Apoenzyme reactivation electrochemical detection method and assay

[0001] This application claims the priority benefit of provisional application 60/549,691, “Apoenzyme reactivation electrochemical detection method and assay”, filed Mar. 3, 2004.

[0002] 1. Field of the Invention

[0003] The field of the invention is improved electrochemical dry reagents useful for instrumented tests for coagulation, immunoassays, and other analytes.

[0004] 2. Description of the Related Art

[0005] There is a wide range of chemical entities (test ligands, test analytes) where rapid identification of the presence and relative levels of the entity are highly important. In medicine, it is often critically important to rapidly identify medical analytes such as hormones, drugs, pathogens, and physiological enzymes. In agricultural areas, it is often important to identify trace levels of contaminants or pathogens, such as harmful bacteria, adulterants, or other undesirable contaminants. In environmental studies, it is often important to rapidly identify trace levels of pollutants. For military applications, identification of trace levels of toxic agents is also important.

[0006] As a result of this common need for rapid identification of test ligands, various different rapid detection schemes have been devised. These include general-purpose detection methodologies, such as chromatography and mass spectrometry, and more specialized detection methodologies, such as the various diagnostic chemical methodologies that employ test reagents designed to produce detectable signals upon chemical reaction with the test analyte. The present application is focused on this latter type of rapid chemical test methods.

[0007] Although complex automated chemical analyzers exist, using liquid chemical reagents, which can rapidly analyze many different types of test ligand, such devices tend to be expensive, delicate, and often require skilled users. As a result, an alternative approach, using premixed reagents stored in a dry form, and reconstituted by the fluid in the test analyte's sample, has become quite popular in recent years. Such tests are referred to generically as “dry reagent tests”.

[0008] There are two basic categories of dry reagent test. Dry reagent tests that produce a detectable change in the electrochemical potential of an electrode are typically referred to as electrochemical dry reagent tests, and dry reagent tests that produce a detectable optical change in the optical characteristics of the reagent (change in color, change in fluorescence, etc.) are typically referred to by the type of optical change used in the assay (e.g. colorimetric tests, fluorescence tests, etc.).

[0009] Due to the high demand for simple blood glucose tests for diabetics, electrochemical dry reagent tests have become increasingly popular in recent years. In contrast to optical dry reagent tests, which require both precise optical measuring equipment, and precise ways to translate the optical signal into a final answer, electrochemical tests usually can function with simpler equipment. The need for a precise optical section is eliminated, and the electrochemical signal generated by the reagent can be converted to a final answer using simple and low cost electronic circuits. As a result, electrochemical blood glucose tests have become a multi-billion dollar a year industry. A wide variety of electrochemical methods have been devised, and due to the high economic activity in this space, the technology is now in a well-developed and mature state.

[0010] At present, not all analytes can be measured by electrochemical means. This is because in many cases, simple and practical ways to transduce the chemical signal produced by the test reagent-test analyte reaction over to an electrochemical signal capable of being detected at a test reagent electrode have not been identified. As a result, many useful assays, such as immunochemical assays, enzyme substrate assays, and the like must currently be performed using older optical dry reagent technology. Because, in many cases, this technology is not as fully developed as modern dry reagent electrochemical technology, many of these assays are currently being performed using the older, more expensive, and less reliable optical format. Additionally, the lower volume of many of these assays has made it uneconomic to develop improvements, creating many “orphan” tests that have not improved much beyond the original, previous generation, optical technology.

[0011] One example of an “orphan” optical dry reagent technology for immunochemical analytes is the Apoenzyme Reactivation Immunoassay (ARIS). The ARIS concept is based upon the formation of a unique type of hybrid molecule. This hybrid molecule consists of an apoenzyme reactivation factor (also called an enzyme “cofactor”, “coenzyme” or “prosthetic group”) that is chemically conjugated to a reagent version of the test ligand (antigen) molecule. This conjugation creates a hybrid molecule containing both an enzyme reactivation factor, and a reagent version of the test ligand (antigen) molecule of interest. The ARIS assay also contains reagent antibodies that bind to this hybrid molecule, and an inactive apoenzyme. In the absence of test analytes, the reagent antibodies bind to the hybrid molecule and prevent the molecule's apoenzyme reactivation factor from reactivating the apoenzyme. In the presence of test analytes, however, the test ligands compete for binding to the reagent antibodies, and displace the hybrid molecules away from the reagent antibodies. The now unbound apoenzyme reactivation factors are now free to reactivate the apoenzyme, which in turn produces a colored reaction product. Although, in some cases, such tests can be observed directly by eye without need of automated instrumentation, direct visual methods have limited accuracy. As a result, such tests are more commonly read by optical meters. However, as previously discussed, optical metering systems tend to be more complex and more susceptible to inaccuracy, relative to electrochemical metering systems, and thus are less economically attractive. Thus methods to translate optical ARIS immunochemical tests to the more mature electrochemical format are desirable.

[0012] A second example of “orphan” dry reagent technology is blood coagulation monitoring assays. Here a variety of dry reagent tests exist, including optical tests, and non-standard electrochemical tests. The later work by principles that are substantially different than the more common enzyme based electrochemical biosensors, and thus are not at the same level of technological maturity as most enzyme based electrochemical biosensors.

[0013] At present, all coagulation tests are significantly more expensive than electrochemical blood glucose tests, and all require more complex and sophisticated metering systems. Thus methods to translate blood coagulation tests to the more mature and standard enzyme based electrochemical biosensors are also desirable.

[0014] Prior art for electrochemically based prothrombin time assays may be found in U.S. Pat. Nos. 6,066,504; 6,060,323; 6,046,051; 6,673,622 by Jina et. al, U.S. Pat. No. 6,352,630 by Frenkel et. al., and U.S. Pat. No. 6,620,310 by O'hara et. al.

[0015] Prior art for thrombin substrate based coagulation assays may be found in U.S. Pat. Nos. 5,580,744 and 5,418,141 by Zweig.

[0016] Prior art for dry reagent homogeneous apoenzyme reactivation (ARIS) chemistry and immunochemistry can be found in U.S. Pat. Nos. 3,817,837; 4,134,792; 4,213,893; 4,238,565; 4,318,983; 4,495,281 and others.

[0017] Prior art for enzyme based electrochemical biosensors for blood glucose can be found a variety of patents, including many assigned to Genetics International, Medisense, E. Heller, & Company, Therasense, Selfcare, Boehringer Mannheim, and others. These include U.S. Pat. Nos. 4,545,382; 4,711,245; 4,758,323; 5,262,035; 5,262,305; 5,264,105; 5,286,362; 5,312,590; 5,320,725; 5,509,410; 5,628,890; 5,682,884; 5,708,247; 5,727,548; 5,820,551; 5,951,836; 6,134,461 and 6,143,164;

[0018] The invention discloses methods in which dry reagent electrochemical technology, which is in a relatively mature form due to the extensive amount of development pioneered by the blood glucose monitoring industry, may be simply adapted to perform tests for blood coagulation, enzymatic activity, or immunochemical assays for antigens present in a fluid sample.

[0019] In the simplest form, the present invention discloses the utility of combining dry reagent electrochemical enzyme based biosensors with apoenzyme reactivation technology to produce a novel combination dry reagent test platform technology capable of detecting a wide range of different analytes.

[0020] Here, enzyme based electrochemical dry reagent biosensors are produced according to essentially normal methods, but with the substitution of an apoenzyme, or otherwise inactivated form of an electrochemical detection enzyme, in place of the normal active form. Thus, for example, in the case where dry reagent electrochemical glucose tests are used as the basis for the assay, apoglucose oxidase may be used in place of glucose oxidase.

[0021] The inactive electrochemical sensor is turned into an analyte specific sensor through the aid of ARIS like enzyme activation factor (cofactor)-test analyte detection moiety conjugates. These conjugates will generally consist of the appropriate enzyme activation factor (for example the FAD prosthetic group in the case of glucose oxidase), typically linked to a test-analyte detection moiety by a covalent link or high affinity non-covalent link.

[0022] In addition to the enzyme activation factor (cofactor), the test analyte detection moiety has two other parts; an analyte detector part (region) and a blocker part (or region). The moiety's “detector” region is the part of the molecule that interacts specifically with the test ligand, and has a state that is altered as a result of interacting with the test ligand. For example, if the test ligand is a protease, the detector region may be a protease substrate peptide. For an immunoassay, the detector region is a reagent antigen-antibody pair that is disrupted by the presence of antigenic test ligands.

[0023] The blocker part of the analyte detection moiety is an entity that, in the absence of interactions between the moiety's detector region and the test ligand, acts to prevent the enzyme activation factor from binding to the inactivated enzyme (apoenzyme) on the sensing electrode. For example, in the case of a protease substrate assay, the “blocker” region may consist of a larger molecule that acts to sterically prevent the enzyme activation factor part of the conjugate from binding to the inactivated enzyme on the electrode. In the case of an immunochemical assay, the “blocker” region may be an antibody that binds to the conjugate's antigenic “detector” region, and acts to sterically block the enzyme activation factor from binding to the inactivated electrode enzyme. In some cases, the blocker regions may also act to physically separate the enzyme cofactors or prosthetic groups from the region of the assay that contains the apoenzymes. For example, in the case of an immunochemical assay, the “blocker” antibody may be bound to a membrane, bead, or other structure that is close to, but physically distinct from, the region of the assay where the apoenzyme is located.

[0024] Most electrochemically active enzymes require cofactors to operate, and the present invention can work with nearly all of the many different varieties of known enzyme electrode designs, including designs with direct electron transfer between the enzyme and the electrode, electron transfer mediated by diffusion mediators, electron transfer by dissolved enzymes at mediator functionalized electrodes, polymer and inorganic matrix immobilized enzymes contacted by co-immobilized mediators, electrochemical transfer by interprotein electron transfer, and other means commonly used to produce enzyme electrodes (see Katz et. al., Mediated electron-transfer between redox-enzymes and electrode supports. In: Encyclopedia of Electrochemistry, Vol. 9: Bioelectrochemistry, G. S. Wilson, (Ed.), A. J. Bard, M. Stratmann (Editors-in-Chief), Wiley-VCH GmbH, Weinheim, Germany 2002, Chapter 17, pp. 559–626),

[0025] In an alternative configuration of the present invention, unbound (not complexed with their corresponding apoenzyme) prosthetic groups (such as FAD) may be electronically “wired” to electrodes via a molecular link that enables electron transport. Again using apoglucose oxidase as an example, in this alternative configuration, the apoglucose oxidase molecules may themselves be conjugated to an ARIS like test analyte detection moiety, which is in turn bound to a blocker entity. In this case, apoglucose oxidase will be released from this blocker by the action of the test analyte. The liberated apoglucose oxidase can then bind to the wired unbound FAD groups, reassociate, and then start generating an electrical signal that increases in response to higher levels of the test analyte. Thus, for an immunoassay, an antigenic test analyte can displace a conjugate of apoglucose-oxidase and reagent-antigen from a bead or blocker bound antibody, allowing the apoglucose oxidase to diffuse over to the unbound “wired” FAD group, reassociate, and generate an electrical signal. Similarly for a coagulation assay, a coagulation protease can cleave an apoglucose oxidase-protease substrate link to a bead or blocker surface, again enabling the apoglucose oxidase to diffuse over to the “wired” unbound FAD group, reassociate and again generate an electrical signal.

[0026] In yet another embodiment of the invention, the electrochemically active enzyme may contain its cofactor or prosthetic group; yet still require an allosteric enzyme-regulating agent (which is not a classical cofactor or prosthetic group) for activation. In this later embodiment, the test analyte will act to release the allosteric regulating molecule from a bound state, and this unbound allosteric regulator will then activate the electrochemically active enzyme. Such allosteric enzyme regulation may be done by binding of an allosteric moiety, by covalent enzyme modification (e.g. phosphorylation, acetylation, myristoylation, ADP-ribosylation, farnesylation, γ-carboxylation, sulfaction, ubiquinitination, glycosylation, etc.), by proteolytic cleavage, or other posttranslational modification.

[0035] FIG. 1 shows an example of an apoenzyme electrochemical protease assay. Here the apoenzyme (1), which may be the apoenzyme form of glucose oxidase, or other enzyme is mounted or otherwise associated with the surface of electrode (5). Apoenzyme (1) contains a binding site for a prosthetic group (2), which, in the case of a glucose oxidase apoenzyme would be a FAD (flavin adenine dinucleotide) group. Apoenzyme (1) additionally contains a substrate-binding site (3) for enzyme substrate (4). In this example, enzyme substrate (4) would be glucose. Note that in the apoenzyme form of the enzyme, substrate-binding site (3) will be in an inactive conformation.

[0036] In this example, the device additionally contains the FAD apoenzyme prosthetic group (6) complexed to a molecule or surface (7) by way of protease peptide (8). Surface (7) makes it sterically infeasible for prosthetic group (6) to bind to the apoenzyme prosthetic group binding site (2). Protease peptide (8) contains a peptide region that serves as a substrate to a proteolytic enzyme of interest (9), and is cleaved by proteolytic enzyme (9). In the case of a coagulation assay, (such as a prothrombin time assay) the enzyme (9) may be thrombin, which is produced by the reaction of thromboplastin in the reagent (not shown) with the various clotting factors present in a patient sample.

[0037] As the test reaction progresses, proteolytic enzyme (19) cleaves the substrate peptide (18) that binds the prosthetic group (16) to the molecule or surface (17) that prevents the prosthetic group (16) from binding to enzyme prosthetic group binding site (12). As a result of this proteolytic cleavage, prosthetic group (16) now is liberated for binding.

[0038] As a result of this liberation, prosthetic group (26) binds to prosthetic group binding site (22), and converts the inactive apoenzyme to an active holoenzyme (enzyme). As a result of this activation, the active site of this enzyme (23) changes conformation, and becomes capable of performing enzymatic activity. In particular, it is now capable of enzymatically altering enzyme substrate (24), in a reaction that produces a detectable electrochemical change.

[0039] As a result of these changes, enzyme (31) activated by the binding of prosthetic group (36) is able to amplify the signal produced by the proteolytic cleavage of peptide substrate (18) many times. The electrochemically active enzyme converts large amounts of its substrate from substrate (34) to product (44) by way of active site (33). In the process, enzyme (31) is the source or sink for a large number of electrons (40), which can, in turn, react with electrode (35) and produce a detectable electrochemical signal. In the case where enzyme (31) is glucose oxidase, the substrate (34) is glucose, the product (44) is gluconolactone, and the prosthetic group (36) is flavin-adenine dinucleotide (FAD).

Apoenzyme Cofactor Discussion:

[0040] Apoenzymes or inactive enzymes may be reactivated (acquire catalytic activity) by many different cofactors, coenzymes, or prosthetic groups. These cofactors vary according to the enzyme in question. As previously discussed, apoglucose oxidase is activated by the prosthetic group FAD. Other apoenzymes and cofactor requiring enzymes require other cofactor molecules. Some of the other cofactors required to produce catalytic activity in other enzymes include 6-hydroxyDOPA, Ammonia, Ascorbate, ATP, Biotin, Cadmium, Calcium, Cobalamin, Cobalt, Coenzyme-A, Copper, Dipyrromethane, Dithiothreitol, F420, FAD, Flavin, Flavoprotein, FMN, Glutathione, Heme, Heme thiolate, Iron, Iron-molybdenum, Iron-sulfur, Lipoyl groups, Magnesium, Manganese, Molybdenum, NAD, NAD(P)H, Nickel, Potassium, PQQ, Protoheme IX, Pterin, Pyridoxal-phosphate, Pyruvate, Reduced flavin, Selenium, Siroheme, Tetrahydropteridine, Thiamine pyrophosphate, Vanadium and Zinc.

Enzyme Substrate Discussion:

[0041] The substrate for the electrode (electrochemically active) enzyme may either be incorporated into the reagent itself, or else can be a normal component of the liquid sample. As an example, glucose is the substrate for glucose oxidase. A common diagnostic fluid is blood, which normally contains glucose, in addition to other analytes of interest. In this case, when the electrode enzyme is the apo form of glucose oxidase, and the reactivated enzyme is glucose oxidase, the glucose substrate may be provided as part of the test reagents, or alternatively may be obtained from the glucose normally present in the blood sample.

[0042] Thus, although the substrate for the electrochemically active enzyme will usually be incorporated as part of the test reagents, when this substrate is expected to be normally present in the liquid sample, this substrate may be omitted.

Electron Transport Mediator Discussion:

[0043] After the apoenzyme or inactive enzyme has been converted to an active form by interacting with the enzyme activation factor portion (such as a prosthetic group) of the test analyte detection moiety conjugates, various means may be used to transfer electrons produced by the catalytic activity of the newly reactivated enzyme back to a reference electrode.

[0044] In one scheme, both the newly reactivated enzyme, and at least some of the means to transport electrons from the enzyme to the electrode surface, both exist in a non-electrode bound form. Here, electron transport occurs by way of a diffusible electron transport mediator, such as hydrogen peroxide (H₂O₂). This electron transport mediator, produced by the newly reactivated enzyme, may diffuse to a transducer enzyme, such as horseradish peroxidase (HRP). The HRP may in turn be bound to an electrode surface, either by a covalent linkage, or by a non-covalent interaction. HRP will in turn react with the diffusible mediator, and produce electrons, which in turn will transfer to the electrode, where the reaction may be detected. A number of possible transducer enzymes exist, including HRP (previously mentioned), cytochrome c, and others. Typically the active electron transport center of the transducer enzyme is relatively exposed to the outside environment, and thus can easily exchange electrons with electrode surfaces.

[0045] In some cases, the mediator molecule may be a soluble artificial electron transport mediator, such as (S)- and (R)-N,N-dimethyl-1-ferrocenyl-ethylamine, methylene blue, and others that can transport electrons directly from the reactivated enzyme to the electrode surface without need of an intermediate transducer enzyme.

[0046] In a second scheme, the newly reactivated enzyme is still free to diffuse in solution, but all other parts of the electron transport mediator system are bound to the electrode surface.

[0047] Here, the mediator molecule may be an electrode bound artificial electron transport mediator, such as (R)- and (S)-2-Methylferrocene carboxylic acid bound to a silver electrode. Other mediator molecules that can be covalently bound to electrodes include C₆₀ (buckyballs), microperoxidase (the active site of cytochrome c), porphyrin rings, and other molecular entities.

[0048] In a third scheme, the apoenzyme or inactive form of the enzyme is chemically modified by binding an electron transport mediator to the surface of the apoenzyme. For example, ferrocene electron transport mediator molecules may be linked to lysine amino acid residues on the apoenzyme via amide bonds. When reactivated by the enzyme activation factor portion of the test analyte detection moiety, the mediator-modified enzyme may then diffuse and transport electrons directly to electrode surfaces.

[0049] In a fourth scheme, the apoenzyme or inactive enzyme itself is bound to the electrode surface, and the mediator is also bound to the electrode surface. Here the enzyme may be imbedded in a conducting polymer matrix, such as ferrocene-containing pyrrole derivatives, hydrophilic epoxy cements derivitized with to contain electrically conducting pyridinium-N-ethylamine polycationic domains, copolymers of allylamine and ferrocene-functionalized acrylic acid, silicon alkoxide sol-gel matrices doped with electron-transfer mediators, and the like. Since the enzyme is immobilized onto the electrode surface, these later schemes have the advantage of often being very stable and very sensitive.

[0050] In general, any electron transport scheme will work, with the possible exception of schemes that attempt to link the enzyme to the electrode surface by way of the same enzyme activation factor (or prosthetic group) needed to activate the enzyme in the first place. For example, attempts to produce this type of assay by linking apoglucose oxidase to electrode surfaces by way of electrode bound FAD molecules, while also attempting to use FAD as the enzyme activation factor portion of the test analyte detection moiety, will tend to encounter problems. The apoglucose oxidase has previously been converted to active glucose oxidase during the initial linking process. As a result, addition of excess FAD groups from the test analyte detection moieties will have no effect on enzyme activity.

[0051] FIG. 2 shows an example of an apoenzyme electrochemical immunochemical assay. Here, as before, the apoenzyme (1), which may be the apoenzyme form of glucose oxidase, or other enzyme, is mounted or otherwise associated with the surface of electrode (5). Apoenzyme (1) contains a binding site for a prosthetic group (2), which, in the case of a glucose oxidase apoenzyme would be a FAD group. Apoenzyme (1) additionally contains a substrate-binding site (3) for enzyme substrate (4). Again in this example, enzyme substrate (4) would be glucose. Note that in the apoenzyme form of the enzyme, substrate-binding site (3) will be in an inactive conformation.

[0052] In this example, the device additionally contains an ARIS type hybrid molecule consisting of apoenzyme prosthetic group (6) coupled to reagent ligand (antigen) (8).

[0053] This hybrid molecule is in turn bound to antibody (7). The coupling between the prosthetic group (6) and the reagent ligand (antigen) (8) will normally be by either a covalent bond, or a tight non-covalent bond such as an avidin-biotin linkage. The binding of the prosthetic group to the antibody (by way of reagent antigen (8)) makes it sterically infeasible for prosthetic group (6) to bind to the apoenzyme prosthetic group binding site (2).

[0054] As the test reaction progresses, excess unbound ligands (antigens) present in the test sample (19) compete with the prosthetic group bound reagent ligand (antigen) (18) for binding to antibody (17). This displaces prosthetic group (16), and makes it available for binding to the prosthetic group binding region (12) of apoenzyme (11).

[0055] As a result of this liberation, prosthetic group (26) binds to prosthetic group binding site (22), and converts the inactive apoenzyme to an active enzyme. As a result of this activation, the active site of this enzyme (23) changes conformation, and becomes capable of performing enzymatic activity. In particular, it is now capable of enzymatically altering enzyme substrate (24), in a reaction that produces a detectable electrochemical change.

[0056] As a result of these changes, enzyme (31) activated by the binding of prosthetic group (36) is able to amplify the signal produced by binding of the sample test ligand (antigen) (19, 29) to the antibody (17, 27) many times. The enzyme converts large amounts of substrate from substrate (34) to product (44) by way of active site (33). In the process, enzyme (31) is the source or sink for a large number of electrons (40), which can, in turn, react with electrode (35) and produce a detectable electrochemical signal. Again, in the case where enzyme (31) is glucose oxidase, the substrate (34) is glucose, the product (44) is gluconolactone, and the prosthetic group (36) is flavin-adenine dinucleotide (FAD).

Application to Prothrombin Time Tests and Other Blood Coagulation Assays:

[0057] Prothrombin time tests: Certain types of patients, such as patients with artificial heart valves, atrial fibrillation, and other cardiovascular disorders have a heightened risk of blood clot formation, which can lead to stroke or pulmonary embolism. To treat these disorders, physicians commonly prescribe oral anticoagulants, such as warfarin. Oral anticoagulants diminish the ability of the bodies natural “extrinsic pathway” of proteolytic enzymes to produce a clot. This pathway consists of several proteolytic enzymes, including factor VII, factor X, and thrombin. In the body, the extrinsic coagulation pathway is triggered when thromboplastin, a natural membrane-tissue factor component of the blood vessel's endothelial lining, is released from the interior of the cells due to cellular damage. The thromboplastin activates factor VII, which in turn activates factor X, which in turn activates thrombin, which in turn converts fibrinogen to fibrin, forming a clot. In the normal state, this system acts to prevent bleeding due to minor wounds and other minor damage, but in under pathological conditions (such as heart or circulatory system disorders) can cause a lethal blood clot.

[0058] To prevent dangerous blood clots, physicians attempt to diminish the activity of this pathway with oral anticoagulants. It is important not to completely block this pathway, however, since doing so can put the patient at high risk of a lethal bleeding event. To determine if a proper amount of anticoagulant has been administered, the functional capability of the extrinsic coagulation pathway is tested. Since the pathway ends up converting an inactive enzyme, prothrombin, to an active enzyme, thrombin, it is called a prothrombin time test.

[0059] In a prothrombin time test, a sample of blood, preferably a single drop of blood from a fingerstick, is exposed to the coagulation initiating chemical, thromboplastin, at a controlled temperature, such as 37° C. The time elapsed between the initial exposure to thromboplastin, and the subsequent development of thrombin activity, is the “prothrombin time” of the sample, and lets the physician know if an adequate dose of anticoagulants has been given. If the prothrombin time is too short, the patient has not been adequately anticoagulated. If the prothrombin time is too long, the patent has been overly anticoagulated.

[0060] The electrochemical apoenzyme reactivation technology of the present disclosure can be readily adapted to produce a prothrombin time test, or other type of blood coagulation test. To do this, two things are required. The first is that the test analyte detection moiety conjugates must contain a detector region that is sensitive to the progress of the coagulation pathway. Usually this detection moiety will be a protease substrate, such as the thrombin substrate peptide Gly-Pro-Arg (or other substrate peptide, which may include spacer peptides on either end to facilitate the reaction), which is cleaved by active thrombin. In the case where the basic electrochemical detector is based upon apoglucose oxidase, the test analyte detection moiety conjugate may consist of: FAD-(thrombin substrate)-anchor. Here the “anchor” group (blocking group) is chosen for its ability (prior to substrate cleavage by the coagulation protease) to sterically hinder the reassociation between the FAD group on the conjugate, and the FAD binding region on apoglucose oxidase.

[0061] The blocking group or “anchor group” can be many different things. It can simply be a larger soluble, and relatively inert protein, such as an antibody or albumin protein, chosen solely on its ability to serve as a steric blocking group, and not otherwise interfere with the assay. Alternatively, the anchor can be a bead, membrane, or region on the electrode surface, spatially separated from the apoglucose oxidase molecule, so that the FAD prosthetic group is kept separate from apoglucose oxidase in the absence of thrombin proteolytic cleavage of the protease peptide substrate.

[0062] In the case of a prothrombin time test, the second thing that is required is that the test should contain thomboplastin. This is required in order to trigger the start of the coagulation cascade. If use with fresh whole blood is desired, no other components need be present. If use with citrate anticoagulated venous blood or plasma is desired, extra calcium to overcome the effects of the citrate anticoagulation, and optionally a buffer to maintain pH control, may also be used.

[0063] Thus the minimal list of materials needed to produce an apoenzyme based electrochemical prothrombin time assay is as follows:

• • [0064] Apoglucose oxidase (or other inactive enzyme that produces an electrochemical change upon reaction)
  • [0065] FAD-[thrombin substrate]-anchor
  • [0066] Glucose (needed as an enzyme substrate for glucose oxidase) (or alternate substrate if an alternative enzyme is being used).
  • [0067] Electron transport mediator
  • [0068] Optional polymer or crosslinker to hold Apoglucose oxidase and the electron transport mediator onto the electrode surface
  • [0069] Electrode and reference electrode
  • [0070] Thromboplastin
  • [0071] Optional calcium (usually present as a few mM CaCl₂)
  • [0072] Optional buffer (used to control the pH of the reaction).

[0073] The components will typically be packaged in the form of a standard point-of-care dry reagent electrochemical test strip. In order to allow instant rehydration of the thromboplastin, and minimal distortion of the coagulation reaction, it may be desirable to position the thromboplastin in the test device so that the sample contacts the thromboplastin slightly before (ahead of when) the sample contacts the electrodes.

[0074] FIG. 3 shows an example of a prothrombin time test adapted to the present format.

[0075] Here the inactive enzyme (apoenzyme) (1) containing a cofactor or prosthetic group binding site (2) and substrate binding site (3) for enzyme substrate (4) is mounted or otherwise associated with the surface of electrode (5).

[0076] In this example, the device additionally contains the apoenzyme prosthetic group (6) complexed to a molecule or surface (7) that makes it sterically infeasible for prosthetic group (6) to bind to the apoenzyme prosthetic group binding site (2). In this example, prosthetic group (6) is bound to molecule or surface (7) by way of a peptide (8) that contains a region that serves as a peptide substrate to a coagulation factor proteolytic enzyme. In this example, the peptide (8) is a thrombin substrate.

[0077] The test device additionally contains thromboplastin (9), which is used to initiate the extrinsic coagulation pathway that leads to blood coagulation.

[0078] In use, a patient test sample (10) (such as blood or plasma) is applied to the test device. The test sample will normally contain unknown levels of a number of different proteases and other factors involved in the extrinsic coagulation pathway, including unknown levels of Factor VII (11), Factor X (13), prothrombin (15), and typically other factors such as fibrinogen (not shown).

[0079] When the thromboplastin present in the test device (9) contacts the Factor VII in the patient sample (11), Factor VII is converted to an activated form (12). The activated form of factor VII (12) in turn converts the inactive form of Factor X (13) to the active form (14). Activated Factor X (14) then converts prothrombin (15) to thrombin (16). Thrombin (16) is a highly active protease enzyme.

[0080] The next steps of the reaction are shown in 21, 24, 25, 26, 27, 28 and 29. Here thrombin (29) starts to cleave the protease substrate region (28) on the test analyte detection moiety conjugate (26, 27, 28). As previous, the apoenzyme or otherwise inactive enzyme (21) remains associated with electrode surface (25), and is not yet reacting with its enzyme substrate (24).

[0081] The final stages of the reaction are shown in 31, 33, 34, 35, 36, 37, 38, and 44. Here, as a result of the action of thrombin (29) in the previous frame, the thrombin substrate peptide (28) has broken. The residual groups from the test analyte detection moiety conjugate, namely the blocking group (37), and the cleaved region of the protease substrate (38) no longer block the binding of the enzyme reactivation group (36) to the enzyme. The liberated enzyme prosthetic group (or other enzyme reactivation factor) (36) can now bind to the prosthetic group region of apoenzyme (31). This activates the apoenzyme (31), thus restoring the enzymatic activity of the enzyme. The newly reactivated enzyme is now able to convert its enzyme substrate (34), by way of the enzyme's active site (33), producing a reacted substrate (44), and electrons (40). The electrons (40) are transferred to electrode (35) by one of the previously discussed electron transport mediators (not shown).

[0082] It should be apparent that by changing the chemistry of the specific coagulation pathway initiator and/or substrate peptide, other coagulation pathways and coagulation tests may be devised using the same principles discussed above for prothrombin time tests. For example, the intrinsic coagulation pathway can be activated using the appropriate initiator, and an Activated Clotting Time (ACT) test for intrinsic coagulation pathway inhibitors, such as heparin, can also be created.

[0083] One drawback of using a soluble antibody system (shown in FIG. 2), or a soluble enzyme substrate blocking molecule system (shown in FIG. 1) is that if the enzyme activation prosthetic group is not tightly held next to the surface of the antibody (FIG. 2 (7)), or tightly held next to the surface of the steric blocking group anchor molecule (FIG. 1 (17)), an unwanted high background signal can result. This background signal can be caused when, due to random Brownian motion, the sterically blocked prosthetic groups bump up against the apoenzyme with enough force to knock the prosthetic group free from the steric blocking group or antibody, causing some background apoenzyme reactivation in the absence of suitable test ligands.

[0084] As previously discussed, this background can be reduced by binding the apoenzyme and the test analyte detection moiety conjugates to bead, membrane, or electrode surface “anchor groups” that physically separate the enzyme activation cofactors or prosthetic groups from the apoenzymes. These anchor group separation concepts are discussed in more detail in FIGS. 4 and 5.

[0085] FIG. 4 shows an apoenzyme electrochemical immunoassay constructed using a porous electrode, microbead bound antibodies (which in turn bind ligand (antigen)-prosthetic group conjugates), microbead (microsphere) bound apoenzymes, and soluble electron transport mediators. This porous electrode (1) contains regions of electron transporting material (2), such as thin metal or carbon fibers, typically arranged in a open but connected three dimensional meshwork configuration that allows electrical transport over the length or width of the electrode, as well as multiple voids (3) of various sizes, at least some of which are in fluid communication with the outside surface of the electrode. Typically porous electrode (1) will be mounted on a carrier (4) that lends mechanical support and protection to the electrode. Electrical conducting traces (5) that allow electrical communication between the porous electrode (1) and outside electrochemical detection equipment are also typically present. Porous electrode (1) also contains a population of antibody-conjugated microspheres (10) and apoenzyme conjugated microspheres (microbeads) (20) within multiple voids (3). These microspheres (microbeads) are typically micron or sub micron sized particles with surface properties that enable proteins (such as antibodies and apoenzymes) to be tightly bound to the microbead surface. The porous electrode (1) typically has a pore size distribution large enough to enable a large percentage of the microbeads to penetrate a substantial distance into the interior of the electrode, but enough electrode structural material (2) as to at least partially hinder the microbeads from moving freely once the microbeads have penetrated into the interior of the electrode.

[0086] Antibody conjugated microsphere (10) contains bound antibodies (11). These antibodies are typically directed against the specific test ligands (antigens) that are the focus of interest for this particular immunoassay (e.g. anti-hCG antibodies for an hCG immunoassay, etc.) and contain binding sites for these test ligands (antigens). Prior to use, reagent complexes consisting of conjugates between the reagent ligand (antigen) (12) and the enzyme prosthetic group (13) are prepared, and are bound to the antibodies (11) bound to microbeads (10).

[0087] Apoenzyme conjugated microspheres (20) contain the apoenzyme itself (21) tightly coupled to microsphere (20). Typically, apoenzyme (21) will contain prosthetic binding site (22) and the enzyme active site (23) which, in the absence of the prosthetic group (13) will be in an inactive state.

[0088] The porous electrode (1) will typically also contain other reaction chemicals, such as the enzyme substrate (30), electron transport mediator (31), apoenzyme stabilizing agents (such as trehalose), polymers (used to modulate the movement of microbeads within the porous electrode, as well as to modulate the flow of test fluids applied to the porous electrode surface), buffers, surfactants (used to encourage test fluid migration flow into the multiple voids (3)) and other ingredients as needed (not shown).

[0089] When fluid containing or suspected of containing test analyte antigens (32) is added to the surface of porous electrode (1), it permeates into the multiple voids (3) carrying test analyte (test antigens) (32). These test analytes (32) displace the binding between the antibody (11) and the reagent-antigen-enzyme prosthetic group conjugates (12), (13). The now liberated enzyme prosthetic groups (13) are now free to diffuse throughout the multiple voids (3) of porous electrode (1). Eventually, these liberated prosthetic groups (13) diffuse (33) to the prosthetic binding region (22) of apoenzyme (21), where they bind, causing apoenzyme (21) to now become a fully active enzyme.

[0090] The net effect of this test analyte induced enzyme reactivation is shown in the lower half of FIG. 4. Within the interior of porous electrode (40), ligands (41) from the test analyte have bound to antibody (42), which is still bound to antibody microbeads (43). The prosthetic group (44), has now bound to the prosthetic binding site and apoenzyme (21) is now holoenzyme (enzyme) (45). Note that in this configuration, enzyme (45) remains attached to former apoenzyme microbead (20), now renamed enzyme microbead (46).

[0091] Reactivated enzyme (45) then converts its substrate (50) to a reaction product (51). This reaction liberates electrons (52), which can flow, by way of electron transport mediator (53) to the electron transporting zones (54) of porous electrode (40). From here, the electrons may in turn pass into electrical conduits or traces (55), where an outside electrical measuring apparatus can then detect the reaction.

[0092] FIG. 5 shows that with some modifications, the microbead-porous electrode design previously shown in FIG. 4 can be configured for coagulation assays or protease assays as well. FIG. 5 shows an apoenzyme electrochemical protease assay (such as a coagulation assay) constructed using a porous electrode, microbead bound protease substrate peptides capped with apoenzyme prosthetic groups, microbead bound apoenzymes, and a soluble electron transport mediator.

[0093] This porous electrode (1) contains regions of electron transporting material (2), such as carbon or metal fibers, arranged in an open but connected three dimensional meshwork configuration that allows electrical transport over the length or width of the electrode, as well as multiple voids (3) of various sizes, at least some of which are in fluid communication with the outside surface of the electrode. Typically porous electrode (1) will be mounted on a carrier (4) that lends mechanical support and protection to the electrode. Electrical conducting traces (5) that allow electrical communication between the porous electrode (1) and outside electrochemical detection equipment are also typically present. Porous electrode (1) also contains a population of protease (proteolytic enzyme) peptide substrate conjugated microspheres (microbeads) (6) and apoenzyme conjugated microspheres (20) within multiple voids (3). These microspheres are typically micron or sub micron sized particles with surface properties that enable proteins (such as peptides and apoenzymes) to be tightly bound to the microbead surface. The porous electrode typically has a pore size distribution large enough to enable a large percentage of the microbeads to penetrate a substantial distance into the interior of the electrode, but enough electrode structural material as to at least partially hinder the microbeads from moving freely once the microbeads have penetrated into the interior of the electrode.

[0094] Protease substrate conjugated microsphere (6) contains peptides (7). These peptides, which are often formed by a solid phase peptide synthesis process using the microsphere (6) as the solid phase for the synthesis process, typically are covalently attached to the microsphere (6) by a peptide spacer group that is not itself a target for the assay protease enzyme, but rather serves to make the peptide substrate region more sterically accessible to the assay protease analyte. These peptides (which may contain a spacer region, a protease substrate region, and optionally another spacer region are, in turned, capped by the assay's apoenzyme prosthetic group (8).

[0095] Apoenzyme conjugated microspheres (20) contain the apoenzyme itself (21) tightly coupled to microsphere (20). Typically, apoenzyme (21) will contain prosthetic binding site (22) and the enzyme active site (23) which, in the absence of the prosthetic group (8) will be in an inactive state.

[0096] The porous electrode (1) will typically also contain other reaction chemicals, such as the substrate (31) for the electrochemically active enzyme, electron transport mediator (32), apoenzyme stabilizing agents (such as trehalose), polymers (used to modulate the movement of microbeads within the porous electrode, as well as to modulate the flow of test fluids applied to the porous electrode surface), buffers, surfactants (used to encourage test fluid flow into multiple voids (3) and appropriate protease initiators or coagulation pathway initiators, such as thromboplastin and calcium for a prothrombin time assay (not shown)).

[0097] When the test fluid (such as whole blood or plasma) containing coagulation test analytes (28) is added to the surface of porous electrode (1), it permeates into the multiple voids (3) carrying the various coagulation factors, exemplified by protease (29) in an active or inactive form. After coagulation factor (protease) (29) is converted to an active form (for coagulation assays, this is usually done by coagulation initiators that are included in the test strip's reaction chemistry), protease (29) cleaves its peptide substrate (7), liberating the bound prosthetic group (8). The now liberated enzyme prosthetic groups (8) are now free to diffuse throughout the multiple voids (3) of porous electrode (1). Eventually, these liberated prosthetic groups (8) diffuse (30) to the prosthetic binding region (22) of apoenzyme (21), where they bind, causing apoenzyme (21) to now become a fully active enzyme.

[0098] The net effect of this test analyte induced enzyme reactivation is shown in the lower half of FIG. 5. Within the interior of porous electrode (40), the cleaved protease substrate peptide from the protease substrate (41) remains still bound to the peptide microbeads (43). The prosthetic group (44), has now bound to the prosthetic binding site and apoenzyme (21) is now enzyme (45). Note that in this configuration, enzyme (45) remains attached to former apoenzyme microbead (20), now renamed enzyme microbead (46).

[0099] Reactivated enzyme (45) then converts its substrate (50) to a reaction product (51). This reaction liberates electrons (52), which can flow, by way of electron transport mediator (53) to the electron transporting zones (54) of porous electrode (40). From here, the electrons may in turn pass into electrical conduits or traces (55), where an outside electrical measuring apparatus can then detect the reaction.

[0100] Typically, for both FIGS. 4 and 5, the apoenzyme will be apoglucose oxidase, the prosthetic group is flavin-adenine dinucleotide (FAD), the substrate is glucose and the product is gluconolactone.

[0101] FIGS. 4 and 5 both show configurations in which the apoenzyme is bound to a first bead population, and the antibody (or peptide substrate) containing the ligand-bound prosthetic group is bound to a second bead population, both bead populations are embedded in a porous electrode, and both populations are interspersed with a diffusible electron transport mediator. Other configurations are also possible, however.

[0102] There are a number of ways in which these two bead populations can be embedded in a porous electrode, and the optimal way may vary according to the specifics of the assay. Although often the two bead populations may be simply be intermixed, for sensitive assays, the background signal caused by spontaneous apoenzyme reactivation brought about by occasional direct bead-bead contact may need to be reduced still further. This can be accomplished by adding additional “spacer” (separation) beads to the mix, by depositing the two bead populations in closely associated, but spatially distinct, regions on the electrode surface, or alternatively coated on different layers, one above the other.

[0103] Another alternative bead deposition pattern takes advantage of lateral flow techniques. This configuration is particularly favored for ultra sensitive immunoassays, where background signals due to stray interactions between the apoenzyme and the prosthetic group should be totally minimized. Here the immobilized antibody (either bound to microbeads or some other immobile test component), which in turn binds the ligand-prosthetic group, is placed upstream from the apoenzyme. When a liquid sample is applied, it flows (usually by capillary action) past a first region containing the immobilized antibody. If the antigenic test ligands are present, the test ligands displace the bound prosthetic group ligands from the antibody. These displaced prosthetic group ligands are carried, by the capillary transport of the liquid sample, into a second region containing the apoenzyme, electrode, an electron transport mediator, and an enzyme substrate. Here, as before, the prosthetic group combines with the apoenzyme creating an active enzyme, and the active enzyme converts the substrate to the product. This produces electrons, which are carried by the electron transport mediator to the electrode.

[0104] One drawback of electrochemical electrodes based upon diffusible (soluble) electron transport mediators is low efficiency. The diffusible mediator shuttles back and forth between the enzyme and the electrode surface by a slow process involving diffusion and random walk style Brownian motion. By contrast, the work of Heller and others has shown that much higher efficiency can be obtained if the enzyme and electron transport mediator are both affixed to the electrode surface, and electrons can flow directly between the enzyme reaction center and the electrode by an electron transport mediator that is continuously attached to both the enzyme and the electrode surface. These techniques are frequently referred to as “wired enzyme” technology.

[0105] FIG. 6 shows an alternate apoenzyme immunoassay configuration which employs some of the “wired enzyme” concepts of Heller et. al., and physical separation between the apoenzyme and the prosthetic group, to improve assay efficiency. In this configuration, a non-enzyme complexed prosthetic group, such as FAD (1) is bound to an electrode surface (2) by bridging means (3) that act to facilitate electron transport. An apoenzyme (4) containing an active site (5) and a prosthetic group binding site (6), and an artificially coupled reagent-ligand group (antigenic group) (9), is bound to an antibody (7). This in turn is bound to a distant surface (8), such as a microbead or different location on the enzyme electrode. Enzyme substrate (10) is also present.

[0106] When antigenic test ligands (11) are added to the system, they compete for binding between the antigenic reagent ligand group (19) previously coupled to the apoenzyme (14), breaking the bond between the antigenic reagent ligand group (19) and the antibody (17). This allows apoenzyme (14) to diffuse away (15) from sterically isolated bead or surface (18), and towards electrode bound prosthetic group (12).

[0107] The prosthetic group binding region (26) of Apoenzyme (24) then binds to the electrode bound prosthetic group (21). Apoenzyme (24) is reconstituted to an active enzyme (24) configuration, and the active site (25) of enzyme (24) changes from an inactive configuration to an active configuration. Note that the ligand binding sites on nearby antibody (27) bound to physically isolated bead or surface (28) remain complexed with test ligand (20).

[0108] After enzyme activation is complete, now reconstituted enzyme (34) converts the enzyme substrate (39) to its reaction product (40), generating electrons (41) that transfer to the electrode surface (32). This electron transfer occurs through a direct link between the enzyme prosthetic group and the enzyme surface.

[0109] Other “wired enzyme” configurations, in which a group other than the prosthetic group binds the apoenzyme very close to the electrode, are also possible. Two of these configurations are shown in FIG. 7. In the top part of FIG. 7, the apoenzyme (1) is bound to an electrode surface (2) or a surface associated hydrogel by a non-prosthetic group link (3). As before, apoenzyme (1) contains prosthetic group binding region (4) and an active site region (5). Typically, enzyme (1) will be closely associated with hydrogel (6) containing electron transport mediator groups (7).

[0110] As before, the proteolytic peptide enzyme substrate (9) is anchored on microbeads (10) or other support that enables the enzyme substrate to be physically separated from the apoenzyme (1) until the peptide protease substrate (9) is cleaved by the appropriate protease (11). As before, the protease peptide enzyme substrate (9) is capped with apoenzyme prosthetic group (12).

[0111] When proteolytic enzyme (protease) (11) cleaves the substrate peptide (9), the prosthetic group (12) is liberated and can diffuse (13) to and bind with the prosthetic group binding region (4) of apoenzyme (1). Typically enzyme substrate (14), as well as other reaction chemicals (coagulation initiators, buffers, viscosity modifying polymers, and apoenzyme stabilizing agents such as trehalose) are also present.

[0112] After the prosthetic group has recombined with the apoenzyme, forming active enzyme (20), the active enzyme converts its substrate (21) to the reaction product (22) producing an electrochemical change. Electrons from this electrochemical change (24) are transferred to electron transport mediators (25) bound to the hydrogel attached to the electrode (26). These electron transport mediators (25) in turn transfer (27) electrons to the electrode (26).

[0113] In an alternate configuration, the proteolytic enzyme peptide substrate itself contains an electron transport mediator as well as an apoenzyme prosthetic group. This configuration is seen in the lower half of FIG. 7.

[0114] In this configuration, the enzyme prosthetic group may be coupled to one or more electron transport mediators by a tether, and this tether in turn coupled to the protease substrate peptide, and this peptide in turn attached to an anchor group. After the protease substrate peptide is cleaved by the coagulation protease, and the liberated prosthetic group-electron transport mediator conjugate diffuses over to reactivate the apoenzyme, these tethered electron transport mediators can then extend the distance to which electrons can easily be transported away from the now reconstituted apoenzyme (enzyme). If this enzyme in turn is bound to an electrode surface, then if the tethered electron transport mediators are sufficiently long, they can transport electrons between the enzyme's reaction centers and the electrode surface with an efficiency that is relatively high.

[0115] As shown in the lower half of FIG. 7, peptide protease substrate groups (31) are again built up around micron sized plastic microspheres (30) with free carboxyl terminal ends using standard solid phase peptide synthesis techniques, such as Fmoc solid phase peptide synthesis (Chan and White editors, “Fmoc Solid Phase Peptide Synthesis, A practical approach”, Oxford University Press, 2000). After the desired spacer and protease substrate site are built up, the peptide chains are then coupled to an electron transport mediator (32), such as Pyrroloquinoline quinine (PQQ). They are in turn coupled with an electrically active enzyme prosthetic group (33), such as N⁶-(2-aminoethyl)-FAD, resulting in microspheres coupled with chains of protease substrate, electron transport mediators, and enzyme prosthetic groups all arranged in a linear order.

[0116] In this configuration, the apoenzyme, such as apoglucose oxidase (35) is bound to an electrode surface (36), by non-prosthetic group linkage (37). This linkage may be a covalent linkage such as a covalent crosslink, a hydrophobic linkage, an electrostatic linkage, an antibody linkage or so on. This apoenzyme will typically have a prosthetic group binding region (38) and an enzyme active site (39), which, in the apo state, will typically have an inactive conformation. The test will also contain excess substrate (40) for the electrochemically active enzyme, as well as other chemicals and cofactors needed to stimulate the desired coagulation protease reaction (such as thromboplastin and calcium for a prothrombin time test, etc.) (not shown).

[0117] After the relevant protease has been generated (41) (thrombin in the case of a prothrombin time test), detection proceeds as follows. Protease (41) cleaves its corresponding synthetic peptide substrate (31), liberating the chain that contains the enzyme prosthetic group (33) and tethered electron transport mediator (32) away from the support (30).

[0118] This liberated chain is then free to diffuse (42) and bind to the prosthetic group binding site (38) of nearby electrode bound enzyme (35). There the prosthetic group-tethered electron transport mediator (32,33) reactivates the enzyme.

[0119] The reactivated enzyme (50) starts converting its substrate (51) to product (52). Electrons flow through the prosthetic group and through the tethered electron transport mediator (53) to electrode surface (54).

[0120] It should be apparent that the general schemes shown in FIGS. 6 and 7 apply to both immunochemical assays and coagulation (protease) assays, and that with minor modification, the scheme of FIG. 6 will work for a protease assay. Likewise, with minor modification, the schemes of FIG. 7 will work for immunoassays.

[0121] FIG. 8 shows a diagram of one possible apoenzyme electrochemical test strip. Here the test strip has a bottom plastic support (1), a first conducting electrode (2), an optional reference electrode (3) and a second conducting electrode (4). The porous electrode configuration containing the apoglucose oxidase, electron transport mediator, test analyte detection moiety conjugate, and glucose (previously shown in FIGS. 4 and 5) is shown as (5). This porous electrode is put into electrical contact with electrodes (2, 3, 4) by a conducting adhesive.

[0122] In the case where the assay is a coagulation assay, such as a prothrombin time test, the test strip additionally contains a dry thromboplastin pellet (6). For other coagulation tests, a different coagulation initiator may be used. For immunochemical tests, pellet (6) may be omitted. For coagulation assays, if the porous electrode is sufficiently coagulation neutral (as defined in U.S. Pat. No. 5,580,744, the contents of which are incorporated herein by reference), then the thromboplastin (or other coagulation initiator) in pellet (6) may be physically located inside porous electrode (5). In an alternative configuration, which is particularly useful if the porous electrode is not sufficiently coagulation neutral, the thromboplastin pellet (6) may be located outside of porous electrode (5). In this later configuration, the test strip configuration may be optimized so as to allow the liquid sample to hydrate the thromboplastin (or other coagulation initiator) pellet (6) first, and then contact the porous electrode (5) after a slight time delay that allows the coagulation process to begin.

[0123] Plastic spacer (7) is present to separate the plastic sheet (1) containing electrodes (2, 3, 4) from the top plastic covering (8). Plastic spacer (7) has a “U” shaped opening that creates a cavity (9). Cavity (9) is used both to hold the pellet (6), as well as to receive the blood sample from the patient. In practice, cavity (9) has a configuration such that it can be totally filled with about 1–50 microliters of sample.

[0124] In some configurations, plastic layer (8) also holds a reference electrode (10). In the operating configuration, layers (1), (7) and (8) are laminated together to form a single unitized structure electrically connected to the outside by conductive paths (2, 3, 4 and 10), and in liquid communication with the outside through cavity (9).

[0125] For temperature sensitive tests, such as coagulation assays, it is often advantageous to employ means to keep the test strip at a constant temperature, such as 37° C., throughout the reaction. This may be done by various heat transport mechanisms. One simple means is to put the test strip into a cavity formed from a heat conductor made of a material with good heat exchange properties (such as copper), and employ electronic means to keep the heat sink at a constant temperature. Here, the tip of the “U” shaped sample receiving end of the test strip (6) sticks out slightly from the cavity to facilitate sample application, but the electrode base of the strip, as well as the back of the “U” shaped cavity remain inside the heat sink cavity, and thus are warmed to a constant temperature.

[0126] For high volume commercial applications, it may be desirable to produce an extremely low cost reagent reader (meter) with minimal onboard temperature regulation means. In such cases, where a somewhat higher cost electrochemical reagent test strip is acceptable, it may be advantageous to embed a temperature sensor and/or electrical heater directly into the reagent test strip itself. This can be done, for example, by embedding a low-cost thermistor and/or electrical resistance heater into or onto the plastic support (for example elements (1) or (8) in FIG. 8) that also holds the other test strip components. In this later configuration, the reagent test strip reader (meter) need only contain electrical means to read the thermistor and electrical means to apply electrical energy to the heater onboard the test strip itself, and thus the meter will have a simplified and lower cost configuration.

[0127] Onboard controls: In some situations, it may be advantageous to include multiple electrodes in a single test strip in order to obtain both positive (high) and negative (low) onboard controls for the reaction. For a coagulation test, the positive (high) control can be obtained by using a control electrode with an alternate peptide substrate that is not sensitive to the particular protease used in the reaction. Similarly a negative (low) control can be obtained by using free FAD (or appropriate prosthetic group). Other control chemistry is also possible.

[0128] For an immunochemical test, positive control (high) control can be obtained by using a control electrode with free FAD (or appropriate prosthetic group), and a negative (low) control can be obtained by using FAD linked to a different antibody using a different ligand. Other control chemistry is also possible.

Preparation of Apoglucose Oxidase Conjugated Microbeads, Antibody Conjugated Microbeads, and Protease Substrate Conjugated Microbeads:

[0129] Apoglucose oxidase conjugated microbeads: Apoglucose oxidase can be prepared by a number of methods. One of the more modern, and particularly favored, methods is according to the methods of Heiss et. al. (Dip-and-read test strips for the determination of trinitrotoluene (TNT) in drinking water; Carola Heiss, Michael G. Weller, Reinhard Niessner: Analytica Chimica Acta 396 (1999) 309–316). Briefly, The FAD groups from Glucose oxidase (Aspergillus niger, Sigma-Aldrich Corporation) can removed by dissolving the Glucose oxidase in a CHAPS, 30% glycerol, HCL/glycine buffer at pH 1.5. The apoglucose oxidase can then be separated from the unbound FAD groups by gel chromatography in Toyopearl HW-50 F columns in 30% glycerol, 70% HCL/glycine buffer at pH 1.5. Residual FAD groups can be removed by suspending the column eluate directly into a 38 mg/ml suspension of stirred charcoal, and then readjusting the pH to 7.0 to avoid protein denaturation. Residual charcoal is removed by centrifugation, and the supernatant filtered through a 0.2 um polysulfone filter to remove remaining aggregates.

[0130] The apoglucose oxidase solution should then be immediately desalted by running the supernatant through a Chemcon spin-OUT™-6000 micro chromatography column equilibrated in 0.1 M sodium phosphate buffer, pH 7.0. The eluate from this column should then be immediately coupled to 1.0 micron diameter carboxyl modified latex microbeads (Bangs Labs) by carbodiimide coupling, following the materials and procedures contained in Polysciences PolyLink Protein Coupling Kit #644. After coupling, reaction quenching, and washing according to the Polysciences protocol, the apoglucose coupled latex microbeads are spun down in a microfuge, and resuspended in a 1.5M trehalose, 5 mg/ml BSA, PBS buffer for storage. Ideally apoglucose oxidase preparation and the bead coupling reaction should be done on the same day to reduce the level of formation of unwanted apoglucose oxidase aggregates.

[0131] Antibody conjugated microbeads: Monoclonal antibodies against the target analyte of interest can usually be obtained from many different commercial sources. These are typically then put into 0.1 M phosphate buffer, pH 7.0 by dialysis or microcolumn separation and suspended at a concentration of about 1 mg protein per ml of buffer. These can then be conjugated onto 1.0 micron COOH latex microspheres (Bangs Labs) by carbodiimide coupling between the carboxyl groups on the surface of the microbead and the primary amines on the protein of interest using the same PolyLink-Protein Coupling Kit for COOH Microparticles (PolySciences, Inc., Warrington Pa.) discussed previously.

[0132] Protease substrate conjugated microbeads: The protease substrate peptide of interest (typically a thrombin substrate peptide) is built up on small (micron diameter) resin microbeads using standard Fmoc solid phase peptide synthesis techniques. (Fmoc Solid Phase Peptide Synthesis, A practical approach, W. Chan and P. White editors, Oxford University Press, New York, 2000). After the desired substrate peptide, which usually will contain a spacer region both before and after the actual substrate region itself to facilitate steric access to enable the protease of interest to later cleave the desired peptide, is constructed, the N terminal blocking group is removed, and a FAD (or other apoenzyme prosthetic group) is placed on the N terminal end of the microbead bound protease substrate peptide using the FAD-peptide conjugation methods discussed below.

FAD-Peptide Conjugation Methods:

[0133] N6-aminohexyl-flavin adenine dinucleotide can be synthesized according to the methods of Morris et. al. (Morris D L, Ellis P B, Carrico R J, et al. Flavin adenine dinucleotide as a label in homogeneous colorimetric immunoassays. Anal Chem 53, 658–665 (1981).).

[0134] FAD can be coupled to peptides and proteins by the methods of Schroeder et. al. (Schroeder H R, Dean C L, Johnson P K, Morris D L, Hurtle R L.; Coupling aminohexyl-FAD to proteins with dimethyladipimidate. Clin. Chem. 1985 September; 31(9): 1432–7; and Morris D L, Buckler R T. Colorimetric immunoassays using flavin adenine dinucleotide as label. Methods Enzymol. 1983; 92: 413–25.). Briefly N6-aminohexyl-flavin adenine dinucleotide is activated with dimethyladipimidate, run through a Sephadex G-10 gel filtration column in 20 mM NaHCO₃ buffer to remove unbound imidate, and then incubated with the target protein. If the target protein in question is a protease substrate peptide bound to a microbead, after conjugation, the now FAD conjugated protease substrate peptide-microbead complex can then be washed by repeated centrifugation and resuspension. If the target molecule in question is a soluble protein antigen (such as the analyte for an immunoassay) the conjugated target proteins (reagent antigens) can be purified by a second gel separation column in Sephadex G-25 in 100 mM phosphate buffer, pH 7.0 (which separates the unbound FAD-imidate from the FAD conjugated protein).

Porous Electrode Methods:

[0135] Porous carbon electrodes (Torayca Carbon paper TGP-H-120) can be obtained from Toray Industries Corporation, Japan, through a US distributor (FuelCellStore.com, Boulder Colo.). This material is a loose meshwork of connected electrically conducting carbon fibers, and has a structure similar to loose weave filter paper, with large (approximately 50 micron) holes and voids in-between the various thin and interconnected carbon filaments. This material is produced in various thicknesses. One exemplary grade (TGP-H-120) is 0.37 mm thick, has an in-plane electrical resistance of about 4.7 mega ohms per centimeter, is 78% porous, and has a bulk density of 0.45 grams per cubic centimeter. It is highly permeable, and allows passage of gas at a level of (1500 ml gas*mm)/(cm²*hour*mmAq). Thinner grades of this material are also available, and may be appropriate when use of lower volumes of analyte sample is desired. The carbon paper is hydrophobic, and can be rendered hydrophilic by various methods including plasma oxidation, or by pre-treatment with appropriate surfactants such as an aqueous solution of 0.1 mg/ml tyloxapol (which is a gentle surfactant that, in low concentrations, does not lyse red cell membranes).

[0136] Note that other workers have found that the efficiency of carbon paper electrodes can be improved by additionally growing carbon nanotubes on the carbon paper base, or by adding additional conducting microparticles to the carbon paper base. Such methods are highly compatible with the apoglucose oxidase reconstitution methods described in this disclosure, as the nanotubes or conducting particles increase the available electrode surface area, and thus increase electron transfer efficiency.

[0137] Electron transport mediator: Although, depending upon the particular configuration, almost any type of electron transport mediator may be used for the present invention, methylene blue has certain advantages for the porous carbon paper-microbead electrode configurations described here. Methylene blue (Calbiochem Corporation, San Diego, Calif.) is an electron transport mediator with good solubility in water, and thus is available in relatively high concentrations in order to shuttle electrons over the comparatively large (up to several microns) distance between the microbead bound glucose oxidase, and the carbon electrode surface of the porous Toray carbon paper electrode. Methylene blue is also inexpensive, known to be compatible with glucose biosensors, relatively non-toxic, and readily available.

Test Strip Production Methods:

[0138] For immunoassays, the antibody conjugated microspheres can be bound to the FAD (or other prosthetic group) conjugated reagent antigen complex by incubating the microspheres with the FAD conjugated reagent antigen for 30 minutes, followed by washing 3× by centrifugation and resuspension before use to in order to remove unbound FAD-reagent antigen conjugates.

[0139] All microspheres should be reconstituted (separately) in a solution of about 1% microspheres, 50 mM phosphate buffer pH 7.0, 0.1 M NaCl, 1.5 M trehalose (to stabilize the apoglucose oxidase), 5 mg/ml protease free bovine serum albumin, 10 mg/ml Polyvinyl alcohol, 0.1 mg/ml Methylene blue, 0.1 mg/ml tyloxapol (to help disperse the microspheres, and also help improve solubility), 50 mM Glucose (enzyme substrate for the reconstituted glucose oxidase).

[0140] To reduce the amount of microsphere aggregation, microsphere solutions should be sonicated briefly, and then immediately applied to the porous carbon paper using a low volume micro airbrush, such as an Iwata HP-A airbrush. Depending upon the specifics of the experiment, various deposition patterns can be used. In one configuration, a first coating of apoglucose oxidase microspheres is applied, the carbon paper dried using a hot air dryer, and then a second coating of antibody microspheres containing the bound FAD-reagent antigen groups is then applied, immediately followed by drying using a hot air dryer. In other configurations, the separation between the apoglucose oxidase microspheres and the antibody microspheres can be increased by coating one side of the carbon paper with the apoglucose oxidase microspheres, air drying, and then coating the other side of the carbon paper with the antibody microspheres, followed by rapid air drying. In a third configuration, unconjugated spacer microspheres may be used. Here the apoglucose oxidase microspheres are applied and air-dried. The spacer microspheres are then lightly applied, and air-dried. Finally the antibody conjugated microspheres (or peptide substrate microspheres are then applied).

[0141] For immunoassays, no further test chemistry is required. For coagulation assays, such as prothrombin time tests, coagulation initiators such as thromboplastin and calcium are required. If suitable coagulation neutral microbeads and electron transport mediators are provided per the methods of Zweig, (U.S. Pat. No. 5,580,744), then the thromboplastin and calcium can be applied to the porous carbon matrix itself, and all steps of the reaction proceed inside the electrode matrix.

[0142] In some cases, such as when the porous electrode, thrombin substrate, apoenzyme, and electron transport mediators are not entirely coagulation neutral, it may be advantageous to begin at least the initial phases of the coagulation reaction immediately outside the porous electrode, and allow the coagulation proteases, such as thrombin, to diffuse into the porous electrode, where they may then be detected. In this later situation, the porous electrode will often be mounted on one side of the test strip chamber, thromboplastin and calcium placed in a different part of the test strip, and small amounts of whole blood allowed to flow into the test strip chamber by capillary action. The blood or plasma first contacts the thromboplastin, starts the coagulation process, and produces soluble coagulation factors, which then migrate into the electrode where the reaction can be detected.

[0143] After impregnation with the appropriate reaction chemistry, the carbon paper electrodes can then be affixed to larger and more mechanically robust electrically conducting surfaces, usually on plastic supports, suitable for relaying the signal to appropriate instrumentation. These conductors are typically vitreous carbon electrodes, carbon paste coated plastic, and silver-silver chloride electrode coated plastic supports. Binding to the porous electrode can be done using various conducting adhesives, such as the conducting adhesives used to adhere materials to electrodes for scanning electron microscopy purposes, which are provide by Structure Probe, Inc (SPI), West Chester, Pa. This includes, SPI Conductive Carbon Paint; SPI LEIT-C™ Conductive Carbon Cement and Thinner; and SPI Supplies® Brand Conductive Double Sided Carbon Adhesive Tape.

[0144] Other electrically conducting traces and electrode components are available from other vendors. For example, silver-carbon screen-printing paste C70709D14, and reference electrode silver-silver chloride polymer paste C61003D7 may be obtained from Gwent Electronic Materials Ltd., Pontypool, UK. This reference silver/silver-chloride paste is a mixture of fine (roughly 10 micron sized) silver and silver-chloride particles present in a roughly 60% silver, 40% silver chloride ratio. These particles are held in a polymeric binder support. The electrode formed from this material acts as a standard silver chloride reference electrode for the reaction.

[0145] The various plastic support layers, containing the transfer electrodes, can then be laminated together with an additional 10 mil (0.254 mm) to 30 mil thick spacer layer to result in a sandwich electrode with 10 mm×10 mm sized electrode surface area, and an internal volume of about 10 ul to 30 ul. This is shown in FIG. 8. Note that the electrodes on surfaces (8) and (1) all face the interior of the cavity.

[0146] Both two electrode and three electrode (using a Ag—Ag—Cl reference electrode) designs are possible with this configuration. In general, three electrode designs incorporating a reference electrode, although more expensive and difficult to produce, are preferred due to their higher sensitivity and accuracy.

Electrochemical Sensing Methods

[0147] The electrochemical activity of the electrodes can be assessed with a series of linearly variant patterns of potential versus time sweeps (Cunningham, “Introduction to Biolanalytical Sensors”, Wiley Interscience, 1998 p 207–259). One low cost device that functions well, with an optional external amplifier to bring up weak signals, is an IBM EC/225 Voltammetric Analyzer (IBM corporation). Because the reconstituted apoenzyme electrodes generate a comparatively weak signal (relative to standard glucose electrodes), more sensitive potentiostats, such as a Bioanalytical Systems LC4B amperometric detector, with sensitivity to current that extends down into the picoamp range, can also be used. The offset voltages that are used typically vary according to the choice of electron transport mediator, but typically are about 0.5 V for methylene blue. The output from these devices can then be either manually recorded, or stored in a computer file as desired.

Experiment 1:

[0148] Immunochemistry experiment using antibody conjugated beads and a porous electrode. This experiment exemplifies the use of indirect “sandwich” immunoassays by constructing an apoenzyme electrochemical immunoassay sensitive to rabbit IgG.

[0149] To do this, 1-micron COOH microbeads should be conjugated with a first monoclonal antibody directed against rabbit IgG, using previously described methods. A second mouse monoclonal antibody, directed against a different epitope on rabbit IgG, should be conjugated with FAD, again using previously described methods.

[0150] The desired detection complex is somewhat like a sandwich with the bead-capture antibody (here the bead antibody is a first anti-rabbit IgG antibody) forming the first layer, the antigen (here rabbit IgG is used as the antigen) forming the middle layer, and the FAD-conjugated anti-antigen antibody (here a second anti-rabbit IgG antibody that binds to a different epitope on rabbit IgG) forming the top layer. This structure is built up in stages, and the beads are washed in-between each stage. First the middle layer is added to the first layer, and then the top layer is added to the middle layer and lower layer.

[0151] Purified rabbit IgG should be suspended 0.1M Phosphate buffered saline, pH 7.0, at a concentration of 0.1 mg/ml, and incubated with a 1% solution of the mouse-anti-rabbit IgG conjugated microbeads for 1 hour, washed 3× in the same buffer by repeated microcentrifugation, and then resuspended to the original 1% concentration.

[0152] To add the top layer to the sandwich, the FAD-conjugated monoclonal mouse anti rabbit IgG should then be added to these washed microbeads to a final concentration of 0.2 mg/ml of antibody, and allowed to bind for 1 hour. The beads should be washed 3× in phosphate buffer, and resuspended in the 1.5M Trehalose buffer at a 1% suspension.

[0153] The net result is to create a sandwich structure of the type:

• [0154] [Bead]-(Anti-rabbit IgG)(rabbit IgG)(Anti-rabbit IgG)-FAD
  in which the FAD-conjugated anti-rabbit IgG monoclonal antibody binds to the rabbit IgG, which in turn binds to the anti-rabbit IgG coupled to the beads.

[0155] These beads should be deposited using an airbrush on porous Toray carbon paper electrodes, along with the apoglucose oxidase microbeads, as described previously. This electrode then should be mounted on a solid support containing leader electrodes using the conductive adhesives described previously.

[0156] A plastic cover should be laminated on top of the porous carbon electrode-support layer with a 10–30 mil thick plastic spacer, creating a lower support, porous carbon electrode, upper plastic support structure as shown in FIG. 8.

[0157] The test strip is attached to the electrochemistry measuring apparatus. When challenged with 0.1 M Phosphate buffered saline at pH 7.0 containing various amounts of rabbit IgG, the liquid sample flows into the hollow chamber by capillary action, where the rabbit IgG in the sample displaces some of the bound FAD conjugated monoclonal mouse anti rabbit IgG from the bound beads. These free FAD-antibodies bind to the apoglucose oxidase in the neighboring beads, creating active glucose oxidase. The resulting electrochemical reaction can then be detected. This is done by monitoring the change in current at a 0.5 v applied potential. Typical results from this type of study are shown in table 1 below.

[0158]

TABLE 1
Immunochemical assay
	Concentration of	Reaction time
	rabbit IgG	10 seconds	1 minute	2 minutes
	0 ug/ml IgG	0 nA	9 nA	15 nA
	1 ug/ml IgG	23 nA	97 nA	354 nA
	10 ug/ml IgG	107 nA	1.2 uA	1.9 uA

[0159] The sensitivity of this assay to increasing levels of rabbit IgG can be seen by the increasing amount of current as a function of time and concentration of analyte (rabbit IgG) in the applied sample.

Experiment 2:

[0160] Coagulation experiment using thrombin substrate microbeads and porous electrodes.

[0161] Depending upon the details of the coagulation assay or protease assay in question, many different suitable FAD-(substrate peptide)-Anchor configurations are possible. In the specific example where the protease (proteolytic enzyme) is thrombin, a FAD-(thrombin substrate)-anchor is desired, and the anchor is chosen to be a solid phase peptide synthesis bead, it will often be advantageous to include amino acid leader sequences on either side of the thrombin recognition and cleavage region. These leader sequences are designed to allow thrombin to get better steric access to the substrate region, and minimize the interfering effects of both the FAD and bead on the ability of thrombin to cleave the thrombin substrate region.

[0162] Such leader groups should thus be designed to promote steric access (allow the protease to physically reach the desired substrate peptide), but otherwise not interfere with the reaction. Although usually some experimentation (computer modeling and/or direct synthesis of candidates) will be required to find the optimum leader combination, such leader sequences can typically be found by using the amino acid sequence that naturally brackets the protease cleavage site in the natural form of the protease substrate. For example, in the case of a coagulation assay, where it is desired to produce a thrombin substrate analogous to the natural thrombin substrate region on human fibrinogen (see Hughes et. al., Biochemistry 2004, 43, 5246–5255, table 1), the thrombin substrate site can be the natural fibrinogen (P3, P2, P1) sequence “G V R” (using the one letter amino acid code), bracketed on the N terminal (P6, P5, P4) side by the naturally occurring leader “E G G”, and bracketed on the C terminal (P1′, P2′, P3′) side by the naturally occurring leader “G P R”. The resulting finished FAD-(peptide substrate)-Anchor will then be:

• [0163] [FAD]-(E G G)-G V R-(G P R)-[peptide synthesis bead]

[0164] The apoenzyme FAD prosthetic group is [FAD], the thrombin cleavage (substrate) site is shown underlined (GVR), and cleavage by thrombin produces the products:

• [0165] [FAD]-(EGGGVR) and GPR-[peptide synthesis bead].

[0166] After thrombin cleavage, the FAD-peptide group is now liberated from the peptide synthesis bead, and is now free to diffuse over to nearby apoglucose oxidase apoenzymes and create active glucose oxidase.

[0167] In this experiment, FAD-EGGGVRGPR-beads can be created by Fmoc solid phase synthesis and FAD conjugation, suspended in the same Trehalose buffer described previously, and deposited using an air brush on the same porous Toray carbon paper electrodes along with the apoglucose oxidase microbeads as described previously. This electrode is then mounted on a solid support containing leader electrodes using the conductive adhesives described previously.

[0168] In the example where a prothrombin time coagulation test is desired, a coagulation initiator, such as a thromboplastin-calcium solution, is made up and a small (approximately 10–30 ul drop) of this solution is applied to a plastic cover. Suitable thromboplastin-calcium solutions include Dade-Behring thromboplastin C plus, Dade Innovin, Biomerieux Simplastin, and others. This thromboplastin solution is allowed to dry, creating a plastic cover with a dried thromboplastin-calcium pellet attached. This plastic cover is then laminated on top of the porous carbon electrode-support layer with a 10–30 mil thick plastic spacer, creating a lower support, porous carbon electrode, thromboplastin pellet, upper plastic support structure as shown in FIG. 8.

[0169] In use, the test strip is normally maintained at a constant physiological temperature, such as 37° C., in order to improve test accuracy. The test strip is attached to the electrochemistry measuring apparatus. When challenged with plasma or whole blood, the sample flows into the hollow chamber by capillary action, where it rehydrates the dry thromboplastin, activating the prothrombin time coagulation cascade. Thrombin, generated by the coagulation cascade, migrates into the porous carbon electrode, where it cleaves the FAD-(thrombin substrate)-bead complex, liberating free FAD. This in turn reactivates the apoglucose oxidase, and the resulting electrochemical reaction can be detected. When used with a slow acting thromboplastin such as Dade Innovin, and the change in current at a 0.5 V applied potential is monitored, results such as table II (below) can be obtained.

[0170]

TABLE II
Prothrombin time assay signal generation
	Reaction time
	Sample type	30 seconds	1 minute	2 minutes
	INR 1 control	2 nA	31 nA	97 nA
	plasma
	INR 3 control	3 nA	3 nA	35 nA
	plasma

[0171] Note that the INR 1 control plasma, which has a high level of coagulation factors and reacts relatively quickly, produces a significant signal by 1 minute of reaction. By contrast, the INR 3 control plasma, which has a lower level of coagulation factors and reacts much slower, takes 2 minutes to start to produce an electrochemical signal that is significantly above the background.