WO2010015812A1

WO2010015812A1 - Enhancement of electrochemical response

Info

Publication number: WO2010015812A1
Application number: PCT/GB2009/001911
Authority: WO
Inventors: Howard James Orman
Original assignee: F. Hoffmann La Roche Ag
Priority date: 2008-08-04
Filing date: 2009-08-04
Publication date: 2010-02-11
Also published as: GB0814238D0

Abstract

The invention relates to an electrochemical method for determining the concentration of an analyte in a sample. The concentration is determined using measurements of the current generated in a characteristic period of enhanced current occurring shortly after a potential has been applied across the electrodes.

Description

ENHANCEMENT OF ELECTROCHEMICAL RESPONSE

Field of the Invention

The present invention relates to methods for determining the concentration of an analyte in a sample by an electrochemical method involving application of a potential and measuring the electrochemical response of the system during a period of enhanced current.

Background to the Invention

Electrochemical methodology is a versatile technique well suited to detecting many parameters of a substance. For example, the presence or concentration of a test analyte in a sample can be detected electrochemically by containing the sample in an electrochemical cell, applying a potential across the cell and probing the resulting electrochemical response.

The concentration of an analyte in a sample can be determined by measuring an electrochemical parameter and comparing that measurement with control measurements obtained on samples having known analyte concentrations. For example, in chronoamperometry a potential difference is applied across an electrochemical cell and the time-dependent current response (the "current transient") of the cell is measured. The current transient measured in an electrochemical test is related to current transients obtained for control samples (i.e., samples comprising known amounts of analyte) and so can be used to determine the concentration of the test analyte. In one such method according to WO2006030170, a time- varying potential is applied to step the potential applied across two electrodes in electrical contact with a target solution between an initial and a final potential. Once the final potential has been substantially attained, the current flowing between the electrodes is sampled. The current flowing between the electrodes is readily predictable using well-known theoretical equations suitable for a particular system under study. Such equations typically include as variables features of the system such as electrode shape and size, cell configuration, nature of the sample, and so on. However, in practice electrochemical measurements of this type typically comprise a contribution to the observed current in a period shortly after application of the potential that is not readily derivable by such equations. One well-known aspect of this contribution at very small times after application of the potential (for example, up to around 0.1 seconds) is the so-called "charging current" which is caused by capacitive electron flow in the electrochemical cell. The shape of the "charging current" response is dependent on a number of factors, such as electrical configuration of the electrochemical cell, electrical configuration of the electronics and solution resistance. These non-faradaic, electrical contributions are discussed, for example, in US 6,730,200.

However, a further, longer- lived enhancement of the observed current (of the order of seconds) has also been observed, a crucial proportion of which is again not readily derivable by well-known theoretical equations. The magnitude of these current enhancements may peak substantially immediately after a potential has been applied or alternatively may increase in value over time, before reaching a maximum value and then decaying. Once the enhancement has decayed, the experimentally measurable current becomes substantially the same as that predictable using the appropriate electrochemical theory.

The time-dependent behaviour of this current enhancement means that it often has a "shoulder" shape in an observed current transient. However, as used herein, the term "shoulder" can refer not only to a clearly visible shoulder in a current transient, but more generally to any enhancement of current that comprises a peak in time and a subsequent decay in magnitude. Furthermore, the "shoulders", or "current enhancements" that are addressed herein are typically not attributable to the non- faradaic charging peaks.

Generally, these current enhancements in a current transient observable after application of a voltage have, as is the case with electrical charging peaks, been viewed as undesirable signal distortions, whose magnitude is unrelated to the chemistry of the system under study (and, in particular, to the concentration of a test analyte in a sample). For this reason, previous electrochemical methods making use of current transient measurements for quantitative sample analysis have focussed on measuring the current response outside the period of current enhancement (i.e., after the shoulder has decayed and the system behaves substantially in accordance with well-known electrochemical theories). However, there are several disadvantages of this approach. For example, it then becomes necessary to delay obtaining measurements on the system until after the shoulder has fully decayed, or to obtain measurements over a sufficiently large time period such that the measurements in the "shoulder period" become quantitatively insignificant. Moreover, this approach involves discarding data corresponding to what is in experimental terms a large (i.e., enhanced) current response. This can be a particularly important consideration when the current response corresponding to the desired chemistry under study (i.e., that relating to the concentration of the test analyte) is relatively small compared to other factors such as contributions from chemical interferents in the sample. Microelectrode systems and/or biosensors can suffer in particular from large relative errors resulting from the small relative magnitude of current response to the analyte.

Accordingly, there is a need for a new technique that provides an improved electrochemical means of determining analyte concentration in a sample after a potential has been applied to an electrochemical cell. Summary of the Invention

The present invention provides a method for determining the concentration of an analyte in a sample, which method comprises: a) contacting said sample with an electrochemical cell comprising at least two electrodes; b) applying a potential across the electrodes to generate a current; c) obtaining one or more measurements of said current under conditions such that at least one of said measurements occurs in a period of enhanced current; and d) determining the concentration of said analyte from said one or more measurements; wherein said period of enhanced current is an enhancement of current for at least a part of the time from zero to ten seconds after application of the potential compared to a predicted current derivable by: i) determining the relationship between the current and time in a period of time beginning at least ten seconds after application of the potential; and ii) using that relationship to extrapolate a predicted current for the period of time from application of the potential to ten seconds after application of the potential.

The present inventors have found, surprisingly, that measurements of current generated during the period of enhanced current following application of the potential, before the system reverts to a theoretically predictable behaviour, can be used to determine the concentration of analyte in a sample in a reliable, reproducible way. This is in direct contrast to previous methods in which the "shoulder period" typically observed in the period immediately after application of the potential was considered to be an undesirable signal distortion and therefore not amenable to quantitative analysis. An advantage of the present invention is that it allows the concentration of analyte in a sample to be determined from current generated following application of a potential in a period of enhanced response. The surprising finding that this enhanced response can be quantitatively related to analyte concentration means that the relative size of the desired signal is substantially increased compared, in particular, to the electrochemical background response (as well as any errors resulting from other sources). This can be particularly important in systems characterised by small current responses, such as microelectrode systems, in particular biosensors and the like. Still further, measurement in the period of enhanced current allows the electrochemical method to be performed more rapidly, since it is not necessary to delay making measurements until the shoulder has decayed to a substantially negligible magnitude.

Brief Description of the Figures

Figure 1 shows a device according to one embodiment of the present invention.

Figure 2 shows experimental transient current responses for two samples containing 7.5 mM NADH in an NADH assay, compared to a theoretical response derived using microband theory. Graph A shows a sample containing n-heptyl-β-D- glucopyranoside surfactant; graph B shows a sample containing Cymal-4 surfactant. Black lines are the observed current responses and broken lines are the theoretical responses.

Figure 3 shows experimental transient current responses for samples containing 2.5 mM NADH (graph A), 5.0 mM NADH (graph B) and 7.5 mM NADH (graph C), compared to a theoretical response derived using microband theory. Black lines are the observed current responses and grey lines are the theoretical responses.

Figure 4 shows current response at specific times in the transient current response as a function of NADH concentration. Graph A shows samples containing Cymal 3 surfactant and graph B shows samples containing HEGA 9 surfactant. Current responses at 1 second are the black lines/black diamonds; current responses at 3 seconds are the broken lines/crosses; current responses at 8 seconds are the grey lines/grey circles.

Figure 5 shows the cumulative difference between the area under the curve of (i) actual current response and (ii) theoretical response derived using microband theory as a function of time after application of potential to NADH assay samples. Black lines show assays at 7.5 mM NADH; grey lines show assays at 5 mM NADH; broken lines show assays at 2.5 mM NADH.

Figure 6 shows the cumulative current response, integrated up to specific times in the transient current response, as a function of NADH concentration, for NADH assay samples containing a Cymal 3 surfactant. The black line/black diamonds show cumulative current response integrated up to 8 seconds; the broken line/crosses show cumulative current response integrated up to 3 seconds; the grey line/grey circles show cumulative current response integrated up to 1 second.

Figure 7 shows the cumulative current response, integrated up to specific times in the transient current response, as a function of NADH concentration, for NADH assay samples containing a HEGA 9 surfactant. The black line/black diamonds show cumulative current response integrated up to 8 seconds; the broken line/crosses show cumulative current response integrated up to 3 seconds; the grey line/grey circles show cumulative current response integrated up to 1 second.

Detailed Description of the Invention

The present invention involves applying a potential to an electrochemical system under conditions such that the current transient generated comprises a period of enhanced current.

The present inventors have found that the end of the period of enhanced current typically does not exceed ten seconds after application of the potential. Accordingly, a convenient means of defining the "period of enhanced current" in the context of the present invention is that it is an enhancement of current for at least a part of the time from zero to ten seconds after application of the potential compared to a predicted current derivable by: determining the relationship between the current and time in a period of time beginning at least ten seconds after application of the potential; and using that relationship to extrapolate a predicted current for the period of time from application of the potential to ten seconds after application of the potential.

The current enhancement has decayed to a negligible level by the period of time beginning at least ten seconds after application of the potential. Therefore, the current response of the system in the period of time beginning at least ten seconds after application of the potential can be used to determine the relationship between the current and time that would be expected for the system based on its physical characteristics (electrode design, size, etc.) and the chemistry occurring in it. By extrapolating this relationship back to give predicted current for the period from application of the potential to ten seconds after application of the potential, one can readily identify the period of time over which the current enhancement occurs.

In a preferred embodiment of the invention, the predicted current is derivable by determining the relationship between the current and time in a period of time beginning at least twelve seconds after application of the potential, for example at least fifteen or at least twenty seconds after application of the potential.

It is also possible that the current enhancement in a particular system will have decayed to a negligible level in a shorter time than ten seconds after application of the potential. In that case, it will be appreciated that the predicted current is derivable by determining the relationship between the current and time in a period of time beginning less than ten seconds after application of the potential. Thus, in a further preferred embodiment, the predicted current is derivable by determining the relationship between the current and time in a period of time beginning at least five seconds after application of the potential, for example at least eight seconds. However, it will also be appreciated that even when the current enhancement decays to a negligible level rapidly (i.e., in under ten seconds after application of the potential), it is still possible to derive the predicted current from the relationship between the current and time in a period of time beginning at least ten seconds after application of the potential.

The relationship between the current and time in a period of time beginning at least ten seconds after application of the potential can be determined entirely empirically based on applying a fit to real experimental data. The experimental data may, for example, correspond to current transients obtained in one or more control experiments using known analyte concentrations.

Alternatively, the relationship between the current and time in a period of time beginning at least ten seconds after application of the potential can be determined using well known theoretical equations that predict the current response of a known system to a known applied potential. For example, according to Electrochemical Methods: Fundamentals and Applications, A. J. Bard and L. R. Faulkner, John Wiley & Sons, New York, 2" Edition, 2001, Chapter 5, page 175 and to Journal of

Electroanalytical Chemistry, Issue 217, 1987, pages 417-423, a simple theoretical equation exists for the amperometric oxidation current observable at a microband electrode at a given experimental time and applied potential: 2ττAnFD_ox[Ox]

I =

where / is the microband current, F is a constant, A is the electrode area, n is the number of electrons involved in the electrochemical reaction, D_0x is the diffusion coefficient of the oxidisable redox agent, [Ox] is the concentration of the oxidisable redox agent, w is the width of the microband electrode and t is the time. The deviation of the current observed in an experiment from this current-time relationship in the period before ten seconds after application of the potential can be used to identify the period of enhanced current. It will also be appreciated it is not necessary to know the concentration of the analyte in advance, since even in the absence of such information the above equation indicates the predicted shape of the current transient, provided that the diffusion coefficient (D_0x) remains substantially constant over all samples. The diffusion coefficient could, for example, be a quantity having a known magnitude from the characteristics of the compounds making up the sample. Alternatively, the diffusion coefficient could be determined empirically by fitting it to the above equation for a particular experimentally observed current-time relationship.

The skilled person would of course recognize that analogous well-known equations can be applied to electrochemical systems other than those comprising a microband electrode. For example, the Cottrell equation relates current to time in the case of a planar working electrode:

^ nFAD_0x[Ox]

where all of the terms have the same definitions as those given above.

It is also possible to derive the predicted current relationship in the presence of decreases in the currents resulting from additional factors, for example chemical reactions of the redox mediator, provided that these decreases can be appropriately characterised (using, for example, well known kinetic theories). The present invention is useful in the electrochemical analysis of a test analyte comprised in a sample. Suitable samples include biological and non-biological substances, including water, beer, wine, blood, plasma, sweat, tears and urine samples. Preferably, the sample is a liquid sample, and more preferably it is an aqueous sample. Suitable test analytes include transition metals and their salts, heavy metals, and physiological species such as enzymes, cholesterol, triglycerides, glucose, cations, anions, biomarkers and biological analytes of clinical interest. In a preferred embodiment, the analyte is cholesterol or triglyceride. Cholesterol can be HDL cholesterol, LDL cholesterol or total cholesterol.

Electrochemical methods to which the present invention can be applied include any electrochemical method where a current enhancement occurs after a potential has been applied across the electrodes. Typically, therefore, the electrochemical method comprises applying a potential across the electrodes and measuring the electrochemical response, namely the current response.

hi one embodiment, the electrochemical method is any method in which a steady- state or a substantially steady state current is expected to be achieved following application of a potential. The present invention can be applied to microelectrode systems and to macroelectrode systems, non-exhaustive examples being thin layer cells, flow cells and rotating disc electrodes. The invention can also be used in non- steady state electrochemical methods.

There are many mechanisms that may generate the observed current enhancements. One possible explanation for the enhancements of the present invention is they arise due to pseudocapacitance effects on the surface of at least one of the electrodes in the cell. Pseudocapacitance is an electrochemical term relating to the electrochemistry of surface-active groups on an electrode surface and may be at least partially responsible for the enhancements observed when a potential is applied to the electrodes. Pseudocapacitance comprises both a capacitive term and a resistive term. In an experiment where the potential is stepped from a first value (which may be zero or non-zero) to a second, non-zero potential, the current resulting from pseudocapacitance can take many seconds to dissipate and so result in a shoulder in a measured transient, for example a current transient. The capacitive term varies according to the material adsorbed on the electrode (for example, surfactant micelles, proteins), while changes in the ionic strength of the solution may result in increases or decreases in the resistance of the solution and thus alter the resistive term of the pseudocapacitance.

A second explanation is that current enhancement could be due in part to the presence of nucleation-growth cycles. Such processes have been extensively studied (see, for example, J. Electroanalytical Chemistry 1966, 11, p.205).

The presence of the current enhancements is thus a complex process involving a variety of factors and their interactions in the electrochemical cell and with the electrodes (see, for example, Instrumental methods in electrochemistry, Horwood publishing, 2001, section 2.4.4, page 67). Such factors involve not only the electrode surfaces, but also the impact of surface-active agents and processes such as freeze drying, laser drilling of the electrodes and reagent mixing.

It is to be understood, however, that the present invention is not bound by any of these theories.

In the present invention, the sample comes into contact an electrochemical cell comprising at least two electrodes. A device according to one embodiment of the invention is depicted in Figure 1. In this embodiment, the device comprises a strip [S] comprising four electrochemical cells [C] and an electronics unit [E], e.g. a handheld portable electronics unit, capable of forming electronic contact with the strip [S]. The electronics unit [E] may, for example, house a power supply for providing a potential to the electrodes, as well as a measuring instrument for detecting an electrochemical response and any other measuring instruments required. One or more of these systems may be operated by a computer program.

The electrochemical cell [C] may be a two-electrode, a three-electrode, a four- electrode or a multiple-electrode system. A two-electrode system comprises a working electrode and a pseudo reference electrode. A three-electrode system comprises a working electrode, an ideal or pseudo reference electrode and a separate counter electrode. As used herein, a pseudo reference electrode is an electrode that is capable of providing a substantially stable reference potential. In a two-electrode system, the pseudo reference electrode also acts as the counter electrode; in this case a current passes through, but does not analytically significantly perturb the reference potential. As used herein, an ideal reference electrode is an ideal non-polarisable electrode through which no current passes.

hi one embodiment of the invention, the electrochemical cell is in the form of a receptacle. The receptacle may be in any shape as long as it is capable of containing a liquid which is placed into it. For example, the receptacle may be cylindrical. Generally, a receptacle will contain a base and a wall or walls that surround the base. Suitable embodiments of electrochemical cells in the form of receptacles are, for example, disclosed in WO03056319.

The electrochemical cell may have at least one microelectrode, for example a microband electrode. If so, typically the working electrode is a microelectrode. For the purposes of this invention, a microelectrode is an electrode having at least one dimension that comes into contact with the sample that does not exceed 50 μm. The r microelectrodes of the invention may have a dimension that contacts with the sample that is macro in size, i.e. which is greater than 50 μm. A typical microelectrode of the invention has one dimension of 50 μm or less and one dimension of greater than 50 μm (where the dimensions referred to are those in contact with the sample). For the purposes of this invention, a microband electrode is defined as having one dimension more than 50 μm and one dimension less than 50 μm (where the dimensions referred to are those in contact with the sample). A microband electrode is present in the cell in the shape of a band.

In the present invention, it is preferable that at least one of the at least two electrodes is a microelectrode. For example, in one specific embodiment one of the electrodes is a microband working electrode.

Further details regarding electrochemical cells that can be used in the devices of the present invention can be found in WO2006000828.

The electronics unit [E] comprises a voltage source arranged to selectively apply a voltage across the cell and a measurement circuit arranged to obtain measurements of an electrochemical parameter on the cell. The unit may also comprise other features, such as a display panel to read out the measured electrochemical parameter.

The devices of the present invention may comprise two or more (e.g. three or four) electrochemical cells. In such an embodiment, a plurality of strips may be used or the strip [S] may itself comprise a plurality of electrochemical cells. This embodiment allows a number of measurements to be taken either substantially simultaneously or in a step-wise fashion. The same or different reagent mixtures can be associated with each of the cells, allowing several identical measurements to be made or, for example, the concentrations of several different analytes in a sample to be measured simultaneously in a single device.

In the present invention, after the sample is contacted with the cell a potential is applied across the electrodes to generate a current. The current generated as a function of time comprises a period of enhanced current, i.e. an enhancement of current for at least a part of the time from zero to ten seconds after application of the potential.

In a preferred embodiment, the applied potential is a substantially constant potential. The potential may be applied in such a way that the potential is raised from zero to a final potential substantially immediately. Alternatively, a time-varying potential can be applied to step the potential applied across two electrodes in electrical contact with a target solution between an initial and a final potential. Once the final potential has been substantially attained, the current flowing between the electrodes can be sampled. Further details on suitable time- varying potentials that may be applied to step the potential across the electrodes in this way are disclosed in WO2006030170, the content of which is herein incorporated by reference in its entirety.

For the avoidance of doubt, references herein to times "after application of the potential" refer to times after the final (constant) potential has been substantially attaine^. As would be understood by those skilled in the art, a substantially constant potential can if desired be attained over a short period of time, the precise period being subject to the specific configuration of the cell.

Prior to application of the potential, the sample may be contacted with one or more other reagents. These one or more other reagents may comprise one or more of a redox mediator, a surfactant, at least one enzyme, a coenzyme, a reductase, a cholesterol ester hydrolysing agent and a triglyceride hydrolysing reagent. The one or more other reagents may be provided separately or in the form of a single reagent mixture. The sample may be contacted with the one or more reagents before being contacted with the electrochemical cell, after being contacted with the electrochemical cell, or at the same time as being contacted with the electrochemical cell. For example, the one or more reagents may be present as a single reagent mixture comprised in the electrochemical cell, so that the sample contacts them at the same time as being contacted with the cell. In a preferred embodiment, the sample is contacted with at least a surfactant. More preferably, the sample is contacted with at least a surfactant and a redox mediator.

The one or more other reagents are typically selected in accordance with the analyte to be detected in a particular embodiment of the invention. In one preferred embodiment, the analyte is cholesterol (HDL, LDL or total) or triglyceride and accordingly the one or more other reagents are reagents suitable for carrying out an electrochemical test to detect either cholesterol (HDL, LDL or total) or triglyceride. Detailed descriptions of reagent mixtures and methods suitable for carrying out cholesterol (HDL, LDL or total) tests are described in WO2007/072013, WO 2006/067424, WO 2007/132223 and WO 2007/132226, the contents of all of which are herein incorporated by reference in their entirety.

The redox mediator is an electroactive substance capable of being oxidised or reduced to form a product, which on contact with the sample interacts with the analyte such that it is present in a concentration that is related to the concentration of the analyte. It therefore acts as a mediator, first being oxidised or reduced by the analyte (either directly, or via one or more intermediate species such as an enzyme) to form a product and then, on application of the potential, being reduced or oxidised to give rise to the electrochemical response of the cell.

The redox mediator may be a molecule or an ionic complex. It may be a naturally occurring electron acceptor such as a protein or may be a synthetic molecule. The redox mediator will have at least two oxidation states.

Preferably, the redox mediator is an inorganic complex. The mediator may comprise a metallic ion and will preferably have at least two valencies. In particular, the mediator may comprise a transition metal ion. Preferred transition metal ions include those of cobalt, copper, iron, chromium, manganese, nickel, osmium and ruthenium. The redox mediator may be charged; for example, it may be cationic or alternatively anionic. An example of a suitable cationic mediator is a ruthenium complex such as Ru(NH₃)₆ ³⁺. An example of a suitable anionic mediator is a ferricyanide complex such as Fe(CN)₆ ^3".

Examples of complexes which may be used include Cu(EDTA) ^", Fe(CN)₆ ^", Fe(CN)₅(O₂CR)^3", Fe(CN)₄(oxalate)^3", Ru(NH₃)₆ ³⁺ and chelating amine ligand derivatives thereof (such as ethylenediamine), Ru(NH₃)₅(py)³⁺, cis-[bis(2,4- dioxopentan-3-ido)bis(3-pyridine carboxylic acid)-Ruthenium (IH)], ferrocenium and derivatives thereof with one or more of groups such as -NH₂, -NHR, -NHC(O)R, for example, ferrocenium monocarboxylic acid (FMCA), and -CO₂H substituted into one or both of the two cyclopentadienyl rings.

Further suitable redox mediators are disclosed in WO2007072018, the contents of which are herein incorporated by reference in their entirety. Examples of such complexes are those of the formula

[M(A)_x(B)_y]^m (X^z)_n wherein M is ruthenium or osmium and has an oxidation state of 0, 1 , 2, 3 or 4; x and n are independently an integer selected from 1 to 6; y is an integer selected from 1 to 5; m is an integer from -5 to +4 and z is an integer from -2 to +1 ;

A is a mono- or bidentate aromatic ligand containing 1 or 2 nitrogen atoms;

B is independently selected to be any suitable ligand other than a heterocyclic nitrogen-containing ligand;

X is any suitable counter ion; wherein A is optionally substituted by 1 to 8 groups independently selected from 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, alkylthio; wherein the number of coordinating atoms is 6. Still further examples of such complexes are those of formula [M(A)_x(B)_y]^m (X^z)_n wherein M is ruthenium or osmium and has an oxidation state of 0, 1, 2, 3 or 4; x and n are independently an integer selected from 1-6; y is an integer selected from 0-5; m is an integer from -5 to +4 and z is an integer from -2 to +1 ; A is a bi-, tri-, tetra-, penta- or hexadentate ligand which can be either linear having the formula R_IRN(C₂H₄NR)WR' or cyclic having the formulae (RNC₂H₄)_V, (RNC₂H₄)_p(RNC₃H₆)_q, or [(RNC₂H₄XRNC₃H_O)]_S, wherein w is an integer from 1-5, v is an integer from 3-6, p and q are integers from 1-3 whereby the sum of p and q is 4, 5 or 6, and s is either 2 or 3, and wherein R and R¹ are either hydrogen or methyl; B is independently selected to be any suitable ligand; X is any suitable counter ion; wherein B is optionally substituted by 1-8 groups independently selected from 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, alkylthio; wherein the number of coordinating atoms is 6.

Particularly preferred redox mediators are Ru(acac)₂(Py-3-CO₂H)(Py-3-CO₂)].H₂O and Ru(m)(Me₃TACN)(acac)(l-MeIm)](NO₃)₂. In these formulae, "acac" is the bidentate ligand acetylacetonate anion, CsH₇O₂ ^", and TACN is the tridentate ligand 1,4,7-triazacyclononane. Furthermore, Py means pyridine and Im means imidazole.

A surfactant can be used in order to break down lipoproteins to which triglycerides, cholesterol or cholesterol esters are incorporated. Examples of surfactants suitable for use in the present invention include polyoxyethylene derivatives such as polyoxyethylene alkylene tribenzyl phenyl ether and polyoxyethylene alkylene phenyl ether, sucrose esters, maltosides, hydroxyethylglucamide derivatives, N-methyl-N- acyl glucamine derivatives and bile acid derivatives (or salts thereof). Preferred surfactants include sucrose nionocaprate ("SMC"), Anameg-7 (Anatrace A340; Methyl-ό-O-CN-heptylcarbamoy^-α-D-glucopyranoside) and bile acid derivatives such as CHAPS.

Anameg-7, Cymal-2, Cymal-3, Cymal-4, Cymal-5, Cymal-6, Cymal-7 (which are 3- Cyclohexyl-1-alkyl-β-D-maltoside, where the "alkyl" moiety is ethyl, propyl, butyl, pentyl, hexyl and heptyl, respectively), Cyglu-3 (3-Cyclohexyl-l-propyl-β-D- glucoside), C-HEGA-10 (Cyclohexylbutanoyl-N-hydroxyethylglucamide), HEGA-9 (Nonanoyl-N-hydroxyethylglucamide), MEGA-8 (Octanoyl-N-methylglucamide), n- decyl-β-D-maltoside, n-undecyl-β-D-maltoside, n-heptyl-β-D-glucoside, n-octyl-β- D-glucoside, octanoyl sucrose, sucrose monocaprate and dodecanyol sucrose are particularly preferred surfactants. Cymal-2, Cymal-3, Cymal-4, Cymal-5, Cymal-6, Anameg-7, HEGA-9, MEGA-8, n-decyl-β-D-maltoside and n-heptyl-β-D-glucoside are most preferred.

The surfactant is typically provided in such an amount that when mixed with the sample to be tested the concentration of surfactant in the mixture of sample with the surfactant and any other reagents used is at least 1OmM, preferably at least 2OmM, for example at least 25mM.

In a particularly preferred embodiment of the present invention, before the step b) the sample is contacted with:

(a) a redox mediator selected from Ru(acac)₂(Py-3-CO₂H)(Py-3-CO₂)].H₂O and Ru(m)(Me₃TACN)(acac)(l-MeIm)](NO₃)₂, most preferably Ru(acac)₂(Py-3- CO₂H)(Py-3-CO₂)].H₂O; and

(b) a surfactant selected from Cymal-2, Cymal-3, Cymal-4, Cymal-5, Cymal-6, Anameg-7, Cyglu-3, C-HEGA-10, HEGA-9, MEGA-8, n-decyl-β-D-maltoside, n- undecyl-β-D-maltoside, n-heptyl-β-D-glucoside, n-octyl-β-D-glucoside, octanoyl sucrose, sucrose monocaprate and dodecanyol sucrose, most preferably selected from Cymal-2, Cymal-3, Cymal-4, Cymal-5, Cymal-6, Anameg-7, HEGA-9, MEGA-8, n- decyl-β-D-maltoside and n-heptyl-β-D-glucoside.

The at least one enzyme is a species that is capable of catalysing a reaction between the analyte and the redox mediator. Enzymes suitable for use in detecting triglyceride in a sample include (i) glycerol dehydrogenase and (ii) glycerol phosphate oxidase in combination with glycerol kinase, which may be used in conjunction in with the redox mediators of the invention and optionally further reagents. An enzyme such as (i) cholesterol oxidase or (ii) cholesterol dehydrogenase is suitable for use in a formulation detecting cholesterol.

Any commercially available forms of glycerol dehydrogenase, glycerol phosphate oxidase, glycerol kinase, cholesterol oxidase and cholesterol dehydrogenase may be employed. For instance, the cholesterol dehydrogenase is, for example, from the Nocardia species. The oxidase or dehydrogenase may be used in an amount of from O.Olmg to lOOmg per ml of reagent mixture, hi one embodiment, the oxidase or dehydrogenase is used in an amount of from 0.1 to 50 mg per ml of reagent mixture, preferably from 0.5 to 25 mg per ml. hi one embodiment, the glycerol kinase is present in an amount of from 450 U/ml reagent mixture to 45000U/ml reagent mixture.

The coenzyme is capable of being reversibly oxidised and reduced. Typically, the coenzyme becomes oxidised or reduced by reducing or oxidising the test analyte in the sample via the cholesterol oxidase or cholesterol dehydrogenase. The coenzyme then oxidises or reduces the redox mediator (either directly or via one or more intermediate species). An example of such an assay is shown below: Reduced

Cholesterol

Cholestenone

where ChD is cholesterol dehydrogenase. Thus, cholesterol is oxidised to cholestenone by cholesterol dehydrogenase, which is oxidised by the coenzyme, which is then oxidised by the redox mediator. The amount of reduced redox mediator produced by the assay (the "product") can then be detected electrochemically, by applying a potential across the cell and measuring the electrochemical response. Cholesterol dehydrogenase could be replaced with cholesterol oxidase in this assay if desired.

Suitable coenzymes include NAD⁺ or an analogue thereof such as APAD (Acetyl pyridine adenine dinucleotide), TNAD (Thio-NAD), AHD (acetyl pyridine hypoxanthine dinucleotide), NaAD (nicotinic acid adenine dinucleotide), NHD (nicotinamide hypoxanthine dinucleotide), or NGD (nicotinamide guanine dinucleotide). The coenzyme is typically present in the reagent mixture in an amount of from 1 to 25 mM, for example from 3 to 15 mM, preferably from 5 to 1OmM.

The reductase typically transfers two electrons from the reduced coenzyme and transfers two electrons to the redox mediator. The use of a reductase therefore provides swift electron transfer. Examples of reductases which can be used include diaphorase and cytochrome P450 reductases, in particular, the putidaredoxin reductase of the cytochrome P450_cam enzyme system from Pseudomonas putida, the flavin (FAD/FMN) domain of the P450_BM-₃ enzyme from Bacillus megaterium, spinach ferrodoxin reductase, rubredoxin reductase, adrenodoxin reductase, nitrate reductase, cytochrome bs reductase, corn nitrate reductase, terpredoxin reductase and yeast, rat, rabbit and human NADPH cytochrome P450 reductases. Where a nitrate reductase is employed preferably com nitrate reductase is used. Preferred reductases for use in the present invention include diaphorase and putidaredoxin reductases. The reductase may be a recombinant protein or a naturally occurring protein which has been purified or isolated. The reductase may have been mutated to improve its performance such as to optimise the speed at which it carries out the electron transfer or its substrate specificity.

The reductase is typically present in the reagent mixture in an amount of from 0.5 to 100mg/ml, for example from 1 to 50mg/ml, 1 to 30 mg/ml or from 5 to 20 mg/ml.

The cholesterol ester hydro lysing reagent may be any reagent capable of hydro lysing cholesterol esters to cholesterol. The reagent should be one which does not interfere with the reaction of cholesterol with cholesterol dehydrogenase and any subsequent steps in the assay. Preferred cholesterol ester hydrolysing reagents are enzymes, for example cholesterol esterase and lipases. A suitable lipase is, for example, a lipase from a pseudomonas or Chromobacterium viscosum species. The cholesterol ester hydrolysing reagent may be used in an amount of from 0.1 to 25 mg per ml of sample, for example from 0.1 to 20mg per ml of sample, and preferably from 0.5 to 25 mg per ml, such as 0.5 to 15 mg per ml.

hi a triglyceride test, a glycerol enzyme is typically used to determine the triglyceride content. The triglycerides which are liberated from the lipoproteins must therefore first be broken down to glycerol before reaction with the glycerol dehydrogenase or glycerol phosphate oxidase in combination with glycerol kinase. This is typically achieved by including a triglyceride hydrolysing reagent. Any reagent which hydrolyses triglycerides to glycerol may be used as long as it does not interfere with the activity of the dehydrogenase enzyme. Lipases and esterases are suitable examples of triglyceride hydrolysing reagents. The lipases described above as the cholesterol ester hydrolysing reagent are also appropriate for use in hydrolysing triglycerides. The triglyceride hydrolysing reagent may be used in an amount of from 0.1 to 100 mg per ml of sample, for example from 0.1 to 70 mg per ml of sample, and preferably from 0.5 to 50 mg per ml, such as 0.5 to 25 mg per ml.

In the method of the present invention, one or more measurements of current are obtained under conditions such that at least one measurement occurs in a period of enhanced current. The period of enhanced current is as defined above.

Each measurement corresponds to a value of the current generated at a particular time after application of the potential. Preferably, a series of measurements are obtained, thus generating a current transient. Preferably, at least two measurements are obtained and more preferably at least ten, for example at least twenty.

At least one measurement occurs in the period of enhanced current, i.e. it corresponds to a value of the current generated at a time after application of the potential when the shoulder, or current enhancement, is present. Preferably, at least half of the measurements occur in said period of enhanced current. More preferably at least three quarters of the measurements occur in said period of enhanced current. Most preferably, substantially all, for example all, of the measurements occur in said period of enhanced current.

In the period before 0.05 seconds after application of the potential, the electrical charging peaks as hereinbefore described are often observed in current transients and these are not thought to be related to the chemistry of the system under study (although it should be noted that the present invention is not any way limited to this theory). It is therefore to be understood that the one or more measurements in the present invention are obtained in a period where the purely non-faradaic "charging peak" contribution to the observed current is negligible. Typically the one or more measurements of current are thus obtained in the period beginning at least 0.05 seconds after application of the potential. Preferably, the one or more measurements of current are obtained in the period beginning at least 0.1 seconds after application of the potential, most preferably at least 0.15 seconds after application of the potential, for example at least 0.2 seconds after application of the potential.

As indicated above, the period of enhanced current is typically finished by ten seconds after application of the potential. Therefore, the least one measurement occurring in the period of enhanced current is/are typically obtained in the period up to a maximum often seconds after application of the potential. In a preferred embodiment, said at least one measurement is/are obtained in the period up to a maximum of eight seconds after application of the potential, for example up to a maximum of five seconds.

After the one or more measurements of current have been obtained, these are used to determine the concentration of the analyte. Methods for deteπnining concentration of an electroactive analyte, or redox agent whose concentration is related to that of an analyte in cases where a particular assay has been used, are well known. Typically, the magnitude of the electrochemical response of the cell can be correlated with the concentration of the analyte. This correlation can, for example, be achieved with reference to calibration data obtained in advance using samples having known analyte concentrations.

The analyte concentration can be obtained, for example, by correlating a single measurement of current response in the period of enhanced current to calibration data. Alternatively, a simple average or appropriately weighted average of two or more current measurements can be used.

In one preferred embodiment, the concentration of the analyte is determined by: calculating from at least two measurements the total charge passed in the period over which the measurements have been obtained; and determining the concentration of the analyte from said total charge passed. The total charge passed is the integral of the current over the period over which said measurements have been obtained. This method is particularly preferred when substantially all of the current measurements have been obtained in the period of enhanced current, because it allows all of the enhanced experimentally observed current to be used in determining the analyte concentration. It will be appreciated that a rigorous determination of the integral of current over time is not necessarily required. For example, a suitable estimate of the total charge passed could be obtained as an area under a graph of current as a function of time, or even as a simple summation of current measurements obtained at a series of time points.

hi another preferred embodiment, the concentration of the analyte is determined by: calculating from at least two measurements the experimental total charge passed in the period over which the measurements have been obtained; determining a predicted current for the period of time from application of the potential to ten seconds after application of the potential; - using the predicted current to calculate the predicted total charge passed in the period over which the measurements have been obtained; and determining the concentration of the analyte from the difference between the experimental and predicted total charge passed. hi this embodiment, the predicted current can be determined, for example, using a suitable well-known theoretical equation, such as those described above adapted appropriately for the specific system under study. Alternatively, it can be determined empirically, by determining the relationship between the current and time in a period of time beginning at least ten seconds after application of the potential and then using that relationship to extrapolate back to a predicted current in the period over which the measurements have been made.

Accordingly, the contribution to the signal from, for example, electrochemical background is substantially reduced.

In a further specific embodiment, the present invention provides a method for determining the concentration of an analyte in a sample, which method comprises: a) contacting said sample with an electrochemical cell comprising at least two electrodes, one of which is a microband electrode; b) applying a potential across the electrodes to generate a peak current, which subsequently decays; c) obtaining one or more measurements of said current under conditions such that at least one of said measurements occurs in a period during which the current is decaying; and d) determining the concentration of said analyte from said one or more measurements.

Within this specific embodiment, it is preferred that step b) comprises applying a potential across the electrodes to generate a peak current, which subsequently decays to a substantially steady state current and that in step c) at least one of said measurements occurs before the current is a substantially steady state current. More preferably, the substantially steady state current is a substantially constant current.

Examples

Handheld biosensor device A device of the type depicted in Figure 1 and described in detail in WO

2007/072013, having four electrochemical cells comprised in the strip [S], was used.

Each electrochemical cell comprised a carbon working electrode and a Ag/ AgCl pseudo reference electrode. The volume of each cell was approximately 0.6μl.

Identical deposition solutions were inserted into all four of the cells.

Deposition solution (0.4 μl of aqueous solution was inserted per electrochemical cell)

0.1M Tris (pH 9.0)

30 mM KOH

30 mM [Ru(acac)₂(Py-3-COOH)(Py-3-COO)] 10% w/v lactose 8.9 mM thionicotinamide adenine dinucleotide

4.2 mg/ml putidaredoxin reductase

3.3 mg/ml lipase (Genzyme)

22 mg/ml cholesterol dehydrogenase, gelatin free 100 mM sugar surfactant (see specific surfactants listed below)

Sugar surfactants used in deposition solutions

(i) Cymal 2

(ϋ) Cymal 3

(iii) Cymal 4

(iv) Cymal 5

(V) Cymal 6

(Vi) Cymal 7

(vii) Anameg 7

(viii) Cyglu 3

(ix) C-Hega 9

(X) C-Hega 10

(Xi) C-Hega 11

(xii) Hega δ

(xiii) Hega 9

(xiv) Mega 7

(XV) Mega 8

(xvi) n-decyl-β-D-maltopyranoside

(xvii) n-dodecyl- β -D-maltopyranoside

(xviii) n-undecyl-β-D-maltopyranoside

(xix) n-hexyl-β-Dglucopyranoside

(XX) n-heptyl- β -D-glucopyranoside

(xxi) n-octyl-β-D-glucopyranoside

(xxii) n-octanoyl sucrose

(xxiii) n-dodecanyol sucrose (xxiv) sucrose monocaprate

The dispensed sensor sheets were then placed into a LS40 freeze drier (Severn Science) for freeze drying.

Test Samples

The following test samples were prepared in delipidated serum (Scipac): (i) 0 mM NADH (ii) 2.5 mM NADH (iii) 5.O mM NADH

(iv) 7.5 mM NADH

Testing Protocol

20μl of sample was used per electrode. On the addition of 20μl of sample the chronoamperometry test was initiated. The oxidation current was measured at 0.15 V at

5 time points (0, 56, 112, 168 and 224 seconds), with a reduction current measured at -

0.45 V at the final time point (280 seconds). At each time point a transient current was measured for 8 seconds, with a data acquisition rate of 100Hz. Each sample was tested with at least one sensor (four electrochemical cells).

Results

i. Demonstration of current shoulder

Each transient current response to 7.5 mM NADH at the final time point (224 seconds) was analysed to determine the magnitude of the shoulder on the transient current response. This was done by curve fitting the observed transient response to that predicted by the microband electrode equation for the quasi-steady state response of a microband electrode. Specifically, the equation used was: 2πAnFD_ox[Ox\

/ =

where / is the microband current, F is the Faraday constant (96485 C/mol), A is the electrode area, n is the number of electrons involved in the electrochemical reaction, D_0x is the diffusion coefficient of the mediator, [Ox] is the concentration of reduced mediator, w is the width of the microband electrode and t is the time.

Curve fitting was performed for each transient by varying the value of D_0x to obtain good agreement between the observed and theoretical current values at long times on the transient current response. Two specific examples showing the period of current enhancement on transient current responses to 7.5 mM NADH are shown in Figure 2 (A used n-heptyl-β-D-glucopyranoside surfactant and B used Cymal-4 surfactant). It will be noted that from about 7 seconds after application of the potential, the relationship between current and time for the experimental data is substantially in accordance with microband theory.

A grade scale for the magnitude of the shoulder was constructed based on the percentage difference between the observed current value and the theoretical current value at 0.5 second time intervals along the transient current response. A shoulder was assigned a grade of 'N', where N is equal to twice the longest time point at which the average difference between the observed and theoretical currents was greater than 10%. A grade of zero would have been assigned if the average percentage difference was less than 10% at the first time point (0.5 seconds).

The grades of the shoulders are given in Table 1.

OmM Cymal-5 time /sec % difference in (i_Ob_S - itheo) grade of shoulder

OmM MEGA- time /sec % difference in (i₀b_s - itheo) grade of shoulder

Table 1: Extent of shoulder for mixes of Example 1, as indicated by excess current in the experimental current transient relative to microband theory. ii. Analysis of measurements in period of enhanced current

As a demonstration of the method of the invention, two specific Examples were selected. These involve samples comprising (A) Cymal 3 surfactant and (B) HEGA 9 surfactant. 2.5 mM, 5 mM and 7.5 mM NADH concentrations were used.

Typical current transients for the HEGA 9 system are shown in Figure 3 for each of the three NADH analyte concentrations: Graph A is for 2.5 mM NADH, Graph B is for 5 mM NADH and graph C is for 7.5 mM NADH. Also shown on these graphs are the fitted theoretical responses obtained using the microband equation (as explained above).

The current response at 1 second, 3 seconds and 8 seconds as a function of NADH concentration is shown in Figure 4 for both Cymal 3 surfactant (Graph A) and HEGA 9 surfactant (Graph B). This clearly shows that the current response at 3 seconds and, particularly, at 1 second is enhanced compared to the current response after the shoulder has decayed (at 8 seconds). Furthermore, in each case the current response varies linearly with NADH concentration, demonstrating that the assay is appropriate as a means of quantifying NADH concentration in an unknown sample. Due to the current enhancement at small times after application of the potential, the undesired contribution to the signal from the background response is reduced in relative size.

Further data analysis was undertaken by assessing the total charge passed in the period of current enhancement compared to that predicted by microband theory. The total charge passed is an integral of current response over time and so could be obtained as the area under the current transient. Data obtained prior to 0.15 seconds were not used to avoid contamination from non-faradaic charging peaks.

Figure 5 shows the cumulative difference between the area under the curve of (i) actual current response and (ii) theoretical current response for the Cymal 3 system. The Figure clearly shows that an enhanced charge is passed as a result of the period of current enhancement. The gradient of the plot of cumulative difference then tends towards zero as the period of current enhancement comes to an end.

Figure 6 shows a plot of cumulative area (i.e., the total integral, starting from 0.15 seconds, of current response up to a particular time) versus NADH concentration where the final time up to which current was integrated was 1 second, 3 seconds or 8 seconds, for the Cymal 3 system. Again, this shows that measurement of current, in this case integrated to obtain charge, in the shoulder period is capable of providing an assay system for quantifying NADH concentration. Figure 7 shows the same data, but for the HEGA 9 system.

The invention has been described with reference to various embodiments and examples. However, it is to be understood that the invention is in no way limited to these embodiments and examples.

Claims

1. A method for determining the concentration of an analyte in a sample, which method comprises: a) contacting said sample with an electrochemical cell comprising at least two electrodes; b) applying a potential across the electrodes to generate a current; c) obtaining one or more measurements of said current under conditions such that at least one of said measurements occurs in a period of enhanced current; and d) determining the concentration of said analyte from said one or more measurements; wherein said period of enhanced current is an enhancement of current for at least a part of the time from zero to ten seconds after application of the potential compared to a predicted current derivable by: (i) determining the relationship between the current and time in a period of time beginning at least ten seconds after application of the potential; and (ii) using that relationship to extrapolate a predicted current for the period of time from application of the potential to ten seconds after application of the potential.

2. A method according to claim 1, wherein said sample is a liquid sample.

3. A method according to claim 1 or 2, wherein said analyte is cholesterol or triglyceride.

4. A method according to any one of claims 1 to 3, wherein before step b) said sample is contacted with one or more of a redox mediator, a surfactant, at least one enzyme, a coenzyme, a reductase, a cholesterol ester hydrolysing agent and a triglyceride hydrolysing reagent.

5. A method according to claim 4, wherein before step b) said sample is contacted with a surfactant.

6. A method according to claim 5, wherein before step b) said sample is further contacted with a redox mediator.

7. A method according to any one of claims 4 to 6, wherein said redox mediator is Ru(acac)₂(Py-3-CO₂H)(Py-3-CO₂)].H₂O or Ru(π±)(Me₃TACN)(acac)(l- MeIm)](NO₃)₂.

8. A method according to any one of claims 4 to 7, wherein said surfactant is Cymal-2, Cymal-3, Cymal-4, Cymal-5, Cymal-6, Anameg-7, Cyglu-3, C-

HEGA-IO, HEGA-9, MEGA-8, n-decyl-β-D-maltoside, n-undecyl-β-D- maltoside, n-heptyl-β-D-glucoside, n-octyl-β-D-glucoside, octanoyl sucrose, sucrose monocaprate or dodecanyol sucrose.

9. A method according to any one of claims 4 to 8, wherein said at least one enzyme is cholesterol oxidase, cholesterol dehydrogenase, glycerol dehydrogenase or glycerol phosphate oxidase in combination with glycerol kinase.

10. A method according to any one of the preceding claims, wherein at least one of said at least two electrodes is a microelectrode.

11. A method according to any one of the preceding claims, wherein step c) comprising obtaining at least two measurements of said current.

12. A method according to claim 11, wherein step c) comprising obtaining at least ten measurements of said current.

13. A method according to claim 11 or 12, wherein in step c) at least half of said measurements occur in said period of enhanced current.

14. A method according to claim 13, wherein in step c) substantially all of said measurements occur in said period of enhanced current.

15. A method according to any one of claims 11 to 14, wherein in step d) the concentration of said analyte is determined by: calculating from said measurements the total charge passed in the period over which said measurements have been obtained; and determining the concentration of said analyte from said total charge passed.

16. A method for determining the concentration of an analyte in a sample, which method comprises: a) contacting said sample with an electrochemical cell comprising at least two electrodes, one of which is a microband electrode; b) applying a potential across the electrodes to generate a peak current, which subsequently decays; c) obtaining one or more measurements of said current under conditions such that at least one of said measurements occurs in a period during which the current is decaying; and d) determining the concentration of said analyte from said one or more measurements.