Small volume in vitro analyte sensor

Abstract

A sensor designed to determine the amount and concentration of analyte in a sample having a volume of less than about 1 μL. The sensor has a working electrode coated with a non-leachable redox mediator. The redox mediator acts as an electron transfer agent between the analyte and the electrode. In addition, a second electron transfer agent, such as an enzyme, can be added to facilitate the electrooxidation or electroreduction of the analyte. The redox mediator is typically a redox compound bound to a polymer. The preferred redox mediators are air-oxidizable. The amount of analyte can be determined by coulometry. One particular coulometric technique includes the measurement of the current between the working electrode and a counter or reference electrode at two or more times. The charge passed by this current to or from the analyte is correlated with the amount of analyte in the sample. Other electrochemical detection methods, such as amperometric, voltammetric, and potentiometric techniques, can also be used. The invention can be used to determine the concentration of a biomolecule, such as glucose or lactate, in a biological fluid, such as blood or serum. An enzyme capable of catalyzing the electrooxidation or electroreduction of the biomolecule is provided as a second electron transfer agent.

We claim:  
     
         1 . A method for determining a concentration of an analyte in a sample, comprising the steps of: 
 contacting a sample with an electrochemical sensor comprising: 
 a facing electrode pair comprising a working electrode and a counter electrode; and  
 a sample chamber for holding the sample in electrolytic contact with the working electrode, the sample chamber comprising a measurement zone positioned between the working and counter electrodes, wherein the measurement zone is sized to contain a volume of less than about 1 μL of sample; and  
   determining the concentration of the analyte in the sample by coulometry.    
     
     
         2 . The method of  claim 1 , wherein the measurement zone is sized to contain a volume of less than about 0.5 μL of sample.  
     
     
         3 . The method of  claim 2 , wherein the measurement zone is sized to contain a volume of less than about 0.2 μL of sample.  
     
     
         4 . The method of  claim 3 , wherein the measurement zone is sized to contain a volume of less than about 0.1 μL of sample.  
     
     
         5 . The method of  claim 1 , wherein the sample chamber is sized to contain a volume of less than about 1 μL of sample.  
     
     
         6 . The method of  claim 5 , wherein the sample chamber is sized to contain a volume of less than about 0.5 μL of sample.  
     
     
         7 . The method of  claim 6 , wherein the sample chamber is sized to contain a volume of less than about 0.2 μL of sample.  
     
     
         8 . The method of  claim 1 , wherein the step of determining the concentration of the analyte comprises: 
 electrolyzing at least 90% of analyte present in the measurement zone by applying a potential across the working and counter electrodes;    determining an electrical charge used to electrolyze the analyte; and    correlating the electrical charge with the concentration of the analyte in the sample.    
     
     
         9 . The method of  claim 8 , wherein at least 90% of the analyte is electrolyzed in less than about 5 minutes.  
     
     
         10 . The method of  claim 9 , wherein at least 90% of the analyte is electrolyzed in less than about 1 minute.  
     
     
         11 . The method of  claim 8 , wherein the step of determining an electrical charge comprises the steps of: 
 measuring a current generated at the working electrode at two or more times as the analyte is electrolyzed; and    integrating the measured currents over time to obtain the electrical charge used to electrolyze the analyte.    
     
     
         12 . The method of  claim 1 , wherein the step of determining the concentration of the analyte by coulometry comprises the steps of: 
 electrolyzing a portion of the analyte by applying a potential across the working and the counter electrode;    measuring a current generated at the working electrode at two or more times during the electrolysis;    extrapolating a current curve based on the measured currents;    integrating the current curve over time to obtain an electrical charge necessary to electrolyze at least 90% of the analyte; and    correlating the electrical charge with the concentration of the analyte in the sample.    
     
     
         13 . The method of  claim 1 , wherein the sensor further comprises a non-leachable enzyme on the working electrode.  
     
     
         14 . The method of  claim 13 , wherein the enzyme is immobilized on the working electrode.  
     
     
         15 . The method of  claim 1 , wherein the sensor further comprises a non-leachable redox mediator on the working electrode.  
     
     
         16 . The method of  claim 15 , wherein the redox mediator is immobilized on the working electrode.  
     
     
         17 . The method of  claim 15 , wherein the redox mediator comprises a polymer and a redox species ionically, covalently, or coordinatively bound to the polymer.  
     
     
         18 . The method of  claim 17 , wherein the redox species is coordinatively bound to the polymer.  
     
     
         19 . The method of  claim 15 , wherein the redox mediator comprises an air-oxidizable redox mediator.  
     
     
         20 . The method of  claim 19 , wherein the air-oxidizable redox mediator comprises Os[4,4′-dimethoxy-2,2′-bipyridine] 2 Cl +/+2  or Os[4,7-dimethoxy-1,10-phenanthroline] 2 Cl +/+2  complexed with poly(1-vinyl imidazole) or poly(4-vinylpyridine).  
     
     
         21 . The method of  claim 15 , wherein at least 90% of the redox mediator is in an oxidized state prior to introduction of the sample in the sensor.  
     
     
         22 . The method of  claim 15 , wherein the sensor further comprises a non-leachable second electron transfer agent on the working electrode.  
     
     
         23 . The method of  claim 22 , wherein the second electron transfer agent is immobilized on the working electrode.  
     
     
         24 . The method of  claim 22 , wherein the second electron transfer agent comprises an enzyme.  
     
     
         25 . The method of  claim 24 , wherein the analyte is glucose.  
     
     
         26 . The method of  claim 25 , wherein the enzyme is a glucose oxidase.  
     
     
         27 . The method of  claim 1 , wherein the sensor further comprises sorbent material disposed in the measurement zone.  
     
     
         28 . The method of  claim 27 , wherein the step of contacting the sample with an electrochemical sensor further comprises contacting the sample with the sorbent material to wick the sample into the measurement zone.  
     
     
         29 . The method of  claim 1 , wherein the working electrode and counter electrode have a separation distance of less than about 0.2 mm.  
     
     
         30 . The method of  claim 29 , wherein the separation distance is less than about 0.1 mm.  
     
     
         31 . The method of  claim 30 , wherein the separation distance is less than about 0.05 mm.  
     
     
         32 . The method of  claim 1 , wherein the sensor comprises two or more facing electrode pairs.  
     
     
         33 . A method of determining the concentration of an analyte in a sample, comprising the steps of: 
 contacting a sample with an electrochemical sensor, wherein the sensor comprises a working electrode, a non-leachable redox mediator on the working electrode, and a sample chamber sized to contain a volume of less than about 1 μL of sample in electrolytic contact with the working electrode; and    determining the concentration of the analyte in the sample by coulometry.    
     
     
         34 . The method of  claim 33 , wherein the sample chamber is sized to contain a volume of less than about 0.5 μL of sample.  
     
     
         35 . The method of  claim 34 , wherein the sample chamber is sized to contain a volume of less than about 0.2 μL of sample.  
     
     
         36 . The method of  claim 33 , wherein the redox mediator is immobilized on the working electrode.  
     
     
         37 . The method of  claim 33 , wherein the sensor further comprises a non-leachable second electron transfer agent on the working electrode.  
     
     
         38 . The method of  claim 37 , wherein the second electron transfer agent is immobilized on the working electrode.  
     
     
         39 . The method of  claim 37 , wherein the second electron transfer agent comprises an enzyme.  
     
     
         40 . The method of  claim 33 , wherein the sensor further comprises a sorbent material within the sample chamber to reduce the volume of sample that the sample chamber is sized to hold.  
     
     
         41 . The method of  claim 33 , wherein the redox mediator comprises a transition metal complex.  
     
     
         42 . The method of  claim 41 , wherein the transition metal complex is an osmium, ruthenium, iron, or cobalt complex.  
     
     
         43 . The method of  claim 42 , wherein the transition metal complex comprises an osmium complex.  
     
     
         44 . The method of  claim 43 , wherein the osmium complex comprises osmium complexed with at least one ligand having a nitrogen-containing heterocycle.  
     
     
         45 . The method of  claim 44 , wherein the ligand having a nitrogen-containing heterocycle comprises 2,2′-bipyridine, 1,10-phenanthroline, or a derivative thereof.  
     
     
         46 . The method of  claim 45 , wherein the ligand having a nitrogen-containing heterocycle comprises a mono-, di-, or polyalkoxy derivative of 2,2′-bipyridine or 1,10-phenanthroline, wherein the carbon to oxygen ratio of the alkoxy functional groups is sufficient to retain solubility of the transition metal complex in water prior to crosslinking.  
     
     
         47 . The method of  claim 45 , wherein the nitrogen-containing heterocycle comprises 2,2′-bipyridine, 4,4′-dimethyl-2,2′-bipyridine, 4,4′-dialkoxy-2,2′-bipyridine, 1,10-phenanthroline, 4,7-dimethyl-1,10-phenanthroline, or 4,7-dialkoxy-1,10-phenanthroline, wherein the carbon to oxygen ratio of the alkoxy functional groups is sufficient to retain solubility of the transition metal complex in water prior to crosslinking.  
     
     
         48 . The method of  claim 43 , wherein the osmium complex comprises osmium complexed with a polymeric ligand.  
     
     
         49 . The method of  claim 48 , wherein the polymeric ligand comprises a nitrogen-containing heterocycle.  
     
     
         50 . The method of  claim 49 , wherein the polymer comprises poly(4-vinyl pyridine) or poly(1-vinyl imidazole).  
     
     
         51 . An electrochemical sensor for determining the concentration of an analyte in a sample, the sensor comprising: 
 a facing electrode pair comprising a working electrode and a counter electrode;    a non-leachable redox mediator on the working electrode; and    a measurement zone positioned between the working electrode and counter electrode, wherein the measurement zone is sized to contain a volume of less than about 1 μL of sample.    
     
     
         52 . The sensor of  claim 51 , wherein the measurement zone is sized to contain a volume of less than about 0.5 μL of sample.  
     
     
         53 . The sensor of  claim 52 , wherein the measurement zone is sized to contain a volume of less than about 0.2 μL of sample.  
     
     
         54 . The sensor of  claim 53 , wherein the measurement zone is sized to contain a volume of less than about 0.1 μL of sample.  
     
     
         55 . The sensor of  claim 51 , wherein the redox mediator is immobilized on the working electrode.  
     
     
         56 . The sensor of  claim 51 , wherein the redox mediator is an air-oxidizable redox mediator.  
     
     
         57 . An electrochemical sensor comprising: 
 two or more facing electrode pairs, each electrode pair comprising a working electrode, a counter electrode and a measurement zone disposed approximately between the working and counter electrodes, wherein the measurement zone is sized to hold less than about 1 μL of sample; and    non-leachable redox mediator on the working electrode of at least one of the electrode pairs.    
     
     
         58 . The sensor of  claim 57 , wherein the redox mediator is immobilized on the working electrode of at least one of the electrode pairs.  
     
     
         59 . The sensor of  claim 57 , wherein the measurement zone of at least one of the electrode pairs is sized to hold less than about 0.5 μL of sample.  
     
     
         60 . The sensor of  claim 59 , wherein the measurement zone of at least one of the electrode pairs is sized to hold less than about 0.2 μL of sample.  
     
     
         61 . The sensor of  claim 60 , wherein the measurement zone of at least one of the electrode pairs is sized to hold less than about 0.1 μL of sample.  
     
     
         62 . The sensor of  claim 57 , wherein at least one electrode pair comprises a working electrode including non-leachable enzyme and redox mediator, and wherein at least one electrode pair comprises a working electrode including non-leachable redox mediator in the absence of the enzyme.  
     
     
         63 . The sensor of  claim 62 , further comprising a third electrode pair having no redox mediator or enzyme on the working electrode.  
     
     
         64 . The sensor of  claim 57 , further comprising sorbent material disposed in the measurement zone of at least one of the electrode pairs.  
     
     
         65 . The sensor of  claim 57 , wherein the redox mediator is an air-oxidizable redox mediator.  
     
     
         66 . An electrochemical sensor for determining the concentration of an analyte in a sample, the sensor comprising: 
 a facing electrode pair comprising a working electrode and a counter electrode;    a measurement zone positioned between the working electrode and counter electrode; and    sorbent material disposed at least partially within the measurement zone to reduce the volume of sample needed to fill the measurement zone;    wherein the measurement zone is sized to contain a volume of less than about 1 μL of sample.    
     
     
         67 . The sensor of  claim 66 , wherein the measurement zone is sized to contain a volume of less than about 0.5 μL of sample.  
     
     
         68 . The sensor of  claim 67 , wherein the measurement zone is sized to contain a volume of less than about 0.2 μL of sample.  
     
     
         69 . The sensor of  claim 68 , wherein the measurement zone is sized to contain a volume of less than about 0.1 μL of sample.  
     
     
         70 . The sensor of  claim 66 , wherein the sensor further comprises non-leachable redox mediator on the working electrode.  
     
     
         71 . The sensor of  claim 70 , wherein the redox mediator is immobilized on the working electrode.  
     
     
         72 . The sensor of  claim 66 , wherein the sorbent material has a void volume of between about 5% and 50% of the total volume of the sorbent material.  
     
     
         73 . The sensor of  claim 72 , wherein the void volume is about 10% to about 25% of the total volume of the sorbent material.  
     
     
         74 . An electrochemical sensor for determining the concentration of an analyte in a sample, the sensor comprising: 
 a working electrode;    a non-leachable redox mediator on the working electrode;    a sample chamber for holding the sample in electrolytic contact with the working electrode; and    sorbent material disposed within the sample chamber;    wherein the sample chamber is sized to contain a volume of less than about 1 μL of sample.    
     
     
         75 . The sensor of  claim 74 , wherein the sample chamber is sized to contain a volume of less than about 0.5 μL of sample.  
     
     
         76 . The sensor of  claim 75 , wherein the sample chamber is sized to contain a volume of less than about 0.2 μL of sample.  
     
     
         77 . A method for determining the concentration of an analyte in a sample, comprising the steps of: 
 contacting the sample with an electrochemical sensor; the sensor comprising: 
 an electrode pair comprising a working electrode and a counter electrode;  
 a sample chamber for holding the sample in electrolytic contact with the working electrode; and  
 a sorbent disposed within the sample chamber;  
 wherein the sample chamber is sized to hold less than about 1 μL of sample; and  
   determining the concentration of the analyte by coulometry.    
     
     
         78 . The method of  claim 77 , wherein the method further comprises wicking the sample into the sample chamber using the sorbent material.  
     
     
         79 . The method of  claim 77 , wherein the electrode pair is a facing electrode pair.  
     
     
         80 . A sensor for the determination of the concentration of an analyte in a sample having a volume of less than about 1 μL, comprising: 
 a support; and  
 an air-oxidizable redox mediator coated on the support to form a working surface for contacting sample;  
 wherein, at least 90% of the air-oxidizable redox mediator is in an oxidized state prior to introduction of sample.  
 
     
     
         81 . The sensor of  claim 80 , wherein the air-oxidizable redox mediator is immobilized on the support.  
     
     
         82 . A method for determining a concentration of an analyte in a sample, comprising the steps of: 
 contacting the sample with a sensor comprising a support; and an air-oxidizable redox mediator coated on the support to form a working surface, wherein, at least 90% of the air-oxidizable redox mediator is in an oxidized state prior to introduction of a sample; and    correlating the concentration of the analyte in the sample to a change in oxidation state of the redox mediator in the presence of analyte.    
     
     
         83 . The method of  claim 82 , wherein the sensor is an optical sensor.  
     
     
         84 . The method of  claim 83 , wherein the step of correlating the concentration of the analyte comprises: 
 irradiating the redox mediator with light;    measuring the response of the redox mediator to irradiation by light; and    correlating the concentration of the analyte to the measured response of the redox mediator.    
     
     
         85 . The method of  claim 84 , wherein the step of measuring the response of the redox mediator to irradiation by light comprises measuring the absorption of the irradiated light by the redox mediator.  
     
     
         86 . The method of  claim 84 , wherein the step of measuring the response of the redox mediator to irradiation by light comprises measuring the transmittance of the irradiated light by the redox mediator.  
     
     
         87 . The method of  claim 84 , wherein the step of measuring the response of the redox mediator to irradiation by light comprises measuring the fluorescence of the redox mediator after irradiation by light.  
     
     
         88 . The method of  claim 84 , wherein the step of measuring the response of the redox mediator to irradiation by light comprises measuring the reflection of light by the redox mediator.  
     
     
         89 . The method of  claim 82 , wherein the sensor is an electrochemical sensor.  
     
     
         90 . The method of  claim 89 , wherein the step of correlating the concentration of the analyte comprises: 
 applying an electrical potential across the redox mediator;    measuring a current at one or more intervals, the current being generated in response to the electrolysis of the redox mediator in the presence of the analyte; and    correlating the concentration of the analyte to the measured current.    
     
     
         91 . The method of  claim 82 , wherein the redox mediator comprises a transition metal complex.  
     
     
         92 . The method of  claim 91 , wherein the transition metal complex comprises an osmium complex.  
     
     
         93 . The method of  claim 92 , wherein the osmium complex comprises osmium complexed with at least one ligand having a nitrogen-containing heterocycle.  
     
     
         94 . The method of  claim 93 , wherein the ligand having a nitrogen-containing heterocycle comprises 2,2′-bipyridine, 1,10-phenanthroline, or a derivative thereof.  
     
     
         95 . The method of  claim 94 , wherein the ligand having a nitrogen-containing heterocycle comprises a mono-, di-, or polyalkoxy derivative of 2,2′-bipyridine or 1,10-phenanthroline, wherein the carbon to oxygen ratio of the alkoxy functional groups is sufficient to retain solubility of the transition metal complex in water prior to crosslinking.  
     
     
         96 . The method of  claim 95 , wherein the nitrogen-containing heterocycle comprises 4,4′-dialkoxy-2,2′-bipyridine or 4,7-dialkoxy-1,10-phenanthroline, wherein the carbon to oxygen ratio of the alkoxy functional groups is sufficient to retain solubility of the transition metal complex in water prior to crosslinking.  
     
     
         97 . The method of  claim 96 , wherein the nitrogen-containing heterocycle comprises 4,4′-dimethoxy-2,2′-bipyridine or 4,7-dimethoxy-1,10-phenanthroline.  
     
     
         98 . The method of  claim 92 , wherein the osmium complex comprises osmium complexed with a polymeric ligand.  
     
     
         99 . The method of  claim 98 , wherein the polymeric ligand comprises a nitrogen-containing heterocycle.  
     
     
         100 . The method of  claim 99 , wherein the polymer comprises poly(4-vinyl pyridine) or poly(1-vinyl imidazole).  
     
     
         101 . The sensor of  claim 100 , wherein the redox mediator comprises Os[4,4′-dimethoxy-2,2′-bipyridine] 2 Cl +/+2  or Os[4,7-dimethoxy-1,10-phenanthroline] 2 Cl +/+2  complexed with poly(1-vinyl imidazole).  
     
     
         102 . The sensor of  claim 82 , wherein the sensor further comprises a second electron transfer agent coated on the support and in contact with the redox mediator.  
     
     
         103 . The sensor of  claim 102 , wherein the second electron transfer agent is an enzyme.  
     
     
         104 . The sensor of  claim 102 , wherein the second electron transfer agent is immobilized on the support.  
     
     
         105 . The sensor of  claim 82 , wherein the air-oxidizable redox mediator is immobilized on the support.  
     
     
         106 . A method for measuring analyte in a patient sample, the method comprising: 
 contacting the patient with an analyte measuring device, the device comprising: 
 sample acquisition means for producing a patient sample; and  
 an electrochemical sensor for measuring analyte in the sample;  
 wherein the electrochemical sensor comprises: 
 a facing electrode pair, comprising a working electrode and a counter electrode; and  
 a measurement zone positioned between the working electrode and counter electrode, wherein the measurement zone is sized to contain a volume of less than about 1 μL of sample;  
 
   acquiring a sample using the sample acquisition means;    transporting a portion of the sample to the measurement zone of the electrochemical sensor; and    determining the concentration of the analyte in the sample by coulometry.    
     
     
         107 . The method of  claim 106 , wherein the sample acquisition means comprises a skin-piercing member and the step of acquiring a sample comprises piercing the patient's skin to produce a sample.  
     
     
         108 . The method of  claim 107 , wherein the skin piercing member comprises a lancet.  
     
     
         109 . The method of  claim 106 , wherein the electrochemical sensor further comprises a sorbent material disposed within the measurement zone of the sensor.  
     
     
         110 . The method of  claim 109 , wherein said transporting comprises: 
 wicking the sample into the measurement zone using the sorbent material.    
     
     
         111 . The method of  claim 106 , wherein the measurement zone is sized to contain a volume of less than about 0.5 μL of sample.  
     
     
         112 . The method of  claim 111 , wherein the measurement zone is sized to contain a volume of less than about 0.2 μL of sample.  
     
     
         113 . The sensor of  claim 112 , wherein the measurement zone is sized to contain a volume of less than about 0.1 μL of sample.  
     
     
         114 . The method of  claim 106 , wherein the sensor further comprises a non-leachable redox mediator.  
     
     
         115 . The method of  claim 114 , wherein the redox mediator is an air-oxidizable redox mediator.  
     
     
         116 . A method for measuring analyte in a patient sample, the method comprising: 
 contacting the patient with an analyte measuring device, the device comprising: 
 sample acquisition means for producing a patient sample; and  
 an electrochemical sensor for measuring analyte in the sample;  
 wherein the electrochemical sensor comprises: 
 a working electrode;  
 non-leachable redox mediator on the working electrode; and  
 a sample chamber for holding the sample in electrolytic contact with the working electrode, wherein the sample chamber is sized to contain a volume of less than about 1 μL of sample;  
 
   acquiring a sample using the sample acquisition means;    transporting a portion of the sample to the sample chamber of the electrochemical sensor; and    determining the concentration of the analyte in the sample by coulometry.    
     
     
         117 . An analyte measurement device comprising: 
 sample acquisition means for producing a patient sample;    an electrochemical sensor comprising: 
 a facing electrode pair comprising a working electrode and a counter electrode;  
 a non-leachable redox mediator on the working electrode; and  
 a measurement zone positioned between the working electrode and counter electrode;  
 wherein the measurement zone is sized to contain a volume of less than about 1 μL of sample; and  
   transport means for transporting the sample produced by the sample acquisition means to the measurement zone of the sensor.    
     
     
         118 . The device of  claim 117 , wherein the sample acquisition means comprises a skin-piercing member.  
     
     
         119 . The device of  claim 118 , wherein the skin-piercing member comprises a lancet.  
     
     
         120 . The device of  claim 117 , wherein the redox mediator is immobilized on the working electrode.  
     
     
         121 . The device of  claim 117 , wherein the redox mediator is an air-oxidizable redox mediator.  
     
     
         122 . The device of  claim 117 , wherein the transport means comprises a wicking material, a capillary chamber, or a pump.  
     
     
         123 . An analyte measurement device comprising: 
 sample acquisition means for producing a patient sample; and    an electrochemical sensor coupled to the sample acquisition means, comprising: 
 a facing electrode pair comprising a working electrode and a counter electrode;  
 a measurement zone positioned between the working electrode and counter electrode; and  
 sorbent material disposed within the measurement zone;  
 wherein the measurement zone is sized to contain a volume of less than about 1 μl of sample; and  
   transport means for transporting the sample produced by the sample acquisition means to the measurement zone of the sensor.    
     
     
         124 . The device of  claim 123 , wherein the transport means is the sorbent material.  
     
     
         125 . A method for determination of a concentration of an analyte in a sample, the method comprising the steps of: 
 contacting a sample with an electrochemical sensor comprising: 
 first and second electrode pairs, each pair comprising a working electrode, and a sample chamber for holding the sample in electrolytic contact with the working electrode, the sample chamber sized to contain a volume of less about 1 μL of sample; wherein said first electrode pair includes a non-leachable redox mediator and a non-leachable enzyme on the working electrode;  
   and wherein said second electrode pair includes non-leachable redox mediator on the working electrode in the absence of enzyme;    measuring substantially simultaneously, and at two or more times, a first current generated at the first electrode pair and a second current generated at the second electrode pair; and    integrating the measured first currents over time to obtain a first charge;    integrating the measured second currents over time to obtain a second charge;    subtracting the second charge from the first charge to obtain a noise-reduced charge; and    correlating the concentration of the analyte to the noise-reduced charge.    
     
     
         126 . The method of  claim 125 , wherein the first and second electrode pairs are facing electrode pairs and further comprise a counter electrode and a measurement zone between the working electrode and counter electrode, the measurement zone sized to contain a volume of less than about 1 μL of sample.  
     
     
         127 . A method for determining a concentration of an analyte in a sample, the method comprising the steps of: 
 providing an electrochemical sensor, the sensor having one or more facing electrode pairs, each electrode pair comprising a working electrode, a counter electrode, and a measurement zone between the working electrode and the counter electrode, the measurement zone having a volume of less than about 1 μL, wherein the measurement zones of the one or more electrode pairs have approximately equal volumes, and wherein at least one of the electrode pairs further comprises redox mediator on its working electrode;    measuring a capacitance of at least one of the electrode pairs;    calculating the volume of the measurement zone from the capacitance measurement;    contacting the sample with the sensor; and    determining the concentration of the analyte in the sample by coulometry.    
     
     
         128 . The method of  claim 127 , wherein the redox mediator is a non-leachable redox mediator.  
     
     
         129 . A method of storing and packaging an analytical sensor comprising the steps of: 
 packaging an analytical sensor in an atmosphere containing molecular oxygen, the sensor comprising an air-oxidizable redox mediator.    
     
     
         130 . The method of  claim 129 , wherein greater than 90% of the redox mediator is in an oxidized state after being stored for more than one month.  
     
     
         131 . A method for determining a concentration of an analyte in a sample comprising the steps of: 
 contacting the sample with an electrochemical sensor, the sensor comprising a working electrode and a non-leachable redox mediator on the working electrode, wherein the molar amount of redox mediator in a reduced form prior to introduction of the sample is less than, on a stoichiometric basis, 5% of the expected molar amount of the analyte to be electrolyzed;    electrolyzing less than about 1 μL of sample; and    determining the concentration of the analyte in the sample by coulometry.    
     
     
         132 . A method of determining a concentration of an analyte in a sample comprising the steps of: 
 contacting the sample with an electrochemical sensor comprising a working electrode, a counter electrode, and a measurement zone bounded on at least two sides by the working and the counter electrodes, wherein the measurement zone is sized to contain less than about 1 μL of sample; and    determining the concentration of the analyte in the sample by coulometry.