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    • 专利摘要:
    A method and apparatus for electrochemical detection of analyte in a sample makes use of a binding interaction and relies on the discovery that asymmetric distribution of a redox enzyme between two electrodes that occurs when a redox enzyme-containing reagent is 5 immobilized at the surface of one electrode can be detected as a chemical potential gradient arising from an asymmetry in the distribution of oxidized or reduced redox substrate. This chemical potential gradient can be detected potentiometrically by observing the potential difference between the electrodes in an open circuit, or amperometrically by observing the current flow between the electrodes when the circuit is closed. In both cases, the observation 10 of asymmetry can be done without the application of an external potential or current to the electrodes. WO 20091015291 PCT!IJS2008107 1073 Ccoo (Q.0 ULJ LUj Ae A du LILuJ % If x LL
    公开号:AU2013204779A1
    申请号:U2013204779
    申请日:2013-04-12
    公开日:2013-05-09
    发明作者:Ian Harding；Sridhar Iyengar；Richard Williams
    申请人:Agamatrix Inc；
    IPC主号:G01N33-53
    专利说明:
    P/00/001 Regulation 3.2 AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Invention title: ELECTROCHEMICAL ANALYFE DETECTION APPARATUS AND METHOD The following statement is a full description of this invention, including the best method of performing it known to us: - la ELECTROCHEMICAL ANALYTE DETECTION APPARATUS AND METHOD Related Application This application is a divisional of Australian patent application no. 2008279042, the entire disclosure of which is incorporated herein by reference. Background of the Invention This application relates to an apparatus and method for electrochemical detection of an analyte in a sample. The invention utilizes a specific binding relationship between the analyte and at least one reagent provided in the apparatus as a means for detecting the analy te. Analysis methods in which specific binding between an analyze and a reagent forms the basis for the assay are known. For example, in enzyme-linked immunoassay (ELA or ELISA) procedures, a sandwich is formed between an immobilized antibody and a mobile enzyme-anti body reagent when analyte is present through the interaction of the analyte with the antibody components. This results in the immobilization of the enzyme. The subsequent detection of immobilized enzyme is therefore indicative the presence of analyte in a test solution. (See US Patents Nos. 3,654,090, 4,169,012, and 4,642,285) Similar sandwich assays are used in chromatographic immunoassays where a colored tag (for example a colored latex bead) on the mobile reagent becomes trapped on a substrate at a defined location to produce a pattern that indicates the presence of analyte in a sample. (See US Patents Nos. 4,943,522, 5,656,503, 5,712,172 and 5,766,961) Analysis methods that depend on specific binding between an analyte and a reagent may also take the fomi of competition assays, in which the formation of a complex involving a labeled reagent is inhibited in the presence of analyte in an analyte concentration dependent manner (See US Patents Nos. 4,868,131, 5,981,298 and 5,989,921), or displacement assays in which a pre-existing complex involving a labeled reagent is disrupted in the presence of analyte in an analyte-concentration dependent manner. (See US Patents Nos. 4,746,631 and 6,020,209). Immunoassays inwhich detection of the analyte depends on an electrochemical measurement are also known. US Patent No. 5,149,630 discloses an assay in which the extent to which the transfer of electrons between an enzyme substrate and an electrode, associated with the substrate reaction, is perturbed by complex formation or by displacement of any ligand complex relative to unbound enzyme-labelled component. This determination WO 2009/015291 PCT/US2008/071073 is made in the presence of an applied potential. An applied potential is also used to measure current in the assay device disclosed in US Patents Nos. 5 198,367, 5,427,912. US Patent No. 5,494,831 discloses the application of a current and the measurement of changes in impedance that result in binding. Summaryof te Ivetion The present invention provides a new method and apparatus for electrochemical detection of analy'e in a sample that makes use of a binding interaction. The invention relies on the discovery that asymmetric distribution of a redox enzyme between two electrodes that occurs when a redox enzyme-containing reagent is irmmobil ed at the surface of one electrode can be detected as a chemical potential gdient arising from an asynnetry in the distribution of oxidized or reduced redox substrate. This c enical potential gradient can be detected potentiornetrically by observing the potential difference between the electrodes in an open circuit, or amnperornetrically by observing the current flow between the elect rodes wh en the circuit is closed, In both cases, the observation of asymmetry can be done without the application of an external potential or current to the electrodes. In a first embodiment of the invention, a sandwich type assay is utilized. In this emnbodinent, a sample to be tested for analyte is introduced to a test cell in the presence of a mobile test reagent. The test cell comprises a first and a second electrode. The first electrode has immobilized on the surface thereof an inunobilized test reagent. The mobile test reagent conprises an analyte-binding portion and a redox enzyme portion, and the immobilized test reagent comprises an analyze binding portion. If analyze is present in the sample in the test cell, at least a portion of the mobile test reagent becomes immobilized on the first electrode thereby creating an asynmetry in redox enzyme condemnation between the first and second electrodes when analyte is present. A redox substrate for the redox enzyme in the test cell is also supplied. The redox substrate is acted upon (oxidized or reduced) by the redox enzyne. If there is an asymmetry in redox enzyme concentration between the first and second electrodes (i.e. when analyte is present in the sample), this results in a chemical potential gradient between the first and second electrodes. This chemical potential gradient is detected to determine analyze in the sample. In this embodiment, the greater the asymmetry and the potential gradient, the greater the amount of analyte in the sample.
    WO 2009/015291 PCT!US2008/07 1073 In a second embodiment of the invention, a competition or displacenent type of assay is utilized. In tis assav the redox enzymre Is coupled to analyte or an analog of the analyte that can bind to a common immobilized test reagent. Optionally. the redox enzyme is provided already bound to the electrode via the immobilized test reagent. When sample is added. analyte present in the sample will compete with the redox enzyme for the binding sites provided by the innobilized test reagent resulting in a reduction in the amount of asymmetry that would occur is all of the binding sites were occupied by redox enzyme. Bief Decrto othe Drawiss Figs' LA and B show schematic representations of reduced mediator distribution between two electrodes When redox enzyme distribution is asynnetric (Fig. IA) or symmet(ri G (g 1B). Figs. 2A and B illustrate the determination of asymm etric enzyme distrn at two electrodes using amperometry. Fig. 3 shows a sandwich assay format useftui in the preset invention. Figs. 4 and 5 shows competition/displacement assay format useful in the present invention. Figs. 6A-C shows construction of a test cell with facing electrodes. Figs. A-C show examples of side-by-side electrode configurations. Fig. 8 shows an external view of a meter in accordance with the invention. Fig. 9 shows a circuit usable in the meter of the invention. Fi g 10 shows potential difference in open circuit as a. function of time for different levels of asymmetry in enzyme distribution between two electrodes. Fig. II shows current in short circuit as a function of time for different levels of asymnmetry in enzyme distributi between two electrodes. Fig. 12 shows the correlation of peak potential difference with ratio of electrode enzyme activities Fig 13 shows the correlation of peak current with ratio of electrode enzyme activities Fig. 14 shows a schematic plot of a known total enzyme E, on x-axis and measured I10-o the V-axis. Fig. 15 shows a schematic plot of a known E, on x-axis and measured 1I on the y-axis.
    WO 2009/015291 PCTIUS2008/071073 Fig. 16 shows a plot of how log (R) affects the slope of L versus E, which is used to derive paramers a and b. Fig. 17 shows a plot of log (R.) affects the intercept of I, versus E which is used to derive parameters c and d. hig. 18 shows the correlation between the actual amount of an enzyme and the estimated amount of enzyme as determined in accordance with the invention. Detailed Description of the Invention I. Definitions As used in the specification and claims of this application, the following detiitions should be applied: (a) "analyte" refers to a material of interest thatrnay be present in a sample. Analytes that are detectable in the present invention are those that can be associated in a specific-bmding interaction with at least one other reagent to that they can participate in a sandwich, competition or displacement assay configuration as described herein. Examples of analytes include antigens or happens such as peptides (for example hormones), proteins (for example, enzymes), drugs, pesticides, microorgamisms, antibodies, and nucleic acids that can participate in sequence specific hybridization reactions with a complementary sequence,. (b) analyte-specific enzyme component" or reagent refers to a reagent that includes both an analyte-binding portion and a redox enzyme portion. An analyte-specific enzyme component is suitably used as a mobile reagent. (c) "deteninaton of an analyze" refers to qualitative, semi-quantitative and quantitative processes for evaluating a sample, In a qualitative evaluation, a result indicates whether or not analyte was detected in the sample. in a serm-quanttative evaluation, the result indicates whether or not analyte is present above sonie pre-defined threshold. In a quantitative evaluation, the result is a numerical indication of the amount of analyte present. (d) the tenn "redox enzyne" refers to an enzyme that oxidizes reduces a substrate. Such enzymes may generally be known s oxidases, peroxidases reductases, or dehydrogenases. Enzymes such as glucose oxidase, and various peroxidases are commonly used in the analytical devices, and therefore the preparation of these nzymes in stable form is wel lnown.
    WO 2009/015291 PCTUS2008/07 1073 (e) the tern "redox substrate" refers to a compound or combination of compounds that interact with the redox enzyme fto produce a chemical potential gradient. In some cases, the enzyme substrate may directly produce a redox active species sufficient to create the chemical potential gradient. In others, a secondary compound may be needed. For example, in the case of lcucose oxidase, the interaction with glucose to produce gluconolactone and reduced enzyme produces the chemical potential gradient when the reduced enzyme is oxidized by a mediator compound, which is the actual redox active species in the chemical potential gradient. Thus, in this case the redoxx substrate" is the combination of the substrate compound glucose and the mediator compound. (f) the term "chemical potential gradient" refers to a concentration gradient of a redox active species. It will be appreciated that more rigorously, the potential gadient arises from a gradient in the ratio or reduced to oxidized species between the electrodes, -however, the idea of a concentration gradient of one species is more easily visualized and is therefore used here. When such a gradient is present between two electrodes, a potential difference is detectable if the circuit is opened, and a current will flow until the gradient dissipates when the circuit is closed. Itwill be appreciated that the chemical potential gradient is transient in the devices of the invention, and that the distribution of the redox active species will even out over time, when new redox active species stops being created. The term "chemical potential gradient" as used herein refers only to this transient gradient that arises from the asmnmetrV of the distribution of redox enzyme and not to any potential gradient that arises from the applicatior of a potent tal difference or current flow between the electrodes. () the prase "detecting the chemical potnial gradient between the first and second ilectrodes" refers to the detection of the chemical potential gradient in either an open or a closed circuit, using either potentiomnetric or amerornetric measurements. (h) the terms "enzyme activity" and enzymee concentration" are used as equivalent terms herein, although it will be appreciated that in ordinary usage they may have different meanings. Activity of an enzyme provides a quantitative measure of the cataivti capabili of an enzyme. This depends not only on the physical amount of the enzyme Present in the volume (i.e. the concentration), but also on the conditions which affect the catalytic etffici-rency of the enzyme. The present invention actually measures asymnetry in enzyme activity, since the presence of inactive enzyme will not produce an asymmetry in redox substrate. However, WO 2009/015291 PCT!US2008107 1073 since it is desirable to control the quality of the enzyme and the conditions, this is in effect also a measurement of asymmetry in enzyme concentration. (i) the term "inmobilized on the first electrode" refers to immobilization directly or indirectly on the surface of the electrode, provided that the material inmnobilized becomes inmobilized in volume associated with the first electrode and closer to the first electrode than the second electrode. For example, in the case of the formation of an electrode-antibody anaiyte-antibody-enzyme sandwich, the enzyme is considered to be "immobilized on" the electrode even though there are several interning moieties in the sandwich. (J) the term "imnobilized test reagent" refers to the component of a sandwich or competition/displacement reaction that is associated with an electrode when performing the assay of the invention. Immobilization may be through the formation of a chemical bond (covalent or non-covalent) between the innobilized test reagent and the surface of the electrode, or it may be a physical association as through the placement of the immobilized test rcagent within a get or membrane disposed on the surface of the electrode. The unmobilized test reagent comprises a binding moiety which interacts with analyte to produce a change in th-e distribution of the redox enzyme (as part of the analyte-binding enzyme component) when analye is present in a sample, [. some embodiments, the immobilized reagent vil be immobilized during initial manufacture of the test apparatus. In other embodiments, immobilization will occur in situ after addition of the sample. Thus, the term immobilized test reagent refers to any circumstance in which the structure: electrod e-(iink>reagent with binding site for analyte is obtained, where ii is 0 or an. integer of 1 or greater. The latter option allows production of one set of generic devices in which the linking agent is not analyze specific, with the addition of recent that binds to the link and tie analyte for any given analyte-specific test. This additional reagent could be added to the test strip at manufacture, or to the sarim.ple prior to application to the test device. iL Theory of the invention For convenience, the theoretical basis for the invention will be discussed in the context of glucose oxidase as the redox enzyme and a combination of glucose and a mediator WO 2009/015291 PCTfIS2008/071073 as the redox substrate. Nothing in this discussion should be taken as an indication, however, that the invention is limited to use with these materials. in common glucose measuremem sys tems, enzyme present in the sample cell oxidizes glucose to gluconolactone. and the enzyme is reduced. Oxidized mediator (for example ferricyanide) reacts with the reduced enzyme to regenerate the oxidized formr of the enzyme, and produc reducedi mediator. This process continues ut i eiher gucose or oxidized mediator is exhausted. If the enzyme is distributed asymmeticay within the sample cell, then the prodction of reduced mediator is also asymnetnc, and the resulting asymmetry in the distribution of reduced mediator persists for a period of time determinedd by diffusion parameters) even after exhaustion of the limiting reagent. When reduced me diator is present in a solution between two electrodes, the potential difference between the two electrodes is given by the Nernst equation RT [nedd ] [med E = ------ X log --- X-r mned, med where E is the potential difference between electrodes I and 25 R, is the gas constant, T is the absolute temperature and F is the Faraday constant. Wen there is no dif rence in the concentration of reduced mediator at the two electrodes (i.e. no chemical potential gradient) because there is no asymmetry, the mediator sterns reduce to 1, log 1 : 0, and so the potential difference is 0. As the asymmetry increases, the potential difference 'increases. Furthermore, if the system is designed such that the oxidized mediator concentration at the time of neasurenent is large compared to the amount of reduced mediator (either due to a large excess of oxidized mediator initially or rapid taking of the measurement or both) then the oxidized mediator concentration at the two electrodes is essentially equal, and the equation car be simplified to: .,E = X1log WO 2009/015291 PCT!US20081071073 Figs. !A and B show schematic representations of reduced mediator distribution between two electrodes (1, 2) when redox enzyme distribution is asymmetric (Fig 1 A) or symiuetric (Fig. 1B). The equations above can be used to quantitate the ratio of concentrations of enzyme at two electrodes 1 and 2 using potentiometry. The two electrodes are connected in open circuit, and the potential difference between them is measured. It enzyme 3 is more active or concentrated at one electrode than the other (i.e., it has an asymmetric distibution as shown in Fig. 1A), the concentration of reduced mediator will be higher at that electrode than the other, giving rise to a electrical potential difference between the electrodes. If eqial activity or concentrations of enzyme are present at both electrodes (iKe., i has a symmetric distribution as shown in Fig. 1 B), the concentration of reduced mediator at each electrode will be equal, and the electrodes will be at equal electrical potential and the m-easu ed potential difference will be 0. If instead of potentiometry, the electrodes are connected in short-circuit, the asynunetric distribution of enzyme activity will result in an asymmetric distribution of reduced mediator, that can be observed by following current when the potential difference between the electrodes is forced to zero by closing the circuit between them., If reduced mediator is generated by enzyme activity and diffuses to an electrode, current will flow and sufficient mediator will be reoxidized, such that the reduced mediator concentration is equal at both electrodes. The current flow will be proportional to the difference in flux of reduced mediator to the two electrodes. Figs. 2A and B illustrate the use of these principles to quantify the ratio of concentrations of enzyne at two electrodes, using amperometry. The two electrodes are connected in short circuit, and the current flowing between them is measured, As electrical potential difference between the electrodes is constrained to zero, mediator will be reduced or oxidized at the electrodes when necessary to maintain an equal chemical potential at each electrode. As reduced mediator is oxidized at one electrode, current will flow and an equivalent quantity of oxidized mediator will be reduced at the other electrode. If the enzyme activity at each electrode is equal, no electron transfer will be necessary to keep the chemical potentials balanced, so no current will flow. If more enzyme activity is present at one electrode than the other, reduced mediator will be consmtned at that electrode and produced at the other electrode to maintain a balanced chemical potential. and current will flow.
    WO 2009/015291 PCTUS2008/07 1073 IIL Practical_4llgictons The theory of the present invention is applicable to the practical aolication of determining analytes in a sample. This can be done in various binding formats. In general, two analyte-specific chemical components are utilized, and these are selected in to correspond to the tanalyte to be determined: In general, it is desired to have at least one of the components (generally the analyte-receptor) be highly specific for the analyte while the other component can be less specific in its binding. Various non-limiting combinations are set forth in 'Fabe 1. I____ ------ --- - 1~-------------- - ----- - ----- -- Analye I Analyte receptor binding component of Analyxte specific enzyme -- -- - - -- -- - --- -- -- --- - -- - -- -- -- - -- -- -- - - - -- - -- ------- - -- ----- -- - -- ----- - -- -- - disease-specific disease associated- antibody that recognizes human antibody, for example antigen, such as peptide antibodies, aptamers an antibody to epitope found on an iuenza, disease in o interest causing organism aptamers K------- - -------- 4 ___ .- - .--. . ...... amplified nucleic acid nucleic acid nucleic acid sequence sequence including a compementary to the complementary to the known primer suspected target and a suspected target known pimer hornone honone receptor, Ihornone receptor, apataimer or apatarmer or antibody antibody drug drug receptor, aptamer honnone receptor apatamer or or antibody antibody pesticides pesticide binding pesticide binding proteins. apatmers proteins, aptamers, antibodies that recognmize the antibodies o pestcides antibodies to pesticides As will be tmderstood hi the art, the "antibody" incorporated in the device can be a complete antibody such as an immunoglobulin or it may be a engineered binding portion of an antibody such as a single-chain antibody (scFv) or Doma-in Antibodies (dAb) as described at www.domatis.com. Immurnoassays for drugs are described in tS Patents Nos. 7,220,842, 5,67 , 132 and 5,618,926. Irmnunoassays for pesticides and pesticide degradation products are described in US Patent No. 6,635,434. Drug and hormone receptors may be native hormone receptors, or they may be engineered species wil similar binding properties WO 2009/015291 PCT!US2008/07 1073 - 10 as are known in the art (See US Patents Nos. 7,214,511, and 6,806,359, Rasmussen et a], j. Blio. Chem, Vol. 276, Issue 7, 4717-4723, February 16, 200 1). US Patent Publication 200501 3020S discloses a cocaine-specific aptamer that could be used as a binding portion of an analyte-specific enzyme component or as an immobilized test reagent. Other aptners are listed on the database of the Ellington research group at http://aptamercmbutexas.edu/. Enzyme-aptamer coupling has been described byMir et al. "Different Strategies To Develop an Electrochemical Thrombin Aptasensor." Electrochemical Communication 8 (2006): 505-511. The conoonents of the reaction are combined in. such a way that they result in a difference in distribution syrrnetry of the redox enzyme that is dependent on the presence or absence of analyte in a sample. Various device formats can achieve this result. A. Sandich format Fig. 3 shows two electrodes 31 and 32. Electrode 31 has analyte-specific receptors 33 inunobilized on the interior surface thereof Analyte 34 associates with the analyte specific receptors 33. Also present is an analyte-specific enzyme reagent 35 which associates with analyze in such as way that the activity of the enzyme is maintained in the bound and the unbound state, and a redox substrate 36, 36' . Capture of the analyte-specific enzyme reagent 35 occurs at the surface of electrode 31 when analyte 34 is present because of the formation of receptor 33-analyte 34-analyte-specic enzyme species 35 sandwich. The activity of the enzyme results in an increased concentration of one formn of the redox substrate (in. the case or Pig. 3, of form 36) near electrode 31, and hence a detectable chemical potential gradient whose magnitude is related to the amount of analyte develops. When the analyte is not present, the sandwich does not form and thus no detectable chemical potential gradient is .produced, B, tromp.tirioWDipj nentFormat Figs 4 and 5 show a competition format for an assay device in accordance with the invention. The sample ce' 1 is defined by two electrodes 41, 42. Electrode 41 has analyte receptors 43 disposed thereon. The redox enzyme 45 comprises the enzyme coupled to an analyte or an analyte mirnetic. In the absence of analyte, as shown in Fig. 4, redox enzyme 45 WO 2009/015291 PCT/US2008/071073 is coupled to analyte receptor 43, resulting in an asymmetry in the distribution of the redox enzyme. Additional redox enzyme 45' may be present in the bulk of the sample depending on the amount of redox enzyme, analyte receptors and the affinity of the analyze receptors for the redox enzym.e. As shown in Fig. 5, when analyze 44 is present, it cornpetes with redox enzyme 45 for the analyze receptor 43, resulting in the displacement of at least some of the redox enzymne 45 from the analyze receptors 43, creating a less asymmetric distribution of the redox enzyme and thus the ability to determine analyte in a sample. C. Devic Co oructo am;e C11vii i r acing Euectcdes Figs, 6A-C show a sample cell for a device in accrdance with te invention, constructed using facing electrodes The device is forced ftrom a top layer 61 and bottom layer 62 , each having a conductive surface (61, 62') facing the nteri of the device, and an insulating spacer layer 63. Contacts 64 and 65 extend from the top layer 61 and bottom layer 62 to allow contact with the conductive surfaces for either potentioietric or amperomietric measurements. As shown in Fig. 63, the conductive layer 63 has an openg therein that together with the facing conductive surfaces 61' and 62' defines a sample cell. his sample cell is open to the exterior for introduction of sample, for example through an opening 66 at the end device as shown in Fig. 6C. Various structures for forming cells of this type, with multiple openings and/or vent holes are known in the art for example as the particular design of the cell is not critical to the practicing of the present invention. Similarly, other designs for comectors are known, and this also is not critical to the practicing of the present invention. ri an embodirmnt of the device of the invention, one of the conductive surfaces (61' or 62') has the analyte receptor (inobilized test reagent) disposed thereon. The remainder of the reagents for determination of analyte may be disposed within the sample cell as a dried reagent layer 67, or may be added to the sample prior to introduction to the sample cell. The sample cell portion as shown in Figs 6A-C is generally constructed as a single use, disposable component. D. D~ke:iiejlectrodes In addition to facing electrodes as discussed above, the first and electrodes may be disposed on the same surface within the sample cell. This configuration is referred to herein WO 2009/015291 PCT/US2008/071073 -12 as side-by-side electrodes. Figs; 7A-C show non-limiting examples of configurations that can be used for side-by-side configurations, including parallel strips (Fig. 7A), interdigitated strips (7B) and concentric rings (Fig. 7C). In each case, asyrirnetry in redox enzyme distribution between one electrode 71 and the other electrode 72 results in a measurable chemical potential gradient between the two electrodes. The sample cell portions as shown in Figs 7A-C are generally constructed as a single use, disposable component. E Device Constmetion -Communiationrof Re-sult In addition to the sample cell, the device of the invention has a means for conununicating the observed potential or current to a user in a meaningful way indicative of the determination of analyte. This can range from a simple qualitative result (analyte present or riot present), or to a specific rmerical value for the amount of analyze. The sophistication of the communication means varies accordingly. In one embodiment of the invention, to provide a qualitative result, the device may have at a visible location a spot of a material that changes color iii response to the passage of current or tie application of the potential difference created by an asymmetry above a threshold level. In this case, the device does not need a separate meter portion, and may be simply a disposable test strip with an indicator spot on the outside. More commonly, the sample cell will be contained within a disposable test strip that is inserted into a reusable meter. The meter Will contain the electronics for measuring the potential difference or current at a defined period of time after introduction of a sarnple or test strip, and fom conversion of the measured value to a displayed value. This conversion may make use of a look-up table that converts specific value of current or potential to values of analyte depending on the calibration values for the specific device geoiretry and analyze. Fig. 8 shows an extemal View of a ireter in accordance with the invention. As shown, the meter has a housing 81 having a display 82 and one or nuimore control interfaces 83 (for example power buttons, or scrolI wheels etc). The housing has a slot 84 for receiving a test device. internal to the hous ng 1 is 1circitry for applying a potential or current to the electrodes of the test device when a sample is applied. This may be done following an WO 2009/015291 PCTIUS2008/071073 3 initialization signal from a user, or following an automated detection of test device insertion and sample application. Suitable circuits usable in the meter of the invention are known in the art, for example from US Pateni Publcation No. US 2005-0265094 Al. which is incorporated herein by reference. One such circuit is shown in Fig. 9. In Fig. 9, an ideal voltmneter 942 is provided which can. measure the potential across the electrodes 941, 938. Switch 944 is provided which is opened when the potential is to be measured or closed for measurement of current. When ope, the cell 939 is "floating" as to at least one of it electrodes, permitting a voltage measurement that is unaffected by signals at the ampliier 935 The switch 944 may be a rmechanical switch (e.g. a relay) or art FET (field-effect transistor) switch, or a solid-state switch. in a simple case the switch opens to an open circuit; more generally it could open to a very hi igh resistance. This circuit can be used to measure either a potential difference or a current difference. As will be appreciated by persons skilled in the art, other circuits including much simpler and more complicated circuits can be used. to achieve e application of either or both of a potential difference or a current. The circuit of Fig. 9 can also be used to apply a potential difference to the test device of the invention. While, as discussed above, such application of potential is not required to perform the measurement of the invention, application of pot ential prior to the mneasureIen. (particularly in a test device with facing electrode construction) car result in a more rapid measurement ine by effectively driving analyte andior analyte-binding enzyme component toward-s the electrode With the immobilized reagent using what is in essence electrophoresis, provided that the analyze or analyte-bindina enzyme con-monent is charged under the conditions (particularly pH) found within the test device. A step function or a sine wave passing f&om negative to Positive potential differences could also effect something similar to washing in situ within the test device. V. Advantages of the Invention The present invention provides the ability to perform binding assays for the detection of analyte with electrochemical detection, without the need to apply an external potential or current, and without the need for washing steps commonly employed in sandwich WO 2009/015291 PCTUS2008/071073 -14 irmunoassay procedures. Because of this, the apparatus used to perform the assay can be much simpler, easy to use, and less expensive. In addition, the device components in an electrochemical assay are more robust than those used for example in optical measurements, facilitating the manufacture of the low cost reusable meter for use with disposable test devices. The present invention can make use of small samples of blood, tears, saliva, or sweat, with minimal invasiveness to the subject. saless of less than 10 ul, preferably less than I ul are particularly desirable for blood tests). Further, the present invention can provide for very rapid test times. Common immunoassay procedures take up to an ihour or more. The small volume of the Lest devices of the invention and the sensitivity of the test to small di Fferences in the distribution of the enzyme means that shorter times (for example less the 30 minutes, more preferably less than 10 minutes, and most preferably less than I minute) are accessible depending on the concentration of analyte to be measured and the binding kinetics of the analyte with the binding elements of the mobile and immobilized reagents. Depending on the nature of the sample and the redox substrate, it may be possible to use materials from the sample as part of the reaction systern. For example, endogenous glucose can be used as a comp.onent of the redox substrate in a gIucose oxidase-based system when the sample is a blood sample. Examples In the following examples, the utility and operability of the invention are demonstrated using a model system in which enzyme is coated onto the surface of one of two electrodes in an electrode pair. This rmodel does not require the capture of the enzyme, and therefore is useful for modeling the after-capture bhavir wih a limited number of variables, although it does not provide a full picture of the binding kinetics of an actual test system. Capture of enzyme using reagents in other enzyme sandwich, competition and displacement assays and the like is well known. fxamle t An electrochemical cell test strip comprising two gold electrodes separated by a double-sided adhesive layer was constructed using the method described in US Patent WO 2009/015291 PCT!US20081071073 Publication No. US-2005-0258035-Ai which is ircorporated herein by reference. An total of 100 nL enzyme solution (27 mg/mL glucose oxidase in 100 mM sodium citrate buffer pH 4.1) was dispensed onto the two electrodes and allowed to dr, Test strips were made with different ratios of electrode enzyme activity by apportioning the 100 nL enzyme solution between the two electrodes, with the balance made up with water. For example to make strips with 75% enzyme activity on electrode I and 25% enzyme activity on electrode 2, 100 nL of a mixture of 3 parts enzyme "olution to I part water was dispensed onto electrode 1, and 100 nL of a mixture of 1 part enzyme solution to 3 parts water was dispensed onto electrode 2. A solution of 100 mM -beta-D-glucose and 100 mM potassium ferricyanide in water was added to the electrochemical cell. Thie potential difference in open circuit (Fi .10), or current in short circuit (Fig.11), was recorded. it is noted that the period of time required to achieve clear differences in the signal at the different levels of asymmetry is short (less than seconds) thus making the invention suitable for rapid measurement. Fig. 12 shows that peak potential difference attained correlated with the ratio of enzyme activity dispensed on each electrode. Fig. 13 shows that peak current attained correlated with the ratio of enzyme activity dispensed on each electrode. Examine 2 By combining the detennination of the ratio of enzyme activity dispensed on each electrode (as in Example 1) with a determination of total enzyme activity, the amount of enzyme (il) present at one electrode can be determined independent of the amount (E2) present at the other electrode. Varing amounts of enzyme were dispensed on 2 surfaces that served as electrodes in a sandwich configuration as described in Exanmple 1. Ratio of enzyme activity present at the two electrodes (R=El /E2) was determined by measurement f current flowing in a short circuit configuration. Then the total enzyme activity (E=::::E1 +E2) was turned by mreasuring the current flowing with an applied potential difference. El and E2 were then calculated from the determined values of R and E,. Test strips containing a range of 0.25 microgram to 1 microgram of glucose oxidase were trade, with the enzyme distributed between the two electrodes such that R was between L5 and 19. These strips were made by dispensing a solution of 0.25 microgram to I WO 2009/015291 PCT!US2008107 1073 - 16 microgram glucose oxidase in 100 nL of 100 rmM sodium citrate buffer pH 4.1 onto electrode 1, and a solution of 0 to 0.4 microgram glucose oxidase in 100 nL of 100 mM sodium citrate buffer pH1 4.1 onto electrode 2, then allowing the dispensed solutions to dry. A solution of 100 nMYv beta-D-glucose and 100 mM potassium temcyanide in water was added to the electrochemical cell and current in short circuit was recorded for 5 seconds. followingg this, 300mn'V was applied for 1 Os. The current immediately prior to 5s was averaged to give To and the current immediately prior to 15s was averaged to give Is, (i.e. the measured I at 5s is L, and measured I at 15s was designated as Measured I (both f0 and 3o0) fio increasing amoni s of E, each with a variable ratio R, was recorded arid plotted, Fig, 14 shows a schematic of known total enzyme E, on the x-axis and measured I0 on the y-axis. The relationship between E, and I5 can be established from this plot, for example the data can be represented by the equation = m x E + n where m and n are the slope and intercept of the line. Fig. 1S shows a schemnatic plot of a known E, on x-axis and measured I on the y-axis. As shown, a farnily of lines is generated, one for each value of the ratio R. From Fig. 15, the linear re ationship of the 1, current vs E, is dependent on R, ic I= f(, g R) To fiuther establish this function, we plotted known log (R) vs slope (Fig. 16) for eacn line shown in Fig. 15. In addition, as shown in Fig. 17 we plotted known log (R.) and intercept for each line in ig. 15. Thus the function fcan fmrther be defined as the relationship 10= (a log R + b) x E, + (c log R - d) The parameters a and b can be calculated from the plots in Fig. 16 and the parameters .c and d from Fig 17. Once the parameters a., b, c, d, m and n have been determined, El can be calculated as follows: WO 2009/015291 PCT!US2008107 1073 1. Calculate E, from I300 E,=('30 - +m 2. Calculate P from I. and E, iog R =l b - x E d) - (a x E, + c) R.=1 " 3. Calculate E, from .E, and R E = (. xE)+ (1 + R) Fig. 18 shows calculated values of E, versus actual values of E!, for strips with varying amounts of enzyme on the two electrodes. Fig. 18 indicates that our estimated values for the enzyme dispensed on one electrode correlated well with the actual amount that was dispensed, and were independent of the amount of enzyne dispensed on the other electrode.
    权利要求:
    Claims (20)
    [1] 2. The method of claim 1, wherein the test cell comprises as one of the reagents that interact wit, the analyte an inmnobilized test reagent disposed or the surface of the first electrode, said immobilized test reagent comprising an analyte binding portion, and the mobile test reagent conprises an analyte-binding portion and the redox enzyme portion, whereby if analyze is present in the sample in the test cell, at least a portion of the mobile test reagent becomes immobilized on the first electrode thereby creating an asyimetrie distribution of redox enzyre concentration. between the first and second electrodes when analyte is present. WO 2009/015291 PCTIUS2008/071073 19 3 '-The method of claim 2 wherein the analyte binding portion of the immobilized test reagent and the analyze are an antibody-antigen pair or antibody/hapten pair.
    [2] 4. The method of clain 3, wherein the redox enzyme and the redox substrate comprises glucose and a mediator compound.
    [3] 5. The method of1 claim 2, wherein the analyte binding poroof the imobilized test reagent is a drug receptor.
    [4] 6. The method of claim 5, wherein the redox enzyme and the redox substrate com rises glucose and a mediator compound.
    [5] 7. The method of claim 2, wherein the analyte binding portion of the immobilized test reagent is a hormone receptor. . The method of claim 7, wherein the redox enzyme and the redox substrate comprises glucose and a mediator compound.
    [6] 9. The method of claim 1, wherein the analyte binding portion of the inunobilized test reagent and the analyte are an antibody-antigen pair or antibody/hapten pair.
    [7] 10. The method of claim 9, wherein the redox enzyme and the redox substrate comprises glucose and a mediator compounId
    [8] 11. The method of claim ,. wherein the analyte binding portion of the immobilized test reagent is a drug receptor.
    [9] 12. The method of claim i1, wherein the redox enzvnme and the redox substrate comprises glucose and a mediator compound, WO 2009/015291 PCT!US2008/07 1073
    [10] 13. The method of claim 1, wherein the analyte binding portion of the imunobilized test reagent is a hormone receptor,
    [11] 14. The method of claim 13, wherein the redox enzyme and the redox substrate comprises glucose and a mediator compound.
    [12] 15. An aparatus for determining an analyte in a sample comprising a test strip comprising first and second electrodes disposed to contact a test cell for receiving sample, said test strip farther a mobile test reagent comprising a redox enzyme portion, and reagents that interact with the mobile test reagent and/or the analyte, such that the mobile test reagent has a first distribution relative to the electrodes when analte is present in the sample and a second distribution relative to the electrodes when analyte is not present sample, one of said first and second distributions being asymmetric with respect to a line between the electrodes, and the other of the first and second distributions being symmetric or less asymmetric with respect to the line between the electrodes, and a circuit for detecting the synunetric or asymmetric distribution of the mobile test reagent in the test cell in the absence of an ap plied external potential or current., and means for communicating the detected distribution to a user.
    [13] 16. The apparatus of clain 15, wherein the circuit detects the potential difference between the first and second electrode.
    [14] 17. The apparatus of claim 15, wherein the circuit detects current flow in a circuit including the electrodes.
    [15] 18. The apparatus of any of claims 15 to 17, wherein the first and second electrodes are in a facing configuration.
    [16] 19. The apparatus of any of claims 15 to 1.7, wherein the first and second electrodes are in a side-by-side configuration. WO 2009/015291 PCT!US2008107 1073 - 21
    [17] 20. The apparatus in accordance with any of claims 15 to 19, wherein, the test cell comprises as one of the reagrents that interact with the analyze an immobilized test reagent disposed on the surface of te first electrode, said innobilized test agent comprising an analyte binding portion, and the mobile test reagent comprises an analyte-binding portion and the redox enzme portion. whereby it analyte is present in the sample in the test cell, at least a portion of the mobile test reagent becomes inrobilized on the first electrode thereby creating an asymnetric distribution of redox enzyme concentration between the first and second electrodes when analyte is present
    [18] 21. The apparatus of any of claims 15 to 20, wherein the analyte binding portion of the immobilized test reagent and the analyte are an antibody-antigen pair or antibody/hapten pair.
    [19] 22. The apparatus of any of claims 15 to 20, wherein the analyte binding portion of the immobilized test reagent is a drug receptor. 3. The apparatus of any of claims 15 to 20, wherein the analyte binding portion of the immobilized test reagent is a honnone receptor.
    [20] 24. The apparatus of any of claims 15 to 23, wherein the redox eyne arid the redox substrate comprises glucose and a mediator compound.
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    同族专利:
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    AU2013204779B2|2015-05-28|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US5413690A|1993-07-23|1995-05-09|Boehringer Mannheim Corporation|Potentiometric biosensor and the method of its use|
    法律状态:
    2015-09-24| FGA| Letters patent sealed or granted (standard patent)|
    优先权:
    申请号 | 申请日 | 专利标题
    US60/952,099||2007-07-26||
    AU2008279042A|AU2008279042B2|2007-07-26|2008-07-24|Electrochemical analyte detection apparatus and method|
    AU2013204779A|AU2013204779B2|2007-07-26|2013-04-12|Electrochemical analyte detection apparatus and method|AU2013204779A| AU2013204779B2|2007-07-26|2013-04-12|Electrochemical analyte detection apparatus and method|
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