WO2010035048A1

WO2010035048A1 - Formulation

Info

Publication number: WO2010035048A1
Application number: PCT/GB2009/051267
Authority: WO
Inventors: Haren Arulanantham; Lindy Murphy
Original assignee: F Hoffmann La Roche AG; Roche Diagnostics GmbH
Current assignee: F Hoffmann La Roche AG; Roche Diagnostics GmbH
Priority date: 2008-09-26
Filing date: 2009-09-28
Publication date: 2010-04-01
Anticipated expiration: 2011-03-26

Abstract

The present invention relates to a formulation for measuring the activity of an enzyme or enzyme-activating ion in a sample by an electrochemical rate assay, to an electrochemical sensor for measuring the activity of an enzyme or enzyme-activating ion, to an electrochemical assay method for measuring the activity of an enzyme or enzyme-activating ion in a sample and to the use of a redox mediator in a kinetic enzyme assay.

Description

Formulation

Many enzymes are biochemical markers for clinical conditions. For example, creatine kinase is a clinical marker of myocardial infarction (heart attack), rhabdomyolysis (severe muscle breakdown), muscular dystrophy and acute renal failure. Elevation of creatine kinase is an indication of muscular damage. For example, lactate dehydrogenase is a clinical marker of tissue damage. Serum lactate dehydrogenase elevation occurs in a variety of clinical conditions including myocardial infarction, haemolysis and disorders of the liver, kidneys, lung and muscle. For example, an elevated level of gamma glutamyl transferase (GGT), alkaline phosphatase (ALP), alanine aminotransferase (ALT) or aspartate aminotransferase (AST) may indicate an abnormality in the liver. For example, lactate dehydrogenase (LDH) is often used as a marker of tissue breakdown.

Conventional enzyme assays for measuring enzyme activity rely on measuring the consumption of a substrate for the enzyme or the production of a product over a temporal range. These measurements are made continuously by spectrophotometry {eg colorimetry), fluorimetry, calorimetry, chemiluminescence or light scattering or discontinuously by radiometry or chromatography.

Liu etal: Sensors and Actuators B (2007), 122: 295-300 discloses a disposable amperometric biosensor based on a trienzyme electrode for the determination of total creatine kinase. An osmium redox polymer modified electrode and glycerol kinase/ glycerol phosphate oxidase/ HRP are used to measure a reduction current. Responses are linear only at short times or low concentrations. G. Davis et a Enzyme Microb. Technol. (1986), 8: 349-352 discloses detection of ATP and creatine kinase using an enzyme electrode. The technique uses HK/glucose dehydrogenase and ferrocene monocarboxylic acid and is in bulk solution. EP-A-0125136 discloses a ferrocene based enzyme assay.

Commercially available point-of-care creatine kinase assays are rare. The commercial assay methods are discontinuous and require sophisticated non-portable instrumentation. One commercially available electrochemical creatine kinase assay (I- Stat) is coupled to an immunoassay and utilises a washing step. A further commercially available assay (Abaxis Piccolo) uses freeze dried reagents and a colour reaction. There is a centrifugal step involved in the assay which limits utility to the laboratory rather than point of care.

The present seeks to improve the measurement of enzyme or enzyme-activating ion activity by exploiting the linear rate of response and stability of a stable redox mediator in an electrochemical kinetic assay. Viewed from a first aspect the present invention provides a formulation for measuring the activity of an enzyme or an enzyme-activating ion in a sample by an electrochemical rate assay comprising: a substrate capable of an enzyme-catalysed reaction; and a redox mediator capable of a measurable change in oxidation state in response to the enzyme-catalysed reaction, wherein the redox mediator is an osmium complex, ruthenium hexamine trichloride or a ruthenium complex of Formula I

[Ru(A)_w(B)_x(C)_y]^m (X^z)_n (I)

(wherein

Ru has an oxidation state of 0, 1, 2, 3 or 4; each of w, x, and y is an integer independently selected from the integers 1 to 4; m is an integer selected from the integers -5 to +4; n is an integer selected from the integers 1 to 5; z is an integer selected from the integers -2 to +1 ;

A is NCS or a monodentate 5- or 6- membered aromatic ligand containing 1 , 2 or 3 nitrogen atoms which is optionally substituted by 1 to 8 substituents each selected from the group consisting of substituted or unsubstituted alkyl, alkenyl, or aryl groups, -F, -Cl, -Br, -I, -NO₂, -CN, -CO₂H, -SO₃H, -NHNH₂, -SH, aryl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, -OH, alkoxy, -NH₂, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino and alkylthio;

B is acetylacetonate (acac) or is a bi-, tri-, tetra-, penta- or hexadentate ligand which is linear having the formula R¹RN(C₂H₄NR)_WR¹ or cyclic having the formula (RNC₂H₄)_V, (RNC₂H₄)_P(RNC₃H₆)C₁ or [(RNC₂H₄XRNC₃H₆)J₅, wherein w is an integer selected from the integers 1-5, v is an integer selected from the integers 3-6, each of p and q is an integer independently selected from the integers 1-3 whereby the sum of p and q is 4, 5 or 6, s is either 2 or 3 and each of R and R¹ is independently hydrogen or alkyl; C is a ligand; and X is a counter ion, wherein the number of coordinating atoms is 6).

The formulation exhibits advantageously a rapid linear response to the enzyme- catalysed reaction which may in some circumstances permit measurement of the activity of the enzyme or the enzyme-activating ion in the sample to be made without correction factors typically within five minutes or less of the onset of reaction.

Typically the rate of the enzyme-catalysed reaction is the rate limiting step. This may be achieved by ensuring that the substrate is present in excess. Since it is the rate limiting step which determines the magnitude of the response to the enzyme-catalysed reaction, the result is a constant rate of production of redox mediator which has undergone a measurable change in oxidation state. By measuring the rate of response (for example nA/min) at two separate time points, the enzyme activity can be determined. The formulation of the invention permits the measurement of activity as a unit which is the amount of enzyme which turns over one micromole of substrate per litre per minute under optimal conditions.

In a preferred embodiment, the enzyme-catalysed reaction is an enzyme-catalysed reaction between the substrate and a reagent.

The reagent may be adenosine diphosphate (ADP).

In a preferred embodiment, the enzyme-catalysed reaction is an enzyme-catalysed transformation in the substrate. The transformation may be decomposition and the substrate may be a decomposable substrate capable of enzyme-catalysed decomposition.

Typically the change in oxidation state is a reduction. For example, the change in oxidation state may be measured by a change in current, voltage or charge at an electrode.

The measurable change in oxidation state of the redox mediator may be in direct response to the enzyme-catalysed reaction. The product of the enzyme-catalysed reaction may be sufficiently electroactive to cause the measurable change in oxidation state of the redox mediator.

The measurable change in oxidation state of the redox mediator may be in indirect response to the enzyme-catalysed reaction. For this purpose, the formulation may comprise auxiliary reagents of one or more sequential coupling reactions. Typically the rate of the (or each) coupling reaction is significantly greater than the rate of the enzyme-catalysed reaction.

Preferably the formulation comprises the auxiliary reagents of one or more kinase coupling reactions.

The auxiliary reagents of a kinase coupling reaction may be one or more of the group consisting of glucose, glycerol and β-NAD⁺ in the presence of a kinase auxiliary enzyme. The kinase auxiliary enzyme may be glucokinase, glycerol kinase, NAD⁺ kinase or hexokinase.

Preferably the formulation comprises the auxiliary reagents of one or more dehydrogenase coupling reactions.

The auxiliary reagents of a dehydrogenase coupling reaction may be NAD⁺, L- glutamate or glycerol-3 -phosphate in the presence of a dehydrogenase auxiliary enzyme. The dehydrogenase auxiliary enzyme may be glucose-6-phosphate dehydrogenase (G6PDH), glycerol 3-phosphate dehydrogenase (G3PDH), diaphorase or glutamate dehydrogenase.

Preferably the formulation comprises the auxiliary reagents of one or more oxidase coupling reactions. The auxiliary reagents of an oxidase coupling reaction may be present with an oxidase auxiliary enzyme. The oxidase auxiliary enzyme may be NADH oxidase.

The formulation may comprise the auxiliary reagents of one or more transferase coupling reactions.

The auxiliary reagents of a transferase coupling reaction may be NAD⁺, glutamate dehydrogenase or α-ketoglutaric acid.

To ensure that the rate of the enzyme-catalysed reaction is the rate limiting step, the auxiliary reagents and auxiliary enzymes are typically present in excess.

Preferably the formulation further comprises: hexokinase, G6PDH and diaphorase.

Hexokinase and G6PDH are auxiliary enzymes of rapid coupling reactions which ensure advantageously that the enzyme-catalysed reaction is the rate limiting reaction.

The formulation may further comprise: NADH oxidase and optionally glutamate dehydrogenase.

The formulation may further comprise: glucose, glycerol, glycerol 3 -phosphate or L- glutamate.

The formulation may further comprise: NADH oxidase, glutamate dehydrogenase and NAD⁺ kinase.

The formulation may further comprise diaphorase or putidaredoxin reductase (PDR).

In a first embodiment, the formulation is capable of measuring the activity of an enzyme.

Preferably the enzyme is a kinase, dehydrogenase, hydrolase, reductase, oxidase, peroxidase or transferase. Particularly preferably the hydrolase acts on esters or sugars (eg a glycoside hydrolase, esterase or phosphatase).

Preferably the enzyme is creatine kinase. Particularly preferably the enzyme is selected from the group consisting of creatine kinase-MM, creatine kinase-BB and creatine kinase-MB. Particularly preferably the creatine kinase contains creatine kinase-MM, creatine kinase-BB and creatine kinase-MB.

The enzyme may be selected from the group consisting of creatine kinase (CK), gamma glutamyl transferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), cholesterol dehydrogenase (ChDH), diaphorase, amylase, alkaline phosphatase (ALP), acid phosphatase (ACP), alanine aminopeptidase (AAP), N-acetyl-beta-d-glucosaminidase (NAG), maltase, glucose-6-phosphate dehydrogenase (G6PDH) and isoenzymes thereof.

The formulation may further comprise an enzyme activator. The enzyme activator (eg ffoorr ccrreeaattiine kinase) may be N-acetyl cysteine or a source of Mg²⁺ (such as magnesium acetate). In a preferred first embodiment (eg where the enzyme is creatine kinase), the substrate is creatine phosphate. Preferably the reagent is an ADP salt.

In a preferred first embodiment (eg where the enzyme is lactate dehydrogenase), the substrate is a lactic acid salt or ester. The reagent may be NAD⁺.

In a preferred first embodiment (eg where the enzyme is hexokinase), the substrate is an ATP salt. The reagent may be a magnesium salt such as magnesium acetate.

In a preferred first embodiment (eg where the enzyme is L-alanine aminotransferase), the substrate is L-alanine.

In a preferred first embodiment (eg where the enzyme is L-aspartate aminotransferase), the substrate is L-aspartic acid.

In a preferred first embodiment (eg where the enzyme is an amylase), the substrate is a nitrophenol linked-maltooligosaccharide (preferably nitrophenol linked - maltotrioside) which is decomposable.

In a preferred first embodiment (eg where the enzyme is alkaline phosphatase), the substrate is a phosphate ester. The reagent may be an aqueous medium (eg water).

In a preferred first embodiment (eg where the enzyme is cholesterol dehydrogenase), the substrate is cholesterol. The reagent may be a nicotinamide such as TNAD.

In a preferred first embodiment (eg where the enzyme is diaphorase), the substrate is NADH.

In a preferred first embodiment (eg where the enzyme is glucose-6-phosphate dehydrogenase), the substrate is a glucose 6-phosphate salt. The reagent may be magnesium acetate.

In a preferred first embodiment (eg where the enzyme is glycerol dehydrogenase), the substrate is glycerol. The reagent may be TNAD salt.

In a second embodiment, the formulation is capable of measuring the activity of an enzyme-activating ion. For this purpose, the formulation further comprises: an enzyme-activating ion-dependent enzyme capable of an enzyme-catalysed reaction with the substrate.

The enzyme-activating ion may be a cofactor. The enzyme-activating ion may be a metal ion (eg a metal ion cofactor).

The enzyme-activating ion may be selected from the group consisting of Na⁺, K⁺, Mg²⁺, Ca²⁺, phosphate, chloride, ammonium and HCO₃ ^".

In a preferred second embodiment (eg where the enzyme-activating ion is Na⁺), the substrate may be a lactose or a nitrophenol linked-sugar (such as o-nitrophenol-β-D- galactopyranoside) and the enzyme-activating ion-dependent enzyme may be a hydrolase (such as β-galactosidase). The formulation may further comprise galactose dehydrogenase and NADH oxidase, β-galactosidase may be used in the presence of a sodium binder to reduce the effective concentration of sodium. The sodium binder may be a cryptand such as Kryptofix K221.

In a preferred second embodiment (eg where the enzyme-activating ion is K⁺), the substrate may be a pyruvate (eg phosphoenolpyruvate) and the enzyme-activating ion- dependent enzyme may be pyruvate kinase. Alternatively the substrate may be urea and the enzyme-activating ion-dependent enzyme may be urea amidolyase.

In a preferred second embodiment (eg where the enzyme-activating ion is Mg²⁺), the substrate may be glucose and the enzyme-activating ion-dependent enzyme may be hexokinase. Alternatively the enzyme-activating ion-dependent enzyme may be NAD+-isocitrate dehydrogenase.

In a preferred second embodiment (eg where the enzyme-activating ion is Ca²⁺), the substrate may be an amylose (eg maltotetraose) and the enzyme-activating ion- dependent enzyme may be an amylase (eg α-amylase). Alternatively the enzyme- activating ion-dependent enzyme may be NAD+-isocitrate dehydrogenase.

In a preferred second embodiment (eg where the enzyme-activating ion is phosphate), the substrate may be sucrose and the enzyme-activating ion-dependent enzyme may be sucrose phosphorylase. The formulation may further comprise phosphoglucomutase, G6PDH and NADH oxidase.

In a preferred second embodiment (eg where the enzyme-activating ion is chloride), the substrate may be an amylose (eg maltotetraose) and the enzyme-activating ion- dependent enzyme may be an amylase (eg α-amylase). The formulation may further comprise hexokinase and G6PDH.

In a preferred second embodiment (eg where the enzyme-activating ion is ammonium), the substrate may be a nicotinamide (eg NAD⁺ or NADPH) and the enzyme-activating ion-dependent enzyme may be glutamate dehydrogenase. The formulation may further comprise L-glutamic acid. The reaction is operable in the reverse direction.

In a preferred second embodiment (eg where the enzyme-activating ion is HCO₃ ^"), the substrate may be a pyruvate (eg phosphoenolpyruvate) and the enzyme-activating ion- dependent enzyme may be phosphoenolpyruvate carboxylase. The reaction is operable in the reverse direction.

Typically the redox mediator is a stable redox mediator which exhibits a current loss one minute after the onset of current flow of 35% or less, preferably 20% or less, particularly preferably 15% or less, most preferably 10% or less.

Preferably the redox mediator is ruthenium hexamine trichloride.

Preferably the redox mediator is a complex of Formula I

[Ru (A)_w(B)_x (C)_y]^m (X^z)_n (I) (wherein

Ru has an oxidation state of 0, 1, 2, 3 or 4; each of w, x, and y is an integer independently selected from the integers 1 to 4; m is an integer selected from the integers -5 to +4; n is an integer selected from the integers 1 to 5 z is an integer selected from the integers -2 to +1 ;

A is NCS or a monodentate 5- or 6- membered aromatic ligand containing 1, 2 or 3 nitrogen atoms which is optionally substituted by 1 to 8 substituents each selected from the group consisting of substituted or unsubstituted alkyl, alkenyl, or aryl groups, -F, -Cl, -Br, -I, -NO₂, -CN, -CO₂H, -SO₃H, -NHNH₂, -SH, aryl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, -OH, alkoxy, -NH₂, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino and alkylthio;

B is acetylacetonate (acac) or is a bi-, tri-, tetra-, penta- or hexadentate ligand which is linear having the formula R¹RN(C₂H₄NR)_WR¹ or cyclic having the formula (RNC₂H₄)_V, (RNC₂H4)p(RNC₃H₆)_q or [(RNC₂H₄XRNC₃H₆)L, wherein w is an integer selected from the integers 1-5, v is an integer selected from the integers 3-6, each of p and q is an integer independently selected from the integers 1-3 whereby the sum of p and q is 4, 5 or 6, s is either 2 or 3 and each of R and R¹ is independently hydrogen or alkyl; C is a ligand; and X is a counter ion, wherein the number of coordinating atoms is 6.

The ligand A in the complex of Formula I may be selected from the group consisting of NCS, optionally substituted imidazole, optionally substituted pyrazole, optionally substituted thiazole, optionally substituted oxazole, optionally substituted isoquinoline, optionally substituted pyridyl (eg 3- and/or 4-substituted pyridyl) and isomers thereof.

Preferably w is 1 or 2.

The ligand A in the complex of Formula I may be substituted by one or more substituents selected from the group consisting of C₁-C₆ alkyl, CpC₆ alkoxy, C₂-C₆ alkenyl, C₂-C₆ alkynyl, halogen, carboxy, amino, Ci_-6-alkylamino, C_1-6-dialkylamino and hydroxyl,

Preferably the ligand A in the complex of Formula I is substituted by one or more substituents selected from the group consisting of Ci-C₆ alkyl, carboxy, Ci_-6- alkylamino and Ci_-6-dialkylamino.

The ligand A in the complex of Formula I may be or contain a 5- or 6-membered aromatic ligand containing 1 or 2 nitrogen heteroatoms. Preferably ligand A is a 5- or 6-membered aromatic ligand containing 1 nitrogen heteroatom. Preferably the ligand A in the complex of Formula I is optionally substituted pyridyl or imidazolyl, particularly preferably optionally substituted pyridyl, more preferably alkylamino-, dialkylamino- or carboxy-substituted pyridyl.

The ligand B in the complex of Formula I may be acetylacetonate (acac) or a bi-, tri- or tetra-dentate ligand which may be linear having the formula R¹RN(C₂H₄NR)_JR¹ or cyclic having the formula (RNC₂H₄)_V, (RNC₂H₄)_p(RNC₃H₆)_q or [(RNC₂H₄)(RNC₃H₆)]_s, wherein r is an integer selected from the integers 1-3, v is 3 or 4, each of p and q is an integer independently selected from the integers 1-3 whereby the sum of p and q is 4 and s is 2 or 3.

Preferably the ligand B in the complex of Formula I is acetylacetonate (acac) or a tri- or tetra-dentate ligand which is cyclic having the formula (RNC₂H₄)_V, wherein v is 3 or 4.

The ligand B in the complex of Formula I may be acetylacetonate (acac), 1,4,7- trirnethyl-l,4,7-triazacyclononane, l ,4,8,l l-tetramethyl-l,4,8,l l -tetra- azacyclotetradecane, 1 , 1 ,4,7, 10, 10-hexamethyltriethylenetetramine, 1 ,2- dimethylethylenediamine or 1,1,2,2-tetramethylethylenediamine.

Preferred ligands B in the complex of Formula I are 1 ,4,7-trimethyl- 1,4,7- triazacyclononane and acetylacetonate (acac).

Preferably C is a ligand other than B. Particularly preferably C is a ligand other than A or B.

The ligand C in the complex of Formula I may be selected from the group consisting of an amine ligand (such as NH₃), CO, CN, NCS, a halogen, acetylacetonate (acac), 3- bromo-acetylacetonate (Bracac), oxalate, troplone, pyridine and 5-chloro-8- hy droxy quinoline .

A preferred ligand C in the complex of Formula I is an acetylacetonate anion (acac).

The oxidation state of Ru in the complex of Formula I may be 2+, 3+ or 4+. The oxidation state of Ru in the complex of Formula I is preferably 3+.

The ligands A, B and C may be selected such that the overall charge on the complex of Formula I is selected from the group consisting of +3, +2, +1, 0, -1, -2 and -3.

The counterion X in the complex of Formula I may be F^", Cl^", Br^", I^", NO₃ ^", NH₄ ⁺, NR₄ ⁺, PF₆ ^", CF₃SO₃ ^", SO₄ ^2", ClO₄ ^", K⁺, Na⁺, Li⁺ or a combination thereof.

Preferably the redox mediator is ruthenium hexamine trichloride, cis-[Ru(acac)₂(Py-3- COOH)(Py-3-COO)], [Ru¹¹¹ (l,4,7-trimethyl-l,4,7-triazacyclononane)(acac) (1- melm)] (NO₃)₂ or Ru¹¹¹ (1,4,7-trimethyl - l,4,7-triazacyclononane)(acac)(4-Me₂N- py)] (NOs)₂.

Particluarly preferably the redox mediator is ruthenium hexamine trichloride, cis- [Ru(acac)₂(Py-3-COOH)(Py-3-COO)] or Ru¹¹¹ (1,4,7-trimethyl - 1,4,7- triazacyclononane)(acac)(4-Me₂N-py)] (NO₃)₂. A formulation containing ruthenium hexamine trichloride exhibits a good electrochemical response to the enzyme-catalysed reaction. cis-[Ru(acac)₂(Py-3-COOH)(Py-3-COO)] has a highly stable reduced form and exhibits an excellent electrochemical response to the enzyme-catalysed reaction.

In a preferred embodiment, the formulation is freeze-dried. The freeze-dried formulation may be freeze-dried in situ. A freeze-dried formulation may be prepared for example according to WO-A-2007/006132, WO-A-03/056319 or PCT/GB2008/001835.

The formulation may further comprise an excipient. The excipient may be a protein (such as BSA or ovalbumin), glycine, sodium glutamate, lysine, glycylglycine, a sugar (such as sodium gluconate, mannitol, lactitol, maltitol, sucrose, maltose, lactose or trehalose), a salt (such as KCl, KNO₃, NaNO₃ or NaCl), an ectoine, a hydroxyectoine, inositol, myo inositol or hydroxyinositol. The excipient is preferably lactose (eg 10%w/v lactose). The formulation may further comprise a buffer.

Viewed from a further aspect the present invention provides an electrochemical sensor for measuring the activity of an enzyme or an enzyme-activating ion in a sample by an electrochemical rate assay comprising: a main body defining one or more electrochemical cells, wherein each electrochemical cell includes a well, a working electrode exposed in the well and a reference or pseudo-reference electrode exposed in the well; and a formulation as hereinbefore defined in the well so as to be in contact or contactable with the working electrode.

The electrochemical sensor according to the invention exhibits advantageously a rapid linear response to the enzyme-catalysed reaction which permits measurement of the enzyme or enzyme-activating ion activity in the sample.

The main body typically has a laminate structure extending through which is the one or more (eg an array of) wells, wherein the (or each) well is at least partly bound by the working electrode. Preferably the laminate structure includes a layer of a material (such as a conductive ink) operable as a working electrode, wherein the internal wall of the (or each) well is at least partly composed of the material (eg in a continuous band).

Preferably the formulation in the electrochemical sensor of the invention is a freeze- dried formulation. Particularly preferably the freeze-dried formulation is contactable with the working electrode only on reconstitution.

The main body may be a well-containing strip or sheet. The strip or sheet is typically portable and disposable. Suitable examples of a main body which may be used in the sensor of the invention are disclosed in GB0809740.4, WO-A-2007/006132, WO-A- 03/056319 and PCT/GB2008/001835.

The freeze-dried formulation of the sensor may be reconstitutable by the sample or by an aqueous solution (eg water). The incubation time before the first reading is low. In a preferred embodiment, the main body is a well-containing strip and the formulation is a freeze-dried formulation.

The sensor typically measures enzyme activity up to 6000U/L (eg in the range 150 to 6000 U/L).

Viewed from a yet further aspect the present invention provides an electrochemical assay method for measuring the activity of an enzyme or an enzyme-activating ion in a sample comprising (or consisting essentially of):

(a) introducing the sample to a formulation as hereinbefore defined in an electrochemical cell;

(b) measuring an electrical parameter at a plurality of times in a temporal range;

(c) determining the rate of response of the electrical parameter; and

(d) relating the rate of response of the electrical parameter to the activity of the enzyme.

The kinetic assay method of the invention is advantageously continuous with no additional steps such as washing or centrifuging.

In a preferred embodiment of the electrochemical assay method there are no washing or centrifuging steps.

The temporal range may be 300 seconds or less, preferably 120 seconds or less, particularly preferably 60 seconds or less. The first measurement may take place immediately after step (a) or after a short incubation period.

The plurality of times may be twice.

The electrical parameter may be current, charge or voltage. The electrical parameter is preferably current.

The sample may be a biological sample such as a bodily fluid sample (eg serum, blood, saliva, interstitial fluid, plasma, dermal fluid, sweat or tears). The sample may be a non-biological sample (eg water or a liquid beverage).

The method of the invention may be carried out at room temperature. Preferably the method is carried out at an elevated temperature (eg about 37⁰C) to achieve optimum current response and optimum enzyme activity.

Typically the method is carried out at a pH in the range 5.5 to 8.5 (eg β.l to 7.8).

The working electrode may be made of palladium, platinum, gold or carbon. The reference or pseudo-reference electrode may be typically carbon, Ag/AgCl, Ag/Ag₂SO₄, palladium, gold, platinum, Cu/CuSO₄, Hg/HgO, Hg/HgCl₂, Hg/HgSO₄ or ZnAZnSO₄. Viewed from a still yet further aspect the present invention provides use of a redox mediator (preferably a ruthenium-based redox mediator) as hereinbefore defined or a formulation as hereinbefore defined in a kinetic enzyme assay.

The present invention will now be described in a non-limitative sense with reference to Examples and the accompanying Figures in which:

Figures IA-D show plots of average current vs time for each CK-MM sample in sensor types A (cis-[Ru(acac)₂(Py-3-COOH)(Py-3-COO)], no excipient), B (cis- [Ru(acac)₂(Py-3-COOH)(Py-3-COO)], 2% BSA), C (cis-[Ru(acac)₂(Py-3 -COOH)(Py- 3-COO)] and 10% lactose) and D (([Ru ^Iπ (1,4,7-trimethyl- 1,4,7- triazacyclononane)(acac) (l-melm)] (NO₃)₂ , 10% lactose); Figures 2A-D show calibration plots for sensor types A-D;

Figures 3A and B show plots of average current vs time for CK-MM with cis- [Ru(acac)₂(Py-3-COOH)(Py-3-COO)] mediator in testing wet sensors and freeze dried sensors respectively;

Figures 4A and B shows calibration plots of rate of response vs CK-MM activity with cis-[Ru(acac)₂(Py-3-COOH)(Py-3-COO)] mediator in testing wet sensors and freeze dried sensors respectively;

Figure 5 shows average current vs time for CK-MM with freeze dried 4OmM ([Ru "' (l,4,7-trimethyl-l,4,7-triazacyclononane)(acac) (l-melm)] (NO₃)₂ mix; Figure 6 shows the CK titration rate with freeze dried 4OmM ([Ru ^iπ (1,4,7-trimethyl- 1 ,4,7-triazacyclononane)(acac) (l-melm)] (NO₃)₂;

Figure 7 shoes the average current vs time for each CK-MM sample with Ru ⁰' (1,4,7- trimethyl - 1 ,4,7-triazacyclononane)(acac)(4-Me₂N-py)] (NO₃)₂ as mediator; Figure 8 shows a calibration plot of rate of response vs CK-MM activity with Ru ^πι (1,4,7-trimethyl - l,4,7-triazacyclononane)(acac)(4-Me₂N-py)] (NO₃)₂ as mediator; Figures 9A, 9B and 9C show plots of average current vs time for CK-MM with Ru(NH₃)₆CI₃, ([Ru ^ffl (1,4,7-trimethyl- 1 ,4,7-triazacyclononane)(acac) (l-melm)] (NO₃)₂ and cis-[Ru(acac)₂(Py-3-COOH)(Py-3-COO)] respectively; Figures 1OA, 1OB and 1OC show calibration plots of rate of response vs CK-MM activity for Ru(NH₃)₆Cl₃, ([Ru¹¹¹ (1,4,7-trimethyl- 1 ,4,7-triazacyclononane)(acac) (1- melm)] (NO₃)₂ and cis-[Ru(acac)₂(Py-3-COOH)(Py-3-COO)] respectively; Figure 11 shows a plot of average current vs time for each LDH sample in example 6; Figure 12 shows a calibration plot of rate of response vs LDH concentration from example 6;

Figure 13 shows a plot of average current vs time for each Mg acetate sample in example 7;

Figure 14 shows a calibration plot of rate of response vs Mg acetate concentration from example 7;

Figure 15 shows a plot of average current vs time for each LDH sample in example 8; Figure 16 shows a calibration plot of rate of response vs LDH concentration from example 8;

Figures 17A to L show plots of average current vs time for each NADH concentration for each Mediator respectively in Example 9;

Figures 18A to 18L show a calibration plot of rate of response vs NADH concentration for each Mediator respectively from example 9; Figure 19 shows the variation in rate for the stable mediators of example 9; Figure 20 shows the variation in rate for the unstable mediators of example 9; Figures 21 A to 211 show plots of average current vs time for each CK-MM concentration for sensors prepared with KCl, KNO₃, NaNO_3, BSA , Ovalbumin,

Lactose, Lactitol, Sucrose and Trehalose respectively in example 10;

Figures 22A to 221 show the rates of response (gradient of current vs time) determined between the time points 22 and 64 seconds for each CK-MM concentration for each excipient in example 10;

Figures 23 A to 23D show plots of average current vs time for each CK concentration for each CK sample (MM or MB) in buffer and plasma respectively in example 11 ;

Figures 24A to 24D show a calibration plot of rate of response vs concentration for

CK-MM in buffer and plasma and CK-MB in buffer and plasma respectively from example 11 ;

Figure 25 shows a plot of average current vs time for each ChDH sample in example

12;

Figure 26 shows a calibration plot of rate of response vs ChDH concentration from example 12;

Figures 27 A-C, 28A-C and 29A-C show plots of average current vs time for each CK-

MM sample for each mediator respectively in example 13;

Figure 3OA, 3OB and 3OC show a calibration plot of rate of response vs CK-MM activity for each mediator respectively in example 13;

Figure 31 shows a plot of average current vs time for each diaphorase sample in example 14;

Figure 32 shows a calibration plot of rate of response vs diaphorase concentration from example 14;

Figure 33 shows a plot of average current vs time for each G6PDH sample in example

15;

Figure 34 shows a calibration plot of rate of response vs G6PDH concentration from example 15;

Figure 35 shows a plot of average current vs time for each GDH sample of example

16;

Figure 36 shows a calibration plot of rate of response vs GDH concentration from example 16;

Figure 37 shows a plot of average current vs time for each HK sample in example 17;

Figure 38 shows a calibration plot of rate of response vs HK concentration from example 17;

Figure 39 shows plots of average current vs time for each CK sample in example 18;

Figure 40 shows a plot of average current vs time for each AST sample in example

19;

Figure 41 shows a calibration plot of rate of response vs AST concentration for each sample between the time points 8 seconds and 50 seconds in example 19;

Figure 42 shows a calibration plot of rate of response vs AST concentration for each sample between the time points 162 seconds and 204 seconds in example 19;

Figure 43 shows a plot of average current vs time for each ALT sample in example

20;

Figure 44 shows a calibration plot of rate of response vs ALT concentration for each sample between the time points 8 seconds and 50 seconds in example 20;

Figure 45 shows a calibration plot of rate of response vs ALT concentration for each sample between the time points 162 seconds and 204 seconds in example 20;

Figures 46a, 47a and 48a show plots of average current vs time for each MgAc concentration for each mediator respectively in example 21 ; Figures 46b, 47b and 48b show a calibration plot of rate of response vs NADH concentration for each mediator respectively in example 21 ;

Figure 49a shows a plot of average current vs time for each MgAc concentration for the mediator of example 22;

Figure 49b shows a calibration plot of rate of response vs NADH concentration for the mediator of example 22;

Figures 50a, 51a, 52a, 53a, 54a, 55a, 56a, 57a and 58a show plots of average current vs time for each MgAc concentration for Lactose, Lactitol, Sucrose, Trehalose,

Ovalbumin, BSA, KCl, KNO₃ and NaNO₃ respectively in example 23;

Figures 50b, 51b, 52b, 53b, 54b, 55b, 56b, 57b and 58b show a calibration plot of rate of response vs MgAc concentration for Lactose, Lactitol, Sucrose, Trehalose,

Ovalbumin, BSA, KCl, KNO₃ and NaNO₃ respectively in example 23;

Figure 59a shows plots of average current vs time for each MgAc concentration in example 24;

Figure 59b shows a calibration plot of rate of response vs MgAc concentration in example 24;

Figure 60 shows plots of average current vs time for each MgAc concentration in example 25;

Figure 61 shows a calibration plot of rate of response vs MgAc concentration from example 25;

Figure 62 shows plots of average current vs time for each MgAc concentration in example 26;

Figure 63 shows plots of average current vs time for each Calcium Lactate concentration in example 27;

Figure 64 shows a calibration plot of rate of response vs Calcium Lactate concentration from example 27;

Figure 65 shows a plot of average current vs time for each KCl concentration in example 28;

Figure 66 shows a calibration plot of rate of response vs KCl concentration from example 28;

Figure 67 shows a plot of average current vs time for each KCl concentration in example 29;

Figure 68 shows a calibration plot of rate of response vs KCl concentration from example 29;

Figure 69 shows a plot of average current vs time for each Ammonium Chloride concentration in example 30;

Figure 70 shows a calibration plot of rate of response vs Ammonium Chloride concentration from example 30;

Figure 71 shows a plot of average current vs time for each Sodium Chloride concentration in example 31 ; and

Figure 72 shows a calibration plot of rate of response vs Sodium Chloride concentration from example 31.

Examples 9-31 refer to

Mediator 2 which is αs-[Ru(acac)₂(Py-3-COOH)(Py-3-COO)]

Mediator 6 which is [Ru ¹¹¹ (l,4,7-trimethyl-l,4,7-triazacyclononane)(acac) (1- melm)] (NO₃)₂

Mediators 1, 3, 4, 8 and 11 which are rutheniumn mediators amongst which are [Ru¹¹¹ (l,4,7-trimethyl-l,4,7-triazacyclononane)(acac)(4-Me₂N-py)](NO₃)₂, [Ru ¹¹¹

(NH₃)₅(py-3-CO₂H)] (PFe)₂(CF₃SO₃), m-[Ru(acac)₂(2,2'-bpy)]Cl, [Ru¹¹¹ (1,4,7- trimethyl-l,4,7-triazacyclononane)(acac)(3-OH-py)](NO3)₂ and [Ru(Tet- Me₆(acac)]Cl₂

Mediators 5, 7, 9 and 10 which are osmium complexes.

Preparation of cis- [Ru(acac)₂(Py-3-COOH)(Py-3-COO)]

The compound was prepared according to the description in WO-A-2007/072018.

Preparation of [Ru . Ill (l,4,7-trimethyl-l,4,7-triazacyclononane)(acac) (l-melm)]

(NO₃)₂

N-methylimidazole (0.5 g, 6.0 mmol) was added to [Ru^π(L)(acac)(OH)]PF₆ (100 mg, 0.19 mmol) in absolute ethanol (5 mL). The solution was refluxed under argon in the presence of a few pieces of Zn amalgam for 24 h. After cooling to room temperature, acetone (15 mL) was added and the solution was then filtered. The filtrate was evaporated to dryness to give an orange solid [Ru^π(l,4,7-trimethyl- 1,4,7- triazacyclononane)(acac)(l -MeIm)]PF₆ which was filtered and washed with diethyl ether. Yield (100 mg). ESI/MS (positive mode) in acetone: m/z = 454.4, [M]⁺.

A solution Of AgCF₃SO₃ (45 mg, 0.17 mmol) in acetone (2 mL) was slowly added to [Ru^π(l,4,7-trimethyl-l,4,7-triazacyclononane)(acac)(l-MeIm)]PF₆ (100 mg, 0.17 mmol) in acetone (3 mL). After 3 minutes the purple solution was filtered to remove the silver. ["Bu₄N]NO₃ (1500 mg, 0.5 mmol) was then added to give a purple precipitate of the target compound which was filtered and washed with acetone. Yield (50 mg). ESI/MS (positive mode) in methanol: m/z = 277.5, [M]²⁺. E_I/2 of Ru^III/!I = + 0.09 V vs NHE in buffer solution (pH 8.20).

Preparation of [Ru Ill (l,4,7-trimethyl-l,4,7-triazacyclononane)(acac)(4-Me₂N- py)](NO₃)₂

4-Dimethylaminopyridine (0.3 g, 2.4 mmol) was added to [Ru^m(l,4,7-trimethyl- 1,4,7- triazacyclononane)(acac)(OH)]PF₆ (150 mg, 0.28 mmol) in absolute ethanol (10 mL). The solution was refluxed under argon in the presence of 10 pieces of Zn amalgam for 24 h. After cooling to room temperature, the orange solid of [Ru¹¹Q, 4, 7-trimethyl- l,4,7-triazacyclononane)(acac)(4-Me₂N-py)]PF₆ was filtered and recrystallized from acetone/diethyl ether. Yield (120 mg). ESI/MS (positive mode) in acetone: m/z = 494.1, [M]⁺. Anal, calcd. for C_2IH₃₈N₅O₂PF₆Ru: C, 39.50; H, 6.00; N, 10.97. Found: C, 39.73; H, 6.05; N, 10.81.

A solution Of AgCF₃SO₃ (50 mg, 0.19 mmol) in acetone (2 mL) was slowly added to the orange solution of [Ru^II(l,4,7-trimethyl-l,4,7-triazacyclononane)(acac)(4-Me₂N- Py)]PF₆ (120 mg, 0.19 mmol) in acetone (5 mL). After 5 min. the purple solution was filtered to remove the silver, and then ["Bu₄N]NO₃ (150 mg, 0.5 mmol) was added to give a purple precipitate of [Ru^iπ(l,4,7-trimethyl-l,4,7-triazacyclononane)(acac)(4- Me₂N-py)](NO₃)₂lΗ₂O which was filtered, washed with acetone and vacuum dried. Yield (80 mg). ESI/MS (positive mode) in methanol: m/z = 247.3, [M]²⁺. E_m of _Ruiii/π _{= + 0 07} Y _{vs NHE in buffer solution} (p_{H 8} 20). Anal_, calcd. for

C₂₁H₃₈N₇O₈Ru-IH₂O: C, 39.68; H, 6.34; N, 15.42. Found: C, 39.95; H, 6.40; N, 15.61.

Example 1: Freeze dried CK sensors

Methods

Solution 1 contained 0.1M imidazole buffer (pH 6.75, balanced with acetic acid). Solution 2 contained 0.1M imidazole buffer (pH 6.75) and 40 mM KOH. Final enzyme mixes A, B and C containing 40 mM cw-[Ru(acac)₂(Py-3 -COOH)(Py- 3-COO)] and no excipient, 2% w/v BSA or 10% w/v lactose respectively were prepared using solution 2 by sequential addition of reagents.

Final enzyme mixture D containing 40 mM [Ru ^m (1,4,7-trimethyl- 1,4,7- triazacyclononane)(acac) (l-melm)] (NO₃)₂ and 10% w/v lactose was prepared using solution 1 by sequential addition of reagents.

Final enzyme mixes A, B, C and D also contained:

2O mM NAD⁺

5 mg/ml diaphorase (Unitika)

5 mg/ml glucose 6-phosphate dehydrogenase (Sigma G8529, from Leuconostoc mesenteroides, recombinant)

20 mg/ml hexokinase (Sigma H6380, from yeast overproducing strain)

20 mM ADP (disodium salt)

2 mM ethylenediaminetetraacetic acid (tetrasodium salt, dehydrate)

20 mM magnesium acetate

20 mM D-glucose (monohydrate) and

30 mM creatine phosphate (disodium salt, tetrahydrate).

Hand Dispense and Freeze Drying

0.4μL/well of solution was dispensed onto the sensors as described in WO200356319, using an electronic pipette. The dispensed sensor sheets were then placed into a freeze drier (Severn Science) for freeze drying.

N-acetyl-L-cysteine solution

Solution 3 was prepared containing 200 mM N-acetyl-L-cysteine (NAC) in solution 1.

CK-MM Samples

Recombinant human CK-MM was obtained from Asahi Kasei (HC-CKII, T-74). A concentrated stock solution of CK-MM was made by dissolving 1 mg in 0.1 mL of solution 1. Dilutions of the stock CK-MM solution were made with solution 1 to obtain samples with lower CK-MM activity. The samples were analysed using a Space clinical analyser for CK activity (U/L).

Testing

Solutions were kept on ice until use. For testing, 10.8 uL of CK-MM sample and 1.2 uL of solution 3 were mixed together in an eppendorf and incubated at 37⁰C for 3 minutes. 12 uL of the final mixture was placed on a sensor for testing. A blank sample was also tested using solution 1 in place of CK-MM sample.

Testing Protocol

Testing was performed at 37°C using a heat block. On the addition of sample the chronoamperometry test was initiated using a multiplexer attached to an Autolab (PGSTAT 12). The oxidation current was measured at 0.15 V at 15 time points (0, 14, 28, 42, 56, 70, 84, 98, 112, 126, 140, 154, 168, 182 and 196 seconds) with a reduction current measured at -0.45 V at the final time point (210 seconds). The transient current was measured for 1 second with a data acquisition rate of 200Hz. Each sample was tested with at least one sensor. These data were then transferred to the data analysis template, along with the CK-MM activities of the samples from the Space analyser.

Results

For each sensor, plots of average current vs time were made for each CK-MM sample. Plots of average current vs time are shown in Figure 1 for each sensor A-D.

The rate of response (gradient of current vs time) was determined for each sample using a 42 second time period (84-126 seconds for A, 56-98 seconds for B, 98-140 seconds for C and 0-42 seconds for D). Different time periods are required for the different chemistries due to different dissolution characteristics.

Calibration plots of rate of response vs CK-MM activity (as measured by the Space analyzer) were made. Calibration plots for rate of response are shown in Figure 2 for each sensor A-D. Linear rate of response vs CK-MM activity was obtained for each sensor.

Example 2: Wet and freeze dried CK sensors

Methods

Solution 1 contained 0.1M imidazole buffer (pH 6.7, balanced with acetic acid).

Solution 2 contained:

0.1M imidazole-acetate buffer (pH 6.7)

40 mM cw-[Ru(acac)₂(Py-3-COOH)(Py-3-COO)]

20 mM NAD⁺

5mg/ml diaphorase (Unitika)

5 mg/ml glucose 6-phosphate dehydrogenase (Sorachim G6D-321, from

Microorganism)

20 mg/ml hexokinase (Sorachim HXK-311, from Saccharomyces sp.)

20 mM ADP (disodium salt)

2 mM ethylenediaminetetraacetic acid (tetrasodium salt, dehydrate)

20 mM magnesium acetate

20 mM D-glucose (monohydrate) and

10% w/v lactose.

Solution 3 contained 0.1M imidazole buffer (pH 7.1, balanced with acetic acid).

Testing sensors for CK response

N-acetyl-L-cvsteine solution

Solution 5 was prepared containing 200 mM N-acetyl-L-cysteine (NAC) in solution 1.

Creatine phosphate solutions

Solution 7 was prepared containing 30OmM creatine phosphate in solution 1.

CK-MM Samples

Recombinant human CK-MM was obtained from Asahi Kasei (HC-CKII, T-74). A concentrated stock solution of CK-MM was made by dissolving 10 mg in 1 mL of solution 1. Dilutions of the stock CK-MM solution were made with solution 1 to obtain samples with lower CK-MM activity. The samples were analysed using a Space clinical analyser for CK activity (U/L).

Testing Procedure

Solutions were kept on ice until use. For testing the non-optimized mix, 9.6 uL of solution 2, 1.2 uL of solution 5 and 1.2 uL of CK-MM sample were mixed together in an eppendorf and incubated at 37°C for 3 minutes. 1.2 uL of solution 7 was then added to initiate the reaction and 12 uL of the final mixture was placed on a sensor for testing. A blank sample was also tested using solution 1 in place of CK-MM sample.

The composition of the final enzyme mixture: 0.1 M imidazole acetate buffer (pH 6.7)

29.2 mM c/5-[Ru(acac)₂(Py-3-COOH)(Py-3-COO)] 14.6 mM NAD⁺

3.7 mg/ml diaphorase

3.7 mg/ml glucose 6-phosphate dehydrogenase

14.6 mg/ml hexokinase

14.6 mM ADP (disodium salt)

1.46 mM EDTA

14.6 mM magnesium acetate

14.6 mM D-glucose

7.3 % w/v lactose

27.3 mM creatine phosphate and 18.2 mM NAC.

Variable CK-MM activity.

Dispense and Freeze Drying

Aliquots of solutions 2 and 4 were used to dissolve creatine phosphate to give solution 2 containing 30 mM creatine phosphate. 0.4μL/well of solution was dispensed and freeze dried as described in example 1.

Testing freeze dried sensors

CK-MM samples were prepared as for wet testing. Solution 9 containing 200 mM NAC was prepared in solution 3. Solutions were kept on ice until use. For testing, 10.8 uL of CK-MM sample and 1.2 uL of solution 9 were mixed together in an eppendorf and incubated at 37°C for 3 minutes. 12 uL of the final mixture was then placed on a sensor for testing. A blank sample was also tested using solution 3 in place of CK-MM sample.

Testing Protocol

Testing was performed as described in example 1.

Results

Plots of average current vs time for each CK-MM sample are shown in Figure 3.

For each set of data, the rate of response (gradient of current vs time) was determined for each sample (between the time points 0-42 seconds for wet testing and 56-98 for freeze dried sensors) using the average current values. Calibration plots of rate of response vs CK-MM activity were made and are shown in Figure 4. A linear rate of response vs CK-MM activity was obtained for each experiment.

Example 3; Freeze dried CK sensors using [Ru ^iπ (l,4,7-trimethyl-l,4,7- triazacyclononaneKacac) (l-melm)l (NCM?

Methods

Preparation of solutions for freeze dried sensors

Solution 1 contained 0.1M imidazole buffer (pH 7.1, balanced with acetic acid).

Solution 2 was prepared by dissolving [Ru ^m (1,4,7-trimethyl- 1,4,7- triazacyclononane)(acac) (l-melm)] (NO₃)₂, NAD⁺, diaphorase, glucose 6-phosphate dehydrogenase, hexokinase, ADP, EDTA, magnesium acetate, D-glucose, creatine phosphate and lactose in solution 1.

The final enzyme mixture for dispense contained:

0.1M imidazole-acetate buffer (pH 7.1)

40 mM [Ru¹¹¹ (l,4,7-trimethyl-l,4,7-triazacyclononane)(acac) (l-melm)] (NO₃)₂

20 mM NAD

5 mg/ml diaphorase (Unitika)

5 mg/ml glucose 6-phosphate dehydrogenase (Sorachim G6D-321, from microorganism)

20 mg/ml hexokinase (Sorachim HXK-311, from Saccharomyces sp.)

6.25 mM ADP (disodium salt)

5 mM ethylenediaminetetraacetic acid (tetrasodium salt, dehydrate)

30 mM magnesium acetate

20 mM D-glucose (monohydrate)

100 mM creatine phosphate and

10% w/v lactose.

Dispense and Freeze Drying

0.4μL/well of solution was dispensed and tested as described in example 1.

Results

A plot of average current vs time for each CK-MM sample is shown in Figure 5. The rate of response (gradient of current vs time) was determined for each sample between the time points 14 seconds and 56 seconds using the average current values. A calibration plot of rate of response vs CK-MM activity (as measured by the Space analyzer) was made and is shown in Figure 6. A linear rate of response vs CK-MM activity was obtained.

Example 4: Wet testing CK sensors using [Ru ^In (1,4,7-trimethyl - 1,4,7- triazacyclononane^*)(acac)(4-MeiN-py)l (NOVh

Methods

Preparation of solutions

Solution 1 contained 0.1M imidazole buffer (pH 6.75, balanced with acetic acid). Solution 2 was prepared by dissolving [Ru¹" (1,4,7-trimethyl - 1,4,7- triazacyclononane)(acac)(4-Me₂N-py)] (NO₃)₂, NAD⁺, diaphorase, glucose 6- phosphate dehydrogenase, hexokinase, ADP, EDTA, magnesium acetate and D- glucose in solution 1.

Solution 3 containing 300 mM creatine phosphate (disodium salt, tetrahydrate) was prepared in solution 1.

Solution 4 was prepared containing 200 mM N-acetyl-L-cysteine (NAC) in solution 1.

CK-MM Samples

Recombinant human CK-MM was obtained from Asahi Kasei (HC-CKII, T-74). A concentrated stock solution of CK-MM was made by dissolving 1.1 mg in 0.1 1 mL of solution 1. Dilutions of the stock CK-MM solution were made with solution 1 to obtain samples with lower CK-MM activity.

Testing

Solutions were kept on ice until use.

For testing, 9.6 uL of solution 2, 1.2 uL of solution 4 and 1.2 uL of CK-MM sample were mixed together in an eppendorf and incubated at 37°C for 3 minutes. 1.2 uL of solution 3 was then added to initiate the reaction and 12 uL of the final mixture placed on a sensor for testing. A blank sample was also tested using solution 1 in place of CK-MM sample.

The final enzyme mixture in the test solution contained: 0.1M imidazole-acetate buffer (pH 6.75)

29.1 mM [Ru¹¹¹ (1,4,7-trimethyl - l,4,7-triazacyclononane)(acac)(4-Me₂N-py)] (NO₃)₂

14.5 mM NAD

3.6 mg/ml diaphorase (Unitika)

3.6 mg/ml glucose 6-phosphate dehydrogenase (Sigma G8529, from Leuconostoc mesenteroides, recombinant)

14.5 mg/ml hexokinase (Sigma H6380, from yeast overproducing strain)

14.5 mM ADP (disodium salt)

1.45 mM ethylenediaminetetraacetic acid (tetrasodium salt, dehydrate)

14.5 mM magnesium acetate

14.5 mM D-glucose (monohydrate)

18.2 mM NAC and

27.3 mM creatine phosphate. Variable activity of CK-MM.

The samples were tested as described in example 1.

Results

A plot of average current vs time for each CK-MM sample is shown in Figure 7. The rate of response (gradient of current vs time) was determined for each sample between the time points 0 seconds and 42 seconds using the average current values. A calibration plot of rate of response vs CK-MM activity was made and is shown in Figure 8. A linear rate of response vs CK-MM activity was obtained.

Methods

Solution 1 contained 0.1M imidazole buffer (pH 6.75, balanced with acetic acid).

Solution 2 was prepared containing 40 mM KOH using solution 1.

Solution 3 was prepared by dissolving Ru(NH₃)₆Cl₃, NAD⁺, diaphorase, glucose 6- phosphate dehydrogenase, hexokinase, ADP, EDTA, magnesium acetate and D- glucose in solution 1.

Solution 4 was prepared by dissolving [Ru ^πI (1,4,7-trimethyl- 1,4,7- triazacyclononane)(acac) (l-melm)] (NO₃ )₂, NAD⁺, diaphorase, glucose 6-phosphate dehydrogenase, hexokinase, ADP, EDTA, magnesium acetate and D-glucose in solution 1.

Solution 5 was prepared by dissolving cw-[Ru(acac)₂(Py-3-COOH)(Py-3-COO)], NAD⁺, diaphorase, glucose 6-phosphate dehydrogenase, hexokinase, ADP, EDTA, magnesium acetate and D-glucose in solution 2.

Solution 6 containing 300 mM creatine phosphate (disodium salt, tetrahydrate) was prepared in solution 1.

Solution 7 was prepared containing 200 mM N-acetyl-L-cysteine (NAC) in solution 1.

CK-MM Samples

Recombinant human CK-MM was obtained from Asahi Kasei (HC-CKII, T-74). A concentrated stock solution of CK-MM was made by dissolving 2.6 mg in 0.26 mL of solution 1. Dilutions of the stock CK-MM solution were made with solution 1. The samples were analysed using a Space clinical analyser for CK activity (U/L).

Testing

Sensors used were as described in WO200356319.

For testing, 9.6 uL of solution 3, 4 or 5, 1.2 uL of solution 7 and 1.2 uL of CK-MM sample were mixed together in an eppendorf and incubated at 37°C for 3 minutes. 1.2 uL of solution 6 was then added to initiate the reaction and 12 uL of the final mixture placed on a sensor for testing. A blank sample was also tested using solution 1 in place of CK-MM sample.

The final mixture in the test solution contained:

0.1 M imidazole-acetate buffer (pH 6.75)

29.1 mM Ru(NH₃)₆Cl₃, [Ru¹¹¹ (l,4,7-trimethyl-l,4,7-triazacyclononane)(acac) (1- melm)] (NO₃)₂ or cw-[Ru(acac)₂(Py-3-COOH)(Py-3-COO)] (including 29.1 mM

KOH for cw-[Ru(acac)₂(Py-3-COOH)(Py-3-COO)])

14.5 mM NAD⁺

3.6 mg/ml diaphorase (Unitika) 3.6 mg/ml glucose 6-phosphate dehydrogenase (Sigma G8529, from Leuconostoc mesenteroides, recombinant)

14.5 mg/ml hexokinase (Sigma H6380, from yeast overproducing strain)

14.5 mM ADP (disodium salt)

1.45 mM ethylenediaminetetraacetic acid (tetrasodium salt, dehydrate)

14.5 mM magnesium acetate

14.5 mM D-glucose (monohydrate)

18.2 mM NAC and

27.3 mM creatine phosphate. Variable activity of CK-MM.

Testing Protocol

Testing was performed as described in example 1.

Results

Plots of average current vs time for each CK-MM sample are shown in Figure 9A, 9B and 9C for Ru(NH₃)₆Cl₃, [Ru ^m (l,4,7-trimethyl-l,4,7-triazacyclononane)(acac) (1- melm)] (NO₃)₂ and cw-[Ru(acac)₂(Py-3-COOH)(Py-3-COO)] respectively.

The rate of response (gradient of current vs time) was determined between the time points 0 and 42 seconds for Ru(NH₃)₆Cl₃ and [Ru ^m (1,4,7-trimethyl- 1,4,7- triazacyclononane)(acac) (l-melm)] (NO₃)₂, and 56 and 98 seconds for cis- [Ru(acac)₂(Py-3-COOH)(Py-3-COO)] using the average current value at each time point. For each mediator, a calibration plot of rate of response vs CK-MM activity was made. These are shown in Figure 1OA, 1OB and 1OC for Ru(NH₃)₆Cl₃, [Ru "' (l,4,7-trimethyl-l,4,7-triazacyclononane)(acac) (l-melm)] (NOs)₂ and cis- [Ru(acac)₂(Py-3-COOH)(Py-3-COO)] respectively. A linear rate of response vs CK- MM activity was obtained with each mediator.

Example 6: Response to lactate dehydrogenase using [Ru ⁿⁱ (1,4,7-trimethyl- l,4,7-triazacyclononane)(acac) (l-melm)i (NOVh

Methods

Preparation of solutions

Solution 1 contained 0.1M Tris buffer (pH 9.1).

Solution 2 was prepared by dissolving [Ru ^m (1,4,7-trimethyl- 1,4,7- triazacyclononane)(acac) (l-melm)] (NO₃)₂, NAD⁺, diaphorase and sodium L-lactate in solution 1.

LDH Samples

Lactate dehydrogenase was obtained from Sigma (product code 61309, from Rabbit muscle, 142 U/mg). A concentrated stock solution of LDH was made by dissolving 0.5 mg in 0.1 mL of solution 1. Dilutions of the stock LDH solution were made with solution 1 to obtain samples with lower LDH concentration.

Testing

Solutions were kept on ice until use. For testing, 12 uL of solution 2 and 1.2 uL of LDH sample were mixed together in an eppendorf and 12 uL of the final mixture placed on a sensor for testing within 10 seconds of mixing. A blank sample was also tested using solution 1 in place of LDH sample.

The final enzyme mixture in the test solution contained:

4.5 mg/ml diaphorase (Unitika) and

545 mM sodium L-lactate.

Variable concentration of LDH.

Testing Protocol

Testing was performed at room temperature. Testing was carried out using the procedure described in example 1.

The data were then transferred to the data analysis template, along with the LDH concentrations of the samples.

Results

A plot of average current vs time for each LDH sample is shown in Figure 1 1. The rate of response (gradient of current vs time) was determined for each sample between the time points 0 seconds and 42 seconds using the average current values. A calibration plot of rate of response vs LDH concentration was made and is shown in Figure 12. A linear rate of response vs LDH concentration was obtained.

Example 7; Testing response to Mg²⁺ ions using hexokinase and glucose 6- phosphate dehydrogenase

Methods

Preparation of solutions

Solution 1 contained 0.1M imidazole buffer (pH 7.1, balanced with acetic acid) and

40 mM KOH.

Solution 2 was prepared by dissolving [Ru(acac)₂(Py-3-COOH)(Py-3-COO)], NAD+, diaphorase, glucose 6-phosphate dehydrogenase, hexokinase, ATP (di-sodium salt) and EDTA (tetra sodium salt dehydrate) in solution 1.

Solution 3 was prepared by dissolving D-glucose in solution 1.

Mg acetate Samples

A concentrated IM stock solution of Mg acetate tetrahydrate was made by dissolving 0.0169 g in 0.0788 mL of solution 1. Dilutions of the stock Mg acetate solution were made with solution 1 to obtain samples with lower Mg acetate concentration.

Testing

Solutions were kept on ice until use. For testing, 9.6 uL of solution 2 and 1.2 uL of MgAcetate sample were mixed together in an eppendorf and incubated at 37⁰C for 3 minutes. 1.2 uL of solution 3 was then added to initiate the reaction and 12 uL of the final mixture placed on a sensor for testing. A blank sample was also tested using solution 1 in place of Mg acetate sample.

The final enzyme mixture in the test solution contained:

0.1M imidazole buffer (pH 7.1, balanced with acetic acid)

32 rnM cis-[Ru(acac)2(Py-3-COOH)(Py-3-COO)]

32 mM KOH

16 mM NAD+

4 mM EDTA (tetra sodium salt, dehydrate)

16 mM ATP (di-sodium salt)

20 mM D-glucose

4 mg/ml diaphorase (Unitika)

4 mg/mL glucose 6-phosphate dehydrogenase (Sigma G8529, recombinant, from

Leuconostoc mesenteroides) and

8 mg/mL hexokinase (Sigma H6380, from Saccharomyces cerevisiae).

Variable concentration of Mg acetate.

Testing Protocol

Testing was performed at 37°C using a heat block. On the addition of sample the chronoamperometry test was initiated using a multiplexer (MX452, Sternhagen design) attached to an Autolab (PGSTAT 12). The oxidation current was measured at 0.15 V at 15 time points (0, 14, 28, 42, 56, 70, 84, 98, 112, 126, 140, 154, 168, 182 and 196 seconds) with a reduction current measured at -0.45 V at the final time point (210 seconds). The transient current was measured for 1 second with a data acquisition rate of 200Hz. Each sample was tested with at least one sensor.

Analysis

The data were then transferred to the data analysis template, along with the Mg²⁺ concentrations of the samples.

Results

A plot of average current vs time for each Mg acetate sample is shown in Figure 13. The rate of response (gradient of current vs time) was determined for each sample between the time points 0 seconds and 28 seconds using the average current values. A calibration plot of rate of response vs Mg acetate concentration was made and is shown in Figure 14. A linear rate of response vs Mg acetate concentration was obtained.

Example 8: Freeze dried testing response to lactate dehydrogenase using fRu^1II(l,4,7-trimethyl-l,4,7-triazacyclononane)(acae)(l-meIm)l (NOi)₂

Methods

Solution 1 contained 0.1M Tris buffer (pH 9.1).

Solution 2 was prepared by dissolving [Ru^iπ(l,4,7-trimethyl- 1,4,7- triazacyclononane)(acac)(l-melm)] (NO3)₂, NAD⁺, diaphorase, sodium L-lactate and lactose in solution 1. The final enzyme mixture in solution 2 contained: (NO₃)₂

5.0mg/ml diaphorase (Unitika)

60OmM sodium L-lactate and

10% w/v lactose.

Solution 2 was dispensed onto sensors as described in WO200356319. These sensors were freeze dried overnight.

LDH Samples

Lactate dehydrogenase was obtained from Sigma (product code 61309, from Rabbit muscle, 142 U/mg). A concentrated stock solution of LDH was made by dissolving 0.45 mg in 1 mL of solution 1. Dilutions of the stock LDH solution were made with solution 1 to obtain samples with lower LDH concentration 0.045, 0.090, 0.180, 0.270 and 0.360 mg/mL). Solutions of LDH were kept on ice until use.

Testing

For testing, 12 μL of each LDH sample was placed on each sensor. A blank sample was also tested using 12μL solution 1 in place of LDH sample. Testing was performed at room temperature.

Testing Protocol

On the addition of sample the chronoamperometry test was initiated. The oxidation current was measured at 0.15 V at 21 time points (8, 22, 36, 50, 64, 78, 92, 106, 120, 134, 148, 162, 176, 190, 204, 218 , 232, 246, 260, 274 and 288 seconds) with a reduction current measured at -0.45 V at the final time point (302 seconds). The transient current was measured for 1 second. Each sample was tested with at least four sensors each having four wells.

Results

A plot of average current vs time for each LDH sample is shown in Figure 15. The rate of response (gradient of current vs time) was determined for each sample between the time points 0 seconds and 42 seconds using the average current values. A calibration plot of rate of response vs LDH concentration is shown in Figure 16. A linear rate of response vs LDH concentration was obtained.

Conclusion

A freeze dried rate assay for LDH has been demonstrated using [Ru^m(l,4,7-trimethyl- 1 ,4,7-triazacyclononane)(acac)(l -melm)] (NO₃)_2.

Example 9: Freeze Dried testing CK sensors using a range of Mediators

Methods

Solution 1 contained 0.1M imidazole buffer (pH 7.1, balanced with acetic acid). Solution 2 was prepared by dissolving NAD⁺, diaphorase, glucose 6-phosphate dehydrogenase, hexokinase, ADP, EDTA, magnesium acetate, creatine phosphate, D- glucose and lactose in solution 1.

Solutions 3-15 were prepared by dissolving a sample of each of the thirteen mediators in solution 2.

Solution 16 containing 10 mM NADH was prepared in solution 1.

Solutions 17-18 containing 5 and 2.5mM NADH were prepared by dissolving solution 16 in solution 1.

The final mixture in the solutions (3-15) freeze-dried on the sensors contained:

0.1 M imidazole-acetate buffer (pH 7.1)

40 mM of the Mediator (including 40 mM KOH for Mediator 2)

4O mM NAD⁺

10 mg/ml diaphorase

10 mg/ml glucose 6-phosphate dehydrogenase

40 mg/ml hexokinase (Sigma H6380, from yeast overproducing strain)

12.5 mM ADP (disodium salt)

10 mM ethylenediaminetetraacetic acid (tetrasodium salt, dehydrate)

40 mM magnesium acetate

100 mM creatine phosphate

40 mM D-glucose (monohydrate) and

10% w/v lactose.

Testing

Sensors were prepared as described in WO200356319. Solutions were kept on ice until use. For testing, 12 μL of each NADH sample was placed on each sensor. A blank sample was also tested using 12μL solution 1 in place of NADH sample. Testing was performed at 37⁰C.

Testing Protocol

On the addition of sample the chronoamperometry test was initiated. The chronoamperometry test was carried out as described in example 8. Data were then transferred to the data analysis template, along with the NADH concentrations of the samples.

Results

Plots of average current vs time for each NADH concentration are shown in Figure 17A to 17L for Mediator 1, Mediator 2, Mediator 3, Mediator 4, Mediator 6, Mediator 8, Mediator 11, Ru(NH₃)₆Cl₃, Mediator 5, Mediator 7, Mediator 9 and Mediator 10 respectively.

The rate of response (gradient of current vs time) was determined between the time points 176 and 218 seconds for each mediator using the average current value at each time point. For each mediator, a calibration plot of rate of response vs NADH concentration was made and these are shown in figures 18 A to 18L for Mediator 1 , Mediator 2, Mediator 3, Mediator 4, Mediator 6, Mediator 8, Mediator 11, Ru(NH₃)₆Cl₃, Mediator 5, Mediator 7, Mediator 9 and Mediator 10 respectively. Conclusions

The linear relationship between rate of response and NADH concentration for the CK assay for a range of different mediators using freeze-dried sensors was demonstrated.

Mediator Stability with Respect to the CK Assay

A mediator was considered to be stable with respect to the CK assay if the rate of change of the average current per second for a sensor tested with 5.OmM NADH was greater than -0.5nA/sec and less than 0.5nA/sec between the time points 100 and 300 seconds. A mediator was considered to be unstable with respect to the CK assay if the rate of average current change per second for a sensor tested with 5.OmM NADH was less than -0.5nA/sec or greater than 0.5nA/sec between the time points 100 and 300 seconds.

Figure 19 shows the variation in rate for the stable mediators [Mediator 2, Mediator 4, Mediator 6, Mediator 7, Mediator 8, Mediator 10 and Mediator H]. Figure 20 shows the variation in rate for the unstable mediators [Mediator 1, Mediator 3, Mediator 5, Mediator 9 and ruthenium hexamine trichloride].

Stable Mediators Unstable Mediators

Ruthenium Osmium Ruthenium Osmium

Mediators Mediators Mediators Mediators

Mediator 2 Mediator 7 Mediator 1 Mediator 5

Mediator 4 Mediator 10 Mediator 3 Mediator 9

Mediator 6 ruthenium hexamine trichloride

Mediator 8

Mediator 11

Example 10: Freeze dried testing CK sensors using a range of excipients

Methods

Preparation of solutions for freeze dried sensors

Solution 1 contained 0.1M imidazole buffer (pH 7.1, balanced with acetic acid).

Solutions 2-4 were prepared by dissolving Mediator 6, NAD⁺, diaphorase, glucose 6- phosphate dehydrogenase, hexokinase, ADP, EDTA, magnesium acetate, D-glucose and creatine phosphate and 7.5% w/v KCl, KNO₃ and NaNO₃ respectively in solution 1.

Solutions 5 and 6 were prepared by dissolving Mediator 6, NAD⁺, diaphorase, glucose 6-phosphate dehydrogenase, hexokinase, ADP, EDTA, magnesium acetate, D-glucose and creatine phosphate and 2.0% w/v BSA and Ovalbumin respectively in solution 1. Solutions 7-10 were prepared by dissolving Mediator 6, NAD⁺, diaphorase, glucose 6- phosphate dehydrogenase, hexokinase, ADP, EDTA, magnesium acetate, D-glucose and creatine phosphate and 10% w/v lactose, lactitol, sucrose and trehalose respectively in solution 1.

Solution 11 contained 200 mM N-acetyl-L-cysteine (NAC) dissolved in solution 1.

The final enzyme mixes 2-10 for dispense contained:

0.1 M imidazole-acetate buffer (pH 7.1)

40 mM Mediator 6

2O mM NAD

5 mg/ml diaphorase

5 mg/ml glucose 6-phosphate dehydrogenase (Sorachim)

10 mg/ml hexokinase (Sorachim)

6.25 mM ADP (disodium salt)

5 mM ethylenediaminetetraacetic acid (tetrasodium salt dehydrate)

30 mM magnesium acetate

20 mM D-glucose (monohydrate)

100 mM creatine phosphate with 10% w/v lactose, lactitol, sucrose and trehalose

2.0% w/v BSA and Ovalbumin and

7.5% w/v KCl, KNO₃ and NaNO₃ respectively.

Recombinant human CK-MM was obtained from Asahi Kasei (HC-CKII, T-74). A concentrated stock solution of CK-MM was made by dissolving 10 mg in ImL of Solution 1. Dilutions of the stock CK-MM solution were made with solution 1 to obtain samples with lower CK-MM activity.

Testing Protocol

Sensors were prepared as described in WO200356319. Solutions were kept on ice until use. For testing, 54μL of each concentration of CK was mixed with 6μL of 200 mM N-acetyl-L-cysteine (NAC) and 12 μL was placed on each sensor. A blank sample was also tested using 12μL solution 1 in place of CK. Testing was performed at 37⁰C.

On the addition of sample the chronoamperometry test was initiated. The chronoamperometry test was carried out as described in example 8.

Results

Figures 21 A to 211 show plots of average current vs time for each CK-MM concentration for sensors prepared with KCl, KNO₃, NaNO₃, BSA , Ovalbumin, lactose, lactitol, sucrose and trehalose respectively.

The rates of response (gradient of current vs time) determined between the time points 22 and 64 seconds for each CK-MM concentration for each excipient are shown in Figure 22A to 221, using the average current value at each time point. Calibration plots of rate of response vs CK-MM concentration are shown in Figures 22A to 221 for each excipient.

Conclusions The salts KCl, KNO₃ and NaNO₃, the sugars lactose, lactitol, sucrose and trehalose and the proteins BSA and Ovalbumin are suitable excipients for the CK assay.

Example 11: Freeze Dried testing CK sensors using a range of CK samples

Methods

Preparation of solutions for freeze dried sensors

Solution 1 contained 0.1M imidazole buffer (pH 7.1, balanced with acetic acid).

Solution 2 was prepared by dissolving Mediator 2, NAD⁺, diaphorase, glucose 6- phosphate dehydrogenase, hexokinase, ADP, EDTA, magnesium acetate, D-glucose, creatine phosphate and lactose in solution 1.

Solution 3 contained 200 mM N-acetyl-L-cysteine (NAC) dissolved in solution 1. Solution 4 contained 200 mM N-acetyl-L-cysteine (NAC) dissolved in plasma.

The final enzyme mixture for dispense contained:

0.1M imidazole-acetate buffer (pH 7.1)

40 mM Mediator 2

2O mM NAD

5 mg/ml diaphorase

5 mg/ml glucose 6-phosphate dehydrogenase (Sorachim)

10 mg/ml hexokinase (Sorachim)

6.25 mM ADP (disodium salt)

5 mM ethylenediaminetetraacetic acid (tetrasodium salt, dehydrate)

30 mM magnesium acetate

20 mM D-glucose (monohydrate)

10% w/v lactose and

100 mM creatine phosphate.

CK-MM & CK-MB Samples

Recombinant human CK-MM was obtained from Asahi Kasei (HC-CKII, T- 74). A concentrated stock solution of CK-MM was made by dissolving 10 mg in ImL of solution 1. Dilutions of the stock CK-MM solution were made with solution 1 to obtain samples with lower CK-MM activity. A second stock solution of CK-MM was made up in plasma and similarly diluted with plasma.

Recombinant human CK-MB was obtained from Sigma. A concentrated stock solution of CK-MB was made by dissolving 10 mg in ImL of solution 1. Dilutions of the stock CK-MB solution were made with solution 1 to obtain samples with lower CK-MB activity. A second stock solution of CK-MB was made up in plasma and similarly diluted with plasma. The samples were analysed using a Konelab analyser for CK activity (WL).

Testing Protocol

Sensors were prepared as described in WO200356319. Solutions were kept on ice until use. For testing, 54μL of each concentration of each CK sample was mixed with 6μL of 200 mM N-acetyl-L-cysteine (NAC) in the correct diluents (solutions 3 and 4 respectively) and 12μL was placed on each sensor. A blank sample was also tested using 54μL solution 1 or plasma in place of CK sample. Testing was performed at 37⁰C.

On the addition of sample the chronoamperometry test was initiated. The chronoamperometry test was carried out as described in example 8. Data were then transferred to the data analysis template, along with the concentrations of the samples of CK-MM and CK-MB.

Results

Plots of average current vs time for each CK concentration for each CK sample (MM or MB) are shown in Figures 23A to 23D for CK-MM in buffer and plasma and CK- MB in buffer and plasma respectively.

The rate of response (gradient of current vs time) was determined between the time points 22 and 64 seconds for each CK sample using the average current value at each time point. For each CK sample, a calibration plot of rate of response vs concentration was made. These are shown in Figures 24A to 24D for CK-MM in buffer and plasma and CK-MB in buffer and plasma respectively.

Conclusions

The suitability of the assay for both CK-MM and CK-MB sample types was demonstrated.

Example 12: Response to cholesterol dehydrogenase (ChDH) using Mediator 2

Methods

Preparation of solutions

Solution 1 contained 0.1M Tris buffer (pH 9.0).

Solution 2 was prepared by dissolving triton X-IOO, cholesterol powder and KOH in solution 1.

Solution 3 was prepared by dissolving lactose, Mediator 2, NaCl, TNAD, putidaredoxin reductase and lipase in solution 2. Lipase does not take part in the measurement sequence. It was included in the reaction mixture since it was desired to measure the activity of ChDH in the full enzyme mixture used for the total cholesterol assay.

ChDH samples

ChDH was obtained from Amano (product code CHDH-6, from Nocardia sp.). A concentrated stock solution of 50 mg/mL ChDH was made by dissolving 0.21 g of ChDH in 4.22 mL of solution 1. Dilutions of the stock ChDH solution were made with solution 1 to obtain samples with lower ChDH concentration.

Testing

Sensors were prepared as bottom fill sensors with X-type flow cells. Solutions were kept on ice until use.

For testing, 10 uL of solution 3 was pipetted into an eppendorf and 1.0 uL of ChDH solution added. A 10 uL aliquot of the final mixture was placed on a sensor for testing within 5 seconds of mixing. A blank sample was also tested using solution 1 in place of ChDH sample.

The final enzyme mixture in the test solution contained: O.lM Tris buffer (pH 9.0)

9.1 % w/v Triton X-100 9.5 mM cholesterol 36.4 mM KOH

9.09 % w/v lactose

36.4 mM Mediator 2

454 mM NaCl

7.9 mM TNAD

3.8 mg/mL putidaredoxin reductase (Biocatalyst) and

3.2 mg/mL lipase (Chromobacterium viscosum, Genzyme LIPA-70-1461). Variable concentration of ChDH.

Testing Protocol

Testing was performed at 22°C. On the addition of sample the chronoamperometry test was initiated. The oxidation current was measured at 0.15 V at 13 time points (8, 42, 76, 110, 144, 178, 212, 246, 280, 314, 348, 382 and 416 seconds), with a reduction current measured at -0.45 V at the final time point (450 seconds). The transient current was measured for 4 second, with a data acquisition rate of 200Hz. Each sample was tested with at least one sensor.

These data were then transferred to the data analysis template, along with the ChDH concentrations of the samples.

Results

A plot of average current vs time for each ChDH sample is shown in Figure 25.

The rate of response (gradient of current vs time) was determined for each sample between the time points 0 seconds and 76 seconds using the average current values. A calibration plot of rate of response vs ChDH concentration was made and is shown in Figure 26. A linear rate of response vs ChDH concentration was obtained.

Conclusion

A rate assay for ChDH has been demonstrated using Mediator 2.

Example 13: Temperature dependence of CK sensors using Ru(NH VtoCh, Mediator 2 or Mediator 6

Methods

Solution 2 was prepared containing 40 mM KOH using solution 1.

Solution 3 was prepared by dissolving Ru(NH₃)₆Cl₃, NAD⁺, diaphorase, glucose 6- phosphate dehydrogenase, hexokinase, ADP, EDTA, magnesium acetate and D- glucose in solution 1. Solution 4 was prepared by dissolving Mediator 6, NAD⁺, diaphorase, glucose 6- phosphate dehydrogenase, hexokinase, ADP, EDTA, magnesium acetate and D- glucose in solution 1.

Solution 5 was prepared by dissolving Mediator 2, NAD⁺, diaphorase, glucose 6- phosphate dehydrogenase, hexokinase, ADP, EDTA, magnesium acetate and D- glucose in solution 2.

CK-MM Samples

Recombinant human CK-MM was obtained from Asahi Kasei (HC-CKII, T- 74). A concentrated stock solution of CK-MM was made by dissolving 2.6 mg in 0.26 mL of solution 1. Dilutions of the stock CK-MM solution were made with solution 1 to obtain samples with lower CK-MM activity. The samples were analysed using a Space clinical analyser for CK activity (U/L).

Testing

Sensors were prepared as described in WO200356319. Solutions were kept on ice until use. For testing, 9.6 uL of solution 3, 4 or 5, 1.2 uL of solution 7 and 1.2 uL of CK-MM sample were mixed together in an eppendorf and incubated at the testing temperature (23, 30 or 37°C) for 3 minutes. 1.2 uL of solution 6 was then added to initiate the reaction and 12 uL of the final mixture placed on a sensor for testing. A blank sample was also tested using solution 1 in place of CK-MM sample.

The final mixture in the test solution contained: 0.1 M imidazole-acetate buffer (pH 6.75)

29.1 mM Ru(NH₃)₆Cl₃, Mediator 6 or Mediator 2 (plus 29.1 mM KOH for Mediator

2)

14.5 mM NAD⁺

3.6 mg/ml diaphorase (Unitika)

14.5 mg/ml hexokinase (Sigma H6380, from yeast overproducing strain)

14.5 mM ADP (disodium salt)

1.45 mM ethylenediaminetetraacetic acid (tetrasodium salt, dehydrate)

14.5 mM magnesium acetate

14.5 mM D-glucose (monohydrate)

18.2 mM NAC and

27.3 mM creatine phosphate. Variable activity of CK-MM.

Testing Protocol

Testing was performed at 23, 30 or 37⁰C using a heat block. On the addition of sample the chronoamperometry test was initiated. The oxidation current is measured at 0.15 V at 15 time points (0, 14, 28, 42, 56, 70, 84, 98, 112, 126, 140, 154, 168, 182 and 196 seconds), with a reduction current measured at -0.45 V at the final time point (210 seconds). The transient current was measured for 1 second with a data acquisition rate of 200Hz. Each sample was tested with at least one sensor. Data were then transferred to the data analysis template, along with the CK-MM activities of the samples from the Space analyser.

Results

Plots of average current vs time for each CK-MM sample are shown in Figures 27A- C, 28A-C and 29A-C for Ru(NH₃)₆Cl₃, Mediator 6 and Mediator 2 respectively at a measurement temperature of 23, 30 or 37°C.

The rate of response (gradient of current vs time) was determined between the time points 0 and 42 seconds for Ru(NH₃)₆Cl₃ and Mediator 6 and between 56 and 98 seconds for Mediator 2 using the average current value at each time point. For each mediator, a calibration plot of rate of response vs CK-MM activity (as measured by the Space analyzer) was made. These are shown in Figure 3OA, 30B and 30C for Ru(NH₃ )_όCl₃, Mediator 6 and Mediator 2 respectively (open circle/ solid line represents 23 °C, closed circle/dashed line represents 30⁰C and open square/dot-dash line represents 37⁰C). A linear rate of response vs CK-MM activity was obtained with each mediator (see table 2 below).

Conclusions

The temperature dependence of the rate of response to CK-MM has been demonstrated using Ru(NH₃)₆Cl₃, Mediator 6 or Mediator 2.

Table 2

Example 14: Response to diaphorase using Mediator 6

Methods

Preparation of solutions

Solution 1 contained 0.1M imidazole buffer (pH 6.7 at 37°C).

Solution 2 was prepared by dissolving Mediator 6 in solution 1. Solution 3 was prepared by dissolving NADH in solution 1.

Diaphorase samples

Diaphorase was obtained from Unitika (product code BlDl I l, from Bacillus stearothermophilus). A concentrated stock solution of 55 mg/mL diaphorase was made by dissolving 3.6 mg in 0.0651 niL of solution 1. Dilutions of the stock diaphorase solution were made with solution 1 to obtain samples with lower diaphorase concentration.

Testing

Sensors were prepared as described in WO200356319. Solutions were kept on ice until use.

For testing, 9 uL of solution 2 and 1.0 uL of solution 3 were mixed together in an eppendorf and 1.0 uL of diaphorase solution added. A 9 uL aliquot of the final mixture was placed on a sensor for testing within 10 seconds of mixing. A blank sample was also tested using solution 1 in place of diaphorase sample.

The final enzyme mixture in the test solution contained:

0.1M imidazole buffer (pH 6.7)

32.7 mM Mediator 6 and

10.O mM NADH.

Variable concentration of diaphorase.

Testing Protocol

Testing was performed at 37°C. On the addition of sample the chronoamperometry test was initiated. The chronoamperometry test was carried out as described in example 13. These data were then transferred to the data analysis template, along with the diaphorase concentrations of the samples.

Results

A plot of average current vs time for each diaphorase sample is shown in Figure 31. The rate of response (gradient of current vs time) was determined for each sample between the time points 0 seconds and 56 seconds using the average current values. A calibration plot of rate of response vs diaphorase concentration was made and is shown in Figure 32. A linear rate of response vs diaphorase concentration was obtained.

Conclusion

A rate assay for diaphorase has been demonstrated using Mediator 6.

Example 15: Response to glucose 6-phosphate dehydrogenase (G6PDH) using Mediator 6

Methods

Preparation of solutions

Solution 1 contained 0.1M imidazole buffer (pH 6.7 at 37°C).

Solution 2 was prepared by dissolving magnesium acetate, Mediator 6, diaphorase and NAD in solution 1.

Solution 3 was prepared by dissolving glucose 6-phosphate (sodium salt) in solution 1.

G6PDH samples G6PDH was obtained from Sorachim (product code G6D-321, from microorganism). A concentrated stock solution of 55 mg/mL G6PDH was made by dissolving 5.0 mg in 0.0909 mL of 20 mM magnesium acetate solution in solution 1. Dilutions of the stock G6PDH solution were made with solution 1 to obtain samples with lower G6PDH concentration.

Testing

For testing, 9 uL of solution 2 and 1.0 uL of solution 3 were mixed together in an eppendorf and 1.0 uL of G6PDH solution added. A 9 uL aliquot of the final mixture was placed on a sensor for testing within 10 seconds of mixing. A blank sample was also tested using solution 1 in place of G6PDH sample.

The final enzyme mixture in the test solution contained:

0.1 M imidazole buffer (pH 6.7)

16.4 mM magnesium acetate

32.7 mM Mediator 6

8.2 mM NAD

4.1 mg/mL diaphorase (Bacillus Stearothermophilus, Unitika BlDl 11) and

10 mM glucose 6-phosphate sodium salt.

Variable concentration of G6PDH

Testing Protocol

Testing was performed at 37⁰C. On the addition of sample the chronoamperometry test was initiated. The chronoamperometry test was carried out as described in example 13. These data were then transferred to the data analysis template, along with the G6PDH concentrations of the samples.

Results

A plot of average current vs time for each G6PDH sample is shown in Figure 33. The rate of response (gradient of current vs time) was determined for each sample between the time points 0 seconds and 56 seconds using the average current values. A calibration plot of rate of response vs G6PDH concentration was made and is shown in Figure 34. A linear rate of response vs G6PDH concentration was obtained.

Conclusion

A rate assay for G6PDH has been demonstrated using Mediator 6.

Example 16: Wet testing response to glycerol dehydrogenase (GDH) using Mediator 2

Methods

Preparation of solutions

Solution 1 contained 0.1M HEPBS buffer (pH 9.0).

Solution 2 was prepared by dissolving lactose, Mediator 2 and KOH in solution 1. Solution 3 was prepared by dissolving TNAD (potassium salt), Anameg-7, diaphorase and lipase in solution 2. Lipase does not take part in the measurement sequence. It was included in the reaction mixture since it was desired to measure the activity of GDH in the full enzyme mixture used for the triglyceride assay.

Solution 4 was prepared by dissolving glycerol in solution 2.

GDH samples

GDH was obtained from Sorachim (product code GYD-301, from Cellulomonas sp.).

Concentrated stock solutions of 5 or 50 mg/mL GDH were made by dissolving 4.7 mg of GDH in 940 uL or 6.4 mg of GDH in 128 uL of solution 1. Dilutions of the stock

GDH solution were made with solution 1 to obtain samples with lower GDH concentration.

Testing

For testing, 18 uL of solution 3 and 2.0 uL of solution 4 were pipetted into an eppendorf and 2.0 uL of GDH sample was added. An 18 uL aliquot of the final mixture was placed on a sensor for testing within 10 seconds of mixing. A blank sample was also tested using solution 1 in place of GDH sample.

The final enzyme mixture in the test solution contained: 0.1M HEPBS buffer (pH 9.0) 27.3 mM KOH

27.3 mM Mediator 2 9.1 % w/v lactose 0.82 % w/v Anameg-7

14.4 mM TNAD (potassium salt)

5.6 mg/mL diaphorase (Stearothermophilus sp., Unitika)

40.9 mg/mL lipase (Chromobacterium viscosum, Genzyme LIPA-70-1461) and

45.5 mM glycerol.

Variable concentration of GDH.

Testing Protocol

Testing was performed at 22°C. On the addition of sample the chronoamperometry test was initiated. The oxidation current was measured at 0.15 V at 13 time points (8, 42, 76, 110, 144, 178, 212, 246, 280, 314, 348, 382 and 416 seconds), with a reduction current measured at -0.45 V at the final time point (450 seconds). The transient current was measured for 4 second with a data acquisition rate of 200Hz. Each sample was tested with at least one sensor. Data were then transferred to the data analysis template, along with the GDH concentrations of the samples.

Results

A plot of average current vs time for each GDH sample is shown in Figure 35. The rate of response (gradient of current vs time) was determined for each sample between the time points 0 seconds and 110 seconds using the average current values. A calibration plot of rate of response vs GDH concentration was made and is shown in Figure 36. A linear rate of response vs GDH concentration was obtained. Conclusion

A rate assay for glycerol dehydrogenase has been demonstrated using Mediator 2.

Example 17; Response to hexokinase (HK) using Mediator 6

Methods

Preparation of solutions

Solution 1 contained 0.1M imidazole buffer (pH 6.7 at 37⁰C).

Solution 2 was prepared by dissolving magnesium acetate, Mediator 6, diaphorase, NAD, glucose 6-phosphate dehydrogenase (G6PDH) and glucose in solution 1.

Solution 3 was prepared by dissolving ATP (disodium salt) in solution 1.

HK samples

HK was obtained from Sorachim (product code HXK-311, from Saccharomyces sp.). A concentrated stock solution of 55 mg/mL HK was made by dissolving 1.6 mg in 29.IuL of 20 mM magnesium acetate solution in solution 1. Dilutions of the stock HK solution were made with solution 1 to obtain samples with lower HK concentration.

Testing

Sensors were prepared as described in WO200356319. Solutions were kept on ice until use. For testing, 9 uL of solution 2 and 1.0 uL of solution 3 were mixed together in an eppendorf and 1.0 uL of HK solution added. A 9 uL aliquot of the final mixture was placed on a sensor for testing within 10 seconds of mixing. A blank sample was also tested using solution 1 in place of HK sample.

The final enzyme mixture in the test solution contained:

0.1M imidazole buffer (pH 6.7)

16.4 mM magnesium acetate

32.7 mM Mediator 6

8.2 mM NAD

4.1 mg/mL diaphorase (Bacillus Stearothermophilus, Unitika BlDl 11)

4.1 mg/mL G6PDH (from microorganism, Sorachim G6D-321),

8.2 mM glucose and

1.8 mM ATP (disodium salt). Variable concentration of HK.

Testing Protocol

Testing was performed at 37°C. On the addition of sample the chronoamperometry test was initiated. The oxidation current was measured at 0.15 V at 15 time points (0, 14, 28, 42, 56, 70, 84, 98, 112, 126, 140, 154, 168, 182 and 196 seconds) with a reduction current measured at -0.45 V at the final time point (210 seconds). The transient current was measured for 1 second with a data acquisition rate of 200Hz. Each sample was tested with at least one sensor.

These data were then transferred to the data analysis template, along with the HK concentrations of the samples. Results

A plot of average current vs time for each HK sample is shown in Figure 37.

The rate of response (gradient of current vs time) was determined for each sample between the time points 0 seconds and 56 seconds using the average current values. A calibration plot of rate of response vs HK concentration is shown in Figure 38. A linear rate of response vs HK concentration was obtained.

Conclusion

A rate assay for hexokinase has been demonstrated using Mediator 6.

Example 18: CK Sensor using Mediator 6 - Macroelectrode

Methods

Solution 1 contained 0.1M imidazole buffer (pH 7.1, balanced with acetic acid).

Solution 2 was prepared by dissolving NAD⁺, diaphorase, glucose 6-phosphate dehydrogenase, hexokinase, lactose, ATP, creatine phosphate, magnesium acetate, EDTA and Mediator 6 in solution 1.

Solution 2 was freeze-dried on prefabricated laminated wells of 80μL volume. The laminated structures were manufactured from 250 micron white PET with a PSA applied to one side. The acrylic adhesive thickness was approximately 50 microns. The base of the structure was manufactured from 125 micron clear PET treated with a print receptive coating. All material cutting was performed using a computer numerically controlled CO₂ laser.

The final mixture in solution 2 freeze-dried in the wells contained:

0.1 M imidazole-acetate buffer (pH 7.1)

4OmM of Mediator 6

2OmM NAD⁺

5 mg/ml diaphorase

5 mg/ml glucose 6-phosphate dehydrogenase

20 mg/ml hexokinase

6.25 mg/ml ATP

3OmM magnesium acetate

10OmM creatine phosphate

5 mM ethylenediaminetetraacetic acid (tetrasodium salt, dehydrate) and

10%w/v lactose.

Solutions 3 and 4 were prepared by dissolving CK in solution 1 at concentrations of 5.5 and l lmg/mL (6.07U/mg) respectively.

Solution 5 was prepared by dissolving N-acetyl-L-cysteine (NAC) in solution 1 at a concentration of 20OmM.

Testing

Solutions were kept on ice until use. For testing, 90μL of each concentration of each CK sample was mixed with lOμL of 20OmM N-acetyl-L-cysteine (NAC) and 90μL was placed in each well for testing. A blank sample was also tested using 90 μL solution 1 or plasma in place of CK sample. Testing was performed at 37⁰C.

The final mixture in solutions 6-8 pippetted on the sensors contained: 0.1M imidazole-acetate buffer (pH 7.1) 2OmM N-acetyl-L-cysteine (NAC) 0, 5 and 10mg/mL CK respectively.

Testing Protocol

On the addition of sample the chronoamperometry test was initiated. The oxidation current was measured at 0.15 V at 21 time points (8, 22, 36, 50, 64, 78, 92, 106, 120, 134, 148, 162, 176, 190, 204, 218 , 232, 246, 260, 274 and 288 seconds) with a reduction current measured at -0.45 V at the final time point (302 seconds). The transient current was measured for 8 seconds. Each sample was tested once.

Results

Plots of average current vs time for each CK concentration is shown in Figure 39.

Conclusions

A CK macroelectrode sensor has been demonstrated.

Example 19: Freeze-Dried testing response to L-aspartate aminotransferase using Mediator 2

Methods

Preparation of solutions

Solution 1 contained 0.1M Tris buffer (pH 7.2).

Solution 2 was prepared by dissolving Mediator 2, potassium hydroxide, NAD⁺, putidaredoxin reductase, glutamate dehydrogenase, α-ketoglutaric acid, L-aspartic acid and lactose in solution 1.

The final enzyme mixture in solution 2 contained:

0.1M Tris (pH 7.2)

4OmM Mediator 2

4OmM KOH

2OmMNAD⁺

10mg/ml putidaredoxin reductase

40mg/ml glutamate dehydrogenase

12mM α-ketoglutaric acid

25OmM L-aspartic acid and

10% w/v lactose.

Solution 2 was dispensed onto sensors as described in WO200356319 and freeze dried overnight. They were tested the following morning.

AST Samples

L-aspartate aminotransferase (AST) was obtained from Asahi Kasei Pharma

Corporation (HC-AST II, 5.11U/mg). A concentrated stock solution of 100kU/mL of AST was prepared using solution 1. Dilutions of the stock ALT solution were made with solution 1 to obtain samples with lower AST concentrations of 25, 50 and 75kU/mL). Solutions of AST were kept on ice until use. A stock solution of 0.1 ImM pyridoxal-5'-phosphate (P-5'-P) in solution 1 was also prepared.

Testing

For testing, 8μL of each AST sample was mixed with 72μL of the P-5'-P stock solution and 20μL of this mixture was placed on each sensor for testing. A blank sample was also tested using 20μL solution 1 in place of the AST sample. Testing was performed at 37⁰C.

Testing was carried out as described in example 8. Data were then transferred to the data analysis template, along with the AST concentrations of the samples.

Results

A plot of average current vs time for each AST sample is shown in Figure 40.

The rate of response (gradient of current vs time) was determined for each sample between the time points 8 seconds and 50 seconds using the average current values. A calibration plot of rate of response vs AST concentration is shown in Figure 41. A linear rate of response vs AST concentration was obtained.

A similar calibration plot of rate of response vs AST concentration for each sample between the time points 162 seconds and 204 seconds is shown in Figure 42. A linear rate of response vs AST concentration was obtained.

Conclusion

A rate assay for AST (L-aspartate aminotransferase, EC 2.6.1.1) has been demonstrated using Mediator 2 in a freeze dried sensor.

Example 20: Freeze-Dried sensor for L-alanine aminotransferase using Mediator

2

Methods

Solution 1 contained 0.1 M Tris buffer (pH 7.2).

Solution 2 was prepared by dissolving Mediator 2, potassium hydroxide, NAD⁺, putidaredoxin reductase, glutamate dehydrogenase, α-ketoglutaric acid, L-alanine and lactose in solution 1.

The final enzyme mixture in the solution 2 contained:

0.1M Tris (pH 7.2)

4OmM Mediator 2

4OmM KOH

2OmMNAD⁺

10mg/ml putidaredoxin reductase

40mg/mL glutamate dehydrogenase

15mM α-ketoglutaric acid

50OmM L-alanine and

10% w/v lactose. Solution 2 was dispensed onto as described in WO200356319 and freeze dried overnight.

ALT Samples

L-alanine aminotransferase (ALT) was obtained from Asahi Kasei Pharma Corporation (HC-ALT II, 11.8 U/mg). A concentrated stock solution of 50kU/mL of ALT was prepared using solution 1. Dilutions of the stock ALT solution were made with solution 1 to obtain samples with lower ALT concentrations of 10, 20, 30 and 40 kU/mL). Solutions of ALT were kept on ice until use. A stock solution of 0.1 ImM pyridoxal-5 '-phosphate (P-5'-P) in solution 1 was also prepared.

Testing

For testing, 8μL of each ALT sample was mixed with 72μL of the P-5'-P stock solution and 20μL of this mixture was placed on each sensor for testing. A blank sample was also tested using 20μL solution 1 in place of the ALT sample. Testing was performed at 37⁰C. Testing and analysis was carried out as described in example 8.

Results

A plot of average current vs time for each ALT sample is shown in Figure 43.

The rate of response (gradient of current vs time) was determined for each sample between the time points 8 seconds and 50 seconds using the average current values. A calibration plot of rate of response vs ALT concentration is shown in Figure 44. A linear rate of response vs ALT concentration was obtained.

A similar calibration plot of rate of response vs ALT concentration for each sample between the time points 162 seconds and 204 seconds is shown in Figure 45. A linear rate of response vs ALT concentration was obtained.

Conclusion

A rate assay has been demonstrated for ALT (L-alanine aminotransferase, EC 2.6.1.2) with Mediator 2 in a freeze dried sensor.

Example 21: Freeze Dried Mg²⁺ sensors using a range of ruthenium mediators

Methods

Solution 1 contained 0.1M imidazole buffer (pH 7.1, balanced with acetic acid).

Solution 2 was prepared by dissolving NAD⁺, diaphorase, glucose 6-phosphate dehydrogenase, hexokinase, ATP, lactose and EDTA in solution 1.

Solutions 3-5 were prepared by dissolving a sample of each of the three mediators, Mediator 2, Mediator 6 and ruthenium hexamine in solution 2.

Solution 6 containing 10 mM magnesium acetate (MgAc) was prepared in solution 1.

Solutions 7-10 containing 8, 6, 4 and 2 mM MgAc were prepared by diluting solution 6 in solution 1. Solution 11 containing 40 mM glucose was prepared in solution 1.

The final mixture in the solutions (3-5) freeze-dried on the sensors contained:

0.1 M imidazole-acetate buffer (pH 7.1)

40 mM of Mediator (plus 40 mM KOH for Mediator 2)

20 mM NAD⁺

5 mg/ml diaphorase

5 mg/ml glucose 6-phosphate dehydrogenase

10 mg/ml hexokinase

20 mM ATP (disodium salt)

5 mM ethylenediaminetetraacetic acid (tetrasodium salt, dehydrate) and

10%w/v lactose.

Testing

Sensors were prepared as described in WO200356319. Solutions were kept on ice until use. 50 μL of each MgAc solution (solutions 6-10) was mixed with 50 μL of the glucose solution (solution 11) and equilibrated to 37⁰C. A 20 μL aliquot of this mixture was placed on the sensors. A blank sample was also tested using 20μL solution 1 in place of MgAc sample. Testing was performed at 37 C. Chronoamperometry and analysis was carried out as described in example 8.

Results

Plots of average current vs time for each MgAc concentration using sensors prepared with each Mediator are shown in Figures 46a, 47a and 48a for Mediator 6, ruthenium hexamine and Mediator 2 respectively.

The rate of response (gradient of current vs time) was determined between the time points 22 and 64 seconds for each mediator using the average current value at each time point. For each mediator, a calibration plot of rate of response vs NADH concentration was made. These are shown in Figure 46b, 47b and 48b for Mediator 6, ruthenium hexamine and Mediator 2 respectively.

Conclusions

The linear relationship between rate of response and MgAc concentration for the Mg²⁺ assay for a range of different Ruthenium-containing mediators using freeze- dried sensors was demonstrated.

Example 22: Freeze Dried Mg²⁺ sensors using an Osmium Mediator

Methods

Solution 1 contained 0.1 M imidazole buffer (pH 7.1, balanced with acetic acid).

Solution 2 was prepared by dissolving NAD⁺, diaphorase, D-glucose, glucose 6- phosphate dehydrogenase, hexokinase, ATP, EDTA, BSA and Mediator 10 in solution 1.

Solution 3 containing 20 mM ATP and 2OmM glucose was prepared in solution 1.

Solution 4 containing 5 mM magnesium acetate (MgAc) was prepared in solution 3. Solutions 5-8 containing 4, 3, 2 and 1 mM MgAc were prepared by diluting solution 4 in solution 3.

The final mixture in the solution (2) freeze-dried on the sensors contained:

0.1 M imidazole-acetate buffer (pH 7.1)

40 mM Mediator 10, 20 mM NAD⁺

5 mg/ml diaphorase

2OmM D-glucose

5 mg/ml glucose 6-phosphate dehydrogenase

10 mg/ml hexokinase

20 mM ATP (disodium salt)

5 mM ethylenediaminetetraacetic acid (tetrasodium salt, dehydrate) and

2%w/v BSA.

Testing and analysis was carried out as described in example 21.

Results

A plot of average current vs time for each MgAc concentration for Mediator 10 is shown in Figure 49a.

The rate of response (gradient of current vs time) was determined between the time points 22 and 64 seconds for Mediator 10 using the average current value at each time point. A calibration plot of rate of response vs NADH concentration is shown in Figure 49b.

Conclusions

A linear relationship between rate of response and magnesium ion concentration (ie a Mg²⁺ assay) is demonstrated using sensors freeze-dried with an osmium mediator, thereby demonstrating the stability of the assay with respect to mediators of different metal centres.

Example 23: Freeze Dried Mg²⁺ sensors using a range of excipients

Methods

Solution 1 contained 0.1M imidazole buffer (pH 7.1, balanced with acetic acid).

Solution 2 was prepared by dissolving NAD⁺, diaphorase, glucose 6-phosphate dehydrogenase, hexokinase, ATP, Mediator 6 and EDTA in solution 1.

Solutions 3-11 were prepared by dissolving a sample of each of the nine excipients, lactose, sucrose, trehalose, lactitol, Ovalbumin, BSA, KCl, KNO₃ and NaNO₃ in solution 2.

Solution 12 containing 10 mM MgAC was prepared in solution 1.

Solutions 13-16 containing 8, 6, 4 and 2 mM MgAc were prepared by diluting solution 12 in solution 1.

Solution 17 containing 40 mM glucose was prepared in solution 1. The final mixture in the solutions (3-11) freeze-dried on the sensors contained:

0.1 M imidazole-acetate buffer (pH 7.1)

40 mM Mediator 6

20 mM NAD⁺

5 mg/ml diaphorase

5 mg/ml glucose 6-phosphate dehydrogenase

10 mg/ml hexokinase

20 mM ATP (disodium salt) and

5 mM ethylenediaminetetraacetic acid (tetrasodium salt, dehydrate).

Solutions 3-11 also contained 10% w/v lactose, 10% w/v sucrose, 10% w/v trehalose, 10% w/v lactitol, 2% w/v Ovalbumin, 2% w/v BSA, 7.5% w/v KCl, 7.5% w/v KNO₃ and 7.5% w/v NaNO₃ respectively.

Testing, chronoamperometry and analysis was carried out as described in example 21.

Results

Plots of average current vs time for each MgAc concentration for each Mediator are shown in Figure 50a, 51a, 52a, 53a, 54a, 55a, 56a, 57a, 58a for lactose, sucrose, trehalose, lactitol, Ovalbumin, BSA, KCl, KNO₃ and NaNO₃ respectively.

The rate of response (gradient of current vs time) was determined between the time points 22 and 64 seconds for each excipient using the average current value at each time point. For each excipient, a calibration plot of rate of response vs MgAc concentration was made. These are shown in Figure 50b, 51b, 52b, 53b, 54b, 55b, 56b, 57b, 58b for lactose, sucrose, trehalose, lactitol, Ovalbumin, BSA, KCl, KNO₃ and NaNO₃ respectively.

Conclusions

The linear relationship between rate of response and MgAc concentration using a range of different excipients and freeze-dried sensors was demonstrated.

Example 24: Temperature dependence of the response of a freeze dried Mg²⁺ sensor

Methods

Solution 1 contained 0.1M imidazole buffer (pH 7.1, balanced with acetic acid).

Solution 2 was prepared by dissolving NAD⁺, diaphorase, glucose 6-phosphate dehydrogenase, hexokinase, ATP, lactose, EDTA and Mediator 6 in solution 1.

Solution 3 containing 10 mM MgAC was prepared in solution 1.

Solutions 4-7 containing 8, 6, 4 and 2 mM MgAc were prepared by diluting solution 3 in solution 1.

Solution 8 containing 40 mM glucose was prepared in solution 1.

The final mixture in the solution (2) freeze-dried on the sensors contained: 0.1 M imidazole-acetate buffer (pH 7.1)

40 mM of Mediator 6

2O mM NAD⁺

5 mg/ml diaphorase

5 mg/ml glucose 6-phosphate dehydrogenase

10 mg/ml hexokinase

20 mM ATP (disodium salt)

5 mM ethylenediaminetetraacetic acid (tetrasodium salt dehydrate) and

10%w/v lactose.

Testing

Sensors were prepared as described in WO200356319. Solutions were kept on ice until use. 50 μL of each MgAc solution was mixed with 50 μL of the glucose solution and equilibrated to 23, 30 and 37⁰C respectively. For testing, 20 μL of each mixture sample was placed on each sensor for testing. A blank sample was also tested using 20μL solution 1 in place of MgAc sample. Testing was performed at 23, 30 and 37⁰C respectively.

The samples were tested and analysed as described in example 8.

Results

Plots of average current vs time for each MgAc concentration for each measured temperature are shown in Figure 59a for 23, 30 and 37⁰C.

The rate of response (gradient of current vs time) was determined between the time points 22 and 64 seconds at each temperature using the average current value at each time point. For each mediator, a calibration plot of rate of response vs MgAc concentration was made. These are shown in Figure 59b for 23, 30 and 37⁰C.

Conclusions

The linear relationship between rate of response and MgAc concentration for the Mg²⁺ assay using freeze-dried sensors was demonstrated for a range of different temperatures thereby demonstrating the stability of the assay with respect to temperature.

Example 25: Mg²⁺ Ion Sensor using Mediator 6 - Plasma

Methods

Solution 1 contained 0.1M imidazole buffer (pH 7.1, balanced with acetic acid).

Solution 2 was prepared by dissolving NAD⁺, diaphorase, glucose 6-phosphate dehydrogenase, hexokinase, lactose, EDTA and Mediator 6 in solution 1. Solution 2 was freeze-dried on sensors.

The final mixture in solution 2 freeze-dried on the sensors contained

0.1 M imidazole-acetate buffer (pH 7.1)

40 mM of Mediator 6

2O mM NAD⁺

5 mg/ml diaphorase

5 mg/ml glucose 6-phosphate dehydrogenase, 20 mg/ml Hexokinase 5 mM ethylenediaminetetraacetic acid (tetrasodium salt, dehydrate) and 10%w/v lactose.

Solution 3 was prepared by dissolving ATP and glucose in plasma.

Solutions 4-7 were prepared by dissolving MgAc in solution 3 at concentrations of 0.01, 0.1, 0.5 and 1.0 MgAc respectively.

Testing

Sensors were prepared as described in WO200356319. Solutions were kept on ice until use. 80 μL of each MgAc solution (solutions 4-7) was equilibrated at 37⁰C.

The final mixture in solutions 4-7 pippetted on the sensors contained 20 mM ATP, 20 mM glucose and 0, 0.01, 0.1, 0.5 and 1.0 MgAC respectively.

A 20μL aliquot of this solution was placed on each of the sensors used for that MgAc concentration. A blank sample was also tested using 80μL of solution 3. Testing was performed at 37⁰C.

Testing and analysis was carried out as described in example 8.

Results

Plots of average current vs time for each MgAc concentration are shown in Figure 60.

The rate of response (gradient of current vs time) was determined between the time points 22 and 64 seconds using the average current value at each time point. A calibration plot of rate of response vs MgAc concentration is shown in Figure 61.

Conclusions

An Mg²⁺ ion sensor for plasma sample has been demonstrated.

Example 26: Mg²⁺ Ion Sensor using Mediator 6 - Macroelectrode

Methods

Solution 2 was prepared by dissolving NAD⁺, diaphorase, glucose 6-phosphate dehydrogenase, hexokinase, lactose, ATP, EDTA and Mediator 6 in solution 1.

Solution 2 was freeze-dried on prefabricated laminated wells of 80μL volume.

The laminated structures were manufactured from 250 micron white PET with a PSA applied to one side. The acrylic adhesive thickness was approximately 50 microns. The base of the structure was manufactured from 125 micron clear PET treated with a print receptive coating. All material cutting was performed using a computer numerically controlled CO₂ laser.

The final mixture in solution 2 freeze-dried in these wells contained 0.1 M imidazole-acetate buffer (pH 7.1) 40 mM of Mediator 6 2O mM NAD⁺

5 mg/ml diaphorase

5 mg/ml Glucose 6-phosphate dehydrogenase

20 mg/ml Hexokinase

2OmM ATP

5 mM Ethylenediaminetetraacetic acid (tetrasodium salt dehydrate) and

10%w/v Lactose.

Solutions 3 and 4 were prepared by dissolving MgAc in solution 1 at concentrations of 2 and 1OmM MgAc respectively.

Solution 5 was prepared by dissolving glucose in solution 1 at a concentration of 4OmM.

Testing

Solutions were kept on ice until use. 50μL of each MgAc solution (solutions 3 and 4) was mixed with 50 μL of 4OmM Glucose (solutions 6 and 7) and each mixture was equilibrated to 37⁰C.

The final mixture in solutions 6 & 7 pippetted on the sensors contained:

0.1 M imidazole-acetate buffer (pH 7.1)

20 mM glucose and

0, 1, 3 and 5mM MgAC respectively.

An 80μL aliquot of this solution was placed on each of the sensors used for that MgAc concentration. A blank sample was also tested using 80μL of Solution 3. Testing was performed at 37⁰C.

Testinfi Protocol

On the addition of sample the chronoamperometry test was initiated. The oxidation current was measured at 0.15 V at 21 time points (8, 22, 36, 50, 64, 78, 92, 106, 120, 134, 148, 162, 176, 190, 204, 218 , 232, 246, 260, 274 and 288 seconds), with a reduction current measured at -0.45 V at the final time point (302 seconds). The transient current was measured for 8 seconds. Each sample was tested once.

Analysis

Data were then transferred to the data analysis template.

Results

Plots of average current vs time for each MgAc concentration is shown in Figure 62.

Conclusions

A Mg²⁺ ion macroelectrode sensor has been demonstrated.

Example 27: Ca²⁺ Ion Sensor using Mediator 6

Methods

Solution 1 contained 0.1M imidazole buffer (pH 7.1, balanced with acetic acid). Solution 2 was prepared by dissolving NAD⁺, diaphorase, glucose 6-phosphate dehydrogenase, hexokinase, lactose, EDTA and Mediator 6 in solution 1.

Solution 3 was prepared by dissolving ATP, magnesium acetate, α-Glucosidase (Sigma- Aldrich, G0660: α-glucosidase saccharomyces cerevisiae, recombinant) and amylase (Sigma-Aldrich, A6380: α-Amylase, from Bacillus sp.) in solution 1.

Solution 4 was prepared by dissolving maltotetraose (Sigma-Aldrich, M8253) in solution 1.

Solution 5 was prepared by dissolving 8mM calcium lactate in solution 4.

Solutions 6 and 7 containing 4mM and 6mM of calcium lactate respectively were prepared by diluting solution 5 in solution 4.

Solution 2 was freeze-dried on bottom-fill sensors with X-type flow cells. The final mixture in solution 2 freeze-dried on the sensors contained:

0.1 M imidazole-acetate buffer (pH 7.1)

40 mM of Mediator 6

2O mM NAD⁺

5 mg/ml diaphorase

5 mg/ml glucose 6-phosphate dehydrogenase

20 mg/ml hexokinase

5 mM ethylenediaminetetraacetic acid (tetrasodium salt dehydrate) and

10%w/v lactose.

Testing

Sensors were prepared as described in WO200356319. Solutions were kept on ice until use. 50 μL of each calcium lactate solution (solutions 4-7) was mixed with 50 μL of solution 3 (giving solutions 8-11) and equilibrated to 37⁰C.

The final mixture in solutions 8-11 pippetted on the sensors contained:

0.1 M imidazole-acetate buffer (pH 7.1)

20 mM ATP

5 mM Mg Acetate

25 mg/ml α-glucosidase

1 mg/ml amylase

25 mM maltotetraose and

0, 2, 3 and 4mM calcium lactate (in solutions 8-11) respectively.

For each calcium lactate solution, a 20 μL aliquot of mixture was placed on at least four sensors (four wells per sensor). A blank sample was also tested using 50μL solution 1 in place of solution 3. Testing was performed at 37 C.

Testing and analysis was carried out as described in example 8.

Results

Plots of average current vs time for each calcium lactate concentration are shown in Figure 63. The rate of response (gradient of current vs time) was determined between the time points 8 and 50 seconds using the average current value at each time point. A calibration plot of rate of response vs calcium lactate concentration is shown in Figure 64.

Conclusions

The linear relationship between rate of response and calcium lactate concentration for the Ca²⁺ assay was demonstrated.

Example 28: K⁺ Ion Sensor using Mediator 6

Methods

Solution 1 contained 0.1M imidazole buffer (pH 7.1, balanced with acetic acid).

The final mixture in solution 2 freeze-dried on the sensors contained:

0.1 M imidazole-acetate buffer (pH 7.1)

40 mM of Mediator 6, 20 mM NAD⁺

5 mg/ml diaphorase

5 mg/ml glucose 6-phosphate dehydrogenase

20 mg/ml hexokinase

5 mM ethylenediaminetetraacetic acid (tetrasodium salt dehydrate) and

10%w/v lactose.

Solution 3 was prepared by dissolving ADP, MgAc, pyruvate kinase (Sigma-Aldrich, P9136 : pyruvate kinase Type III from rabbit muscle) in solution 1.

Solution 4 was prepared by dissolving phospho(enol)pyruvic acid monosodium salt hydrate (Sigma-Aldrich, P0564) in solution 1.

Solutions 5-9 were prepared by dissolving KCl in solution 3 at concentrations of 2, 4, 6, 8 and 1OmM KCl respectively.

Testing

Sensors were prepared as described in WO200356319. Solutions were kept on ice until use. 50 μL of each KCl solution (solutions 5-9) was mixed with 50 μL of solution 4 (giving solutions 10-14) and equilibrated at 37⁰C.

The final mixture in solutions 10-14 pippetted on the sensors contained:

0.1 M imidazole-acetate buffer (pH 7.1)

6.25 mM ADP

1O mM MgAc

0.009 mg/ml pyruvate kinase

20 mM phospho(enol)pyruvic acid monosodium salt hydrate and

0, 1, 2, 3, 4 and 5mM KCl respectively. For each KCl solution, a 20 μL aliquot of mixture was placed on at least four sensors (four wells per sensor). A blank sample was also tested using 50μL solution 1 in place of solution 4. Testing was performed at 37⁰C.

Testing and analysis was carried out as described in example 8.

Results

A plot of average current vs time for each KCl concentration is shown in Figure 65.

The rate of response (gradient of current vs time) was determined between the time points 8 and 50 seconds using the average current value at each time point. A calibration plot of rate of response vs KCl concentration is shown in Figure 66.

Conclusions

A linear relationship between rate of response and KCl concentration for the K⁺ assay was demonstrated.

Example 29 : Cl^" Ion Sensor using Mediator 6

Methods

Solution 1 contained 0.1M imidazole buffer (pH 7.1, balanced with acetic acid).

Solution 2 was prepared by dissolving NAD⁺, diaphorase, glucose 6-phosphate dehydrogenase, hexokinase, lactose, EDTA and Mediator 6 in solution 1.

Solution 5 was prepared by dissolving 28OmM KCl in solution 4.

Solutions 6 and 7 containing 12OmM and 20OmM of KCl respectively were prepared by diluting solution 5 in solution 4.

Solution 2 was freeze-dried on sensors which were prepared as described in WO200356319.

The final mixture in solution 2 freeze-dried on the sensors contained:

0.1 M imidazole-acetate buffer (pH 7.1)

40 mM of Mediator 6

20 mM NAD⁺

5 mg/ml diaphorase

5 mg/ml glucose 6-phosphate dehydrogenase

20 mg/ml hexokinase

5 mM ethylenediaminetetraacetic acid (tetrasodium salt dehydrate) and

10%w/v lactose. Testing

Solutions were kept on ice until use. 50 μL of each KCl solution (solutions 4-7) was mixed with 50 μL of solution 3 (giving solutions 8-11) and equilibrated at 37⁰C.

The final mixture in solutions 8-11 pippetted on the sensors contained:

0.1 M imidazole-acetate buffer (pH 7.1)

20 niM ATP

5 mM MgAc

25 mg/ml α-glucosidase

1 mg/ml amylase

25 mM maltotetraose and

0, 60, 100 and 14OmM KCl (in solutions 8-11) respectively.

For each KCl solution, a 20 μL aliquot of mixture was placed on at least four sensors (four wells per sensor). A blank sample was also tested using 50μL solution 1 in place of solution 3. Testing was performed at 37⁰C.

Testing and analysis was performed as described in example 8.

Results

A plot of average current vs time for each KCl concentration is shown in Figure 67.

The rate of response (gradient of current vs time) was determined between the time points 22 and 64 seconds using the average current value at each time point. A calibration plot of rate of response vs KCl concentration is shown in Figure 68.

Conclusions

A linear relationship between rate of response and KCl concentration for the Cl^" assay was demonstrated.

Example 30: NHj⁺ Ion Sensor using Mediator 2

Methods

Preparation of solutions

Solution 1 contained 0.1M imidazole buffer (pH 7.1, balanced with acetic acid).

Solution 2 was prepared by dissolving Mediator 2, potassium hydroxide, NAD⁺, putidaredoxin reductase and glutamate dehydrogenase in solution 1.

Solution 3 was prepared by dissolving L-glutamic acid in solution 1.

Solutions 4-9 were prepared by dissolving 100, 200, 500, 1000, 1500 and 200OmM ammonium chloride respectively in solution 3.

Testing

Sensors were prepared as described in WO200356319. Solutions were kept on ice until use. 50 μL of each ammonium chloride solution (solutions 4-9) was mixed with 50 μL of solution 2 (giving solutions 10-15) and equilibrated to 37⁰C.

The final mixture in solutions 10-15 pippetted on the sensors contained: 0.1M imidazole-acetate buffer (pH 7.1)

4OmM Mediator 2

4OmM KOH

2OmM NAD⁺

10mg/ml putidaredoxin reductase

40mg/mL glutamate dehydrogenase

20 raM L-glutamic acid and

50, 100, 250, 500, 750 and 100OmM ammonium chloride (in solutions 10-15) respectively.

For each ammonium chloride solution, a 20 μL aliquot of mixture was placed on at least four sensors (four wells per sensor). A blank sample was also tested using 50μL solution 3 in place of solutions 4-9. Testing was performed at 37⁰C.

Testing and analysis was carried out as described in example 8.

Results

A plot of average current vs time for each ammonium chloride concentration is shown in Figure 69.

The rate of response (gradient of current vs time) was determined between the time points 8 and 50 seconds using the average current value at each time point. A calibration plot of rate of response vs ammonium chloride concentration is shown in Figure 70.

Conclusions

A linear relationship between rate of response and ammonium chloride concentration for the NH₄ ⁺ assay was demonstrated.

Example 31: Na⁺ Ion Sensor using Mediator 6

Methods

Preparation of solutions

Solution 1 contained 0.1M Tris buffer (pH 7.6).

Solution 2 was prepared by dissolving Mediator 6, NAD⁺, diaphorase and o- nitrophenyl-β-D-galactoside in solution 1.

Solution 3 was prepared by dissolving β-galactosidase in solution 1.

Solution 4 was prepared by dissolving galactose dehydrogenase in solution 3.

Solutions 5-7 were prepared by dissolving 200, 300 and 35OmM sodium chloride in solution 2.

Testing

Sensors were prepared as described in WO200356319. Solutions were kept on ice until use. 30 μL of each sodium chloride solution (solutions 5-7) was mixed with 30 μL of solution 4 (giving solutions 8-10) and equilibrated at 37⁰C. The final mixture in solutions 8-10 pippetted on the sensors contained:

O.lM Tris buffer (pH 7.6)

2OmM Mediator 6

1OmM NAD⁺

2.5mg/ml diaphorase

25mM o-nitrophenyl-β-D-galactoside

0.5mg/mL β-galactosidase,

1.15mg/mL galactose dehydrogenase and

100, 150 and 175mM sodium chloride (in solutions 8 -10) respectively.

For each sodium chloride solution, a 20 μL aliquot of mixture was placed on at least four sensors (four wells per sensor). A blank sample was also tested using 50μL solution 2 in place of solutions 5-7. Testing was performed at 37⁰C.

Testing and analysis was carried out as described in example 8.

Results

A plot of average current vs time for each sodium chloride concentration is shown in Figure 71.

The rate of response (gradient of current vs time) was determined between the time points 8 and 50 seconds using the average current value at each time point. A calibration plot of rate of response vs sodium chloride concentration is shown in Figure 72.

Conclusions

A linear relationship between rate of response and sodium chloride concentration for the Na⁺ assay was demonstrated.

Claims

1. A formulation for measuring the activity of an enzyme or an enzyme- activating ion in a sample by an electrochemical rate assay comprising: a substrate capable of an enzyme-catalysed reaction; and a redox mediator capable of a measurable change in oxidation state in response to the enzyme-catalysed reaction, wherein the redox mediator is an osmium complex, ruthenium hexamine trichloride or a ruthenium complex of Formula I

[Ru(A)_w (B)_x (C)_y]^m (X^z)π (I)

(wherein

Ru has an oxidation state of 0, 1, 2, 3 or 4; each of w, x, and y is an integer independently selected from the integers 1 to 4; m is an integer selected from the integers -5 to +4; n is an integer selected from the integers 1 to 5; z is an integer selected from the integers -2 to +1;

B is acetylacetonate (acac) or is a bi-, tri-, tetra-, penta- or hexadentate ligand which is linear having the formula R¹RN(C₂H₄NR)_WR¹ or cyclic having the formula (RNC₂H₄)V, (RNC₂H₄)_P(RNC₃H₆)_C or [(RNC₂H₄)(RNC₃H₆)]s, wherein w is an integer selected from the integers 1-5, v is an integer selected from the integers 3-6, each of p and q is an integer independently selected from the integers 1-3 whereby the sum of p and q is 4, 5 or 6, s is either 2 or 3 and each of R and R¹ is independently hydrogen or alkyl; C is a ligand; and X is a counter ion, wherein the number of coordinating atoms is 6).

2. A formulation as claimed in claim 1 further comprising auxiliary reagents of one or more kinase coupling reactions, of one or more dehydrogenase coupling reactions, of one or more oxidase coupling reactions or of one or more transferase coupling reactions.

3. A formulation as claimed in claim 1 wherein the enzyme is a kinase, dehydrogenase, hydrolase, reductase, oxidase, peroxidase or transferase.

4. A formulation as claimed in any preceding claim wherein the enzyme is selected from the group consisting of creatine kinase (CK), gamma glutamyl transferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), cholesterol dehydrogenase (ChDH), diaphorase, amylase, alkaline phosphatase (ALP), acid phosphatase (ACP), alanine aminopeptidase (AAP), N-acetyl-beta-d-glucosaminidase (NAG), maltase, glucose-6- phosphate dehydrogenase (G6PDH) and isoenzymes thereof.

5. A formulation as claimed in any preceding claim wherein the enzyme is creatine kinase.

6. A formulation as claimed in claim 1 wherein the substrate is selected from the group consisting of creatine phosphate, a lactic acid salt or ester, an ATP salt, L- alanine, L-aspartic acid, a nitrophenol linked-maltooligosaccharide, a phosphate ester, cholesterol, NADH, a glucose 6-phosphate salt and glycerol.

7. A formulation as claimed in claim 1 wherein the substrate is creatine phosphate.

8. A formulation as claimed in claim 1 or 2 further comprising: an enzyme-activating ion-dependent enzyme capable of an enzyme-catalysed reaction with the substrate.

9. A formulation as claimed in claim 8 wherein the enzyme-activating ion is selected from the group consisting OfNa⁺, K⁺, Mg²⁺, Ca²⁺, phosphate, chloride, ammonium and HCO₃ ^".

10. A formulation as claimed in claim 8 or 9 wherein the substrate is lactose or a nitrophenol linked-sugar and the enzyme-activating ion-dependent enzyme is a hydrolase.

11. A formulation as claimed in claim 8 or 9 wherein the substrate is a pyruvate and the enzyme-activating ion-dependent enzyme is pyruvate kinase.

12. A formulation as claimed in claim 8 or 9 wherein the substrate is glucose and the enzyme-activating ion-dependent enzyme is hexokinase.

13. A formulation as claimed in claim 8 or 9 wherein the substrate is an amylose and the enzyme-activating ion-dependent enzyme is an amylase.

14. A formulation as claimed in claim 8 or 9 wherein the substrate is a nicotinamide and the enzyme-activating ion-dependent enzyme is glutamate dehydrogenase.

15. A formulation as claimed in any preceding claim which is freeze-dried.

16. An electrochemical sensor for measuring the activity of an enzyme or an enzyme-activating ion in a sample by an electrochemical rate assay comprising: a main body defining one or more electrochemical cells, wherein each electrochemical cell includes a well, a working electrode exposed in the well and a reference electrode exposed in the well; and a formulation as defined in any preceding claim in the well so as to be in contact or contactable with the working electrode.

17. An electrochemical assay method for measuring the activity of an enzyme or an enzyme-activating ion in a sample comprising:

(a) introducing the sample to a formulation as defined in any preceding claim in an electrochemical cell;

(c) determining the rate of response of the electrical parameter; and

(d) relating the rate of response of the electrical parameter to the activity of the enzyme

18. Use of a redox mediator as defined in any preceding claim or a formulation as defined in any preceding claim in a kinetic enzyme assay.