WO2015140575A1

WO2015140575A1 - Enzyme detection method

Info

Publication number: WO2015140575A1
Application number: PCT/GB2015/050836
Authority: WO
Inventors: Barrie J. MARSH; Christopher G. FROST; Sean GOGGINS
Original assignee: Atlas Genetics Limited
Priority date: 2014-03-20
Filing date: 2015-03-20
Publication date: 2015-09-24
Also published as: GB201405003D0

Abstract

A method for detecting the presence of an enzyme in a composition. The method involves incubating the composition with a pro-ligand, a proto-catalyst and a substrate. Any enzyme present can cause conversion of the pro-ligand to a ligand. The ligand can activate the proto-catalyst to provide an active catalyst. The active catalyst can cause conversion of the substrate to a detectable product and the detectable product may be detected distinguishably from the substrate. The presence of enzyme is detected by detecting the detectable product. The method may be used to detect the presence of an analyte in a composition.

Description

ENZYME DETECTION METHOD

This application claims the benefit of United Kingdom patent application 1405003.3 (filed March 20 2014), the complete contents of which are hereby incorporated herein by reference for all purposes.

TECHNICAL FIELD

This invention is in the field of enzyme detection and its use in enzyme-linked assays. BACKGROUND ART

Amplification and detection methods are well known in the field of assays for target analytes. Amplification methods may involve steps of target amplification and signal amplification. Target amplification involves the amplification of the analyte to be detected. An example of a method of target amplification is the polymerase chain reaction.

Signal amplification involves the production of a detectable signal which can be visualised that corresponds to the presence of the analyte. The signal is amplified by virtue of each target analyte molecule being capable of giving rise to multiple detectable signals. Examples of methods of signal amplification are enzyme-linked assays including enzyme-linked immunosorbent assays (ELISA) of which many examples are well known in the art. Such assays are generally based on the principle that an enzyme is capable of specifically binding an analyte (usually via an antibody). In the presence of the analyte in a composition, the enzyme becomes bound to the analyte. The analyte can be immobilised to a solid support so that the enzyme becomes bound to the solid support via the analyte. Enzyme substrate is then added. The presence of enzyme in the composition (which signifies the presence of analyte) causes the conversion of substrate into detectable product which can be distinguishably detected from substrate. In the absence of enzyme in the composition (and therefore absence of analyte), detectable product is not formed and therefore cannot be detected.

Alternatively, the presence of enzyme in the composition due to the presence of analyte may activate an auto-catalytic system which generates a detectable signal.

Enzyme amplification methods can be slow to generate a detectable signal and auto-catalytic protocols often suffer from high-background rates which lead to a poor signal to noise ratio.

It is an object of the present invention to provide a method for detecting the presence of an enzyme in a composition which allows for improved signal amplification with a low background rate. It is consequently a further object of the invention to provide method for detecting an enzyme in a composition with an improved signal to noise ratio. These methods can be used for detecting analytes in enzyme-linked assays.

Additionally, it is an object of the invention to provide a method comprising an enzyme-linked assay in a manner which allows for the result of the assay to be provided as an electronic read-out.

Additionally, it is an object of the present invention to provide a detection method which allows for quantitative detection of an enzyme, and optionally quantitative detection of an analyte. DISCLOSURE OF THE INVENTION

The inventors have surprisingly found that an amplified signal can be produced by additionally linking an enzyme-linked assay to a catalysed reaction. Tight linkage of the catalysed reaction and the enzyme-linked assay allows for accurate and quantitative detection of the signal. The enzyme to be detected catalyses a reaction involving the conversion of a pro-ligand to a ligand, and in turn the ligand is capable of activating a proto-catalyst to form an active catalyst. The greater the amount of enzyme that is present in the composition, the greater the amount of active catalyst that is produced. The active catalyst itself catalyses a second reaction which involves the conversion of a substrate to a detectable product, and the greater the amount of active catalyst that is produced due to the presence of the enzyme, the greater the amount of detectable product that is produced. The use of an additional catalysis step which is controlled by the level of enzyme present in the composition allows an additional level of signal amplification to occur in the detection method. The conversion of pro- ligand to ligand requires the presence of enzyme, the conversion of proto-catalyst to active catalyst requires the presence of ligand, and the conversion of substrate to detectable product requires the presence of active catalyst under the conditions of the method. The tight linkage of the presence of enzyme to conversion of pro-ligand to ligand means that the pro-ligand system provides a high signal to noise ratio.

The presence of enzyme in a composition may subsequently be detected by detecting the presence of the detectable product.

The presence of an additional catalysed step (in comparison to known signal amplification methods) which links the presence of the enzyme to the production of the detectable product allows for the signal produced by the presence of enzyme to be further amplified. The fact that the conversion of pro-ligand to ligand, proto-catalyst to active catalyst, and substrate to detectable product are all specific to the presence of the product of the previous step means that a very high signal to noise ratio is provided. The tight linkage of the reactions to the presence of the product of the previous step also allows for quantitative detection to occur.

Accordingly, the invention provides a method for detecting the presence of an enzyme in a composition comprising:

a) incubating the composition with a pro-ligand, a proto-catalyst and a substrate, wherein any enzyme present can cause conversion of the pro-ligand to a ligand, whereby the ligand can activate the proto-catalyst to provide an active catalyst, and whereby the active catalyst can cause conversion of the substrate to a detectable product, wherein the detectable product may be detected distinguishably from the substrate; and

b) detecting the detectable product and thereby detecting the presence of the enzyme. The invention also provides a method for detecting the presence of an analyte in a composition comprising the method of the invention, wherein the enzyme is capable of specifically binding to the analyte. The invention also provides a method for diagnosing an infection comprising the method discussed above, wherein the composition comprises a sample obtained from a subject suspected of being infected with a pathogen, and detection of the presence of enzyme in the composition indicates that the patient has been infected with the pathogen.

The invention also provides a method for diagnosing infection with Treponema pallidum comprising the method discussed above, wherein the composition comprises a sample obtained from a subject suspected of being infected with Treponema pallidum, and detection of the presence of enzyme in the composition indicates that the patient has been infected with Treponema pallidum.

The invention also provides a compound having the formula I, IA, IB, or IC.

The invention also provides a cartridge comprising a composition inlet, a proto- catalyst, a pro-ligand and a substrate.

The invention also provides a method for detecting the presence of an analyte in a composition comprising:

a. contacting the composition with an enzyme to permit the binding of enzyme to any analyte; b. incubating the resulting composition with a proto-catalyst, a pro-ligand and a substrate, wherein the enzyme can cause conversion of the pro-ligand to a ligand, the ligand can cause activation of the proto-catalyst to provide an active catalyst, and the active catalyst can cause conversion of the substrate to a detectable product, wherein the detectable product may be detected distinguishably from the substrate;

c. detecting the detectable product and thereby detecting the presence of the analyte.

Methods for detecting enzymes

A method for detecting the presence of an enzyme may qualitatively or quantitatively detect the presence of enzyme. Detecting the presence of the enzyme may be the primary purpose of the method, or the method may be used to detect the presence of an analyte to which the enzyme specifically binds, either directly or indirectly. Examples of how methods for detecting the presence of an enzyme can be linked to the detection of an analyte are described in more detail below.

Methods for detecting the presence of an enzyme or an analyte in a composition include methods that are performed on compositions which are known or suspected to contain the enzyme or analyte, as well as compositions in which the enzyme or analyte are only potentially present. Even if a method is performed on a composition in which no enzyme or analyte is actually present, the method is still considered to be a method for detecting the presence of an enzyme or analyte if the method was performed to detect the presence (or confirm the absence) of any enzyme or analyte that might have been present.

Compositions on which the methods of the invention are performed

The methods of the invention may be performed on any composition in order to detect the enzyme or analyte of interest. The composition may be one in which the enzyme to be detected is suspected to be present, or a composition in which the enzyme to be detected is known to be present, or a composition in which the enzyme to be detected is potentially present. Similarly, the composition may be one in which an analyte is suspected to be present, or a composition in which the analyte is known to be present, or a composition in which the analyte is potentially present. Where the enzyme is not endogenous to a composition being analysed then it will have been added so that it can interact with analyte, directly or indirectly. Enzyme that has not interacted with analyte is removed from the composition prior to the methods of the invention being performed.

The composition may comprise a sample. The composition may therefore comprise the sample and any enzyme which has become bound to analyte present in the sample during contacting of the sample with the enzyme.

The invention is applicable to a wide variety of samples. For example, the sample may be material obtained from a living organism, such as an animal or plant. The sample may be a cellular sample. The sample may be obtained with minimal invasiveness or non-invasively, e.g., it may be obtained from an animal using a swab, or it may be a bodily fluid, e.g. blood or urine. In some embodiments the sample can be material obtained from food or water. Samples may be treated, e.g. diluted, concentrated, purified or incubated with additional components, prior to the methods of the invention being performed. For example, a blood sample may be treated to provide serum or plasma.

An animal from which a sample is obtained may be a vertebrate or non-vertebrate. Vertebrate animals may be mammals, including humans. Examples of non-human mammals include but are not limited to mouse, rat, pig, dog, cat, rabbit, primates, etc. Preferably the subject is human, such that methods of the invention are useful for detecting the presence of analytes in samples taken from humans.

Enzyme

The enzyme to be detected may be any enzyme which is capable of catalysing a reaction which involves the conversion of a pro-ligand to a ligand, wherein the ligand can subsequently activate a proto-catalyst to form an active catalyst. The active catalyst is capable of catalysing a reaction which involves the conversion of a substrate to a detectable product.

Various enzymes can be used, but preferred enzymes are those commonly used in enzyme-linked assays, as these reagents are already available. This allows well-known enzyme-linked assays to be applied to a cartridge on which the result of the assay is provided as an electronic read-out via electrochemical detection. Any enzyme capable of cleaving a functional group to release the ligand from the pro-ligand can be used within the present invention. Preferably, the enzyme is an enzyme that catalyses the hydrolysis of a chemical bond, such as a hydrolase. Suitable hydrolases are those in class 3 of the 'EC enzyme classification system (as implemented by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology) or in patent IPC class C12N 9/14. For example, the enzyme may be selected from the group consisting of phosphatases {e.g. alkaline phosphatase), sulfatases, glycosylases {e.g. beta-galactosidase), peptidases, esterases, and beta- lactamases. Alternatively, the enzyme may be a peroxidase (e.g. horseradish peroxidase). These enzymes are known to be capable of specific catalysis of reactions which will not take place at a detectable level in the absence of the enzyme, and specific enzyme catalysis of this type permits increased signal to noise ratio.

As explained above, the enzyme may be detected using a method of the invention as part of a method for detecting the presence of an analyte. In such situations, the methods for detecting the presence of the enzyme may be utilised because the presence of the enzyme can be tightly linked to both the presence of analyte and the conversion of substrate to detectable product. The presence of the enzyme in the composition is used a marker of the presence of the analyte. Further details of the analyte and how the detection of the presence of the enzyme in a composition is linked to the detection of the presence of analyte are provided below.

Detection of anafytes

The methods of the invention may be used to detect the presence of an analyte in a composition via detection of the enzyme. Where the composition is suspected of comprising analyte, the presence of enzyme is linked to the presence of analyte. Optionally, the amount of enzyme present may be linked to the amount of analyte present (e.g. in the same way as the known ELISA format).

The analyte may be any compound which is suspected to be present, known to be present or is potentially present in the composition. The analyte may be specific to a particular pathogen, e.g. the analyte may be an antigen from the pathogen or may be an antibody produced by the subject in the presence of a pathogen. The analyte may be, for example, a small molecule, a nucleic acid, a protein, an antigen, or an antibody.

The composition may be contacted with enzyme in order for the enzyme to bind any analyte present either directly or indirectly. Any analyte present in the composition may be immobilised on a solid support. If analyte is present the enzyme is able to bind to the solid support via the analyte. Conversely, the enzyme does not become bound to the solid support in the absence of analyte. Enzyme which does not become bound to the solid support is removed from the composition prior to incubation with pro-ligand, e.g. by washing.

The solid support may form part of a cartridge in which the methods of the invention are conducted.

The analyte may be immobilised on the solid support using any means. The means by which the analyte is immobilised on the solid support will depend on the nature of the analyte. Methods of immobilising analytes are well known in the art (e.g. based on years of experience with ELISA).

For example, where the analyte is an antigen it may be immobilised on the solid support via a capture antibody. The capture antibody may be attached to the solid support using various methods. The solid support coated with antibodies may be contacted with the composition, and antigen present in the composition may bind to the capture antibody causing it to become immobilised on the solid support. Once the antigen is immobilised, the solid support may be contacted with enzyme such that enzyme binds to any antigen present (usually via an antibody). In an alternative format, where the analyte is a target antibody, an antigen to which the target antibody specifically binds may be immobilised on a solid support either directly, or via a capture antibody as described above. As a further alternative, a capture antibody which specifically binds to the target antibody may be immobilised on a solid support. The solid support coated with antigen or capture antibody may be contacted with the composition, and target antibody present in the composition may bind to the immobilised antigen or capture antibody, thereby causing the target antibody analyte to become immobilised on the solid support. Once the target antibody is immobilised on the solid support, the solid support may be contacted with enzyme such that enzyme binds to any target antibody present (again, usually via an antibody).

The enzyme may be capable of specifically binding to the analyte either directly or indirectly. The enzyme may be attached to a binding partner which is capable of specifically binding to the analyte. The binding partner may be a peptide or a protein, e.g. an antibody. Where the enzyme is capable of binding to the analyte via a binding partner, the enzyme is capable of specifically binding the analyte indirectly. As an alternative, the enzyme may be linked to a binding partner which is capable of binding to a secondary binding partner which is capable of specifically binding to the analyte. The secondary binding partner may be a secondary peptide or a secondary protein, e.g. a secondary antibody. Multiple binding partners may be used, each of which is capable of binding the previous binding partner. The skilled person would understand how to identify and select binding partners for different analytes, as such methods of identification are well known in the art of enzyme-linked assays.

As a further alternative, where the enzyme has a structure which is capable of specifically binding the analyte, the enzyme is capable of specifically binding the analyte directly.

Overall, therefore, when the analyte to be detected is present in the sample, the enzyme specifically binds to that analyte. The enzyme may subsequently be detected using the methods described herein. Quantitative detection of the enzyme also therefore provides quantitative detection of the analyte.

Pro-ligands

The pro-ligand is incubated with the composition as part of the method of the invention. In the presence of the enzyme to be detected the pro-ligand is converted to the ligand.

The specific nature of the pro-ligand and the ligand is dependent on the enzyme to be detected. The activity of the enzyme should be capable of specifically converting the pro-ligand into the ligand.

Preferably, the pro-ligand comprises an enzyme sensitive moiety (ESM). An ESM is a functional group that acts as a substrate for an enzyme, making the pro-ligand particularly useful with a particular enzyme or class of enzyme. Preferably, the ESM is selected to be similar or identical to a natural substrate upon which the enzyme naturally acts. In an embodiment, the ESM is selected from phosphate, sulphate, glycosyl, an amide group or an ester group. For example, if the enzyme is phosphatase the ESM may be a phosphate group; if the enzyme is sulfatase the ESM may be a sulfate group; if the enzyme is gylcosylase the ESM may be a glycosyl group; if the enzyme is peptidase the ESM may be an amide group; if the enzyme is esterase the ESM may be an ester group. In another embodiment, when the enzyme is a peroxidase, the pro-ligand may comprise an azodicarboxylate moiety.

General classes of hydrolases and their mode of action upon examples of their typical functional groups to give the corresponding phenolate/aniline intermediate product are shown below.

Preferably, the ESM is attached to a self-immolative moiety (SIM), which is attached to the ligand (L), i.e. the pro-ligand typically has the structure ESM-SIM-L.

SEVIs (self-immolative moieties) have been described in WO2013/106434. The term self-immolative moiety refers to a bifunctional chemical moiety that is capable of covalently linking the ESM to the ligand. The SEVI is capable of spontaneously separating from the ligand if the bond to the ESM is cleaved. On spontaneous separation of the SEVI, a gaseous side-product may be produced, which makes the separation entropically favourable.

In the present invention, the SIM links an ESM to the ligand. The general reactivity of the proligand to form the ligand via the intermediate is shown below. Upon exposure to enzyme, the ESM is removed. The resulting intermediate is a derivative of the proligand, in which the ESM has been removed, but with the SEVI still attached to the ligand. This intermediate spontaneously degrades by separation of the SIM to form the ligand. In this way the ligand is formed only in the presence of the enzyme. An example of the mechanism is shown in Figure 4.

aspect, the pro-ligand is a compound of formula I:

(I)

wherein:

Z¹ is NH, N(alkyl), O, S, S(O), PH or P(alkyl);

Z² is N, O, S, S(O) or P;

X is alkylene, alkynylene or alkenylene;

Y is S0₂;

R¹ is alkyl or aryl, which may be substituted with any number of R² substituents;

each R² is independently selected from alkyl, alkenyl, alkynyl, aryl, OH, O(alkyl), NH₂,

N(H)(alkyl), N(alkyl)₂, SH, S(alkyl), S(0)alkyl, S(0)₂(alkyl), P(alkyl)₂, Si(alkyl)₃, F, CI, Br, I, CF₃, CN, NC(alkyl), COOalkyl, CONH₂, CONH(alkyl), CON(alkyl)₂, COOH, and N0₂;

G¹ is H or G³;

G² is H or G³;

G³ is a SIM bonded to an ESM; and

X may be substituted with 1, 2, 3, 4 or 5 substituents independently selected from alkyl, alkenyl, alkynyl, aryl, OH, O(alkyl), NH₂, N(H)(alkyl), N(alkyl)₂, SH, S(alkyl), S(0)alkyl, S(0)₂(alkyl), P(alkyl)₂, Si(alkyl)₃, F, CI, Br, I, CF₃, CN, NC(alkyl), COOalkyl, CONH₂, CONH(alkyl), CON(alkyl)₂, COOH, and N0₂.

Preferably in the compound of formula (I), Z¹ is NH, N(alkyl) or O and Z² is N or O. More preferably:

when Z¹ is O and Z² is N, G¹ is H and G² is G³;

when Z¹ is NH and Z² is N, either: G¹ is G³, and G² is H; or G¹ is H and G² is G³; or G¹ is G³ and G² is G³;

when Z¹ is NH and Z² is O, G¹ is G³, and G² is absent; and when Z¹ is O and Z² is O, G¹ is G³, and G² is absent.

Preferably Z¹ is NH. Preferably Z² is N. More preferably Z¹ is NH and Z² is N.

Preferably X is alkylene. More preferably X is - CH₂CH₂-.

Preferably R¹ is aryl substituted with 0, 1, 2, 3, 4 or 5 R² substituents.

The SIM typically comprises a functionalised phenol or aniline, such that, on removal of the ESM, the intermediate is a phenolate anion or an aniline anion. A variety of possible SIMs can be used in the present invention.

G³ may be selected from the following structures:

wherein

\ indicates the position of attachment of G to the remainder of the pro-ligand of formula I; n is an integer;

p is an integer;

W is NH or O;

each V is independently S or O; each R is independently H, alkyl, alkenyl, alkynyl, or aryl;

each R^q is independently selected from alkyl, alkenyl, alkynyl, aryl, OH, O(alkyl), NH₂, N(H)(alkyl), N(alkyl)₂, SH, S(alkyl), S(0)alkyl, S(0)₂(alkyl), P(alkyl)₂, Si(alkyl)₃, F, CI, Br, I, CF₃, CN, NC(alkyl), COOalkyl, CONH₂, CONH(alkyl), CON(alkyl)₂, COOH, and N0₂; and

r is 0, 1, 2, 3 or 4.

In some embodiments n is an integer from 1-20 and p is an integer from 1-20.

Preferably n is 1. Preferably p is 1. Preferably each V is O.

Alternatively, R^q and r may be chosen such that the benzene ring is substituted up to 3 times {ortho, ortho and para to W) with the same linker and repeated in a dendritic fashion, so that G³ is as shown below.

wherein each indicates attachment of G to a compound of formula I and 11 other groups are as defined above.

In the above pro-ligands, the intermediate produced after the ESM is removed by an enzyme is an aniline or aniline anion when W is N and is a phenol or phenolate anion when W is O. Subsequent 1,6-elimination of the SEVI, made further reactive by the entropically favourable release of carbon dioxide (or carbonyl sulfide or carbon disulfide), results in formation of the ligand. A potential mechanism for this elimination is proposed in figure 4.

Selection of R^q can be used to affect the rate of this elimination. For example, use of an electron withdrawing group (such as N0₂, CI, Br, I) may decrease the pK_a of the aniline/phenol proton, to increase the elimination rate. Use of an electron- donating group (such as OH, NH₂, alkyl) may increase the pK_a of the aniline/phenol proton, to decrease the elimination rate.

Furthermore, the intermediate may undergo elimination only when the phenol or aniline is deprotonated. Therefore, the selection of the pH of the reaction medium may be used to affect the rate of this elimination. For example, use of alkaline conditions (i.e. pH > 7) may increase the breakdown rate by making removal of the aniline/phenol proton easier. Use of acidic conditions (i.e. pH < 7) may decrease the breakdown rate by making removal of the aniline/phenol proton more difficult.

In a preferred embodiment, the pro-ligand has the structure of formula IA:

wherein m is 0, 1, 2, 3, 4 or 5 R² and G³ are as defined above.

In an even more preferred embodiment, the pro-ligand has the structure of formula IB:

wherein W, ESM, R^q, r, R² and m are as defined above.

In some embodiments, the compound of formula IA or IB may be chosen such that m is 3 and R² is alkyl positioned ortho, ortho and para to the SO₂ group. In other embodiments, m is 1 and R² is at the position para to the SO₂ group.

Most particularly, the proli and is the compound of formula IC:

where the phosphate group may interact with any cations, e.g. Na⁺ or K⁺ Ligand

The ligand allows for conversion of the proto-catalyst to an active catalyst. The ligand may be of any structure such that a) when it is combined with the proto-catalyst under the conditions of the method active catalyst is formed which is capable of converting substrate into detectable product; b) it is sufficiently different from the structure of the pro-ligand such that when the pro-ligand is combined with the proto-catalyst under the conditions of the method active catalyst is not formed; and c) it does not interfere with the reaction catalysed by the active catalyst.

In an aspect, the ligand has the structure of formula II:

wherein Z¹ is NH₂, N(alkyl)₂, NH(alkyl), OH, O(alkyl), SH, S(alkyl), S(0)(alkyl), PH(alkyl), PH₂ or P(alkyl);

Z² is NH, N(alkyl), O, S, S(O), PH or P(alkyl);

X is alkylene, alkynylene or alkenylene;

Y is S0₂;

X may be substituted with 1, 2, 3, 4 or 5 substituents independently selected from alkyl, alkenyl, alkynyl, aryl, OH, O(alkyl), NH₂, N(H)(alkyl), N(alkyl)₂, SH, S(alkyl), S(0)alkyl, S(0)₂(alkyl), P(alkyl)₂, Si(alkyl)₃, F, CI, Br, I, CF₃, CN, NC(alkyl), COOalkyl, CONH₂, CONH(alkyl), CON(alkyl)₂, COOH, and N0₂;

R¹ is alkyl or aryl, which may be substituted with any number of R² substituents; and each R² is independently selected from alkyl, alkenyl, alkynyl, aryl, OH, O(alkyl), NH₂, N(H)(alkyl), N(alkyl)₂, SH, S(alkyl), S(0)alkyl, S(0)₂(alkyl), P(alkyl)₂, Si(alkyl)₃, F, CI, Br, I, CF₃, CN, NC(alkyl), COOalkyl, CONH₂, CONH(alkyl), CON(alkyl)₂, COOH, and N0₂.

Preferably Z¹ is NH₂. Preferably Z² is NH. More preferably Z¹ is NH₂ and Z² is NH.

Preferably X is alkylene. More preferably X is CH₂CH₂.

Preferably R¹ is aryl substituted with 0, 1, 2, 3, 4 or 5 R² substituents.

Substituents on X may be chosen such that the resulting ligand is chiral. Chiral ligands may induce chiral information onto the detectable product. Examples of chiral ligands include

A preferred subset of formula II is a compound of formula IIA

wherein m is 0, 1, 2, 3, 4 or 5 and R² is defined above.

In one aspect m is 3 and R² is alkyl positioned at the positions ortho, ortho and para to the SO₂ group. In another aspect, m is 1 and R² is positioned at the position para to the SO₂ group.

In one embodiment the ligand is selected from one of the following structures:

In a preferred embodiment the ligand is the compound of formula IIB:

As described above, selection of the enzyme may depend on the design of the pro-ligand, and vice versa. For example, the enzyme may be a phosphatase. Where the enzyme is a phosphatase, the pro- ligand comprises the ligand and at least one additional phosphate group. The pro-ligand has a structure such that the phosphate group of the pro-ligand is removed specifically by the phosphatase enzyme to either directly or indirectly form the ligand, and may not be removed in the absence of the phosphatase enzyme. Where removal of the phosphate group from the pro-ligand directly forms the ligand, the pro-ligand and the ligand differ only by the presence of at least one additional phosphate group on the pro-ligand. Where the removal of the phosphate group causes an effective leaving group on the pro-ligand to be exposed, this group is also removed from the pro-ligand to form the ligand. In such embodiments the removal of the phosphate group indirectly forms the ligand and the pro-ligand and the ligand differ by the presence of at least one additional phosphate group and at least one additional leaving group on the pro-ligand. Where the ligand has formula ΙΓΒ, and optionally the enzyme is alkaline phosphatase, the pro-ligand may have formula IC.

Preferably the conversion of a single molecule of pro-ligand to ligand is adequate to activate the proto-catalyst to provide an active catalyst. Therefore, even very low levels of enzyme are detectable using the method of the invention.

Proto-catalyst

The proto-catalyst is incubated with the composition as part of the method of the invention. The proto-catalyst may be of any structure such that when it is combined with the ligand under the conditions of the method it forms an active catalyst which is capable of converting substrate into detectable product. In preferred aspects, the proto-catalyst can be any transition metal complex M_mL_n, comprising at least one transition metal atom, M, coordinated to any number of ligands, L_n, which is able to undergo ligand exchange with the ligand of the invention, described above, and a hydride (IT).

It is to be understood that the number of possible transition metal complexes is very large, and that one skilled in the art of catalytic compounds will be able to utilize a number of compounds in the present invention. Ru, Rh and Ir are preferred transition metals for use in the present invention.

Ligands on the proto-catalyst are ions or molecules that bind to the central metal atom to form a coordination complex. These ligands may be mono or multi dentate. Examples include, but are not limited to, hydride (H^~), halides (iodide (Γ), bromide (Br ), chloride (CL) and fluoride (F^~)), sulfurous ligands (sulfide (S^2~), thiocyanate (SCN^~) and isothiocyanate (NCS^~)), phosphorus ligands (phosphine (R₃P)), nitrogen ligands (azide (N₃ ^~), nitrile (RCN), cyanide (CN^~), ammonia (NH₃, ethylenediamine (en)), and pyridine (py, bipy, phen), oxygen ligands (hydroxide (OH^"), water (H₂0), oxalate (C₂O₄ ² ), nitrate (N0₃ ^~), and nitrite (N0₂ ^~)) and carbon ligands (carbon monoxide (CO) and carbenes). The ligands may also display multiple hapticity, such that the ligand is coordinated through an uninterrupted and contiguous series of atoms. Examples include, but are not limited to allyl, allenyl, butadienyl, cyclopentadienyl, benzene, cycloheptatrienyl and cyclooctatetraenyl.

Preferably, the proto-catalyst comprises at least one η⁶- or η⁵- coordinated aromatic ring. For example the aromatic ring may be n⁵-cyclopentadiene (cp), n⁶-benzene (ph), which may be substituted, or η⁵- pentamethylcyclopentadiene (cp*).

In particular embodiments, the proto-catalyst may have the formula Ir(cp*)Ci₃ or [Ir(cp*)Cl₂]₂. Preferably, the proto-catalyst is [Ir(cp*)Cl₂]₂. Active catalyst

The active catalyst catalyses a reaction which converts substrate into detectable product. The active catalyst may be any catalyst which catalyses a reaction which involves the conversion of substrate to detectable product. The reaction that is catalysed by the active catalyst is dependent on the nature of the substrate and the nature of the desired detectable product. For example, if the substrate is an aldehyde or ketone and the detectable product is an alcohol, the active catalyst may catalyse a transfer hydrogenation or reduction reaction. Alternatively, if the substrate is an alcohol or an aldehyde and the detectable product is a carboxylic acid, the active catalyst may catalyse an oxidation reaction.

Where the active catalyst catalyses a transfer hydrogenation reaction, the active catalyst may be a compound of formula III:

where M is a transition metal and wherein the identity and number of the ligands (L_a) are chosen such that the metal is coordinatively saturated;

X is alkylene, alkynylene or alkenylene;

Z¹ is NH, N(alkyl), O, S, S(O), PH or P(alkyl);

Z² is O, S, P or N;

Y is S0₂;

each R² is independently selected from alkyl, alkenyl, alkynyl, aryl, OH, O(alkyl), NH₂, N(H)(alkyl), N(alkyl)₂, SH, S(alkyl), S(0)alkyl, S(0)₂(alkyl), P(alkyl)₂, Si(alkyl)₃, F, CI, Br, I, CF₃, CN, NC(alkyl), COOalkyl, CONH₂, CONH(alkyl), CON(alkyl)₂, COOH, and N0₂; and

For example, the active catalyst may have the structure of formula (IIIA)

In an embodiment, when M is Ir, L is an η⁵- ligand such as n⁵-cp or n⁵-cp*.

In a particular embodiment, where the ligand has the formula IIB and/or the proto-catalyst has the formula Ir(cp*)Cl3 or [Ir(cp*)Ci2]2, the active catalyst may have the formula MB:

It is preferred that the active catalyst is formed from the proto-catalyst by a chemical change rather than by assembly and conversion of the proto-catalyst into a physical structure with catalytic activity. Thus the active catalyst is preferably not a palladium nanostructure (particularly on the surface of gold nanoparticles) and preferably does not include palladium particles or gold particles.

Substrate

The substrate is incubated with the composition as part of the method of the invention. In the presence of active catalyst the substrate is converted into detectable product. In order for the methods of the invention to be quantitative, the amount of detectable product produced may be proportional to the amount of enzyme present in the sample.

The substrate may comprise a metallocene group. The metallocene group may be a ferrocene group, with any amount of substitution upon the cyclopentadienyl rings.

Particularly suited for the present invention are those ferrocenyl compounds comprising a carbonyl group or a carbonyl derivative group. Such groups include aldehydes, ketones and imines. More preferably, the substrate is an aldehyde or a ketone and comprises a ferrocene group.

The substrate may be ferrocenecarboxaldehyde having the formula IV:

The ferrocenecarboxaldehyde may optionally have additionally substituted cyclopentadienyl rings. In another embodiment the substrate is a ferrocenyl compound comprising a halide group.

Detectable product

The detectable product is produced in the presence of substrate and an active catalyst. The detectable product must be distinguishably detectable from the substrate. For example, the detectable product may be electrochemically distinguishable from the substrate. The structure of the detectable product is dependent on the structure of the substrate and the specific activity of the active catalyst. The detectable product may comprise a metallocene group. The metallocene group may be a ferrocene group with any amount of substitution upon the cyclopentadienyl rings.

Preferably, the detectable product comprises a primary or secondary alcohol group or a primary or secondary amine group. More preferably, the detectable product is an alcohol and comprises a ferrocene group.

The detectable product may be ferrocenemethanol having the formula V:

The ferrocenemethanol may optionally have additionally substituted cyclopentadienyl rings. For example, the detectable product may be a compound of formula VA

(VA)

wherein:

each X substituent is independently selected from halo, vinyl, alkyl, cycloalkyl, SiR, SnR₃, PR₂, P(0)R₂, SR, S(0)R, S0₂R, aryl, heteroaryl, CHO, C0₂R, CN and CF₃;

each R is independently selected from alkyl, aryl, cycloalkyl or heteroaryl;

a is 0, 1, 2, 3 or 4;

b is 0, 1, 2, 3, 4 or 5;

and vinyl, alkyl, cycloalkyl, alkylene, aryl and heteroaryl may optionally be substituted with 1, 2 or 3 substituents independently selected from unsubstituted alkyl, OH, CN, fluorine, chlorine, bromine and iodine.

In another embodiment the detectable product is a ferrocene compound possessing an aryl group. These detectable products may be useful when the substrate is a ferrocenyl compound comprising a halide group and the active catalyst catalyses a cross-coupling reaction, such as a Suzuki reaction. Cross coupling reactions between ferrocenyl halides and boronic acids can be catalysed by palladium in the presence of water, as illustrated in the scheme below. To accelerate this catalysis, the presence of a phosphine ligand is typically used (S. Parisot, R. Kolodziuk, C. Goux-Henry, A. Iourtchenko, D. Sinou, Tetrahedron Letters, 43 (2002), 7397-7400).

In another embodiment the detectable product is a ferrocene possessing a beta-hydroxy-ketone moiety. These detectable products may be useful when the substrate is a compound of formula IV and the active catalyst catalyses an aldol reaction. Suitable active catalyst include organocatalysts based on proline, which have been shown to catalyse aldol reactions within aqueous media

(T.J.Dickerson, K.D. Janda, J. Am. Chem. Soc, 2002, 124 (13), 3220-3221), as illustrated in the scheme below.

Detecting the detectable product

As mentioned above, the detectable product must be distinguishably detectable from the substrate. The detectable product may be detected using any means. For example, the detectable product may be detected using electrochemical detection, chemiluminescent detection, fluorescent detection or colorimetric detection. Preferably, the detectable product is electrochemically active and is detected using electrochemical detection. For example, where the detectable product is detected using electrochemical detection, on application of a current the substrate and detectable product may produce signals at different voltages which can be distinguished.

Combinations of reagents

The pro-ligand, proto-catalyst and substrate may be mixed prior to step a). In such embodiments, the composition is incubated with pro-ligand, proto-catalyst and substrate at the same time. As an alternative, the pro-ligand, proto-catalyst and substrate may be provided separately. For example, the pro-ligand, proto-catalyst and substrate may be provided sequentially.

The pro-ligand, proto-catalyst and substrate should be stable in the absence of the enzyme under the conditions of the method. The conversion of pro-ligand to ligand, proto-catalyst to active catalyst and substrate to detectable product should not occur at greater than background levels under the conditions of the method in the absence of the enzyme and in the presence of the other reagents used in the methods of the invention. The conversion of pro-ligand to ligand should occur only in the presence of the enzyme under the conditions of the method. The conversion of the proto-catalyst to active catalyst should occur only in the presence of the ligand under the conditions of the method. The conversion of the substrate to detectable product should occur only in the presence of the active catalyst under the conditions of the method. It should not be possible for any of the conversions mentioned above to occur under the conditions of the method in the presence of any of the other reagents of the method of the invention or any enzyme or other compound that may be present naturally in the composition, other than the enzyme to be detected.

None of the reagents or entities described above, i.e. analyte, enzyme, pro-ligand, ligand, proto- catalyst, active catalyst, substrate and detectable product, should interfere with any of the reactions mentioned above, except where stated. If any of these entities do interfere with any of the reactions other than those stated above, then they should be kept separate from the reaction with which they interfere, e.g. by adding the reagents sequentially. For example, pro-ligand does not interfere with the conversion of substrate to detectable product, and substrate does not interfere the conversion of proto-catalyst to active catalyst.

Any compounds or enzymes that are naturally present in the composition (other than any analyte and enzyme) do not interfere with any of the reactions mentioned above.

Methods for diagnosis

The methods of the invention may be performed as part of a method for diagnosing an infection. In such a method, the composition comprises a sample obtained from a subject suspected of being infected with a pathogen, and detection of the presence of enzyme in the composition indicates that the patient has been infected with the pathogen. Any pathogen may be detected using the methods of the invention.

In such methods for diagnosis, an analyte may be detected that is specific to the pathogen. The enzyme may be linked to an antibody which specifically binds to an analyte specific to the pathogen. As an alternative, the enzyme may be linked to an antibody which specifically binds to a secondary antibody that specifically binds to an analyte specific to the pathogen. The analyte specific to the pathogen may be an antibody.

For example, the methods of the invention may be performed as part of a method for diagnosing infection with Treponema pallidum. In this example, the composition comprises a sample obtained from a subject suspected of being infected with Treponema pallidum, and detection of the presence of enzyme in the composition indicates that the patient has been infected with Treponema pallidum.

The enzyme may be linked to an antibody which specifically binds to a Treponema pallidum specific analyte. As an alternative, the enzyme may be linked to an antibody which specifically binds to a secondary antibody that specifically binds to a Treponema pallidum specific analyte. The Treponema pallidum specific analyte may be an antibody.

Cartridge

The methods of the invention may be performed in a cartridge which contains all of the necessary reagents to perform the method other than the composition or sample.

The cartridge may be capable of being inserted into a cartridge reader. The cartridge reader may be capable of performing the methods of the invention in the cartridge and may be capable of providing an electronic read-out result as to whether detectable product is present following the performance of the method of the invention.

For example, the cartridge may comprising a composition inlet, a proto-catalyst, a pro-ligand and a substrate, wherein in the presence of an enzyme in the composition to be tested, the enzyme causes conversion of the pro-ligand to a ligand, the ligand causes activation of the inactive catalyst to provide an active catalyst, and the active catalyst causes conversion of the substrate to a detectable product.

The cartridge may additionally comprise enzyme. This may be useful when the cartridge is to be used to detect the presence of analyte in a composition. The enzyme may be contained in the cartridge separately from the proto-catalyst, pro-ligand and substrate. The composition may be contacted with the enzyme prior to being contacted with the proto-catalyst, pro-ligand and substrate. This may be achieved by the cartridge reader which can allow for release of reagents that are stored separately to be released in a particular order. Where the cartridge comprises enzyme, any enzyme that is not bound to analyte is removed from the composition prior to incubation with the pro-ligand. In some embodiments, a method for detecting the presence of analyte in a composition may be performed on a cartridge in the following way. A composition is applied to the composition inlet, the cartridge is inserted into a cartridge reader, the cartridge reader causes the composition to be contacted with enzyme in the cartridge, the composition is washed to remove any enzyme which is not bound to analyte, the cartridge reader causes the composition to be contacted with a pro-ligand, a proto-catalyst and a substrate, and the cartridge reader detects the presence of detectable product. The detectable product may be detected by application of a current to the composition by the cartridge reader, and detection of the voltage produced. If the presence of the detectable product is detected by the cartridge reader, a positive result for analyte in the composition is provided.

General

The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y.

The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

Unless specifically stated otherwise, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.

The term "alkyl" as used herein refers to a linear, branched, or cyclic saturated hydrocarbon group containing 1 to about 20 carbon atoms, preferably 1 to about 12 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like. Cycloalkyl groups may optionally include one or two heteroatomsselected from O, NH, N and S. The term "alkylene" as used herein refers to a difunctional linear, branched, or cyclic alkyl group, where "alkyl" is as defined above.

The term "alkenyl" as used herein refers to a linear, branched, or cyclic hydrocarbon group of 2 to about 20 carbon atoms, preferably 1 to about 12 carbon atoms, containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, and the like. The term "alkenylene" as used herein refers to a difunctional linear, branched, or cyclic alkenyl group, where "alkenyl" is as defined above.

The term "alkynyl" as used herein refers to a linear or branched hydrocarbon group of 2 to about 20 carbon atoms, preferably 1 to about 12 carbon atoms, containing at least one triple bond, such as ethynyl, n-propynyl, and the like. The term "alkynylene" as used herein refers to a difunctional alkynyl group, where "alkynyl" is as defined above.

The term "aryl" means any single, fused or heteroaromatic ring system, preferably having up to 12 carbon atoms, more preferably up to 6 carbon atoms. Illustrative aryl groups include, for example, phenyl, naphthyl, pyrryl, furyl and pyridyl. The term "arylene" as used herein refers to a difunctional aryl group, where "aryl" is as defined above.

The term "transition metal" means metals whose atoms have a partial or completed shell of electrons. Suitable transition metals for use in the invention include, but are not limited to, cadmium (Cd), copper (Cu), cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru), rhodium (Rh), osmium (Os), rhenium (Re), platinium (Pt), scandium (Sc), titanium (Ti), vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni), molybdenum (Mo), technetium (Tc), tungsten (W), and iridium (Ir).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an overview of the scheme and the way in which the enzyme, pro-ligand, ligand, proto-catalyst, active catalyst, substrate and detectable product are linked.

Figure 2 shows the scheme of a specific embodiment of the invention in which the enzyme is alkaline phosphatase and the substrate and detectable product are ferrocene compounds.

Figure 3 is a voltammogram showing that the ferrocene compounds which are the substrate and detectable product of the scheme in Figure 2 can be detected as providing distinguishably different voltages when a current is applied to them. The y-axis shows current in amps; the x-axis shows potential in volts.

Figure 4 shows a proposed mechanism for the conversion of the pro-ligand to ligand. The mechanism involves a 1,6-elimination and decarboxylation step.

Figure 5 shows a dual signal-amplification protocol used for the detection of CRP

Figure 6 is a graph showing the percentage conversion of electroactive substrate to electroactive product over time (minutes) in the presence of 0 (lower line) or 100 μg/L (upper line) of CRP. Figure 7 is a graph showing the percentage conversion of electroactive substrate to electroactive product over time (minutes) in the presence of known amounts of alkaline phosphatase.

Figure 8 is a graph showing the percentage conversion of electroactive substrate to electroactive product over time (minutes) in the presence of known amounts of alkaline phosphatase-streptavidin conjugate.

MODES FOR CARRYING OUT THE INVENTION

Scheme for dual signal-amplified enzyme linked assay

The inventors have demonstrated that the claimed methods provide effective detection of the presence of an enzyme in a sample using the scheme shown in figure 2.

Substrate and detectable product

The inventors have used an active catalyst which catalyses a transfer hydrogenation reaction. The transfer hydrogenation reaction that is catalysed causes the specific conversion of ferrocenecarboxaldehyde to ferrocenemethanol. Ferrocenecarboxaldehyde was found not to convert into ferrocenemethanol in the absence of the active catalyst. Ferrocenecarboxaldehyde was also found not to convert into ferrocenemethanol in the absence of enzyme and in the presence of proto- catalyst and pro-ligand or any other compounds or enzymes that may naturally occur in the composition.

Ferrocenemethanol is electrochemically distinguishable from ferrocenecarboxaldehyde. This is shown in Figure 3. The voltages provided by the two compounds when a current is applied across them differ detectably.

Identification of an appropriate active catalyst

The active catalyst was required to have the transfer hydrogenation activity in order to be able to catalyse the reaction from ferrocenecarboxaldehyde to ferrocenemethanol. The inventors investigated catalysts which may be used to catalyse such transfer hydrogenation reactions. Active catalysts were identified with the property that they could be formed by reaction of a proto-catalyst with a ligand. It was also necessary that the active catalyst is not formed when the proto-catalyst is present in a composition with the pro-ligand in the absence of the ligand. Therefore, the active catalyst needed to be such that it only forms when the specific ligand is present.

In order for this to be possible, the ligand and pro-ligand needed to be designed such that the proto- catalyst is capable of combining with the ligand to form the active catalyst, but is not capable of combining with the pro-ligand.

The enzyme's activity was considered and the ligand and pro-ligand was designed in such a way that the conversion of pro-ligand to ligand is catalysed by the enzyme.

The inventors found that an active catalyst having the formula ΙΠΒ:

could be produced by reacting an proto-catalyst having the formula [Ir(cp*)Ci2]2 with a ligand having the formula IIB :

In contrast, a pro-ligand having the formula IC:

does not react with the proto-catalyst with the formula [Ir(cp*)Ci2]2 to form the active catalyst. The pro-ligand having the formula IC is capable of being converted into the ligand having the formula IIB by the enzyme alkaline phosphatase. Dephosphorylation of the pro-ligand by alkaline phosphatase forms an unstable phenolate intermediate. Under alkaline conditions, this intermediate undergoes 1,6-elimination made favourable by the entropic release of carbon dioxide, and the ligand is produced along with an equivalent of para-quinone methide.

The scheme therefore provides an additional catalysed step to link the presence of the enzyme to the production of the detectable product. This additional catalysed step allows for the signal produced by the presence of enzyme to be amplified. The fact that the conversion of pro-ligand to ligand, proto- catalyst to active catalyst and substrate to detectable product are all specific to the presence of the product of the previous step means that a very high signal to noise ratio is provided. The tight linkage of the reactions to the presence of the product of the previous step also allows for quantitative detection to occur.

EXPERIMENTAL PROCEDURES

Example 1: Synthesis of pro-ligand of formula IC

Synthesis of the pro-ligand of formula IC was performed in accordance with the scheme and procedures provided below.

Synthesis ofdiallyl (4-(hydroxymethyl)phenyl) phosphate (I)

To an oven-dried 250 mL round-bottom flask, was added phosphorous trichloride (PCI₃) (8.7 mL, 100 mmol, 2 eq.) to tetrahydrofuran (THF) (70 mL) under nitrogen. The solution was then cooled to 0 °C using a water-ice bath. A solution of allyl alcohol (13.6 mL, 200 mmol, 4 eq.) and anhydrous triethylamine (TEA) (31 mL, 220 mmol, 4.4 eq.) in anhydrous THF (30 mL) was then added dropwise via a dropping funnel under nitrogen. After complete addition, the reaction mixture was allowed to warm to room temperature (-21 °C) and stirred for 1 hour. The reaction mixture was then cooled to 0 °C using a water-ice bath and 50 mL of de-ionised water was added slowly. The reaction mixture was allowed to warm to room temperature (-21 °C) and stirred for 30 minutes. The THF was removed under reduced pressure and the residue was transferred to a 500 mL separating funnel. Ethyl acetate (EtOAc) (100 mL) was then added and the mixture was thoroughly shaken. The layers were then separated and the aqueous layer was extracted a further two times with EtOAc (100 mL). The organic layers were combined and washed three times with de-ionised water (100 mL), dried with sodium sulphate (Na₂SC>4) and concentrated under reduced pressure. The residue was then taken up in anhydrous toluene (75 mL) and added, at 0 °C, to an oven-dried 250 mL round-bottom flask equipped with a Schlenk tap, containing a stirring solution of N-chlorosuccinimide (11.7 g, 87.5 mmol, 1.75 eq.) in anhydrous toluene (75 mL) under argon.

The reaction mixture was allowed to warm to room temperature and stirred overnight (-16 hours). The precipitate formed was the filtered and the filtrate concentrated under reduced pressure. To the residue was added diethyl ether (Et^O) (25 mL), the precipitate formed was filtered and the filtrate concentrated under reduced pressure. This process was repeated until no precipitate was seen upon the addition of Et^O. The residue was then taken up in anhydrous THF (50 mL) and added dropwise via a dropping funnel to a stirring solution containing 4-hydroxybenzaldehyde (6.1 g, 50 mmol, 1 eq.), anhydrous triethylamine (10.5 mL, 75 mmol, 1.5 eq.) in anhydrous THF (50 mL) under nitrogen at 0 °C. After complete addition, the reaction mixture was allowed to warm to room temperature and stirred for 2 hours. The reaction mixture was then concentrated to approximately a quarter of the initial volume and cooled to 0 °C. A saturated solution of sodium hydrogen carbonate (NaHC03 (_Sat.)) (50 mL) was added and allowed to warm to room temperature before being transferred to a 250 mL separating funnel and extracted three times with EtOAc (50 mL). The combined organics were then washed three times with de-ionised water (50 mL), dried with Na₂S0₄ and concentrated under reduced pressure. The residue was taken up in THF (50 mL) and sodium borohydride (3.8 g, 100 mmol, 2 eq.) was added portion-wise under nitrogen at 0 °C.

De-ionised water (-0.5 mL) was added dropwise until the reaction mixture became homogeneous, after which, the reaction mixture was allowed to warm to room temperature and stirred for 2 hours. The reaction mixture was cooled to 0 °C before NaHC03 (_Sat.) (50 mL) was added slowly. The reaction was then concentrated under reduced pressure before being transferred to a 250 mL separating funnel and extracted three times with EtOAc (50 mL). The combined organics were then washed twice with 1M sodium hydroxide (NaOH _(aq)) solution (50 mL) and twice with de-ionised water (50 mL) before being dried with Na₂S0₄ and concentrated under reduced pressure. The residue was then purified by silica gel column chromatography (EtOAc 1 :1 hexane (R_f = 0.20, visualised with KMn0₄ at room temperature)) to yield compound 1 as a colourless liquid; (3.965 g, 28%). Ή NMR (300 MHz, CDCI₃); δ 7.22 (2H, d, J= 8.6 Hz), 7.08 (2H, d, J = 8.6 Hz), 5.85 (2H, m), 5.29 (4H, dq, J = 17.1, 1.5 Hz), 4.53 (6H, m), 2.90 (1H, br s).

¹³C NMR (75.5 MHz, CDCI₃); δ 149.7 (d, J_c-P = 7 Hz), 138.3 (d, J_c-P = 1 Hz), 132.0 (d, J_c-P = 7 Hz), 128.2, 119.9 (d, J_c-P = 5 Hz), 118.8, 68.9 (d, J_c-P = 6 Hz), 64.2.

3¹P NMR (121.5 MHz, CDCI₃); δ -5.52

IR (film, cm^"1); v 3419, 2881, 1651, 1608, 1506, 1459, 1425, 1365, 1267, 1210, 1164, 1097, 1013, 988, 931, 874, 824, 733, 693, 638.

HRMS (ESI); calc'd for Ci₃Hi₇0₅P [M+Na]⁺ : m/z 307.0706, found 307.0760.

Synthesis of N-(2-aminoethyl)-2, 4, 6-trimethylbenzene sulfonamide (2)

To an oven-dried 1 L round-bottom flask was added ethylenediamine (66.8 mL, 1 mol, 10 eq.) to anhydrous dichloromethane (250 mL) under nitrogen. The solution was then cooled to 0 °C using a water-ice bath. A solution of 2,4,6-trimethylbenzenesulfonyl chloride (21.9 g, 100 mmol, 1 eq.) in anhydrous dichloromethane (250 mL) was then added slowly via a dropping funnel. Once addition was complete, the reaction mixture was allowed to warm to room temperature and stirred for an hour.

The volume of the reaction mixture was then halved under reduced pressure and transferred to a 1 L separating funnel. The organics were then washed twice with de-ionised water (250 mL), twice with NaHC03 _(sat.) (250 mL), twice again with de-ionised water (250 mL) and once with brine (250 mL). The organics were then separated, dried with magnesium sulfate (MgSC^) and concentrated to yield compound 2 as a white solid; (20.681 g, 85%). The solid is usually of sufficient quality to use in subsequent reactions but an analytically pure sample can be obtained by silica gel column chromatography (dichloromethane 9:1 methanol + 1% triethylamine (R_f = 0.08, visualised with ninhydrin under heating)).

Ή NMR (300 MHz, </₆-DMSO) δ 7.01 (2H, s), 3.31 (2H, br s), 2.68 (2H, t, J= 6.5 Hz), 2.54 (6H, s), 2.47 (2H, t, J= 6.5 Hz), 2.24 (3H, s).

¹³C NMR (75.5 MHz, </₆-DMSO); δ 141.6, 138.6, 134.9, 132.0, 45.7, 41.6, 22.9, 20.7.

Data obtained was in accordance with literature values (J. Tan, W. Tangm Y. Sun, Z. Jiang, F. Chen, L. Xu, Q. Fan, J. Xiao, Tetrahedron, 2011, 67, 6206-6213). Synthesis of 4-((bis(allyloxy)phosphoryl)oxy)benzyl (2-((2,4, 6-trimethylphenyl)sulfonamido) ethyl) carbamate (3)

To an oven-dried 250 mL round-bottom flask was added carbodiimidazole (1.36 g, 8.4 mmol, 1.3 eq.) under nitrogen. Anhydrous acetonitrile (25 mL) was added and the solution was stirred until dissolved. A solution of diallyl (4-(hydroxymethyl)phenyl) phosphate (1) (1.86 g, 6.5 mmol, 1 eq.) in anhydrous acetonitrile (25 mL) was then added slowly via dropping funnel. After complete addition, the reaction mixture was allowed to stir at room temperature overnight (-16 hours). The solvent was then removed under reduced pressure before de-ionised water (50 mL) and chloroform (50 mL) was added and then transferred to a 250 mL separating funnel. The organics were separated and the aqueous layer was extracted twice with chloroform (50 mL). The combined organics were then washed three times with de-ionised water (50 mL), dried with MgSC>4 and concentrated under reduced pressure. The residue was then taken up in anhydrous acetonitrile (50 mL) under nitrogen.

To this solution was added N-(2-aminoethyl)-2,4,6-trimethylbenzenesulfonamide (2) (2.71 g, 8.4 mmol, 1.3 eq.) and the reaction mixture was allow to stir at room temperature for 4 hours. The solvent was then removed under reduced pressure and the residue partitioned between chloroform (50 mL) and de-ionised water (50 mL). The organic layer was separated and the aqueous layer was extracted a further two times with chloroform (50 mL). The combined organics were then washed twice with de-ionised water (50 mL) and once with brine (50 mL) before being dried with MgSC>4 and concentrated under reduced pressure. The residue was then purified by silica gel column chromatography (EtOAc 1 :1 hexane (R_f = 0.10, visualised with KMn0₄ at room temperature)) to yield compound 3 as a colourless oil; (2.709 g, 76%).

Ή NMR (300 MHz, CDC1₃); δ 7.03 (2H, d, J = 8.3 Hz), 6.91 (2H, d, J = 8.3 Hz), 6.66 (2H, s), 5.64 (4H, m), 5.12 (2H, dq, J = 17.2, 1.3 Hz), 5.01 (2H, ap dd, J = 10.4, 1.3 Hz), 4.74 (2H, s), 4.38 (4H, ddd, J = 8.4, 5.7, 1.3 Hz), 2.97 (2H, ap dd, J = 10.9, 5.5 Hz), 2.71 (2H, ap dd, J = 10.9, 5.5 Hz), 2.33 (6H, s), 2.02 (3H, s).

¹³C NMR (75.5 MHz, CDC1₃); δ 156.8, 150.2 (d, J_c-P = 7 Hz), 142.1, 139.0, 133.6, 132.0, 132.0, 131.9, 129.6, 121.1 (d, J_c-P = 5 Hz), 118.8, 69.0 (d, J_c-P = 5 Hz), 65.9, 42.5, 40.7, 22.9, 20.9.

³¹P NMR (121.5 MHz, CDC1₃); δ -5.65.

IR (film, cm^"1); v 3299, 2942, 1707, 1605, 1508, 1456, 1425, 1382, 1324, 1255, 1218, 1153, 1095, 1015, 989, 936, 853, 828, 776, 734, 654.

HRMS (ESI); calc'd for C25H₃₃N2O₈PS [M+H]⁺ : m/z 553.1773, found 553.1765 Synthesis of 4-((((2-((2,4, 6-trimethylphenyl) sulfonamide) 'ethyl) carbamoyl) oxy)methyl)phenyl phosphate (4)

To an oven-dried 25 mL round-bottom flask was added 4-((bis(allyloxy)phosphoryl)oxy)benzyl (2- ((2,4,6-trimethylphenyl)sulfonamido)ethyl)carbamate (3) (0.276 g, 0.5 mmol, 1 eq.) in anhydrous THF (5 mL) under nitrogen and then cooled to 0 °C using a water-ice bath. Polymer-bound tetrakis(triphenylphosphine)palladium (0.014 g, 0.01 mmol, 0.02 eq.) was then added followed by formic acid (0.3 mL, 7.5 mmol, 15 eq.) and anhydrous triethylamine (0.7 mL, 5 mmol, 10 eq.). The reaction mixture was allowed to warm to room temperature and left to stir overnight (-16 hours). The reaction was then filtered through filter paper and washed through with THF (15 mL). The filtrate was then concentrated under reduced pressure and excess formic acid was removed via its hexane azeotrope. The residue was then cooled to 0 °C using a water-ice bath, and 1M NaOH _(aq) (6 mL, 6 mmol, 12 eq.) was slowly added. The reaction was allowed to warm to room temperature and stirred for 1 hour. The solvent was then removed under reduced pressure and excess triethylamine was removed via its toluene azeotrope to give compound 4 as a white powder; (0.257 g, quant).

Ή NMR (300 MHz, D₂0/NaOD); δ 7.07 (2H, d, J= 8.3 Hz), 6.98 (2H, d, J = 8.3 Hz), 6.72 (2H, s), 4.61 (2H, s), 2.77 (2H, m), 2.57 (2H, m), 2.33 (6H, s), 1.97 (3H, s).

³¹P NMR (121.5 MHz, D₂0/NaOD); δ 1.14.

IR (solid, cm^"1); v 3299, 1707, 1605, 1508, 1456, 1425, 1382, 1324, 1255, 1218, 1153, 1095, 1015, 989, 936, 853, 828, 776, 734, 654.

HRMS (ESI); calc'd for C1₉H24N2O₈PS [M-H]^" : m/z 471.0991, found 471.1025.

It is to be understood that one skilled in the art will be able to synthesise other novel ligands and pro- ligands of the invention, using analogous methods to those discussed above.

Example 2: Detection of CRP following the general scheme shown in figure 5

Figure 5 illustrates an assay for detecting C-reactive protein (CRP). A first sheep anti-CRP antibody is immobilised on a microtitre well surface, where it can capture CRP. Then a second sheep anti-CRP antibody, conjugated to alkaline phosphatase (ALP) binds to the captured CRP. The ALP then catalyses breakdown of a pro-ligand as shown. This was implemented as follows:

To a microtitre well coated with affinity purified sheep anti-CRP antibodies, is added 50 μΐ_^ of a CRP standard and left to stand at room temperature (20 °C) for 1 hour. The well is then washed 4 times with 350 μΐ_^ of a buffered saline and surfactant wash solution before 100 μΐ_^ of affinity purified sheep anti-CRP labelled with alkaline phosphatase is added and left to stand at room temperature (20 °C) for 1 hour. The well is then washed 4 times with 350 μΐ_^ of a buffered saline and surfactant wash solution. 100 μΐ_^ of a 6.34 mM solution of the pro-ligand (4) in a pH 9.8 carbonate buffer is added and left to stand at room temperature for 30 minutes. The solution is then added to a reaction mixture containing 10 μΐ_^ of a 38 mM solution of [Ir(cp*)Cl₂]₂ solution in DMSO, 7 mg of ferrocenecarboxaldehyde in 90 μΐ_^ of ethanol and 11 mg of sodium formate. The mixture is then agitated and heated to 37 °C. Every 3 minutes thereafter, a 1 μΐ_^ aliquot is taken and diluted into a 999 μΐ_^ of carbonate buffer. After complete mixing, a 100 μΐ_^ solution of this diluted sample is diluted further into 900 μΐ_^ of carbonate buffer. 20 μΐ_^ of this 1/10000 solution is applied to an electrode and read using a potentiostat. The conversion is then calculated by taking the integral of the peak on the voltammogram (an example of which is shown in figure 3) correlating with the electroactive product (ferrocenemethanol) and dividing it by the integral of both peaks corresponding to the electroactive product (ferrocenemethanol) and the electroactive substrate (ferrocenecarboxaldehyde) and multiplying the result by 100 to obtain the %conversion. This gives a more accurate description of the reaction profile, in comparison to using peak heights (current), as it reduces the accumulative error seen during serial dilution. Roughly 1% conversion corresponds to a peak height for the electroactive product (ferrocenemethanol) of approximately 10 nA. 10% would therefore correspond to approximately 100 nA and 100% would correspond to 1000 nA. However, full conversion is not necessary to indicate a positive result.

A graph showing the conversion of electroactive substrate to electroactive product over time in the presence of 0 and 100 μg/L of CRP is shown in figure 6.

Example 3: Quantitative detection of enzyme

Compositions comprising OU/mL, O.OlU/mL, O. lU/mL, lU/mL, lOU/mL and lOOU/mL alkaline phosphatase or Og/mL, lOng/mL, lOOng/mL, ^g/mL, 10μg.mL, or 100μg/mL alkaline phosphatase -streptavidin conjugate were assayed using pro-ligand (4) solution, [Ir(cp*)Ci₂]₂ solution in DMSO and ferrocenecarboxaldehyde as described in Example 2 above. The mixture was agitated, heated and sampled as described in Example 2 above.

Graphs showing the conversion of electroactive substrate to electroactive product over time in the presence of the different concentrations of enzyme are shown in figure 7 (alkaline phosphatase) and figure 8 (alkaline phosphatase-streptavidin conjugate). These graphs show that the concentration of enzyme present in a composition can be determined quantitatively using the claimed methods. It will be understood that the invention is described above by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. EMBODIMENTS OF THE INVENTION

I . A method for detecting the presence of an enzyme in a composition comprising:

b) detecting the detectable product and thereby detecting the presence of the enzyme. 2. The method of embodiment 1, wherein the enzyme is a hydrolase enzyme, for instance selected from the group consisting of phosphatases (e.g. alkaline phosphatase), sulfatases, glycosylases, peptidases and esterases.

3. The method of embodiment 1 or embodiment 2, wherein the detectable product is an alcohol.

4. The method of any preceding embodiment, wherein the substrate is an aldehyde or a ketone. 5. The method of any preceding embodiment, wherein the detectable product is detected using electrochemical detection and is electrochemically distinguishable from the substrate.

6. The method of any preceding embodiment, wherein the active catalyst catalyses a transfer hydrogenation reaction.

7. The method of any preceding embodiment, wherein the active catalyst has formula III.

8. The method of embodiment 7, wherein M of formula III is selected from the group consisting of iridium, ruthenium and rhodium.

9. The method of embodiment 8, wherein the active catalyst has formula IIIB.

10. The method of any preceding embodiment, wherein the pro-ligand has formula I.

I I . The method of embodiment 10, wherein the ligand has formula IA, preferably formula IB, more preferably formula IC.

12. The method of any preceding embodiment, wherein the presence of the enzyme is indicative of the presence of an analyte in a composition.

13. The method of embodiment 12, wherein the enzyme to be detected is linked to a binding partner which is capable of specifically binding to the analyte.

14. The method of embodiment 12 or embodiment 13, wherein the analyte is an antigen or an antibody.

15. The method of any one of embodiments 13-14, wherein the analyte is selected from the group consisting of a small molecule, a nucleic acid, a protein, an antigen or an antibody. 16. The method of any preceding embodiment, wherein the pro-ligand, proto-catalyst and substrate are mixed prior to step a).

17. The method of any one of embodiments 1-15, wherein the pro-ligand, proto-catalyst and substrate are provided sequentially.

18. The method of any preceding embodiment, wherein the method provides quantitative detection of the enzyme.

19. A method for detecting the presence of an analyte in a composition comprising the method of any one of the preceding embodiments, wherein the enzyme is capable of specifically binding to the analyte.

20. The method of embodiment 19, wherein the analyte is an antigen.

21. The method of embodiment 20, wherein the enzyme is linked to a binding partner, wherein the binding partner is capable of specifically binding to the antigen.

22. The method of embodiment 21, wherein the binding partner is an antibody.

23. The method of embodiment 20, wherein the enzyme is linked to a binding partner, the binding partner is an antibody, the antibody is capable of specifically binding to a secondary binding partner, the secondary binding partner is a secondary antibody, and the secondary antibody is capable of specifically binding to the antigen.

24. The method of any one of embodiments 20-23, wherein the antigen is immobilised on a solid support.

25. The method of embodiment 24, wherein the antigen is immobilised on the solid support via a capture antibody.

26. The method of embodiment 19, wherein the analyte is a target antibody.

27. The method of embodiment 26, wherein the enzyme is linked to a binding partner, wherein the binding partner is capable of specifically binding to the target antibody.

28. The method of embodiment 27, wherein the binding partner is an antibody.

29. The method of embodiment 28, wherein the antigen to which the target antibody specifically binds is immobilised on a solid support.

30. The method of embodiment 29, wherein the antigen is immobilised on the solid support via a capture antibody.

31. A method for diagnosing an infection comprising the method of any one of the preceding embodiments, wherein the composition comprises a sample obtained from a subject suspected of being infected with a pathogen, and detection of the presence of enzyme in the composition indicates that the patient has been infected with the pathogen. 32. The method of embodiment 31, wherein the enzyme is linked to an antibody which specifically binds to an analyte specific to the pathogen.

33. The method of embodiment 31, wherein the enzyme is linked to an antibody which specifically binds to a secondary antibody that specifically binds to an analyte specific to the pathogen.

34. The method of embodiment 32 or embodiment 33, wherein the analyte specific to the pathogen is an antibody.

35. A method for diagnosing infection with Treponema pallidum comprising the method of any one of embodiments 1-30, wherein the composition comprises a sample obtained from a subject suspected of being infected with Treponema pallidum, and detection of the presence of enzyme in the composition indicates that the patient has been infected with Treponema pallidum.

36. The method of embodiment 35, wherein the enzyme is linked to an antibody which specifically binds to a Treponema pallidum specific analyte.

37. The method of embodiment 35, wherein the enzyme is linked to an antibody which specifically binds to a secondary antibody that specifically binds to a Treponema pallidum specific analyte. 38. The method of embodiment 36 or embodiment 37, wherein the Treponema pallidum specific analyte is an antibody.

39. A compound having the formula I, IA, IB, or IC.

40. A cartridge comprising a composition inlet, a proto-catalyst, a pro-ligand and a substrate.

41. The cartridge of embodiment 40, further comprising an enzyme, wherein when a composition is applied to the cartridge, the composition is contacted with enzyme prior to being contacted with the pro-ligand in order to bind to any analyte present in the composition, wherein any unbound enzyme is removed from the composition prior to being contacted with the pro-ligand.

42. The cartridge of embodiment 40, wherein the detectable product is electrochemically distinguishable from the substrate.

43. A method for detecting the presence of an analyte in a composition comprising:

44. The method of embodiment 43, wherein after step a) and prior to step b) any enzyme which is not bound to analyte is removed from the composition.

45. The method of embodiment 44, wherein the enzyme is removed by washing. 46. The method of any one of embodiments 43-45, wherein the enzyme is linked to a binding partner, wherein the binding partner is capable of specifically binding to the analyte.

47. The method of embodiment 46, wherein the binding partner is an antibody.

48. The method of any one of embodiments 43-45, wherein the enzyme is linked to a binding partner, the binding partner is an antibody, the antibody is capable of specifically binding to a secondary binding partner, the secondary binding partner is a secondary antibody, and the secondary antibody is capable of specifically binding to the analyte.

49. The method of any one of embodiments 43-48, wherein the analyte is immobilised on a solid support.

50. The method of embodiment 49, wherein the analyte is immobilised on the solid support via a capture antibody.

Claims

1. A method for detecting the presence of an enzyme in a composition comprising:

b) detecting the detectable product and thereby detecting the presence of the enzyme.

2. The method of claim 1, wherein the enzyme is a hydrolase enzyme, for instance selected from the group consisting of phosphatases (e.g. alkaline phosphatase), sulfatases, glycosylases, peptidases and esterases.

3. The method of claim 1 or claim 2, wherein the detectable product is an alcohol.

4. The method of any preceding claim, wherein the substrate is an aldehyde or a ketone.

5. The method of any preceding claim, wherein the detectable product is detected using

electrochemical detection and is electrochemically distinguishable from the substrate.

6. The method of any preceding claim, wherein the active catalyst catalyses a transfer

hydrogenation reaction.

7. The method of any preceding claim, wherein (i) the active catalyst has formula III or formula IIIB; and/or (ii) the pro-ligand has formula I.

8. The method of any preceding claim, wherein the presence of the enzyme is indicative of the presence of an analyte in a composition.

9. The method of claim 8, wherein the enzyme to be detected is linked to a binding partner which is capable of specifically binding to the analyte.

10. The method of claim 8 or claim 9, wherein the analyte is an antigen or an antibody.

11. The method of any preceding claim, wherein the method provides quantitative detection of the enzyme.

12. A method for detecting the presence of an analyte in a composition comprising the method of any one of the preceding claims, wherein the enzyme is capable of specifically binding to the analyte.

13. The method of claim 12, wherein the analyte is (i) an antigen or (ii) a target antibody.

14. A method for diagnosing an infection comprising the method of any one of the preceding claims, wherein the composition comprises a sample obtained from a subject suspected of being infected with a pathogen, and detection of the presence of enzyme in the composition indicates that the patient has been infected with the pathogen.

15. The method of claim 14, wherein the enzyme is linked to a) an antibody which specifically binds to an analyte specific to the pathogen; or b) an antibody which specifically binds to a secondary antibody that specifically binds to an analyte specific to the pathogen.

16. The method of claim 15, wherein the analyte specific to the pathogen is an antibody.

17. A method for diagnosing infection with Treponema pallidum comprising the method of any one of claims 1-15, wherein the composition comprises a sample obtained from a subject suspected of being infected with Treponema pallidum, and detection of the presence of enzyme in the composition indicates that the patient has been infected with Treponema pallidum.

18. The method of claim 17, wherein the enzyme is linked to an antibody which specifically binds to a Treponema pallidum specific analyte.

19. A compound having the formula I, IA, IB, or IC.

20. A cartridge comprising a composition inlet, a proto-catalyst, a pro-ligand and a substrate.

21. The cartridge of claim 20, further comprising an enzyme, wherein when a composition is applied to the cartridge, the composition is contacted with enzyme prior to being contacted with the pro-ligand in order to bind to any analyte present in the composition, wherein any unbound enzyme is removed from the composition prior to being contacted with the pro-ligand; and/or the detectable product is electrochemically distinguishable from the substrate.

22. A method for detecting the presence of an analyte in a composition comprising: