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Publication numberUS20090026075 A1
Publication typeApplication
Application numberUS 12/179,446
Publication dateJan 29, 2009
Filing dateJul 24, 2008
Priority dateJul 26, 2007
Also published asCA2694045A1, CA2694045C, CN101802612A, EP2174135A1, EP2174135A4, EP2174135B1, EP2584044A2, EP2584044A3, EP2584044B1, WO2009015291A1
Publication number12179446, 179446, US 2009/0026075 A1, US 2009/026075 A1, US 20090026075 A1, US 20090026075A1, US 2009026075 A1, US 2009026075A1, US-A1-20090026075, US-A1-2009026075, US2009/0026075A1, US2009/026075A1, US20090026075 A1, US20090026075A1, US2009026075 A1, US2009026075A1
InventorsIan Harding, Sridhar Iyengar, Richard Williams
Original AssigneeAgamatrix, Inc.
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Electrochemical Analyte Detection Apparatus and Method
US 20090026075 A1
Abstract
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 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 of asymmetry can be done without the application of an external potential or current to the electrodes.
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Claims(24)
1. A method for determining an analyte in a sample, comprising the steps of:
(a) introducing the sample to a test cell comprising first and second electrodes, 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 analyte 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,
(b) supplying a redox substrate for the redox enzyme in the test cell, said redox substrate being acted upon by the redox enzyme to produce a chemical potential gradient between the first and second electrodes, wherein the magnitude of the chemical potential gradient is determined by the distribution of the mobile test reagent and thus on the presence of analyte, and
(c) detecting the chemical potential gradient between the first and second electrodes to determine analyte in the sample, wherein the formation and detection of the chemical potential gradient are performed without application of an external potential or current.
2. The method of claim 1, wherein
the test cell comprises as one of the reagents that interact with the analyte an immobilized test reagent disposed on the surface of the first electrode, said immobilized test reagent comprising an analyte binding portion, and
the mobile test reagent comprises an analyte-binding portion and the redox enzyme portion,
whereby if analyte 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 asymmetric distribution of redox enzyme concentration between the first and second electrodes when analyte is present.
3. The method of claim 2, wherein the analyte binding portion of the immobilized test reagent and the analyte are an antibody-antigen pair or antibody/hapten pair.
4. The method of claim 3, wherein the redox enzyme and the redox substrate comprises glucose and a mediator compound.
5. The method of claim 2, wherein the analyte binding portion of the immobilized test reagent is a drug receptor.
6. The method of claim 5, wherein the redox enzyme and the redox substrate comprises glucose and a mediator compound.
7. The method of claim 2, wherein the analyte binding portion of the immobilized test reagent is a hormone receptor.
8. The method of claim 7, wherein the redox enzyme and the redox substrate comprises glucose and a mediator compound.
9. The method of claim 1, wherein the analyte binding portion of the immobilized test reagent and the analyte are an antibody-antigen pair or antibody/hapten pair.
10. The method of claim 9, wherein the redox enzyme and the redox substrate comprises glucose and a mediator compound.
11. The method of claim 1 wherein the analyte binding portion of the immobilized test reagent is a drug receptor.
12. The method of claim 11, wherein the redox enzyme and the redox substrate comprises glucose and a mediator compound.
13. The method of claim 1, wherein the analyte binding portion of the immobilized test reagent is a hormone receptor.
14. The method of claim 13, wherein the redox enzyme and the redox substrate comprises glucose and a mediator compound.
15. An apparatus 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 further 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 analyte 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 symmetric or asymmetric distribution of the mobile test reagent in the test cell in the absence of an applied external potential or current,
and means for communicating the detected distribution to a user.
16. The apparatus of claim 15, wherein the circuit detects the potential difference between the first and second electrode.
17. The apparatus of claim 15, wherein the circuit detects current flow in a circuit including the electrodes.
18. The apparatus of claim 15, wherein the first and second electrodes are in a facing configuration.
19. The apparatus of claim 15, wherein the first and second electrodes are in a side-by-side configuration.
20. The apparatus in accordance with claim 15, wherein,
the test cell comprises as one of the reagents that interact with the analyte an immobilized test reagent disposed on the surface of the first electrode, said immobilized test reagent comprising an analyte binding portion, and
the mobile test reagent comprises an analyte-binding portion and the redox enzyme portion,
whereby if analyte 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 asymmetric distribution of redox enzyme concentration between the first and second electrodes when analyte is present.
21. The apparatus of claim 15, wherein the analyte binding portion of the immobilized test reagent and the analyte are an antibody-antigen pair or antibody/hapten pair.
22. The apparatus of claim 15, wherein the analyte binding portion of the immobilized test reagent is a drug receptor.
23. The apparatus of claim 15, wherein the analyte binding portion of the immobilized test reagent is a hormone receptor.
24. The apparatus of claim 15, wherein the redox enzyme and the redox substrate comprises glucose and a mediator compound.
Description
  • [0001]
    This application claims the benefit of U.S. Provisional Application No. 60/952,099 filed Jul. 26, 2007, which application is incorporated herein by reference for all purposes.
  • BACKGROUND OF THE INVENTION
  • [0002]
    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 analyte.
  • [0003]
    Analysis methods in which specific binding between an analyte and a reagent forms the basis for the assay are known. For example, in enzyme-linked immunoassay (EIA or ELISA) procedures, a sandwich is formed between an immobilized antibody and a mobile enzyme-antibody 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 U.S. Pat. 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 U.S. Pat. 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 form 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 mariner (See U.S. Pat. Nos. 4,868,131, 5,981,2798 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 U.S. Pat. Nos. 4,746,631 and 6,020,209).
  • [0004]
    Immunoassays in which detection of the analyte depends on an electrochemical measurement are also known. U.S. Pat. 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 is made in the presence of an applied potential. An applied potential is also used to measure current in the assay device disclosed in U.S. Pat. Nos. 5,198,367, 5,427,912. U.S. Pat. No. 5,494,831 discloses the application of a current and the measurement of changes in impedance that result in binding.
  • SUMMARY OF THE INVENTION
  • [0005]
    The present invention provides a new method and apparatus for electrochemical detection of analyte 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 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 of asymmetry can be done without the application of an external potential or current to the electrodes.
  • [0006]
    In a first embodiment of the invention, a sandwich type assay is utilized. In this embodiment, 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 immobilized test reagent. The mobile test reagent comprises an analyte-binding portion and a redox enzyme portion, and the immobilized test reagent comprises an analyte binding portion. If analyte 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 asymmetry in redox enzyme concentration 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 enzyme. 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 analyte in the sample. In this embodiment, the greater the asymmetry and the resulting potential gradient, the greater the amount of analyte in the sample.
  • [0007]
    In a second embodiment of the invention, a competition or displacement type of assay is utilized. In this assay, the redox enzyme 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 immobilized 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.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • [0008]
    FIGS. 1A and B show schematic representations of reduced mediator distribution between two electrodes when redox enzyme distribution is asymmetric (FIG. 1 A) or asymmetric (FIG. 1B).
  • [0009]
    FIGS. 2A and B illustrate the determination of asymmetric enzyme distribution at two electrodes using amperometry.
  • [0010]
    FIG. 3 shows a sandwich assay format useful in the present invention.
  • [0011]
    FIGS. 4 and 5 shows competition/displacement assay format useful in the present invention.
  • [0012]
    FIGS. 6A-C shows construction of a test cell with facing electrodes.
  • [0013]
    FIGS. 7A-C show examples of side-by-side electrode configurations.
  • [0014]
    FIG. 8 shows an external view of a meter in accordance with the invention.
  • [0015]
    FIG. 9 shows a circuit usable in the meter of the invention.
  • [0016]
    FIG. 10 shows potential difference in open circuit as a function of time for different levels of asymmetry in enzyme distribution between two electrodes.
  • [0017]
    FIG. 11 shows current in short circuit as a function of time for different levels of asymmetry in enzyme distribution between two electrodes.
  • [0018]
    FIG. 12 shows the correlation of peak potential difference with ratio of electrode enzyme activities
  • [0019]
    FIG. 13 shows the correlation of peak current with ratio of electrode enzyme activities
  • [0020]
    FIG. 14 shows a schematic plot of a known total enzyme Et on x-axis and measured I300 on the y-axis.
  • [0021]
    FIG. 15 shows a schematic plot of a known Et on x-axis and measured I300 on the v-axis.
  • [0022]
    FIG. 16 shows a plot of how log(R) affects the slope of I0 versus Et which is used to derive parameters a and b.
  • [0023]
    FIG. 17 shows a plot of log(R) affects the intercept of I0 versus Et which is used to derive parameters c and d.
  • [0024]
    FIG. 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
  • [0025]
    As used in the specification and claims of this application, the following definitions should be applied:
  • [0026]
    (a) “analyte” refers to a material of interest that may be present in a sample. Analytes that are detectable in the present invention are those that can be associated in a specific-binding 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 haptens such as peptides (for example hormones), proteins (for example, enzymes), drugs, pesticides, microorganisms, antibodies, and nucleic acids that can participate in sequence specific hybridization reactions with a complementary sequence.
  • [0027]
    (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.
  • [0028]
    (c) “determination of an analyte” 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 semi-quantitative evaluation, the result indicates whether or not analyte is present above some pre-defined threshold. In a quantitative evaluation, the result is a numerical indication of the amount of analyte present.
  • [0029]
    (d) the term “redox enzyme” refers to an enzyme that oxidizes or reduces a substrate. Such enzymes may generally be known as 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 enzymes in stable form is well known.
  • [0030]
    (e) the term “redox substrate” refers to a compound or combination of compounds that interact with the redox enzyme to 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 glucose 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 “redox substrate” is the combination of the substrate compound glucose and the mediator compound.
  • [0031]
    (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 gradient arises from a gradient in the ratio of 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. It will 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 asymmetry of the distribution of redox enzyme and not to any potential gradient that arises from the application of a potential difference or current flow between the electrodes.
  • [0032]
    (g) the phrase “detecting the chemical potential gradient between the first and second electrodes” refers to the detection of the chemical potential gradient in either an open or a closed circuit, using either potentiometric or amperometric measurements.
  • [0033]
    (h) the terms “enzyme activity” and “enzyme 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 catalytic capability 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 efficiency of the enzyme. The present invention actually measures asymmetry in enzyme activity, since the presence of inactive enzyme will not produce an asymmetry in redox substrate. However, 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.
  • [0034]
    (i) the term “immobilized on the first electrode” refers to immobilization directly or indirectly on the surface of the electrode, provided that the material immobilized becomes immobilized 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-analyte-antibody-enzyme sandwich, the enzyme is considered to be “immobilized on” the electrode even though there are several intervening moieties in the sandwich.
  • [0035]
    (j) the term “immobilized 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 immobilized test reagent and the surface of the electrode, or it may be a physical association as through the placement of the immobilized test reagent within a gel or membrane disposed on the surface of the electrode. The immobilized test reagent comprises a binding moiety which interacts with analyte to produce a change in the distribution of the redox enzyme (as part of the analyte-binding enzyme component) when analyte is present in a sample. In some embodiments, the immobilized reagent will 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:
      • electrode-(link)n-reagent with binding site for analyte
        is obtained, where n 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 analyte specific, with the addition of reagent that binds to the link and the analyte for any given analyte-specific test. This additional reagent could be added to the test strip at manufacture, or to the sample prior to application to the test device.
  • II. Theory of the Invention
  • [0037]
    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 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.
  • [0038]
    In common glucose measurement systems, 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 form of the enzyme, and produce reduced mediator. This process continues until either glucose or oxidized mediator is exhausted. If the enzyme is distributed asymmetrically within the sample cell, then the production of reduced mediator is also asymmetric, and the resulting asymmetry, in the distribution of reduced mediator persists for a period of time (determined by diffusion parameters) even after exhaustion of the limiting reagent.
  • [0039]
    When reduced mediator is present in a solution between two electrodes, the potential difference between the two electrodes is given by the Nernst equation
  • [0000]
    E = RT F log ( [ med red ] electrode 1 [ med ox ] electrode 1 [ med ox ] electrode 2 [ med red ] electrode 2 )
  • [0000]
    where E is the potential difference between electrodes 1 and 2, R is the gas constant, T is the absolute temperature and F is the Faraday constant. When there is no difference in the concentration of reduced mediator at the two electrodes (i.e. no chemical potential gradient) because there is no asymmetry; the mediator terms 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 measurement 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 can be simplified to:
  • [0000]
    E = RT F log ( [ med red ] electrode 1 [ med ox ] electrode 2 )
  • [0040]
    FIGS. 1A and B show schematic representations of reduced mediator distribution between two electrodes (1, 2) when redox enzyme distribution is asymmetric (FIG. 1 A) or asymmetric (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. If enzyme 3 is more active or concentrated at one electrode than the other (i.e., it has an asymmetric distribution 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 equal activity or concentrations of enzyme are present at both electrodes (i.e., it has a symmetric distribution as shown in FIG. 1B), the concentration of reduced mediator at each electrode will be equal, and the electrodes will be at equal electrical potential and the measured potential difference will be 0.
  • [0041]
    If, instead of potentiometry, the electrodes are connected in short-circuit, the asymmetric 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.
  • [0042]
    FIGS. 2A and B illustrate the use of these principles to quantify the ratio of concentrations of enzyme 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 consumed at that electrode and produced at the other electrode to maintain a balanced chemical potential, and current will flow.
  • III. Practical Applications
  • [0043]
    The theory of the present invention is applicable to the practical application 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 analyte 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 Table 1.
  • [0000]
    Analyte Analyte receptor binding component
    of Analyte-
    specific enzyme
    disease-specific disease associated- antibody that
    antibody, for example antigen, such as recognizes human
    an antibody to peptide epitope found antibodies,
    influenza, disease on an influenza of aptamers
    causing organism interest, aptamers
    amplified nucleic nucleic acid nucleic acid sequence
    acid sequence complementary to the complementary
    including a suspected target to the known primer
    suspected target
    and a known primer
    hormone hormone receptor, hormone receptor,
    apatamer or antibody apatamer or antibody
    drug drug receptor, hormone receptor,
    aptamer or antibody apatamer or antibody
    pesticides pesticide binding pesticide binding
    proteins, aptamers, proteins, apatmers
    antibodies to pesticides antibodies that recognize
    the antibodies to
    pesticides
  • [0044]
    As will be understood in 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 Domain Antibodies (dAb) as described at www.domantis.com. Immunoassays for drugs are described in U.S. Pat. Nos. 7,220,842, 5,677,132 and 5,618,926. Immunoassays for pesticides and pesticide degradation products are described in U.S. Pat. No. 6,635,434. Drug and hormone receptors may be native hormone receptors, or they may be engineered species with similar binding properties as are known in the art. (See U.S. Pat. Nos. 7,214,511, and 6,806,359, Rasmussen et al, J. Biol. Chem., Vol. 276, Issue 7, 4717-4723, Feb. 16, 2001). US Patent Publication 20050130208 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 aptmers are listed on the database of the Ellington research group at http://aptamer.icmb.utexas.edu/. Enzyme-aptamer coupling has been described by Mir et al. “Different Strategies To Develop an Electrochemical Thrombin Aptasensor.” Electrochemical Communication 8 (9006): 505-511.
  • [0045]
    The components of the reaction are combined in such a way that they result in a difference in distribution symmetry of the redox enzyme that is dependent on the presence or absence of analyte in a sample. Various device formats can achieve this result.
  • [0046]
    A. Sandwich Format
  • [0047]
    FIG. 3 shows two electrodes 31 and 32. Electrode 31 has analyte-specific receptors 33 immobilized 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 analyte 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-specific enzyme species 35 sandwich. The activity of the enzyme results in an increased concentration of one form of the redox substrate (in the case of FIG. 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.
  • [0048]
    B. Competition/Displacement Format
  • [0049]
    FIGS. 4 and 5 show a competition format for an assay device in accordance with the invention. The sample cell 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 mimetic. In the absence of analyte, as shown in FIG. 4, redox enzyme 45 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 analyte receptors for the redox enzyme. As shown in FIG. 5, when analyte 44 is present, it competes with redox enzyme 45 for the analyte receptor 43, resulting in the displacement of at least some of the redox enzyme 45 from the analyte receptors 43, creating a less asymmetric distribution of the redox enzyme and thus the ability to determine analyte in a sample.
  • [0050]
    C. Device Construction—Sample Cell with Facing Electrodes
  • [0051]
    FIGS. 6A-C show a sample cell for a device in accordance with the invention, constructed using facing electrodes. The device is formed from a top layer 61 and bottom layer 62, each having a conductive surface (61′, 62′) facing the interior 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 potentiometric or amperometric measurements. As shown in FIG. 6B, the conductive layer 63 has an opening therein that together with the facing conductive surfaces 61′ and 62′ defines a sample cell. This 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 connectors are known, and this also is not critical to the practicing of the present invention.
  • [0052]
    In an embodiment of the device of the invention, one of the conductive surfaces (61′ or 62′) has the analyte receptor (immobilized 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.
  • [0053]
    The sample cell portion as shown in FIGS. 6A-C is generally constructed as a single use, disposable component.
  • [0054]
    D. Device Construction—Side-by-Side Electrodes
  • [0055]
    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 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, asymmetry in redox enzyme distribution between one electrode 71 and the other electrode 72 results in a measurable chemical potential gradient between the two electrodes.
  • [0056]
    The sample cell portions as shown in FIGS. 7A-C are generally constructed as a single use, disposable component.
  • [0057]
    E. Device Construction—Communication of Results
  • [0058]
    In addition to the sample cell, the device of the invention has a means for communicating 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 not present), or to a specific numerical value for the amount of analyte. The sophistication of the communication means varies accordingly.
  • [0059]
    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 in response to the passage of current or the 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.
  • [0060]
    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 sample or test strip, and for 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 geometry and analyte.
  • [0061]
    FIG. 8 shows an external view of a meter in accordance with the invention. As shown, the meter has a housing 81 having a display 82 and one or more control interfaces 83 (for example power buttons, or scroll wheels etc). The housing has a slot 84 for receiving a test device. Internal to the housing 81 is circuitry for applying a potential or current to the electrodes of the test device when a sample is applied. This may be done following an initialization signal from a user, or following an automated detection of test device insertion and sample application.
  • [0062]
    Suitable circuits usable in the meter of the invention are known in the art, for example from US Patent Publication No. US 2005-0265094 A1, which is incorporated herein by reference. One such circuit is shown in FIG. 9. In FIG. 9, an ideal voltmeter 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 open the cell 939 is “floating” as to at least one of its electrodes, permitting a voltage measurement that is unaffected by signals at the amplifier 935. The switch 944 may be a mechanical switch (e.g. a relay) or an 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 high resistance.
  • [0063]
    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 application of either or both of a potential difference or a current.
  • [0064]
    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 potential prior to the measurement (particularly in a test device with facing electrode construction) can result in a more rapid measurement time by effectively driving analyte and/or analyte-binding enzyme component towards the electrode with the immobilized reagent using what is in essence electrophoresis, provided that the analyte or analyte-binding enzyme component is charged under the conditions (particularly pH) found within the test device. A step function or a sine wave passing from negative to positive potential differences could also effect something similar to washing in situ within the test device.
  • VI. Advantages of the Invention
  • [0065]
    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 immunoassay 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.
  • [0066]
    The present invention can make use of small samples of blood, tears, saliva, or sweat, with minimal invasiveness to the subject. (samples of less than 10 ul, preferably less than 1 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 hour or more. The small volume of the test devices of the invention and the sensitivity of the test to small differences 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 1 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.
  • [0067]
    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 system. For example, endogenous glucose can be used as a component of the redox substrate in a glucose oxidase-based system when the sample is a blood sample.
  • EXAMPLES
  • [0068]
    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 model does not require the capture of the enzyme, and therefore is useful for modeling the after-capture behavior with 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.
  • Example 1
  • [0069]
    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 Publication No. US-2005-0258035-A1 which is incorporated 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 dry. 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 1 and 25% enzyme activity on electrode 2, 100 nL of a mixture of 3 parts enzyme solution to 1 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. The potential difference in open circuit (FIG. 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 15 seconds) thus making the invention suitable for rapid measurement.
  • [0070]
    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.
  • Example 2
  • [0071]
    By combining the determination 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 (E1) present at one electrode can be determined independent of the amount (E2) present at the other electrode.
  • [0072]
    Varying amounts of enzyme were dispensed on 2 surfaces that served as electrodes in a sandwich configuration as described in Example 1. Ratio of enzyme activity present at the two electrodes (R=E1/E2) was determined by measurement of the current flowing in a short circuit configuration. Then the total enzyme activity (Et=E1+E2) was determined by measuring the current flowing with an applied potential difference. E1 and E2 were then calculated from the determined values of R and Et.
  • [0073]
    Test strips containing a range of 0.25 microgram to 1 microgram of glucose oxidase were made, with the enzyme distributed between the two electrodes such that R was between 1.5 and 19. These strips were made by dispensing a solution of 0.25 microgram to 1 microgram glucose oxidase in 100 nL of 100 mM 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 pH 4.1 onto electrode 2, then allowing the dispensed solutions to dry.
  • [0074]
    A solution of 100 mM beta-D-glucose and 100 mM potassium ferricyanide in water was added to the electrochemical cell and current in short circuit was recorded for 5 seconds. Following this, 300 mV was applied for 10 s. The current immediately prior to 5 s was averaged to give I0 and the current immediately prior to 15 s was averaged to give I300 (i.e. the measured I at 5 s is I0 and measured I at 15 s was designated as I300).
  • [0075]
    Measured I (both I0 and I300) for increasing amounts of Et each with a variable ratio R, was recorded and plotted.
  • [0076]
    FIG. 14 shows a schematic of known total enzyme Et on the x-axis and measured I300 on the y-axis. The relationship between Et and I300 can be established from this plot, for example the data can be represented by the equation
  • [0000]

    I 300 =mE t +n
  • [0000]
    where m and n are the slope and intercept of the line.
  • [0077]
    FIG. 15 shows a schematic plot of a known Et on x-axis and measured I0 on the y-axis. As shown, a family of lines is generated, one for each value of the ratio R. From FIG. 15, the linear relationship of the I0 current vs Et is dependent on R, i.e.
  • [0000]

    I 0 =f(E t,log R)
  • [0078]
    To further establish this function, we plotted known log(R) vs slope (FIG. 16) for each line shown in FIG. 15. In addition, as shown in FIG. 17 we plotted known log(R) and intercept for each line in FIG. 15.
  • [0000]
    Thus the function f can further be defined as the relationship
  • [0000]

    I 0=(a log R+b)E t+(c log R+d)
  • [0079]
    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, E1 can be calculated as follows:
  • [0000]
    1. Calculate Et from I300
  • [0000]

    E t=(I 300 −n)m.
  • [0000]
    2. Calculate R from I0 and Et
  • [0000]

    log R=(I 0 −bE t −d)(aE t +c)
  • [0000]

    R=10log R
  • [0000]
    3. Calculate E1 from Et and R
  • [0000]

    E 1=(RE t)(1+R)
  • [0080]
    FIG. 18 shows calculated values of E1 versus actual values of E1, 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 enzyme dispensed on the other electrode.
Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US3654090 *Sep 24, 1968Jul 20, 1982 Title not available
US4169012 *Sep 9, 1977Sep 25, 1979Akzona IncorporatedStabilized peroxidase reagent for enzyme immunoassay
US4642285 *May 2, 1984Feb 10, 1987Diamedix CorporationSandwich EIA for antigen
US4746631 *May 9, 1985May 24, 1988Ultra Diagnostics CorporationImmunoassay method, device, and test kit
US4868131 *Jul 13, 1987Sep 19, 1989Fuji Photo Film Co., Ltd.Competitive binding immunoassay process
US4943522 *Aug 10, 1988Jul 24, 1990QuidelLateral flow, non-bibulous membrane assay protocols
US5149630 *Feb 9, 1988Sep 22, 1992Forrest Gordon CMethods of assay
US5198367 *Jun 9, 1989Mar 30, 1993Masuo AizawaHomogeneous amperometric immunoassay
US5413690 *Jul 23, 1993May 9, 1995Boehringer Mannheim CorporationPotentiometric biosensor and the method of its use
US5427912 *Aug 27, 1993Jun 27, 1995Boehringer Mannheim CorporationElectrochemical enzymatic complementation immunoassay
US5494831 *Aug 30, 1993Feb 27, 1996Hughes Aircraft CompanyElectrochemical immunosensor system and methods
US5618926 *May 26, 1994Apr 8, 1997Hoffmann-La Roche Inc.Immunoassay reagents
US5656503 *Sep 15, 1994Aug 12, 1997Unilever Patent Holdings B.V.Test device for detecting analytes in biological samples
US5677132 *Jun 5, 1995Oct 14, 1997Strahilevitz; MeirImmunological assay reagent package
US5708247 *Feb 14, 1996Jan 13, 1998Selfcare, Inc.Disposable glucose test strips, and methods and compositions for making same
US5712172 *May 18, 1995Jan 27, 1998Wyntek Diagnostics, Inc.One step immunochromatographic device and method of use
US5766961 *Nov 23, 1993Jun 16, 1998Quidel CorporationOne-step lateral flow nonbibulous assay
US5981298 *Dec 13, 1995Nov 9, 1999Surmodics, Inc.Immunoassay device and method
US5989921 *Jul 2, 1997Nov 23, 1999Carter Wallace, Inc.Test device and method for colored particle immunoassay
US6020209 *Apr 28, 1997Feb 1, 2000The United States Of America As Represented By The Secretary Of The NavyMicrocapillary-based flow-through immunosensor and displacement immunoassay using the same
US6110696 *Jun 26, 1995Aug 29, 2000Roche Diagnostics CorporationElectrochemical enzyme assay
US6478938 *Jun 14, 2000Nov 12, 2002Bio Digit Laboratories CorporationElectrochemical membrane strip biosensor
US6635434 *Jul 21, 2000Oct 21, 2003Exiqon A/SImmunoassay for pesticides and their degradation products
US6806359 *Jun 5, 1995Oct 19, 2004The Salk Institute For Biological StudiesHormone receptor compositions and methods
US7214511 *Jun 5, 1995May 8, 2007The Salk Institute For Biological StudiesHormone receptor compositions and methods
US7220842 *Apr 5, 2004May 22, 2007Dade Behring Inc.Immunoassays for buprenorphine and norbuprenorphine
US20020090738 *Aug 30, 2001Jul 11, 2002I-Stat CorporationSystem and method of microdispensing and arrays of biolayers provided by same
US20030143598 *Nov 8, 2002Jul 31, 2003Viswanadham GarimellaBioconjugate-nanoparticle probes
US20040245101 *Aug 12, 2002Dec 9, 2004Itamar WillnerSelf-powered biosensor
US20050130208 *Nov 16, 2004Jun 16, 2005The Trustees Of Columbia UniversityDisplacement assay for detection of small molecules
US20050258035 *May 20, 2005Nov 24, 2005Agamatrix, Inc.Electrochemical Cell and Method of Making an Electrochemical Cell
US20050265094 *Apr 15, 2005Dec 1, 2005Agamatrix, Inc.Measuring device and methods for use therewith
US20060160100 *Jan 19, 2005Jul 20, 2006Agency For Science, Technology And ResearchEnzymatic electrochemical detection assay using protective monolayer and device therefor
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US7875047Jan 25, 2007Jan 25, 2011Pelikan Technologies, Inc.Method and apparatus for a multi-use body fluid sampling device with sterility barrier release
US7892183Feb 22, 2011Pelikan Technologies, Inc.Method and apparatus for body fluid sampling and analyte sensing
US7901365Mar 8, 2011Pelikan Technologies, Inc.Method and apparatus for penetrating tissue
US7909774Mar 22, 2011Pelikan Technologies, Inc.Method and apparatus for penetrating tissue
US7909775Mar 22, 2011Pelikan Technologies, Inc.Method and apparatus for lancet launching device integrated onto a blood-sampling cartridge
US7909777Sep 29, 2006Mar 22, 2011Pelikan Technologies, IncMethod and apparatus for penetrating tissue
US7909778Apr 20, 2007Mar 22, 2011Pelikan Technologies, Inc.Method and apparatus for penetrating tissue
US7914465Feb 8, 2007Mar 29, 2011Pelikan Technologies, Inc.Method and apparatus for penetrating tissue
US7938787May 10, 2011Pelikan Technologies, Inc.Method and apparatus for penetrating tissue
US7959582Mar 21, 2007Jun 14, 2011Pelikan Technologies, Inc.Method and apparatus for penetrating tissue
US7976476Jul 12, 2011Pelikan Technologies, Inc.Device and method for variable speed lancet
US7981055Dec 22, 2005Jul 19, 2011Pelikan Technologies, Inc.Tissue penetration device
US7981056Jun 18, 2007Jul 19, 2011Pelikan Technologies, Inc.Methods and apparatus for lancet actuation
US7988644Aug 2, 2011Pelikan Technologies, Inc.Method and apparatus for a multi-use body fluid sampling device with sterility barrier release
US7988645Aug 2, 2011Pelikan Technologies, Inc.Self optimizing lancing device with adaptation means to temporal variations in cutaneous properties
US8007446Aug 30, 2011Pelikan Technologies, Inc.Method and apparatus for penetrating tissue
US8016774Dec 22, 2005Sep 13, 2011Pelikan Technologies, Inc.Tissue penetration device
US8062231Nov 22, 2011Pelikan Technologies, Inc.Method and apparatus for penetrating tissue
US8079960Oct 10, 2006Dec 20, 2011Pelikan Technologies, Inc.Methods and apparatus for lancet actuation
US8123700Jun 26, 2007Feb 28, 2012Pelikan Technologies, Inc.Method and apparatus for lancet launching device integrated onto a blood-sampling cartridge
US8157748Jan 10, 2008Apr 17, 2012Pelikan Technologies, Inc.Methods and apparatus for lancet actuation
US8162853Apr 24, 2012Pelikan Technologies, Inc.Tissue penetration device
US8197421Jul 16, 2007Jun 12, 2012Pelikan Technologies, Inc.Method and apparatus for penetrating tissue
US8197423Dec 14, 2010Jun 12, 2012Pelikan Technologies, Inc.Method and apparatus for penetrating tissue
US8202231Apr 23, 2007Jun 19, 2012Sanofi-Aventis Deutschland GmbhMethod and apparatus for penetrating tissue
US8206317Dec 22, 2005Jun 26, 2012Sanofi-Aventis Deutschland GmbhTissue penetration device
US8206319Jun 26, 2012Sanofi-Aventis Deutschland GmbhTissue penetration device
US8211037Jul 3, 2012Pelikan Technologies, Inc.Tissue penetration device
US8216154Jul 10, 2012Sanofi-Aventis Deutschland GmbhTissue penetration device
US8221334Jul 17, 2012Sanofi-Aventis Deutschland GmbhMethod and apparatus for penetrating tissue
US8235915Dec 18, 2008Aug 7, 2012Sanofi-Aventis Deutschland GmbhMethod and apparatus for penetrating tissue
US8251921Jun 10, 2010Aug 28, 2012Sanofi-Aventis Deutschland GmbhMethod and apparatus for body fluid sampling and analyte sensing
US8262614Jun 1, 2004Sep 11, 2012Pelikan Technologies, Inc.Method and apparatus for fluid injection
US8267870May 30, 2003Sep 18, 2012Sanofi-Aventis Deutschland GmbhMethod and apparatus for body fluid sampling with hybrid actuation
US8282576Sep 29, 2004Oct 9, 2012Sanofi-Aventis Deutschland GmbhMethod and apparatus for an improved sample capture device
US8282577Oct 9, 2012Sanofi-Aventis Deutschland GmbhMethod and apparatus for lancet launching device integrated onto a blood-sampling cartridge
US8296918Aug 23, 2010Oct 30, 2012Sanofi-Aventis Deutschland GmbhMethod of manufacturing a fluid sampling device with improved analyte detecting member configuration
US8333710Dec 18, 2012Sanofi-Aventis Deutschland GmbhTissue penetration device
US8337419Oct 4, 2005Dec 25, 2012Sanofi-Aventis Deutschland GmbhTissue penetration device
US8337420Mar 24, 2006Dec 25, 2012Sanofi-Aventis Deutschland GmbhTissue penetration device
US8337421Dec 16, 2008Dec 25, 2012Sanofi-Aventis Deutschland GmbhTissue penetration device
US8343075Dec 23, 2005Jan 1, 2013Sanofi-Aventis Deutschland GmbhTissue penetration device
US8360991Dec 23, 2005Jan 29, 2013Sanofi-Aventis Deutschland GmbhTissue penetration device
US8360992Nov 25, 2008Jan 29, 2013Sanofi-Aventis Deutschland GmbhMethod and apparatus for penetrating tissue
US8366637Feb 5, 2013Sanofi-Aventis Deutschland GmbhMethod and apparatus for penetrating tissue
US8372016Feb 12, 2013Sanofi-Aventis Deutschland GmbhMethod and apparatus for body fluid sampling and analyte sensing
US8382682Feb 26, 2013Sanofi-Aventis Deutschland GmbhMethod and apparatus for penetrating tissue
US8382683Feb 26, 2013Sanofi-Aventis Deutschland GmbhTissue penetration device
US8388551May 27, 2008Mar 5, 2013Sanofi-Aventis Deutschland GmbhMethod and apparatus for multi-use body fluid sampling device with sterility barrier release
US8403864May 1, 2006Mar 26, 2013Sanofi-Aventis Deutschland GmbhMethod and apparatus for penetrating tissue
US8414503Apr 9, 2013Sanofi-Aventis Deutschland GmbhMethods and apparatus for lancet actuation
US8430828Jan 26, 2007Apr 30, 2013Sanofi-Aventis Deutschland GmbhMethod and apparatus for a multi-use body fluid sampling device with sterility barrier release
US8435190Jan 19, 2007May 7, 2013Sanofi-Aventis Deutschland GmbhMethod and apparatus for penetrating tissue
US8439872Apr 26, 2010May 14, 2013Sanofi-Aventis Deutschland GmbhApparatus and method for penetration with shaft having a sensor for sensing penetration depth
US8491500Apr 16, 2007Jul 23, 2013Sanofi-Aventis Deutschland GmbhMethods and apparatus for lancet actuation
US8496601Apr 16, 2007Jul 30, 2013Sanofi-Aventis Deutschland GmbhMethods and apparatus for lancet actuation
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US8562545Dec 16, 2008Oct 22, 2013Sanofi-Aventis Deutschland GmbhTissue penetration device
US8574168Mar 26, 2007Nov 5, 2013Sanofi-Aventis Deutschland GmbhMethod and apparatus for a multi-use body fluid sampling device with analyte sensing
US8574895Dec 30, 2003Nov 5, 2013Sanofi-Aventis Deutschland GmbhMethod and apparatus using optical techniques to measure analyte levels
US8579831Oct 6, 2006Nov 12, 2013Sanofi-Aventis Deutschland GmbhMethod and apparatus for penetrating tissue
US8622930Jul 18, 2011Jan 7, 2014Sanofi-Aventis Deutschland GmbhTissue penetration device
US8636673Dec 1, 2008Jan 28, 2014Sanofi-Aventis Deutschland GmbhTissue penetration device
US8641643Apr 27, 2006Feb 4, 2014Sanofi-Aventis Deutschland GmbhSampling module device and method
US8641644Apr 23, 2008Feb 4, 2014Sanofi-Aventis Deutschland GmbhBlood testing apparatus having a rotatable cartridge with multiple lancing elements and testing means
US8652831Mar 26, 2008Feb 18, 2014Sanofi-Aventis Deutschland GmbhMethod and apparatus for analyte measurement test time
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US8845549Dec 2, 2008Sep 30, 2014Sanofi-Aventis Deutschland GmbhMethod for penetrating tissue
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Classifications
U.S. Classification204/403.14, 205/777.5
International ClassificationG01N33/50
Cooperative ClassificationG01N33/5438, G01N2333/902, C12Q1/001
European ClassificationC12Q1/00B, G01N33/543K2B
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