable across the sample. By measuring and presenting this voltage and/or current change, an indication is provided of the amount of iodine formed. This in turn provides an indication of the amount of nitrite (or nitrate once it has been reduced to nitrite) in a sample. Preferably, the sample (containing nitrite) reacts with the source of iodide in the presence of a suitable buffer (such as a citrate buffer) and/or a source of chloride (such as potassium chloride). As the skilled person would appreciate, the source of chloride is used as a buffer to keep the reference electrode voltage constant.

In the embodiment above wherein an agent for oxidation is used, the agent may comprise potassium iodide (which is converted to iodine as previously discussed) or, for example, hydroquinone. In the presence of nitrite, hydroquinone is oxidised to form parabenzoquinone (otherwise known as quinone), which similariy causes a change in the electrochemical properties of the sample, i.e. a change in the voltage andlor current measurable across the sample. By measuring and presenting this voltage and/or current change, an indication is provided of the amount of parabenzoquinone formed, which in turn provides an indication of the amount of nitrite (or nitrate) in a sample. * **

* In order that the present invention may be more readily understood and so that S...

further details thereof may be appreciated, embodiments of the invention will now be * described, by way of example, with reference to the accompanying drawings in a..... * S

which: *., . S. * a * Figure 1 is a perspective view of a nitrate detection product in accordance with one embodiment of the present invention; *5** Figure 2 is a perspective view of a nitrate detection product in accordance with an alternative embodiment of the present invention; Figure 3 is a cross-sectional view of the nitrate detection product of Figure 2 along the line A-A; Figure 4 is a cross-sectional view of the nitrate detection product of Figure 2 along the line B-B; Figure 5 is a perspective view of a nitrate detection product in accordance with an alternative embodiment of the present invention; Figure 6 is a perspective view of a metabolite (for example nitrite) detection product in accordance with an alternative embodiment of the present invention;

V

Figure 7 shows the results of a series of tests as conducted in a vertical flow product

in Example 1 below;

Figure 8 shows a booklet style product as prepared in Example 2 below; and Figure 9 shows the results of a series of tests as conducted in a booklet style product

in Example 2 below.

Referring to Figure 1, a nitrate detection product (1) comprises a transparent cylindrical casing (2) made from a plastics material. This represents a vertical flow device. The casing (2) is open at a first end (3) and at a second end (4). A cap (not shown) may be included to close off either end, as required.

Contained within the casing (2), at the first end (3), is a reaction module (5) which, in : *. use, initially holds the sample to be tested. The reaction module (5) comprises a reduction disc or frit (6), which is made from a porous, fluid conducting membrane such as Vyon (details of which are discussed above) or a cofoam such as a * : polyurethane foam. The reduction disc or frit (6) comprises zinc (i.e. the reducing agent), potassium iodide (i.e. the source of iodide), a suitable acid and a suitable a.....

* buffer embedded therein. All of the aforementioned reagents are embedded in the : . * reduction disc or frit (6) in solid form. *.*

Beneath the reduction disc or frit (6), away from the first end (3), is provided a control disc or fnt (7). The control disc or frit (7) is capable of substantially preventing the flow of zinc though the flow path and is made from a material through which the zinc cannot pass, such as blocked nitrocellulose or polyethersulfone. Beneath the control disc or frit (7), away from the first end (3), is

provided a neutralisation disc or frit (8). The neutralisation disc or frit (8) is made from a porous, fluid conducting membrane such as Vyon (details of which are discussed above) or a cofoam such as a polyurethane foam and has sodium hydroxide embedded therein. The sodium hydroxide is embedded in the neutralisation disc or frit (8) in solid form. (

Beneath the neutratisation disc or frit (8), away from the first end (3), is provided a first barrier disc or frit (9). The first barrier disc or frit (9) is capable of slowing the progress of the sample along the flow path and is made from a material that is soluble in water, such as cellulose, pectin or gelatine. The first barrier disc or fnt (9) must be dissolved before the sample can resume its progress through the flow path.

Beneath the first barrier disc or frit (9), away from the first end (3), is provided a detection module (10) comprising a detection disc or frit (11), which is made from a porous, fluid conducting membrane such as Vyon (details of which are discussed above) or a cofoam such as a polyurethane foam. The detection disc or frit (11) comprises polyvinyl alcohol (i.e. the organic polymer capable of forming a complex with a component of the reaction product formed in the reaction zone (5)), sodium tetraborate, a suitable acid and a suitable buffer embedded therein. All of the aforementioned reagents are embedded in the detection disc or frit (11) in solid form. a... S...

Beneath the detection disc or frit (11), away from the first end (3), is provided a second barrier disc or frit (12) similar to the first barrier disc or frit (9) discussed as....

* above.

*.S... * .

: * Beneath the second barrier disc or frit (12) is provided an absorbent sump (13). The absorbent sump (13) sits against the second end (4) of the casing (2). The as..

absorbent sump (13) is made from a material such as a porous, fluid conducting membrane such as Vyon (details of which are discussed above) or a cofoam such as a polyurethane foam.

It is to be appreciated that the control disc or frit (7) ensures that the zinc remains in the reaction module (5), before the nitrate detection product (1) is used and during use of the nitrate detection product (1).

It also is to be appreciated that the first and second barrier discs or frits (9, 12) regulate the speed at which the sample flows through the nitrate detection product.

Whilst Figure 1 shows two barrier discs or frits, any suitable number of barrier discs or frits may included and located at suitable locations along the flow path so as to regulate the flow.

It also is to be appreciated that whilst the nitrate detection product (1) shown in Figure 1 includes a control disc or fit (7), first and second barrier discs or fits (9, 12) and a neutralisation disc or frit (8), these are not essential and a person skilled in the art would readily be able to determine whether they should be included.

The reduction disc or frit (6), control disc or frit (7), first and second barrier discs or frits (9, 12), neutralisation disc or frit (8), detection disc or frit (11) and absorbent sump (13) define a liquid flow path through which a liquid may be absorbed.

In use, a sample in which it is desired to detect the presence of nitrate is deposited in the reaction module (5) on the reduction disc or frit (6), via the first (open) end (3) of the casing (2). The fluid in the sample is absorbed into the reduction disc or frit (6) and the desired pH is attained by release of the acid and buffer from the reduction * ,* disc or fnt (6). Any nitrate in the sample reacts with the zinc, at the desired pH, and * is reduced to nitrite. As the nitrite is formed, it reacts with the potassium iodide to S...

liberate iodine.

: 15 The sample (including any iodine produced) then passes through the reduction disc or tnt (6), the control disc or frit (7) and onto the neutralisation disc or frit (8), which releases sodium hydroxide and adjusts the pH as desired. The adjustment of the pH aids in the formation of iodine in the presence of nitrite. .. * * * S..

The sample (including any iodine produced) then passes through the first barrier disc or tnt (9) to the detection module (10) and onto the detection disc or frit (11).

The sample is then flushed through the product (1) with water and the product is allowed to develop for an appropriate period of time, such as for 5 minutes.

Once the sample has been deposited on the detection disc or tnt (11), the polyvinyl alcohol, sodium tetraborate, acid and buffer are released and any iodine in the sample binds to the polyvinyl alcohol so as to form a red/brown coloured complex.

The sample then passes through the second barrier disc or frit (12) and into the absorbent sump (13).

Consequently, if there is no nitrate in the sample then the sample passes all of the way through the nitrate detection product (1), into the absorbent sump (13) and no colour is produced on the detection disc or tnt (11) or in the absorbent sump (13). If, on the other hand, nitrate is present in the sample then iodine is produced by the reactions discussed above as the sample passes through the nitrate detection product (1) and a coloured complex is produced, which passes to the absorbent sump (13). The presence of such a colour after completion of the assay, is thus indicative of the presence of the nitrate in the sample. The absence of such a colour after completion of the assay, is indicative of the absence of the nitrate in the sample.

The presence or absence of nitrate in the sample is in turn indicative of the presence or absence of nitric oxide in the sample, i.e. prior to it being metabolised to nitrate.

It is to be appreciated that the detection disc or tnt (11) and/or absorbent sump (13) : *** therefore act as a detection zone or module which is indicative of the outcome of the test. The presence of a colour at this detection zone or module indicates the * * S..

presence of nitrate in the sample whereas the absence of a colour at the detection zone or module indicates the absence of nitrate in the sample. When a colour is * : *..: observed at the detection zone or module, the intensity of the colour is indicative of the amount of nitrate in the sample; the darker the colour produced, the higher the :::. amount of nitrate in the sample. S...

*" 20 It is also to be noted that each of the reduction disc or frit (6), control disc or frit (7), first and second barrier discs or fits (9, 12), neutralisation disc or fnit (8), detection disc or tnt (11) and absorbent sump (13) are effectively independent modules.

Modules can be added, moved or removed with ease in order to generate a nitrate detection product with a different arrangement of modules.

In a variant of the embodiment depicted in Figure 1, a cap (not shown) may be provided over the first end (3) of the casing (2). The cap contains a small tank with a frangible lid. The tank is filled with a wash fluid. A sharp edge protrudes from (he first end (3) of the casing (2) in proximity to the frangible lid. In order to use the nitrate detection product (1) of this embodiment, the cap is first removed from the casing (2) and the sample is deposited in the nitrate detection product (I) as in the above-described embodiment. Subsequently, the cap is returned to the rest of the ( nitrate detection product (1) and the lid of the cap is broken, by pressing down hard on the cap such that the sharp edge punctures the frangible lid. This releases the wash fluid to follow the sample through the flow path. This embodiment ensures that there is sufficient fluid to carry the sample all the way through the liquid flow path to the absorbent sump (13).

Referring to Figure 2, a nitrate detection product (20) comprises a paper or cardboard booklet (21) comprising first and second leaves (22, 23) of identical shape and size connected at a spine (24). This represents a booklet style product. The spine (24) acts as a hinge between the first and second leaves (22, 23).

The first leaf (22) includes a reaction zone (25) located in its centre. Covering the reaction zone (25) is a removable foil wrapper (not shown). * .* * . S S...

* *15 The second leaf (23) includes a detection zone (26) located in its centre. In the S...

centre of the detection zone (26), there is provided a first aperture (27). The * . . detection zone (26) is located on the second leaf (23) facing the reaction zone (25) of the first leaf (22). Covering the detection zone (26) is a removable foil wrapper (not : shown). I...

* 20 Referring now to Figure 3, the reaction zone (25) will be described in further detail.

The reaction zone (25) comprises, attached to the internal face of the first leaf (22), a first adhesive film (29). On top of the first adhesive film (29) (although of a smaller size than the first adhesive film (29)) is provided an absorbent sump (30). On top of the absorbent sump (30) is provided an absorbent reaction film (31) made from gelatine or pectin and having impregnated into it a gelatine gel containing zinc. The reaction film (31) is smaller than the absorbent sump (30) such that the edges (32) of the first adhesive film (29) extend outwardly of the reaction film (31).

Referring now to Figure 4, the detection zone (26) will be described in further detail.

The detection zone (26) comprises, attached to an internal face of the second leaf (23), a second adhesive film (33), in which there is an aperture (34) aligned with, but larger than, the first aperture (27). On top of the second adhesive film (33) is

S

provided a visualisation film (35) made from polyvinyl alcohol and impregnated with a gelatine gel containing a citrate buffer. The vsuahsation film (33), though centred on the aperture (27), is smaller than the second adhesive film (33). The visualisation film (35) is optically clear, carries no inherent colour and allows uniform colour development. It is non-porous before use.

On top of the visualisation film (35) is provided a membrane (36), which is bigger than the film (35) and thus overlaps the visualisation film (35) and connects at its edges onto the second adhesive film (33). Nonetheless, the membrane (36) is smaller than the second adhesive film (33) so that the edges (37) of the second adhesive film (33) extend outwardly from the membrane (36). The membrane (36) is opaque and is preferably white in colour. It contains a plurality of pores (not shown).

The pores render the membrane selective of the molecules which may pass through it, such as iodine molecules. *... * . ***

In use, the booklet (21) is opened to separate the first and second leaves (22, 23) * and the protective films (not shown) covering the reaction zone (25) and the detection zone (26) are removed. A 2M aqueous solution of potassium iodide is * applied to the reaction zone (25), i.e. by spreading the solution over the surface of * :. 20 the absorbent reaction film (31) with the tip of a pipette. The sample to be tested is S...

deposited on the absorbent reaction film (31). Any run-off from the sample is absorbed by the sump (30).

As the sample contacts the zinc in the absorbent reaction film (31), any nitrate in the sample reacts with the zinc and is reduced to nitrite. As the nitrite is formed, it reacts with the potassium iodide to liberate iodine.

The first and second leaves (22, 23) of the booklet (21) then are pressed together after an appropriate reaction time (for example 5 minutes), such that the edges (32) of the first adhesive film (29) meet the edges (37) of the second adhesive film (33) and cause adhesion between the first and second leaves (22, 23). The adhesion of the first and second leaves (22, 23) to each other seals the sample within the booklet (21), which thus hygienically contains the sample.

S

Once the leaves (22, 23) are brought together, any iodine formed passes through the membrane (36) and onto the visualisation film (35). Other components of the sample are unable to pass through the membrane (36) and remain on the absorbent reaction film (31).

When the iodine reaches the visualisation film (35), it binds to the polyvinyl alcohol of the visualisation film so as to form a red/brown coloured complex. The remainder of the sample passes into the absorbent sump (30). The visualisation film (35) is visible through the first and second apertures (27, 34) in the second leaf (23) and the second adhesive film (33) and thus an observer may see the colour change that takes place. Because the membrane (36) is preferably white in colour, opaque and forms a background to the visualisation film (35), it assists an observer in making an unbiased assessment of the colour change, regardless of sample-derived interfering a..' 15 colour. As discussed above, the colour change is indicative of the presence of nitrate in the sample. The darkness of the colour produced is dependent on amount of * : nitrate in the sample.

p....' * S In a sample that does not contain any nitrate, no iodine is formed, so when the two leaves (22, 23) of the booklet (21) are brought into contact, there is no iodine present S..

*,. 20 to form a complex with the polyvinyl alcohol and no colour is observed.

In an alternative embodiment of the booklet device (not shown), a transparent slide or window is attached to the second leaf (23), covering the first aperture (27), on the opposite side of the second leaf (23) from the second adhesive film (33). The transparent slide permits viewing of the visualisation film (35), but does not permit ingress of contaminants or egress of the sample.

In alternative embodiments, the reaction film (31) is instead made from other materials which hydrate but do not dissolve in water such as acrylamide, agarose, pululan or carrageenans.

Referring to Figure 5, an alternative booklet style nitrate detection product (30) comprises a paper or cardboard booklet (31) comprising first, second and third leaves (32, 33, 34) of identical shape and size connected at a spine (35). The spine (35) acts as a hinge between the first, second and third leaves (32, 33, 34).

The first and second leaves (32, 33) each include a reaction zone (36, 36A) located in their centre. Covering each of the reaction zones (36, 36A) is a removable foil wrapper (not shown).

The third leaf (34) includes a detection zone (37) located in its centre. Covering the detection zone (37) is a removable foil wrapper (not shown).

* The reaction zone (36) of the first leaf (32) comprises, attached to the internal face of * * the first leaf (32), a first adhesive film (38). On top of the first adhesive film (38) * 15 (although of a smaller size than the first adhesive film (38)) is provided an absorbent * reaction film (39) made from gelatine or pectin and having impregnated into it a * S,*..

gelatirie gel containing zinc and a suitable acid and buffer. * .

: * The reaction zone (36A) of the second leaf (33) includes a first aperture (40) in its S...

centre. Attached to a face of the first second leaf (33) and covering the first aperture (40) is a second adhesive film (41). On top of the second adhesive film (41) (although of a smaller size than the second adhesive film (41)) is provided an absorbent reaction film (42) made from gelatine or pectin and having impregnated into it a gelatine gel containing potassium iodide.

In the centre of the detection zone (37), there is provided a second aperture (43).

Attached to a face of the third leaf (34) and covering the second aperture (43) is a third adhesive film (44). On top of the third adhesive film (44) (although of a smaller size than the third adhesive film (44)) is provided a visualisation film (45) made from polyvinyl alcohol and impregnated with a gelatine gel containing sodium tetraborate and a citrate buffer. The visualisation film (45) is optically clear, carries no inherent colour and allows uniform colour development. It is non-porous before use.

In use, the booklet (31) is opened to separate the first, second and third leaves (32, 33, 34) and the protective films (not shown) covering the reaction zones (36, 36A) and the detection zone (37) are removed.

The sample to be tested is deposited on the absorbent reaction film (39). As the sample contacts the zinc in the absorbent reaction film (39), any nitrate in the sample reacts with the zinc and is reduced to nitrite.

The first and second leaves (32, 33) of the booklet (31) then are pressed together (in the direction of the arrow (46)) after an appropriate reaction time (for example 5 minutes), such that the absorbent reaction film (39) is located in the first aperture (40) in contact with the absorbent reaction film (42). The edges of the first adhesive film * (38) meet a surface of the second leaf (33) and cause adhesion between the first and second leaves (32, 33), securely holding the absorbent reaction film (39) in the first *.* aperture (40) in contact with the absorbent reaction film (42). The nitrite formed in the sample held in the absorbent reaction film (39) then contacts the potassium * iodide in the absorbent reaction film (42) and the nitrite reacts with the potassium * iodide to liberate iodine. * * I. * *S..

The third leaf (34) is then pressed to the adhered first and second leaves (32, 33) of the booklet (31) (in the direction of the arrow (47)) after an appropriate reaction time (for example 5 minutes), such that the visualisation film (45) contacts the absorbent reaction film (42). Any iodine formed passes onto the visualisation film (45) and binds to the polyvinyl alcohol of the visualisation film so as to form a red/brown coloured complex. The visualisation film (45) is visible through the second aperture (43) and the third adhesive film (44) in the third leaf (33) and thus an observer may see the colour change that takes place. As discussed above, the colour change is indicative of the presence of nitrate in the sample. The darkness of the colour produced is dependent on amount of nitrate in the sample.

In a sample that does not contain any nitrate, no iodine is formed, so when the leaves (32, 33, 34) of the booklet (31) are brought into contact, there is no iodine present to form a complex with the polyvinyl alcohol and no colour is observed.

F

Figure 6 shows an embodiment of a metabolite detection product (50) for detecting a metabolite of nitric oxide in a sample, for example a nitrite detection product, wherein the sample is contacted with an agent for oxidation.

The metabolite detection product (50) comprises a reaction zone or module that comprises a test module (51). The test module (51) includes a working electrode (52), a reference electrode (53) and a counter electrode (54). The test module (51) further includes an insulated region (55). In use, a suitable agent for oxidation (such as potassium iodide or hydroquinone) is added to the insulated region (55) of the test module (51). A reference voltage and/or current is measured at the reference electrode (53). The sample to be tested is then added to the insulated region (55) of * the test module (51) and any nitrite metabolite in the sample acts to oxidise the agent for oxidation. The extent of oxidation is measured by measuring a voltage and/or **..

current at the working electrode (52) and the change in voltage and/or current is indicative of the extent of oxidation. In turn, the extent of oxidation is indicative of the **....

* amount of nitrite in the sample being tested. * S

: ** * As a skilled person would appreciate, a test module (51) could be incorporated into S...

or associated with a vertical flow product as described above. * . S...

It is to be understood that the present invention is not limited to embodiments in which the reducing agent is zinc, the source of iodide is potassium iodide and/or the reactant capable of forming a complex with a component of the reaction product is polyvinyl alcohol. Any alternative suitable reducing agent, iodide source and/or reactant may be used, as discussed above. For example, polyvinyl alcohol may be replaced by starch.

The invention will now be illustrated by the following non-limiting examples in which, unless stated otherwise: (a) operations were conducted at room or ambient temperature, that is a temperature in the range of from 18 to 25 C; (b) chemical symbols have their usual meanings; and

L

(c) SI symbols and units are used.

Example 1

A vertical flow product comprising a polypropylene column was prepared using Vyon HP frits supplied by Porvair. Reagents were applied to the frits and dried.

The fnt for use in the reaction module (referred to as "Frit 1" below) was prepared suspending powdered zinc (1 g) in water (5 ml) and then shaking to prepare a suspension. 10 frits were then added to the suspension in a bijou bottle and put on a rotary mixer for 1 hour. The fnts were then removed from the zinc suspension and dried. * .* * * 0

The frit for use in the detection module (referred to as "Frit 2" below) was prepared by .... 15 preparing a solution comprising 5% weight/volume polyvinyl alcohol in water (5 ml), 2M aqueous citrate solution (500 p1) and 50 mM aqueous sodium tetraborate solution * I...b * (1 ml). The components of the solution were added to one another in the order in * which they are listed. The solution (30 p1) was contacted with the frit until all of the : * * solution had soaked into the fit and then the frit was left to dry at room temperature foraboutl7to24hours. * * Ca..

The prepared column materials were stacked according to the plan below in a holder (top to bottom): Frit 1 (top) Nitrocellulose membrane (blocked with 5% Marvel in water for 1 hour and dried before use) Barrier (blank) frit Frit 2 Barrier (blank) frit The columns were run according to the method below: (a) a sample (potassium nitrate, 500 tM) was diluted into 0.5 M HCI to a pH of from 2to 3; (b) 100 p1 of the diluted sample was applied to the top of the column; (c) 100 uI of 0.2M potassium iodide was applied to the column; (ci) 100 iii more of the diluted sample was applied to the column; (e) 100 p1 of water was added to flush the sample through the column; and (f) the columns were then left to develop for 5 minutes. * **

A series of columns were run using samples containing varying levels of nitrate. The *., *. 15 results are shown in Figure 7.

S

* *.. .

* A dark colour in fit 2 (the detection module) indicates a positive result, i.e. the S.....

* presence of nitrate in the sample. The higher the nitrate concentration, the darker : ,* * the colour produced. *SS* * .

Example 2

A booklet style product was assembled by using double-sided sticky tape to attach a gelatine gel containing zinc to the right hand side of the booklet and using double-sided sticky tape to attach a polyvinyl alcohol film to the left hand side of the booklet (as shown in Figure 8).

A 10 p1 of sample (2M potassium iodide) was added to a gelatine/zinc gel of the reaction zone and spread gently over the film surface with the tip of a pipette. A 30 p1 of sample (potassium nitrate) was then added to the gelatine/zinc gel and spread gently over the film surface with the tip of a pipette.

The booklet was then closed to bring the polyvinyl alcohol film gently into contact with the gelatine/zinc gel. The edges of the booklet were sealed by gently pressing sticky sides of the tape together. The two gels mixed by gently pressing on the window of the detection zone. Incubation of the booklet was 5 minutes.

A series of booklet tests were run using samples containing varying levels of nitrate.

The results are shown in Figure 9. The higher the concentration of nitrate in the sample, the darker the colour produced. * ** * . * *.. * *

I

* I.... * I

I

I..... * * * I * I * **S. **II * I

Claims

Claims 1. A nitrate detection product for detecting nitrate in a

sample, the product comprising: (I) a reaction zone containing a reducing agent and adapted so as to receive the sample and contact it with the reducing agent in the presence of a source of iodide to produce a reaction product; and (ii) a detection zone for presenting a signal in response to the detection of a component of the reaction product.
2. A nitrate detection product according to claim 1, wherein the reaction zone is further adapted so as to contact the sample with the reducing agent at a pH in the range of from 1 to9.

.... 15
3. A nitrate detection product according to claim 1 or 2, wherein the reducing * agent is selected from any one or more of zinc, cadmium, iron and vanadium. * .

****** *
4. A nitrate detection product according to claim 3, wherein the reducing agent is : ** * Zinc.
5. A nitrate detection product according to any one or more of the preceding claims, wherein the source of iodide is selected from any one or more of potassium iodide and sodium iodide.
6. A nitrate detection product according to claim 5, wherein the source of iodide is potassium iodide.
7. A nitrate detection product according to any one or more of the preceding claims, wherein the component of the reaction product is iodine.
8. A nitrate detection product according to any one or more of the preceding claims, wherein the signal presented in the detection zone is a colouration.
9. A nitrate detection product according to any one or more of the preceding claims, wherein the detection zone comprises a reactant capable of reacting with the component of the reaction product to present the signal.
10. A nitrate detection product according to claim 9, wherein reactant is an organic polymer capable of forming a complex with the component of the reaction product.
11. A nitrate detection product according to claim 10, wherein the organic polymer is selected from any one or more of starch and polyvinyl alcohol.
12. A nitrate detection product according to any one or more of claims I to 11, : which comprises a cylindrical casing including the reaction zone and the detection zone.
13. A nitrate detection product according to claim 12, wherein the reaction zone * comprises a reduction disc or frit in which the reducing agent is embedded.

*.* S. * S : ** *
14. A nitrate detection product according to claim 12 or 13, wherein the detection -.3.

zone comprises a detection disc or frit in which a reactant capable of reacting with the component of the reaction product is embedded.
15. A nitrate detection product according to any one or more of claims I to 11, wherein the reaction zone and the detection zone are hingedly connected to each other.
16. A nitrate detection product according to claim 15, wherein the reaction zone comprises a reaction film or pad that contains a gel comprising the reducing agent.
17. A nitrate detection product according to claim 15 or 16. wherein the detection zone comprises a visualisation film or pad comprising a reactant capable of reacting with the component of the reaction product to generate the signal.
18. A nitrate detection product according to any one or more of claims 1 to 7, wherein the signal presented in the detection zone is an electrochemical signal.
19. A method of detecting nitrate in a sample, the method comprising the steps of: (I) contacting the sample with a reducing agent in the presence of a source of iodide in a reaction zone to produce a reaction product; and (ii) presenting a signal in response to the detection of a component of the reaction product in a detection zone.
20. A method according to claim 19, further including the step of passing the reaction product from step (i) directly to the detection zone.
21. A method according to claim 19 or 20, wherein step (i) is conducted at a pH in the range of from 1 to9.

*
22. A method according to any one or more of claims 19 to 21, wherein the I..,..

* reducing agent is selected from any one or more of zinc, cadmium, iron and * vanadium. S. a *5
23. A method according to claim 22, wherein the reducing agent is zinc.
24. A method according to any one or more of claims 19 to 23, wherein the source of iodide is selected from any one or more of potassium iodide and sodium iodide.
25. A method according to claim 24, wherein the source of iodide is potassium iodide.
26. A method according to any one or more of claims 19 to 25, wherein the component of the reaction product is iodine.

C
27. A method according to any one or more of claims 19 to 26, wherein the signal presented in the detection zone is a colouration.
28. A method according to claim 27, wherein step (ii) comprises contacting the reaction product with a reactant capable of reacting with the component of the reaction product to present the signal.
29. A method according to claim 28, wherein the reactant is an organic polymer selected from any one or more of starch and polyvinyl alcohol.
30. A method according to claim 27, wherein the signal presented in the detection zone is an electrochemical signal.
31. The use of a product according to any one or more of claims 1 to 1810 detect *...

.... 15 nitrate in a sample.

*
32. The use of a product according to claim 31 in the diagnosis of impaired 1U1 S * wound healing in an animal. * I *. I S..
33. A nitrite detection product for detecting nitrite in a sample, the product comprising: (i) a reaction zone adapted so as to receive the sample and contact it with a source of iodide to produce a reaction product; and (ii) a detection zone for presenting a signal in response to the detection of a component of the reaction product, which detection zone comprises (a) a reactant capable of reacting with the component of the reaction product to present a visual signal or (b) an electrochemical device to present an electrochemical signal.
34. A method of detecting nitrite in a sample, the method comprising the steps of: (i) contacting the sample with a source of iodide in a reaction zone to produce a reaction product; and p (ii) presenting a signal in response to the detection of a component of the reaction product in a detection zone, wherein the signal is presented (a) by contacting the reaction product with a reactant capable of reacting with the component of the reaction product to generate a visual signal or (b) by measuring a voltage and/or current to generate an electrochemical signal.
35. A product or method according to claim 33 or 34, wherein the visual signal is a colouration.
36. A metabolite detection product for detecting a metabolite of nitric oxide in a sample, the product comprising: : ** (i) a reaction zone adapted so as to receive the sample and contact it I...

with a source of iodide to produce a reaction product; and S... 15 (ii) a detection zone for presenting a signal in response to the detection of * : a component of the reaction product, which detection zone comprises * : * (a) a reactant capable of reacting with the component of the reaction product to present a visual signal or (b) an electrochemical device to : * * present an electrochemical signal. *1** * . S...
37. A metabolite detection product according to claim 36, wherein the reaction zone further contains a reducing agent, so as to receive the sample and contact it with the reducing agent in the presence of the source of iodide to produce the reaction product.
38. A method of detecting a metabolite of nitric oxide in a sample, the method comprising the steps of: (i) contacting the sample with a source of iodide in a reaction zone to produce a reaction product; and (ii) presenting a signal in response to the detection of a component of the reaction product in a detection zone, wherein the signal is presented (a) by contacting the reaction product with a reactant capable of reacting with the component of the reaction product to present a visual signal or (b) by measuring a voltage and/or current to present an electrochemical signal.
39. A method according to claim 38, wherein step (i) further comprises contacting the sample with a reducing agent in the presence of the source of iodide to produce the reaction product.
40. A metabolite detection product for detecting a metabolite of nitric oxide in a sample, the product comprising: (i) an oxidation zone adapted so as to receive the sample and contact it with an agent for oxidation to produce a reaction product; and (ii) a detection zone comprising an electrochemical device for measuring * ** the extent of oxidation of the agent for oxidation upon contact with the sample. S... 15
41. A metabolite detection product according to claim 40, further comprising a reaction zone, the reaction zone comprising a reducing agent so as to receive the S.....

* sample and contact it with the reducing agent prior to addition to the oxidation zone. * . * * S *..*
42. A metabolite detection product according to claim 40 or 41, wherein the agent S...

for oxidation is selected from a source of iodide and hydroquinone.
43. A method of detecting a metabolite of nitric oxide in a sample, the method comprising the steps of: (i) contacting the sample with an agent for oxidation in an oxidation zone to produce a reaction product; and (ii) measuring the extent of oxidation of the agent for oxidation upon contact with the sample.
44. A method according to claim 43, further comprising the step of contacting the sample with a reducing agent in a reaction zone prior to addition to the oxidation zone. (
45. A method according to claim 43 or 44, wherein the agent for oxidation is selected from a source of iodide and hydroquinone.
46. A product substantially as herein described with reference to and as shown in the accompanying figures.
47. A method substantially as herein described with reference to the accompanying figures. * ** * S S S... * . *.

S

*SS.S. * S

S..... * .

:.. 20 *SSS * * *...