Multi-electrode test method

[0001] Biosensors provide analysis of a biological fluid, such as whole blood, serum, plasma, urine, saliva, interstitial or intracellular fluid. Usually, biosensors contain a measuring device that analyzes the sample present in the test sensor. The sample is usually in liquid form and, in addition to being a biological fluid, may be a derivative of a biological fluid, such as an extract, solution, filtrate, or re-precipitated precipitate. The analysis made by the biosensor determines the presence and / or concentration of one or more reagents such as alcohol, glucose, uric acid, lactate, cholesterol, bilirubin, free fatty acids, triglycerides, proteins, ketones, phenylalanine or enzymes in the biological fluid. The analysis can be useful in the diagnosis and treatment of physiological disorders. For example, a person with diabetes may use a biosensor to determine the level of glucose in whole blood to regulate diet and / or drug.

[0002] Many biosensors analyze a single reagent and use various techniques to improve the accuracy and / or precision of the analysis. Accuracy can be expressed in terms of the deviation of the reagent reading by the sensor system compared to the reference reagent reading, with higher deviation values ​​representing a lower accuracy, while precision can be expressed in terms of distribution or variability among many measurements. Calibration information can be used to improve the accuracy and / or precision of the analysis and can be read from the test sensor of the measuring device prior to analysis. The measuring device uses calibration information to regulate biological fluid analysis in response to one or more parameters, such as the type of biological fluid, specific reagent (s) and test sensor production variations. Biosensors can be implemented using stationary, portable and similar measuring devices. Portable measuring devices may be manual and may allow the identification and / or quantification of a reagent in a sample. Examples of portable measuring systems include Ascensia Breeze® and Elite® meters from Bayer Healthcare in Tarrytown, New York, while examples of stationary measuring systems include Electrochemical Workstation available from CH Instruments in Austin, Texas.

[0003] The electrical signal input of the test sensor of the measuring device may be a potential or current and may be constant, variable or a combination thereof, such as when the AC signal is used with the DC signal offset. The input signal can be used as a single pulse or in multiple pulses, sequences or cycles. The reagent or measurable type undergoes a redox reaction when an input signal is applied to the sample. The redox reaction produces an output signal that can be measured continuously or periodically during a transient output and / or in a solid state. Unlike the transient output signal, which is variable, the solid state output is observed,

-2 when the signal change with respect to its independent input variable (time, etc.) is essentially constant, such as within ± 10 or ± 5%.

[0004] Various electrochemical processes can be used, such as colometry, amperometry, voltometry and the like. In contrast to colometry, amperometry and voltometry generally measure the rate at which a reagent is oxidized or reduced to determine the reagent concentration in a sample. In amperometry, a constant potential electrical signal (voltage) is applied to the electrical conductors of the test sensor, while the measured output signal is current. In voltometry, variable potential is applied to a biological fluid sample. Gated amperometry and gated voltmeter methods involving alternating cycles of excitement and relaxation can also be used.

[0005] The "hematocrit effect" is a factor that may reduce the accuracy and / or precision of an analysis performed on a whole blood sample. In addition to water, glucose, proteins, ketones and other biological molecules, whole blood samples contain red blood cells. Hematocrit is the volume of the whole blood sample occupied by red blood cells relative to the total volume of the whole blood sample and is often expressed as a percentage. The more the percentage of hematocrit deviates from the calibration of the% -matocrit system for a whole blood sample, the greater the deviation (error) in the reagent readings obtained from the biosensor. For example, a conventional biosensor system having one set of calibration constants (e.g., slope and intersection for a whole blood sample containing 40% hematocrit) will provide three different glucose concentrations for whole blood samples having identical glucose concentrations, however, a hematocrit percentage of 20%, 40% and 60%. Therefore, although whole blood glucose levels are the same, the system will report that a 20% hematocrit full blood sample contains more glucose than a 40% hematocrit whole blood sample and a 60% hematocrit whole blood sample contains less glucose than 40% hematocrit whole blood sample. Since conventional biosensors are generally configured for glucose reporting, assuming a 40% hematocrit content for a whole blood sample, any glucose measurement performed on a blood sample containing less or more than 40% hematocrit will contain some load error attributable to hematocrit effects.

[0006] The hematocrit error can be expressed by the following equation:

% Hct-Bias = 100% χ (Gm - Gf / Gref, where Gm and Gref are glucose and glucose reference readings, respectively, for any level of hematocrit. The higher the absolute value of the error% Hct, the greater the effect of hematocrit.

[0007] In addition to the effect of hematocrit, measurement inaccuracies may also arise when the concentration of the type being measured does not correspond to the concentration of the reagent. For example, when the biosensor determines the concentration of the reduced mediator produced in response to reagent oxidation, any reduced mediator not produced by reagent oxidation

-3 will indicate that more reagent is present in the sample than normal due to the background of the mediator. A sensor for measuring hematocrit is known from EP 1707953.

[0008] Knowing the output signal attributable to reagents insensitive to the reagent concentration, a false portion of the output signal can be subtracted. Conventional systems have attempted to isolate insensitive portions of the output signal by placing multiple pairs of working electrodes and counter electrodes in a common sample container. By changing the reagents used to form the electrodes, these systems attempted to separate the sensitive and insensitive parts of the reagent by subtracting the two output signals.

[0009] For example, conventional sensor systems may have multiple detection areas in an undivided sample chamber, with each working electrode facing the reference electrode. In another aspect, these systems have a single reference electrode. These types of systems may provide a sensor calibration system for the test, with two known standards, or may provide, for example, separate electrode systems for reagent, interference and hematocrit determination. A common disadvantage of these systems is the single sample chamber, where adjacent electrode systems / detection areas may be chemically contaminated by each other due to diffusion and / or liquid displacement. This defect can be particularly problematic when the reagent system requires a longer test time than another and / or if the test sensor is mechanically disturbed after filling with the sample.

[0010] A biosensor comprising three pairs of working electrodes and counter electrodes in which the output signals are combined to measure the reagent concentration is known from EP1742045. A biosensor containing four pairs of working and counter electrodes is known from DE202005020335U.

[0011] As more information is needed on diagnosis regarding the reagent present in biological samples, there is an increasing need for routine monitoring of many biological types of medical significance. Therefore, there is a continuing need for improved biosensors, especially those that can provide increasingly accurate and / or precise concentration measurements for many reagents. The methods of the invention prevent or correct at least one of the disadvantages associated with conventional biosensors.

Summary [0012] The invention relates to a method according to claim 1. The method of measuring at least one reagent in a sample is characterized by introducing the sample into a test sensor containing four working electrodes and four counter electrodes, each working electrode and counter electrode being essentially chemically isolated in one of eight secondary analysis areas, in which substantially no diffusive or convective mixing of reagents occurs between the secondary analysis areas of each electrode during one or more analyzes, where four working electrodes are independently electrically addressable and four counter electrodes are independently electrically addressable; chemical or biochemical oxidation or reduction

-4 reagent in the sample; providing a gated input signal to a sample on working electrodes and counter electrodes, wherein the gated input signal comprises at least one of the gated amperometry signal and the gated voltometry signal, wherein the gated amperometry signal and the gated voltameter signal have alternating excitation and relaxation cycles; generating at least one output signal from each pair of electrodes; combining at least two of the output signals; and measuring the concentration of at least one sample reagent based on the output signals.

Brief Description of the Drawing [0013] The invention can be better understood with reference to the following drawing and description. The ingredients in the figures are not necessarily to scale, instead the emphasis is on illustrating the principles of the invention. In addition, in the figures, similar reference numbers indicate the respective parts through different views.

Fig. 1A illustrates a test sensor system not according to the invention in which a sample is introduced into the upper part of the main area through the sample opening and flows in a substantially symmetrical manner to fill secondary analysis areas.

Fig. 1B shows the test sensor of Fig. 1A with the addition of a reference electrode.

Fig. 1C shows the test sensor of Fig. 1A with separate counter electrodes.

Fig. 1D shows the test sensor of Fig. 1C with the addition of a reference electrode.

Fig. 2A shows a test sensor system not according to the invention in which the sample is introduced through the sample opening in the side part of the test sensor into the main area, which then flows in an asymmetrical manner to fill the secondary analysis area.

Fig. 2B illustrates a test sensor having the electrode system of Fig. 2A, but a different arrangement of secondary analysis regions.

Fig. 3A illustrates the construction of a rectangular test sensor in which a sample flows from the main area through a first potential electrode position to reach a second potential electrode position (not included in claim 1).

Figures 3B to 3J show alternative designs of secondary analysis regions where the sample does not flow through more than one potential electrode site (while the embodiments of Figs. 3B-3H and 3J are not covered by claim 1).

Fig. 4A is a cyclic voltamogram of the design of a straight channel test sensor as shown in Fig. 3A.

Fig. 4B is a cyclic voltamogram of the Y channel design as shown in Fig. 3E.

-5Fig. 5A depicts chemoamperometric current as a function of time, determining that for a rectangular test sensor of the type used in Fig. 4A, a cyanoferrate peak was observed with the electrode operating within about 5 seconds of sample introduction.

Fig. 5B shows the chemoamperometric current as a function of time, determining that for the Y-channel test sensor of the type used in Fig. 4B, substantially no ferrocyanide reached the working electrode 30 seconds after sample introduction.

Fig. 5C shows the chemoamperometric current as a function of time, specifying that the Y channel design provides better chemical insulation between potential electrode sites than the T channel design.

Fig. 5D determines that three Y-channel designs were resistant to such mixing due to mechanical disturbances.

Fig. 6A shows a test sensor having a stepped arrangement of secondary analysis areas in which the sample is introduced through the sample opening into a primary area in the form of a channel from which two secondary analysis areas branch (not included in claim 1).

Fig. 6B illustrates a test sensor arrangement in which a sample is introduced into a sample hole, into a main area in the form of a channel from which three secondary analysis areas (not included in claim 1) branch off.

Figures 7A and 7B show the test sensors used in the method of the invention, the test sensors having stepped arrays of secondary analysis areas.

Fig. 8A shows a version not according to the invention in which many working electrodes are electrically connected.

Fig. 8B shows a version not according to the invention in which a plurality of counter electrodes are electrically connected.

Fig. 9A shows one electron transfer mediator transferring one electron.

Fig. 9B shows a multiple electron transfer mediator that carries two electrons.

Fig. 10A shows a system not according to the invention having three independently addressable counter electrodes, each working on a different potential, and three electrically connected working electrodes, each having a mediator system that works on a different potential.

Fig. 10B shows the cyclic voltograms of ruthenium (III) hexamine, ferrocyanide and an electroactive organic molecule.

Fig. 10C is a graph of the relationship of the anti-electrode potential and the ratio of the redox conjugate pair.

-6Fig. 10D illustrates the charge transfer systems of many independently addressable counter electrodes, not according to the invention.

Fig. 10E shows cyclical voltamograms determining different action potentials that can be provided for one or more electrodes operating through a plurality of independently addressable counter electrodes.

Fig. 11A specifies that the charge transfer systems of Fig. 10E can be replaced by multiple redox conjugate vapor coefficients to provide system potentials.

Fig. 11B shows the current profiles obtained when the potential at one substantially chemically isolated working electrode is repetitively controlled in sequence by three essentially chemically isolated and independently addressable counter electrodes, each having a different potential provided by different charge transfer systems.

Fig. 12A schematically illustrates a biosensor system not according to the invention that determines the reagent concentration in a biological fluid sample.

Fig. 12B to Fig. 12F show many versions of the potentiostat not according to the invention that can be used with the signal generator of fig. 12A.

Fig. 13 shows an electrochemical analysis not according to the invention for determining the presence and / or concentration of at least one reagent in a sample.

Fig. 14A shows an input signal from a sequential, gated, amperometric pulse sequence, used in conjunction with a test sensor having independently addressable counter electrodes and operating electrodes, according to an embodiment of the invention.

Fig. 14B shows an input from a simultaneous gated amperometric pulse sequence used in combination with a test sensor having independently addressable counter electrodes and operating electrodes in accordance with an embodiment of the present invention.

Fig. 15 shows the results of averaging the results of up to four separate analyzes for the same reagent to determine the reagent concentration in the sample.

Fig. 16 shows the current attenuations obtained from the signal averaging experiment.

Detailed Description [0014] Fig. 1A shows a test sensor system 100 where the sample is introduced to the top of primary area 110 through sample port 115 and flows in a substantially symmetrical manner to fill the four secondary analysis regions 150. Each of the secondary analysis regions 150 includes an outlet 120 to allow the sample to evacuate air from secondary analysis regions 150 during filling. The outlet 120 may be of any shape that is compatible with the shape of the secondary analysis regions 150, such as circular or polygonal. The maximum diameter or width of the outlet 120 can be any size that provides the desired flow

- samples for secondary analysis regions 150, with preferred values ​​from about 0.02 mm to about 1.5 mm.

[0015] A single counter electrode 130 occupies a primary area, while a working electrode 141-144 is present in each secondary analysis region 150. When shown with a counter electrode 130 in primary area 110 and working electrodes 141-144 in secondary analysis regions 150, the position of the working electrodes and counter electrodes could be inverted so that multiple counter electrodes surround a single working electrode (not shown). In another aspect, the electrodes may not occupy the same plane. For example, some electrodes can be arranged horizontally, while others are arranged vertically. In another example, some electrodes may be placed higher than others so that the biological fluid first reaches the lower electrodes. Other electrode configurations can also be used. For example, Fig. 1B shows the test sensor of Fig. 1A with the addition of reference electrode 170 to provide unchanging potential.

[0016] Fig. 1C shows a test sensor 100 where instead of a single counter electrode 130, four independent counter electrodes 131-134 are provided in the central primary area 110. When shown with counter electrodes in the primary area and electrodes operating from secondary analysis regions, the position of any working electrode and any counter electrode may be inverted (not shown). Other electrode configurations can be used.

[0017] Fig. 1D illustrates the test sensor of Fig. 1C with the addition of reference electrode 170 for each secondary analysis region to ensure invariable potential. One or more reference electrodes 170 can operate at one or more potentials to provide invariable potential for each analysis. Because the counter electrode's operating potential may vary, one or more reference electrodes can be used to reference the potential of the counter electrodes in addition to the reference of the working electrode potential, as is common in conventional systems.

[0018] Although not shown in the figure, for test sensors implemented in continuous monitoring applications, such as for electrodes implanted in a living organism or otherwise in contact with biological fluid, the use of multiple reference electrodes may provide increased accuracy and / or precision of specific reagent concentrations. The increase may be due to the reduction of problems associated with the changing potential of working electrodes, implanted in a living organism or otherwise in constant contact with the biological fluid.

[0019] Figs. 1A and 1B, conductors 160 lead from each electrode towards the rear of the test sensor 100, where each conductor 160 can be connected to a measuring device, allowing each working electrode 141-144 to address independently. Therefore, when the conductor 160 is attached to a single electrode, the electrode is independently addressable. Conductors 160 may remain independently addressable or any two or more may be electrically connected (not shown). In relation with

With this, when more than one electrode is electrically connected to the same conductor, the electrodes are not independently addressable because they are electrically addressed together. For example, by electrically connecting two working electrodes 141-144, such as 141 and 144, the resulting test sensor 100 would have three independently addressable working electrodes and one counter electrode 130.

[0020] When configured with a single counter electrode 130 and four independently addressable working electrodes 141-144, the test sensor 100 of Fig. 1A and Fig. 1B can potentially perform a different analysis for each of the working electrodes 141-144. A single counter electrode 130 can provide a single potential for a system by using a charge transfer system that operates at a single potential. Depending on the measuring device, a single counter electrode 130 can provide more than one potential for the system.

[0021] If the electrode types were inverted for test sensor 100 of 1A and Fig. 1B so that there were four independently addressable counter electrodes and a single working electrode, the electrochemistry at the working electrode could potentially be measured at four different potentials. The independent addressability of the counter electrodes allows each counter electrode to be molded with a different charge transfer system, thus changing the potential provided to the working electrode during the analysis. If the working electrode contains reagents that interact with one or more reagents at four different potentials, each reagent interaction can be independently measured by electric addressing of the appropriate counter electrode. Preferably, each independently addressable counter electrode operates at a single potential or potential range.

[0022] In Figs. 1C and 1D, conductors 160 lead from each electrode toward the rear of the test sensor 100, where each conductor 160 can be connected to a measuring device. This setting allows each working electrode 141-144 and each counter electrode 131-134 to address independently. Conductors 160 may remain electrically insulated or any two or more may be electrically connected (not shown). For example, by electrically connecting two counter electrodes, such as 132 and 133, the resulting test sensor would have four independently addressable working electrodes and three independently addressable counter electrodes. Any combination of electrodes can be electrically connected.

[0023] Independently addressable working electrodes potentially allow measurement of a different chemical reaction at each working electrode 141-144. Having independently addressable counter electrodes 131-134 with different action potentials allows the electrode to work against more than one counter electrode potential. Therefore, two charge transfer chemicals present at the same working electrode can be measured independently by two independently addressable counter electrodes, where the first counter electrode operates at the potential of the first charge transfer chemical and the second counter electrode works at the potential of the second charge transfer chemical .

[0024] The test sensor 100 of Fig. 1C provides independent addressability to four working electrodes 141-144 and four counter electrodes 131-134. Due to the fact that each counter electrode can provide a different potential, sixteen different analyzes can potentially be performed. Therefore, the electrochemistry of a single working electrode can be measured at four different potentials, and the potential of a single counter electrode can be used for the chemistry of four different working electrodes. The test sensor of Figure 1D, having four independently addressable reference electrodes 170, can provide the system with up to four different invariant potentials. The measuring device may use one or more immutable potentials to control or determine the action potential at working electrodes 141-144 and at counter electrodes 131-134.

[0025] For the test sensor 100 of Figs. 1A to Fig. 1D, secondary analysis regions 150 may have areas of about 0.5 mm² and a height of about 0.125 mm to provide internal volumes of about 62 nL each. Preferably, the secondary analysis regions have internal volumes of 100 nL and less, with internal volumes of 70 nL and less being more preferred. Larger and smaller secondary analysis areas can be used.

[0026] Fig. 2A illustrates a test sensor system 200 where sample is introduced through sample port 215 at the leading edge 214 of test sensor 200 into primary area 210 and then occurs asymmetrically to fill the first secondary analysis region 251 and the second secondary analysis region 252 Sample flow is asymmetrical because the second secondary analysis region 252 is longer than the first secondary analysis regions 251. Secondary analysis regions 251, 252 may include an outlet 220 to allow the sample to evacuate air from the region during filling.

[0027] At the entrance, the sample passes through the first electrode pair, defined by: working electrode 241 and counter electrode 231. Continuing to pass through the first pair of electrodes, the sample flows towards the second and third electrode pairs, defined by the working electrode 242 and the counter electrode 232 (second pair ) and working electrode 243 and counter electrode 233 (third pair). The sample flowing through the first and third electrode pairs then continues until it passes through the fourth electrode pair, defined by the working electrode 244 and counter electrode 234. Therefore, the sample passes through the fourth pair of electrodes after the first and third pair of electrodes. When passing through the sample, the reagent composition 280 provides electrical conductivity between the pairs of working electrodes and counter electrodes. The independent addressability of the electrode pairs allows filling of secondary analysis regions 251, 252 for monitoring. Other electrode configurations may be used, for example, the position of any working electrode and any counter electrode may be inverted (not shown).

[0028] By monitoring the filling of secondary analysis regions 251, 252, the test sensor 200 provides an underfill detection system to prevent or screen analyzes

-10 related to sample sizes that are insufficient in volume. Because concentration values ​​obtained from an incomplete test sensor may be inaccurate, the ability to prevent or screen these inaccurate analyzes may increase the accuracy of the concentration values ​​obtained. Conventional underfill detection systems have one or more indicators, such as an electrode or conductor, that detect partial and / or complete filling of the sample container within the test sensor. Having the ability to monitor filling between multiple secondary analysis areas, it is possible to more accurately determine the fill level of test sensor 200. An electrical signal can be used to indicate whether a sample is present and whether the sample partially or completely fills a specific region of analysis.

[0029] Fig. 2B illustrates a test sensor 200 having the electrode system of Fig. 2A, but with a different arrangement of secondary analysis regions. The primary area 210, containing the first pair of electrodes, is provided with three symmetrically filled, secondary analysis regions 253, 254, 255. At the entrance, the sample passes through the first pair of electrodes and then independently moves to pass the second, third and fourth pair of electrodes. Generally, the fluid remains asymmetrical due to the first pair of electrodes occupying the primary area, thus filling before the secondary regions of analysis. Each of the secondary analysis regions 253, 254, 255 may include an outlet 220 to allow the sample to evacuate air while filling the test sensor 200.

[0030] A single reagent composition 280 may extend between each of the four pairs of working electrodes and counter electrodes, as shown. The guide 260 leads from each electrode towards the back of the test sensor 200, which can be connected to a measuring device, enabling each electrode to address independently. Although each electrode is independently addressable, each pair of electrodes share the same chemical environment due to the same reagent layer in contact with both the working electrode and the counter electrode of each pair. The electrodes may remain electrically insulated as well as any two or more may be electrically connected (not shown). One or more reference electrodes may be added to ensure invariable potential (not shown).

[0031] Despite the representation with centrally grouped counter electrodes and electrodes operating around the perimeter, the position of any working electrode and counter electrode can be reversed. Four independent working electrodes allow four different reagent compositions to potentially perform four different analyzes. Although four independent counter electrodes can be operated each with a different potential to provide 16 possible analyzes, 90 ° separation between each pair of electrodes may make this impractical.

[0032] Fig. 3A is a design of a rectangular test sensor where the sample flows from primary area 310 through the first potential electrode site 320 to reach the second potential electrode site 330; and Figures 3B to 3G show alternative designs for secondary analysis regions, with the sample

-11 does not flow through more than one potential electrode site. Fig. 3B illustrates the design of the T channel used in some conventional sensors. Fig. 3C illustrates a design with multiple T channels, with additional potential electrode locations 340 and 350 shown. Additional "T" portions may be added if additional potential electrode locations are desired.

[0033] Fig. 3H shows a multi-channel T test sensor 300 having both an independently addressable working electrode 331 and an independently addressable counter electrode 332 in each of the four secondary analysis regions 333. Therefore, each pair of working electrode and counter electrode divides this chemical environment alone, however, each pair of electrodes is essentially chemically isolated from each other pair. The 336 charge transfer system of the combined reagent composition is deposited on each pair of electrodes. Each of the working electrodes 331 and each of the counter electrodes 332 are formed from a conductor 334, which terminates in contact 335. Contact 335a and contact 335b correspond respectively to the working electrodes and counter electrode from the secondary analysis region 333a. The width of each of the secondary analysis regions 333 is 1.2 mm, while the width of primary area 310 is 1.5 mm. The distance in a straight line between the electrode pairs in opposite secondary analysis areas is 3.46 mm. The working electrode width of each pair is defined as 0.50 mm separated from the counter electrode by about 0.05 mm to about 0.25 mm. The circles drawn on each of the 331 working electrodes are the predicted coverage area of ​​the reagent composition. Other widths of the secondary analysis region, electrode widths and separations, and reagent composition coverage areas may be used.

[0034] Fig. 3I illustrates a multi-channel T test sensor 300 having an independently addressable working electrode 331 in each of four essentially chemically isolated secondary analysis regions, and an independently addressable counter electrode 332 in each of four opposing secondary analysis regions 333. Therefore, each electrode is generally chemically insulated from each other electrode. Each electrode is formed from a conductor 334 that terminates at contact 335.

[0035] Fig. 3D illustrates the departure from T-channel designs because the secondary analysis regions are staggered so that the straight line 370 passing through the secondary analysis regions and the primary area cannot be drawn between any two potential electrode locations. A potential advantage of such a staggered design is the resistance to mixing between opposing secondary analysis regions if the test sensor is mechanically disturbed during sample filling. Mechanically disturbed means applying sufficient force to cause the test sensor to move the fluid sample.

[0036] In addition to the failure in the straight-line test, the Y-channel designs of Fig. 3E to Fig. 3G resist mixing between potential electrode sites that are closer together than compared to the designs of Figures 3B and 3C, because the separation of the secondary analysis regions does not is based solely on the distance between potential electrode sites for basic chemical insulation. Chemical separation in the Y channel can

-12 also benefit from a sample that must flow around the "v" portion of the "Y" for mixing. Because the electrodes can be separated closer together, but still resist sample mixing, the total sample volume of the Y channel design sample may be smaller compared to the T channel design having similar chemical separation.

[0037] Preferred sample tank designs have secondary analysis regions branching off from primary area 310 at an angle of less than 90 °, as shown in Figure 3F. In this method, the fluid can enter the test sensor and reach potential electrode locations without turning 90 °. This can allow the sample to quickly enter the sensor, reducing the potential for mixing reagents from sample convection due to vibration. More preferred designs do not include a straight line 370 as shown in Figures 3B and 3C between the electrodes passing through the secondary analysis regions and the primary area and have secondary analysis regions branching from the primary area at an angle of less than 90 °. Other projects, such as those having one or more bends in the primary and / or secondary area in the analysis areas and those where the secondary analysis regions branch off from the primary area at an angle greater than 90 °, may also be used; however, increasing sample size requirements and slower sample filling rates can be limiting factors.

[0038] Fig. 3J illustrates a Y-channel test sensor 300 having both an independently addressable working electrode 331 and an independently addressable counter electrode 332 in each of the two secondary analysis regions 333. Therefore, each pair of working electrode and counter electrode share the same chemical environment, however, each pair of electrodes is essentially chemically isolated from the opposite pair. When the working electrode 331 passes through the secondary analysis region 333, counter-electrode 332 is defined by the edge of the boundary of the secondary analysis region 333, which in effect is formed from the guide 334. The secondary analysis regions 333 branch from the primary region 310 at an angle of about 45 °. Each of the 334 guides terminates in contact area 335. Other electrode designs may be used, such as those in which a single electrode is formed in one or more secondary analysis regions. Other branching angles for secondary analysis regions can also be used.

[0039] The substrate of the test sensor 300 has a width of 11.8 mm and a length of 30 mm. The width of the primary area 310 is 1.2 mm. The distance between the predicted outer edges of the two sediments of the reagent composition is 0.8 mm. Contact areas 335 have a width of 2.9 mm each and the diameter of the precipitate in the reagent composition in each of the two secondary analysis regions 333 is 1.8 mm. Other substrate dimensions, primary area width and contact area as well as sediment diameters of the reagent composition may be used.

[0040] In addition to the number and type of electrodes and the degree of independent electrical addressability of the electrodes, the degree of chemical insulation provided by the secondary regions of the sample tank analysis affects the number of analyzes that can be performed with the test sensor. Basically chemically isolated means that it is diffusive or convective

Mixing of reagents does not generally occur between secondary analysis regions during one or more analyzes.

[0041] If the pair of working electrode and counter electrode are substantially chemically insulated from other pairs of working electrodes and counter electrodes, but not from each other, the pair may perform analyzes compatible with the chemical compound present in the pair. This configuration can enable rapid diffusion mixing of reagents present at the working electrodes and counter electrode in pair. Conversely, if the working electrodes and the counter electrode are essentially chemically isolated from other working and counter electrodes and from each other, each electrode can potentially participate in the analysis with any other electrode if it is independently addressable. Therefore, with substantially chemical isolation, different reagent compositions can be used to provide the electrode with a chemical analysis environment that is different from other electrodes. In combination, basic chemical isolation between analysis areas allows different reagents to be used for each working electrode and / or counter electrode, while independent electrical addressability allows each working electrode to measure independently.

[0042] The secondary analysis regions can generally be chemically isolated depending on the cross-sectional area of ​​the entrances to the secondary regions, the distance between any two electrodes within the secondary analysis regions, the physical arrangement of the secondary analysis regions relative to each other and relative to the primary area and this like. In addition to these concerns, substantial initial chemical isolation may be lost due to mixing of reagents as the sample flows through the counter electrode / counter electrodes (Fig. 1A to Fig. 1D) or electrode pairs at the entrance and on the sides of the test sensor (Fig. 2A and Fig. 2B). In this way, the reagent composition can be transported through the sample to multiple electrode pairs. Conversely, such flow mixing can be substantially eliminated when the sample does not flow more than one electrode (Fig. 3B - Fig. 3J).

[0043] Fig. 4A shows the cyclic voltometry function of a straight-channel design as shown in Fig. 3A. The first pair of electrodes closest to the sample port used a reagent composition containing 0.5M potassium cyanoferrate, while the second pair of electrodes closest to the end of the channel used a reagent composition containing an electroactive organic molecule as depicted in Structure I below. Within about seven seconds or less, two peaks were observed, with the left peak showing the oxidation of the reduced state of the Structure I molecule and the right peak showing the oxidation of cyanoferrate, which was initially removed at the first pair of electrodes. Within approximately 20 full cycles, the molecular peak of Structure I disappeared, suggesting that the cyanoferrate oxidized the Structure I molecule.

[0044] During the analysis, it is believed that the (III) cyanoferrate from the first pair of electrodes was oxidized at the second electrode to form the (II) cyanoferrate. The resulting (II) cyanoferrate then oxidized chemically reduced Structure I particles with a second pair of electrodes. These results determined that chemical contamination between electrode pairs appears quickly in a straight-channel design. The experiment shows that

The stronger oxidizing agent such as (III) cyanoferrate in this case will take over mediation from other mediators such as the Structure I molecule if the electrodes are not substantially chemically isolated. This contamination is considered to be attributed to the combination of passing the sample through the counter electrode before reaching the working electrode, diffusion and convection within the rectangular tank.

[0045] On the other hand, Fig. 4B shows cyclic voltammograms of the Y channel design as shown in Fig. 3E. The electrode was placed near the end of each secondary analysis region. Only oxidation of the Structure I molecule is observed after 20 full cycles (over 20 minutes), specifying that substantial chemical isolation has been achieved for at least 10 minutes with the design of the Y-channel secondary analysis region. These experiments were performed using CH Instruments Electrochemical Workstation, model CHI 660A, supporting version 2.05 software, at about 22 ° C and relative humidity of about 45%. The sample was a pH 7.0 phosphate buffer containing 0.1 M sodium phosphate and about 16% (w / w) PVP polymer having an average molecular weight of about 2000.

[0046] A similar effect was observed for chemoamperometry testing, where current is measured as a function of time. In Fig. 5A, the current versus time graph indicates that for a rectangular sensor of the type used in Fig. 4A, a second peak was observed with an operating potential of 400 mV with the electrode operating within about 5 seconds of sample introduction. Sample introduction generated the first peak in the graph. The second peak corresponds to the second voltometric wave ferrocyanide (II) in Fig. 4A. Figure 5B shows that substantially no ferrocyanide (II) reached the working electrode after 30 seconds, determining that substantial chemical isolation was achieved with the Y-channel secondary analysis region test sensor. In these experiments, the initial sharp peak represented the sample, first establishing electrical connectivity between the electrodes. Amperometry testing was performed using CH Instruments Electrochemical Workstation at about 22 ° C and relative humidity of about 45%. The sample was a pH 7.0 phosphate buffer containing 0.1 M sodium phosphate and approximately 16% (w / w) PVP polymer. having an average molecular weight of about 2000.

[0047] Fig. 5C is a graph of amperometric current, specifying that the Y channel design provides better chemical insulation between potential electrode sites than the T channel design. As illustrated by the Y channel line 501, substantial chemical isolation has been observed up to 1000 seconds between potential electrode locations as shown by positions 320 and 330 in Figure 3E. In contrast, as shown by the T-channel peaks 502, 503, failure of chemical isolation and oxidation of the Structure I molecule was observed after about 84 to about 650 seconds for two Fabric Test sensors as shown in Figure 3B. The large variability between the 84 and 650 seconds time variables can be attributed to the susceptibility of the T channel design to mixing by convection from mechanical disturbance during analysis. FIG. 5D specifies that three Y-channel designs were resistant to such mixing from mechanical disturbances.

-15 The slow current increase observed after approximately 800 seconds may indicate slow mixing by diffusion.

[0048] Fig. 6A shows a test sensor 600 having a staggered arrangement of secondary analysis regions 651, 652 where the sample enters sample port 615 into primary area 610 in the form of a channel from which two secondary analysis regions 650 branch off. Guide 690 can extend from the primary area 610 to provide the ability to detect underflow in the test sensor 600. Similarly, FIG. 6B illustrates a test sensor arrangement wherein the sample enters sample port 615 into primary area 610 in the form of a channel from which secondary regions 651-653 branch off. Each of secondary regions 651-653 includes an independently addressable electrode or conductor.

[0049] In Fig. 6A, the sample fills the first secondary area 651 to the right, then the second secondary area 652 to the left. In Fig. 6B, the sample fills the third secondary area 653 to the left, then the first secondary area 651 to the right, and then the second secondary area 652 to the left.

[0050] The total volume of the sample held by the test sensor 600 having at least two or three secondary analysis regions may be 210 nL or less. Each of the secondary analysis regions and the end of the primary area 610 opposite the sample port 615 may include an outlet 620 to allow the sample to evacuate air during filling. By dividing the sample reservoir defined by primary area 610 and secondary analysis regions 651-653 into one or more primary areas that fill multiple secondary regions, the test sensor 600 may fill faster than the substantially undivided sample reservoir, such as the rectangular design shown in Figure 3A of the same or similar volume due to the effect of capillary action driven by surface tension. Therefore, re-dividing the sample container into smaller secondary analysis areas, each of which may contain an electrode, a pair of electrodes, one or more conductors or a combination thereof, can increase the filling rate for test sensor 600. Essential chemical isolation between secondary regions during filling and during analysis can be ensured by filling the secondary areas from the primary area in this way.

[0051] Because the sample flows mainly to the nearest mouth 620, secondary regions 651-653 are filled in a substantially sequential manner from primary area 610. Because of the sequential filling of secondary regions 651-653, the measuring device can monitor the rate and flow of the sample as as secondary analysis areas 651-653 are filled. Sample flow can also be monitored by equipping test sensor 600 with an electrode or conductor near sample port 615 and / or near outlet 620 of primary area 610. Therefore, one or more conductors and / or electrodes can be monitored by a measuring device to determine the filling level of the test sensor 600. The filling of non-sequential filling designs can also be monitored in this way,

16 however, the system may or may not be able to monitor the filling of each secondary analysis region independently.

[0052] Although not shown in the figure, primary area 610 may be provided with a plurality of sample ports 615 to allow the sample to be introduced from more than one location, such as at the border and at its peak. Similarly, test sensor 600 may be provided with two or more separate sample tanks, each having a primary area and two or more secondary areas, to allow analysis of multiple samples. By changing the structure of the mouth of the tank, different samples can be introduced through multiple sample ports into the same tank, however, remaining essentially chemically isolated during analysis. Other relationships between the primary area or areas and the secondary areas may be used.

[0053] Primary area 610 and / or one or more secondary regions 651653 may contain flow-changing materials that modify the flow of the sample as it spreads in the sample reservoir. For example, hydrophilic and / or hydrophobic treatments, coatings or materials can be used to advantageously direct the flow path and / or rate of filling of aqueous samples. In another aspect, primary area 610 and / or secondary areas 651-653 may include structural features such as walls, grooves, or channels that preferably direct the flow path and / or sample fill rate. In another aspect, materials that chemically or physically alter the composition of the sample may be placed on primary area 610 and / or secondary areas 651-653. For example, material that filters red blood cells from the sample may be placed in a portion of the primary area to remove cells before the sample reaches the secondary area.

[0054] Figs. 7A and 7B show test sensors having designs of staggered secondary analysis regions as discussed previously. The design of Fig. 7A includes eight secondary analysis regions containing angles approximately 90 ° relative to the primary area 710, while Fig. 7B is a similar design of the Y channel. The test sensor 700 contains a total of nine secondary analysis regions, comprising a region at the end of the primary area 710, each occupied by an electrode or conductor. The figure shows four independently addressable counter electrodes 731-734 and four working electrodes 741-744, each present in one of eight secondary regions. Although counter electrodes 731-734 are on one side of primary area 710 and working electrodes 741-744 are on the other side, the system may be mixed up. For example, the first two secondary analysis regions filled with the sample may be working electrodes, while the second two secondary analysis regions filled with the sample may be counter electrodes.

[0055] An optional electrode, such as reference electrode 770 occurs at the end of the primary area 710 opposite the sample port 715. Reference electrode 770 may also be located in the rearmost secondary region relative to where the sample is introduced or, for example, near port 715 of the sample. In this regard, one or more reference electrodes may be placed

In the 710 primary area and / or secondary areas to ensure the system's unchanging potential. When in an environment that is generally chemically insulated from secondary areas, optional electrodes can provide fill information or sample information.

[0056] The conductor 790 electrically connected to the counter electrode 731 is extended to the primary area 710 near the sample port 715. Although not independently addressable, guide 790 can provide fill information for a measuring device. Other electrode and / or conductor configurations are possible. Each secondary region and end of primary area 710 may include an estuary (not shown).

[0057] Eight electrodes 731-734 and 741-744 can be addressed independently by the measuring device. Since the secondary regions are essentially chemically isolated, each may contain a reagent composition providing other chemical compounds for interacting with the sample components. Because the reagent composition may be different for each of the working electrodes 741-744, the charge transfer system may be different for each of the counter electrodes 731-734 and each electrode can be addressed independently, four different analyzes may be possible when a single reagent composition is present at each of the working electrodes 741-744. In this way, any working electrode reagent composition can be used with a dedicated counter electrode. Similarly, if each of the working electrodes 741-744 were provided with two reagent compositions having different redox potentials, a total of eight different analyzes may be possible. Finally, providing each working electrode with four reagent compositions having different redox potentials can provide up to sixteen different analyzes because each working electrode can be independently addressed with each of the four counter electrodes. Practical considerations, such as unwanted interaction between more than one reactant composition on a working electrode, can limit the actual number of analyzes that can be performed by the system. Other sample reservoir designs and electrode configurations can be used.

[0058] Fig. 8A shows a variation of the test sensor of Fig. 7A according to the invention, wherein a plurality of working electrodes 841-844 are electrically connected. Counter electrodes remain independently addressable. In this way, each counter electrode can provide different potential for electrically connected working electrodes. By electrically connecting one or more working electrodes, a working electrode having the redox potential closest to the potential of the selected counter electrode can work. In this mode of operation, each working electrode may have a different mediator system, each mediator system having a different redox potential. By changing the system operating potential from low to high using different counter electrode potentials, different working electrode mediator systems can be addressed progressively. Other sample reservoir designs and electrode configurations can be used.

[0059] Fig. 8B illustrates a variation of Fig. 7A, wherein a plurality of counter electrodes 831-834 are electrically connected. Working electrodes can remain addressable independently. By

-18 electrical connection of one or more counter electrodes, a counter electrode having a charge transfer system with the highest potential can supply the potential to the system. In this way, the electrochemical response to the reagent with each working electrode can be measured. Other sample reservoir designs and electrode configurations may be used.

[0060] Regarding the previously described test sensors, working electrodes and counter electrodes present in secondary analysis regions may be spaced 1000 micrometers or more. Electrode separation distances of less than 1000 microns can also be used. The electrode pattern is not limited to those shown in the figures, instead it is any pattern compatible with the primary area and secondary areas of the test sensor analysis. Preferably, the electrodes are formed by rectangular deposition of the reagent composition and / or charge transfer system. Deposition can be created by screen printing, inkjet printing, micropipette, needle deposition or other methods.

[0061] Reagent layers are formed when the reagent composition is applied to the conductor. For example, the reagent layer forming the working electrode may contain an enzyme, mediator and linker, while the reagent layer forming the counter electrode may include a mediator and linker. Reagents undergo an electrochemical reaction at the working electrode, while the opposite electrochemical reaction occurs on the counter electrode and allows current to flow between the electrodes. For example, if the reagent undergoes oxidation at the working electrode, the reduction occurs at the counter electrode.

[0062] In addition to the working electrodes and counter electrodes, the test sensors may include reference electrodes that ensure the system's constant reference potential. Although many reference electrode materials are known, the composition of silver (Ag) and silver chloride (AgCl) is typical due to the insolubility of the metal and its corresponding salt in the aqueous sample environment. Because the ratio of Ag metal to Cl * does not change significantly in the sample, the electrode potential does not change significantly. If the size and / or modification of the conductive metal is increased, the reference electrode can be used as a counter electrode because it will transfer current. However, a counter electrode cannot serve as a reference electrode because it does not have the ability to isolate half the cell that provides reference potential from the sample solution.

[0063] The conductors that make up the electrodes may be on one or more substrates, depending on the arrangement of the electrodes. The substrate can be made of any material that is compatible with the formation and operation of the biosensor. Preferred materials for the substrate include polytetylene terephthalate (PET), polycarbonate (PC), polyimide (PI), polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), polyoxymethylene (POM), polymer cast nylon (MC), polybutylene teraphthalate (PBT), polymethacrylic resin (PMMA), ABS resin (ABS) and glass. More preferred materials for forming one or more substrates include polyethylene terephthalate (PET), polycarbonate (PC) and polyimide (PI), with currently preferred polyethylene terephthalate

-19 (PET). For forming the test sensor, two substrates in the form of a base and lid can be combined to form a sample container having at least one sample port and at least one outlet. Conductors, splitters and other components may rest between substrates.

[0064] The material or materials used to form conductors on one or more substrates may include any electrical conductor. Preferred electrical conductors are non-ionizing so that the material does not undergo net oxidation or net reduction during sample analysis. Conductors may be made of materials such as solid metals, metal pastes, conductive carbon, conductive carbon pastes, conductive polymers and the like. The conductors preferably contain a thin layer of metal paste or a metal such as gold, silver, platinum, palladium, copper or tungsten. The surface conductor can be embedded on all or part of the conductor. The conductive surface material preferably includes carbon, gold, platinum, palladium or combinations thereof. If the surface conductor is not present on the conductor, the conductor is preferably made of non-ionizing material.

[0065] The conductor and the optional conductive surface material may be deposited onto the substrate by any means compatible with the test sensor, including film deposition, chemical vapor deposition, suspension deposition, metallization and the like. In another aspect, conductors can be formed by processing a conductive layer into a pattern using laser and / or mask techniques.

[0066] The reagent composition or compositions used to form the electrodes may be deposited in solid, semi-solid, liquid, gel, gel, colloidal or other form and may include reagents and optionally a linker. The reagent compositions may contain viscosities in the range of from about 1 cp to about 100 cp. More preferably, the reagent compositions have viscosities in the range of from about 1 cp to about 20 cp or from about 4 cp to about 10 cp. Reagent compositions with other viscosities can be used. Viscosities were determined using a Brookfield Model DV3 Viscometer equipped with a ULA assembly for measuring reagent compositions having viscosities lower than 300 cp. Viscosity measurements were carried out at room temperatures with the instrument temperature set at 25 ° C. Measurements were performed at 50, 100, 200 and 300 cps shear rates (cycles per second) to provide an indication of whether the composition is thin or thick shear. A 100 mM phosphate buffer solution was used as a control, which usually gave viscosity readings in the range of about 1 to about 1.3 cp at different shear rates.

[0067] The linker is preferably a polymeric material that is at least partially water-soluble. The connector may form a gel or gel-like material when hydrated. Suitable partially water-soluble polymer materials for use as a linker may include polyethylene oxide (PEO), carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA), hydroxyethylene cellulose (HEC), hydroxypropyl cellulose (HPC), methyl cellulose, ethyl cellulose, ethyl cellulose hydroxyethyl carboxymethyl ethyl cellulose, polyvinyl pyrrolidone (PVP), acids

Polyamine, such as polylysine, polystyrene sulfate, gelatin and their derivatives, polyacrylic acid and its derivatives and salts, polymethacrylic acid and its derivatives and salts, starch and their derivatives, maleic anhydrides and their salts, agarose gels and their derivatives. The connector may contain one or more of these materials in combination. Among the above connector materials, PEO, PVA, CMC and HEC are preferred, with CMC being more preferred for biosensors today. Other connectors may be used.

[0068] Linkers having molecular weights from 10,000 to 900,000 and preferably from 30,000 to 300,000 (weight / average) are preferred. Couplers having other molecular weights may be used. Molecular weights can be determined by size exclusion chromatography (SEC) and are generally expressed as weight average or number average.

[0069] The reagent composition used to form the working electrode preferably contains a biomolecule responsive to the reagent of interest. Biomolecules may contain active enzyme systems such as oxidoreductases. The biomolecules can also contain biopolymers such as nucleic acids, proteins and peptides. Other biomolecules can be used.

[0070] Oxidoreductases catalyze the transfer of electrons and enable the oxidation or reduction of a reagent and contain "oxidases" which allow oxidation reactions, wherein the oxygen molecule is an electron acceptor; "Reductases" which allow reduction reactions, whereby the reagent is reduced and the molecular flax is not the reagent; and "dehydrogenases" that facilitate oxidation reactions in which molecular oxygen is not an electron acceptor. See, for example, Oxford Dictionary of Biochemistry and Molecular Biology, Revised Edition, AD Smith, ed., New York: Oxford University Press (1997) pages 161, 476, 477 and 560. For example, Table I below provides oxidoreductases useful in the analysis of these reagents .

Table I

<td>oxidoreductase</td><td>Reagent</td>

<td>Glucose dehydrogenase</td><td>Beta-glucose</td>

<td>Glucose oxidase</td><td>Beta-glucose</td>

<td>Cholesterol esterase; cholesterol oxidase</td><td>cholesterol</td>

<td>Lipoprotein lipase, glycerol kinase, glycerol-3-phosphate oxidase</td><td>triglycerides</td>

<td>Lactate oxidase; lactate dehydrogenase;</td><td>lactate</td>

<td>diaphorase</td><td></td>

<td>Pyruvate oxidase</td><td>pyruvate</td>

<td>Alcohol oxidase</td><td>Alcohol</td>

<td>Bilirubin oxidase</td><td>bilirubin</td>

<td>uricase</td><td>Uric acid</td>

<td>Glutathione reductase</td><td>NAD (P) H</td>

<td>Carbon monoxide oxidoreductase</td><td>Carbon monoxide</td>

[0071] Biomolecules may contain amine functional groups capable of hydrogen binding interactions. Biomolecules having a weight / average molecular weight from 10,000 to 500,000 and preferably from 100,000 to 400,000 that retain biological activity after deposition are preferred. In the case of oxidoreductases, from 0.01 to 100 units (U), preferably from 0.05 to 10 U and more preferably from 0.1 to 5 U can be used for a test sensor or analysis. In another aspect, a maximum of 1.3 U of oxidoreductase is used.

[0072] The reagent layer, prepared by depositing the reagent composition on a conductor, may include a reagent-specific enzyme system that can facilitate the reagent reaction, while enhancing the specificity of the reagent sensor system, especially in complex biological samples. The enzyme system may contain one or more enzymes, a cofactor and / or a molecule that participates in the redox reaction with the reagent. For example, alcohol oxidase can be used to provide a biosensor that is sensitive to the presence of alcohol in a sample. Such a system may be useful in measuring blood alcohol levels. In another example, glucose dehydrogenase or glucose oxidase may be used to provide a biosensor that is sensitive to the presence of glucose in the sample. This system may be useful in measuring blood glucose levels, for example in patients who are known or suspected to have diabetes.

[0073] Preferred enzyme systems are independent of oxygen and are therefore not substantially oxidized by oxygen. One such family of oxygen-independent enzymes is glucose dehydrogenase (GDH). Using different coenzymes or cofactors, GDH can be mediated in a different way by different mediators. Depending on their association with GDH, a cofactor such as flavinoadenine dinucleotide (FAD) can be closely maintained by the host enzyme, as is the case with FAD-GDH; or a cofactor, such as pyrroloquinoline quinone (PQQ), can be covalently linked to the host enzyme, as in the case of PQQ-GDH. The cofactor in each of these enzyme systems can either be permanently maintained by the host enzyme or the coenzyme and apoenzyme can be regenerated again before the enzyme system is added to the reagent composition. Coenzyme may also be independently added to the host enzyme molecule in the reagent composition to assist in the catalytic function of the host enzyme, as in the case of NAD / NADH + nitotinamidoadenine dinucleotides or NADP / NADPH nicotinamidoadenine dinucleotide phosphate. Other useful dehydrogenase enzyme systems include alcohol dehydrogenase, lactate dehydrogenase, beta-hydroxybutyrate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase, formaldehyde dehydrogenase, maleate dehydrogenase, and 3-hydroxysteroid dehydrogenase.

[0074] The reagent layer may also include a mediator for communicating the results of the reagent with the conductor. Mediators can be oxidized or reduced and can carry one or more electrons. The mediator is a reagent in electrochemical analysis and is not a reagent of interest, but provides indirect reagent measurement. In a simple system, the mediator goes through a redox reaction

-22 in response to reagent oxidation or reduction. An oxidized or reduced mediator that passes through the opposite reaction at the working electrode of the test sensor and can be reconstituted to its original oxidation number. Therefore, the mediator may enable the transfer of electrons from the reagent to the working electrode.

[0075] Mediators can be separated into two groups based on their electrochemical activity. One electron transfer mediators are chemical molecules capable of receiving one additional electron under electrochemical reaction conditions. Multi-electron transfer mediators are chemical molecules capable of receiving more than one electron under reaction conditions. As shown in fig. 9A, one electron transfer mediators can transfer one electron from an enzyme to a working electrode, while the multi-electron transfer mediator shown in FIG. 9B can transfer two electrons.

[0076] Examples of one electron transfer mediators include compounds such as 1,1'-dimethylferocene, ferrocyanide (II) and ferrocyanide (III) and ruthenium (III) and ruthenium (II) hexamines. Two electron mediators include organic quinones and hydroquinones such as phenanthroline quinone; phenothiazine and fenoxazine derivatives; 3 (phenylamino) -3H-phenoxazine; phenothiazines; and 7-hydroxy-9,9-dimethyl-9H-acridin-2-one and its derivatives. An example of additional two-electron mediators includes electroactive organic molecules as described, for example, in US Pat. Nose. 5,393,615; 5,498,542 and 5,520,786.

[0077] Preferred two electron transfer mediators include 3-phenylamine-3H-phenothiazines (PIPT) and 3-phenylimine-3H-phenoxazine (PIPO). More preferred two-electron mediators contain a carboxylic acid or a salt such as ammonium salts or phenothiazine derivatives. Currently particularly preferred two electron mediators include (E) 2- (3H-phenothiazine-3-ylideneamino) benzene-1,4-disulfonic acid (Structure I), (E) -5- (3H-phenothiazine-3-ilideneamino) isophthalic acid ( Structure II), ammonium (3H-phenothiazine-3-ylideneamino) -5-carboxybenzoate (Structure III) and combinations thereof. The structural formulas of these mediators are presented below. Although only the diacid form of the Structure I mediator is shown, mono and di-alkali metal salts of the acid have been included. Currently, the sodium salt of the acid is preferred for the Structure I mediator. The alkali metal salts of the Structure II mediator can also be used.

<img file="PL2535705T3_D0001.tif" />

Structure I

<img file="PL2535705T3_D0002.tif" />

Structure II

<img file="PL2535705T3_D0003.tif" />

Structure III

In another aspect, preferred two-electron mediators have a redox potential that is at least 100 mV lower, more preferably at least 150 mV lower than ferrocyanide.

[0078] The charge transfer system is any or a combination of electrochemically active types that can transfer one or more electrons from a counter electrode or to a counter electrode. For example, if the working electrode of the system transfers electrons to the counter electrode through the measuring device, the counter electrode charge transfer system adopts electrons from the counter electrode to allow the measurement of current flow through the system. By accepting electrons at a specific potential or potential range, the charge transfer system affects the potential at which the working electrode can transfer the electrodes for measurement. The charge transfer system may or may not include a mediator present at the working electrode; however, if it includes at least a portion of the mediator at the counter electrode, it preferably has a different oxidation state than the mediator at the working electrode.

[0079] Because the electrochemical reaction with the lowest potential occurs first, by supplying working electrodes with one or more reagent sensitive biomolecules, such as oxidoreductase and / or mediators, which transport charge at increasing potentials, the electrochemistry of many working electrodes can be analyzed sequentially from the lowest to highest potential for action. If the working electrodes of the counter electrodes can be addressed independently, the working electrode having a specific redox potential with the reagent can be selectively paired with the counter electrode having the desired potential. If the redox potentials of the reagent, reagent sensitive biomolecule and / or mediator with different addressing working electrodes are different, separate output signals for individual analysis can be measured using electrically connected counter electrodes. Conversely, if redox potentials of types

Charge transfer with independently addressable counter electrodes are different, separate output signals for individual analysis can be measured using electrically connected working electrodes. When multiple counter electrodes have different types of charge transfer, but are electrically connected, the counter electrode with the highest potential will provide the operating potential of the working electrode until the potential of the system drops to the potential of the next counter electrode with the highest potential.

[0080] Fig. 10A shows a system having three independently addressable counter electrodes (CE1-CE3), each operating at a different potential, and three electrically connected working electrodes, each having a mediator system that operates at a different potential. Since the system's operating potential is increased with counter electrodes CE1 to CE3, the redox characteristics of mediators (Med1-Med3) with electrically connected working electrodes can be measured independently. For example, when CE1 is coupled to a working electrode, Med1 reacts at the electrode. When CE2 is coupled to a working electrode, Med1 and Med2 react at the electrode. Finally, when CE3 is coupled to a working electrode, all three mediator systems can respond to the working electrode.

[0081] Numerous operating potentials can be provided to the system by changing the charge transfer system embedded in various conductors to form counter electrodes. The potential provided by the specific counter electrode can be changed with charge transfer systems containing different types of redox (molecules that can be oxidized and / or reduced) and / or different vapor coefficients of redox conjugates (reduced and oxidized molecules of the same redox species) of redox types, such like ferrocyanide (II) / ferrocyanide (III). Examples of different types of redox for use in charge transfer systems include soluble or insoluble types of redox, where soluble types of redox are water-soluble (pH 7, 25 ° C) at a level of at least 1.0 gram per liter and exclude basic and solitary metals metal ions that are insoluble or sparingly soluble in water. Useful types of redox include electroactive organic molecules, organotra- tive metal complexes, and transition metal coordination complexes. Unlike metal-containing organotransmission metal complexes and coordination complexes, electroactive organic molecules do not contain metal capable of oxidation or reduction. Preferred types of redox for use in charge transfer systems include ruthenium (III) hexamine, ferrocyanide (III) and electroactive organic molecules such as PIPT and PIPO. Fig. 10B shows the cyclic voltammograms of ruthenium (III) hexacyanoferrate (III) and the electroactive organic molecule depicted above in Structure I / II / III. As can be seen in the graph, the relative potential positions of each type of redox are separated by about 200 mV.

[0082] Examples of different pair ratio of redox conjugates are the ratio of cyanoferrate (II) to cyanoferrate (III) in the charge transfer system. For example, a ratio of 9.5: 0.5 can be used for a counter electrode with the lowest potential, while a ratio of 8: 2, 5: 5, 2: 8, and 0.5: 9.5 can be used for

-25 providing counter electrodes with progressively increasing potentials of action. Pure ferrocyanide (III) can be used to provide a counter electrode having the highest potential for six counter electrodes. In this way, six independently addressable counter electrodes can be created using different redox conjugate pair ratios, each providing different potential for the system. Therefore, potential differences smaller than those obtained with different types of redox, such as at least 50 mV or at least 100 mV, can be obtained using different redox conjugate coefficients.

[0083] The relationship of counter electrode action potential as a function of (vs.) the coefficient of a pair of redox conjugates is characterized by the Nernst equation and is shown in Figure 10C. Depending on whether oxidation or reduction occurs at the counter electrode during the analysis, the desired potential can be provided to the counter electrode by selecting the appropriate pair ratio of redox conjugates for the embedded charge transfer system. By choosing different pair ratio of redox conjugates for charge transfer systems, the potential of the charge transfer system may differ by approximately ± 150 mV for different ratios of ferrocyanide (II) / ferrocyanide (III). Therefore, in addition to using different types of redox to provide different potentials for numerous counter electrodes, different redox conjugate coefficients can be used. The basic chemical isolation that can be provided by physical separation between the secondary regions allows a different charge transfer system from each counter electrode to provide different system potentials during analysis.

[0084] Fig. 10D illustrates the circumstances where the charge transfer systems of multiple independently addressable counter electrodes (CE1-CE3) provide different absolute action potentials, such as -200 mV, 0 mV, and +200 mV, while maintaining essentially the same relative action potential 0.4 V between counter electrodes and working electrodes. A central redox pair can arbitrarily be assigned a fixed zero potential relative to a Standard Hydrogen Electrode, a Saturated Calomel Electrode or the like. Therefore, ruthenium hexaamine has an erdox potential, which is about 200 mV lower, and cyanoferrate (III) has an erdox potential, which is about 200 mV higher than the potential of the Structure I / II / III molecule. By operating counter electrodes at different absolute action potentials relative to known potential, the system can independently analyze different mediator systems (Med1-Med3) with electrically connected working electrodes WE1 to WE3.

[0085] Fig. 10E shows cyclic voltammograms determining different action potentials that can be provided for one or more electrodes operating through a plurality of independently addressable counter electrodes. A test sensor having a multi-T design with eight secondary analysis regions was produced as previously shown in Figure 3I. Four of the secondary analysis regions were equipped with independently addressing working electrodes and four of the secondary analysis regions were equipped with independently addressable counter electrodes. Each working electrode was manufactured with a reagent composition containing a 0.5% HEC connector

-26 weight / weight (w / w), 50 mM molecule of Structure I and 2 U / uL of the PQQ-GDH enzyme system in phosphate buffer at pH 7. The first counter-electrode was formed with a charge transfer system containing a 0.5% HEC connector (w / w) and 100 mM ruthenium hexaamine in pH 7 phosphate buffer. A second counter electrode was formed with a charge transfer system containing a 0.5% HEC connector (w / w) and 100 mM Structure I molecule in pH 7 phosphate buffer. The third and fourth counter electrodes were formed with a charge transfer system containing a 0.5% HEC connector (w / w) and 100 mM ferrocyanide (III) in pH 7 phosphate buffer.

[0086] After introducing the 300 mg / dL glucose sample, the CH instrument was scanned at 25 mV / s for one of the working electrodes and each of the first, second and third counter electrodes. As shown in Figure 10B, the potential of the ruthenium hexamine counter electrode, line 1010, has a peak at a potential about 400 mV higher than (III) cyanoferrate, line 1030, with a Structure I molecule having a peak approximately in the middle, line 1020. In this way, the results observed for the cyclic voltammograms of Fig. 10B were reproduced in a multi-T test sensor design having a plurality of secondary and analysis regions. Accordingly, the test sensor's ability to operate at multiple potentials has been demonstrated using multiple counter electrodes with various charge transfer systems.

[0087] Fig. 11A specifies that the charge transfer systems of Fig. 10E can be replaced by a plurality of redox conjugate vapor ratios to provide multiple system potentials. The test sensor has been prepared as in fig. 10E, but the first counter electrode was created with a charge transfer system containing a 0.5% HEC connector (w / w) and a ratio of 1: 9 cyanoferrate (III): 200 mM cyanoferrate in phosphate buffer at pH 7, the second counter electrode was formed with a charge transfer system containing a HEC connector 0.5% (w / w) and a 1: 1 ratio of ferrocyanide (III): cyanoferrate (II) 200 mM in phosphate buffer at pH7, the third counter-electrode was created with a charge transfer system containing a 0.5% HEC connector (w / w) and a 9: 1 ratio of ferrous (III) cyanoferrate (II) 200 mM in phosphate buffer at pH 7 and a fourth counter-electrode was created with the transfer system charge, containing a 0.5% HEC linker (w / w) and substantially pure 200 mM cyanoferrate in phosphate buffer at pH 7.

[0088] After introducing the 300 mg / dL glucose sample, the instrument was scanned at 25 mV / s for one of the working electrodes and each of the first, second, third and fourth counter electrodes. FIG. 11A shows a first counter electrode having a peak potential of about 0.149 V (W1 - C1), a second counter electrode having a peak potential of about 0.060 V (W2 - C2), a third counter electrode having a peak potential of about -0.007 V (W3 - C3) and a fourth counter electrode having a peak potential about -0.047 V (W4 - C4). Therefore, the test sensor's ability to operate at multiple potentials has been demonstrated using multiple counter electrodes with charge transfer systems based on different redox conjugate coefficients.

[0089] Fig. 11B shows the current profiles obtained when the potential at one substantially chemically isolated working electrode is repetitively controlled in sequence by three substantially chemically isolated and independently addressable counter electrodes, each having a different potential provided by different charge transfer systems. The test sensor was prepared as in Figure 10E, however, many working electrodes were replaced by a single working electrode. The first peak 1110 in each of the six series of three peaks was obtained from the first counter-electrode, the second peak 1120 in each of the six series of three peaks was obtained from the second counter-electrode and the third peak 1130 in each of the six series of three peaks was obtained from the third counter-electrode. The first peaks of 1110 presented the current level obtained from the use of ruthenium haxaamine as a charge transfer system at the first counter electrode. Second peaks 1120 show the current levels obtained from the use of Structure 1 as a charge transfer system at the second counter electrode. The third peaks 1130 present the current level obtained from the use of the cyanoferrate (III) molecule as the charge transfer system at the third counter electrode. In this way, for the same potential, different counter electrode potentials will correspond to different oxidation points of the same oxidation wave. Therefore, in addition to demonstrating the ability of many counter electrodes to control the working potential of the working electrode, the system's ability to conduct three separate analyzes with the electrode working with the gated input signal was determined.

[0090] Fig. 12A is a schematic representation of a biosensor system 1200 that determines the reagent concentration in a biological fluid sample using an input signal. The biosensor system 1200 includes a measurement device 1202 and a test sensor 1204 that can be implemented in an analytical instrument, including a stationary device, a portable or hand-held device or the like. The biosensor 1200 system can be used to determine reagent concentrations, including glucose, uric acid, lactate, cholesterol, bilirubin and the like.

[0091] Although a specific configuration has been demonstrated, the biosensor 1200 system may have other configurations, including those with additional components. For example, test sensor 1204 may be adapted for use outside, inside or partially inside a living organism. When used outdoors, a sample of biological fluid is introduced into the sample reservoir in test sensor 1204. The test sensor 1204 may be placed in the measuring device before, during or after the sample has been introduced for analysis. When inside or partially inside a living organism, the test sensor may be constantly immersed in the sample or the sample may be intermittently introduced into the sensor.

[0092] The test sensor 1204 has a base 1206 that forms a reservoir 1208 with an opening 1212. The reservoir 1208 may be formed through the lid and outlet. Tank 1208 defines a partially closed volume, but may be open to sample (not shown). Therefore, the sample can continuously flow through the test sensor or be interrupted for analysis.

[0093] Tank 1208 may contain a composition that assists in maintaining a fluid sample, such as water swellable polymers or porous polymer matrices. Reagents can be deposited in reservoir 1208. Reagents can include one or more enzymes, enzyme systems, mediators, linkers and similar types. The linker may include various types and molecular weights of polymers such as HEC (hydroxyethyl cellulose), CMC (carboxymethyl cellulose) and / or PEO (polyethylene oxide). In addition to binding reagents together, the connector may assist in filtering red blood cells by preventing them from coating the electrode 1211 surface. Test sensor 1204 may also include a sample connector 1214 tangentially positioned to reservoir 1208. Sample connector 1214 may partially or completely surround container 1208. Test sensor 1204 may have other configurations. For example, test sensor 1204 may be adapted for transdermal use by forming a reservoir 1208 of porous material or downstream of the porous material in which the sample is held.

[0094] The sample connector 1214 includes conductors 1290 connected to at least one working electrode and at least two counter electrodes. The electrodes may be substantially in the same plane or in more than one plane, such as being returned. The electrodes may be placed on the surface of the base 1206 that forms the reservoir 1208. The electrodes may extend or extend into the reservoir 1208. One or more conductors 1290 may also extend into reservoir 1208 to provide functionality not provided by the electrodes. The dielectric layer may partially cover conductors and / or electrodes. Counter electrodes can be used to balance the potential with one or more electrodes working during the analysis. The balancing potential can be provided by forming a counter electrode from an inert material, such as carbon, and including soluble redox species, such as (III) cyanoferrate, within 1208 tank. Alternatively, the balancing potential may be the reference potential achieved by forming the counter electrode from the reference redox pair, such as Ag / AgCl to provide a reference electrode - counter electrode. The sample connector 1214 may have other electrodes and conductors.

[0095] Measuring device 1202 includes an electrical circuit 1216 attached to sensor connector 1218 and display 1220. Electrical circuit 1216 includes a processor 1222 connected to a signal generator 1224, an optional temperature sensor 1226, and Storage Medium 1228.

[0096] Signal generator 1224 provides the electric input signal of the sensor connector 1218 in response to the processor signal 1222. The electric input signal can be transmitted through the sensor connector 1218 to the sample connector 1214 to apply the electric input signal to the biological fluid sample. The electrical input signal can be transmitted through all or some of the 1290 conductors at the sample connector 1214. The electric input signal may be potential or current and may be constant, variable or a combination thereof, such as when an AC signal is applied with a DC shift. The input signal can be used as a single pulse or in many

- 29 pulses, sequences or cycles. Signal generator 1224 can save the output signal from the sensor connector as a generator-recorder.

[0097] The signal generator 1224 may include the potentiostat of Figure 12B, which may switch between a plurality of independently addressable working electrodes and counter electrodes, or may include a plurality of potentiostat systems of Figure 12C. Fig. 12D shows a potentiostat that can be implemented in a signal generator for switching between four counter electrodes and an electrically connected working electrode. FIG. 12E shows a potentiostat implemented for switching between four working electrodes and electrically connected counter electrodes. Fig. 12F shows a potentiostat implemented for switching between four reference electrodes and an electrically connected working electrode. One or more potentiostats may provide different action potentials for the sample connector 1214. Signal generator 1224 can be configured, whereby the function generator calls the missing waveform inputs to the potentiostat. Signal generator 1224 may have different configurations.

[0098] The optional temperature sensor 1226 determines the sample temperature in the reservoir of test sensor 1204. The sample temperature can be measured, calculated from the output signal, or assumed to be identical or similar to the measurement of the ambient temperature or temperature of the biosensor system device. The temperature can be measured using a thermistor, thermometer or other temperature measuring device. Other techniques may be used to determine the sample temperature.

[0099] Storage medium 1228 may be magnetic, optical or semiconductor memories, other storage devices, or the like. Storage medium 1228 may be a permanent storage device, a removable storage device, such as a memory card, remotely accessible or the like.

[0100] Processor 1222 performs reagent analysis and data processing using software code for computer readout and data stored in storage medium 1228. Processor 1222 can start reagent analysis in response to the presence of test sensor 1204 at sensor connector 1218, sample application to test sensor 1204, in response to user input or the like. Processor 1222 directs the signal generator 1224 to provide an electrical input signal to sensor connector 1218. Processor 1222 can receive sample temperature from optional temperature sensor 1226.

[0101] Processor 1222 receives an output signal from the sensor connector 1218. The output signal is generated in response to the redox reaction of the measurable types in the sample. The electrical output from the test sensor can be current (as generated by amperometry or voltometry), potential (as produced by potentiometry / galvanometry), or cumulative charge (as generated by coulometry). The output signal is associated with the concentration of one or more reagents in the sample using one or more correlation equations in the 1222 processor. Results

-30 reagent analyzes can be shown on the 1220 display and can be stored in 1228 storage medium.

[0102] Equation correlations between reagent concentrations and output signals can be represented graphically, mathematically, by their combination or the like. Correlation equations can be represented by a program number table (PNA), another search table or the like stored in 1228 storage medium. Instructions for carrying out reagent analysis may be provided by a software code readable by a computer stored in 1228 storage medium. The code may be an object code or any other code describing or controlling the functionality described herein. Reagent analysis data may undergo one or more data processing, including the determination of weakness factors, K constants, factors and the like on a 1222 processor.

[0103] Sensor connector 1218 has contacts 1295 that connect or communicate electrically with conductors 1290 in test connector sample 1214 connector 1204. Sensor connector 1218 carries an electrical input signal from generator signal 1224 through a connector in connector 1218 of sensor to contacts 1295 in connector 1214 sample. Sensor connector 1218 also carries the sample output signal through contacts 1295 to processor 1222 and / or signal generator 1224.

[0104] Display 1220 may be analog or digital. The display can be an LCD screen adapted to display numerical readings.

[0105] When used, the liquid sample for analysis is transferred to reservoir 1208 by introducing fluid into port 1212 of the sample. The fluid sample flows through sample port 1212, filling tank 1208 while removing previously contained air. The liquid sample reacts chemically with reagents embedded in secondary regions of vessel analysis 1208.

[0106] The test sensor 1202 is positioned adjacent to the measuring device 1202. The adjacent includes positions where the straight sample connector 1214 is an electrical connection with the sensor connector 1208. Electrical communication includes the transfer of input and / or output signals between the contacts in the sensor connector 1218 and the conductors 1290 in the sample connector 1214. ##### [0107] Fig. 13 presents electrochemical analysis 1300 to determine the presence and / or concentration of at least one reagent in a sample. In sample introduction 1310, the sample is introduced into the test sensor. In the 1320 redox reaction, some of the reagent in the sample undergoes the redox reaction. In 1330 electron transfer, electrons are optionally transferred from the reagent to the mediator. In the first application of the input signal 1340, the input signal is used between the working electrode and the first counter electrode. In the second application of the 1350 input signal, a different potential input signal is used between the working electrode and the second counter electrode. When specifying sample 1350, the presence and / or concentration of one or more types in a sample are determined from one or more output signals, and when transmitting a concentration of sample 1360, a specific concentration of measurable types is displayed, stored, further processed, and the like.

[0108] When inserting sample 1310, the sample is introduced into the sensor portion of the system, such as a test sensor. The test sensor contains at least one working electrode and at least two counter electrodes. The electrodes may contain one or more layers of reagent composition. The working electrode may include a diffusion barrier layer that is integral to the reagent composition layer or is separate from the reagent composition layer. The diffusion barrier layer provides a porous space having an internal volume, the type being measured may be present. The pores of the diffusion barrier layer can be selected so that the type being measured can diffuse into the diffusion barrier layer, while physically larger components of the sample, such as red blood cells, are essentially excluded. When the working electrode includes a separate diffusion barrier layer, the reagent layer may or may not be placed on the diffusion barrier layer. Depending on the nature of the 1300 analysis, conductors can serve as electrodes. In this aspect, reagents may be present in the sample, as when deposited tangentially to the electrodes.

[0109] In the redox reaction 1320 of Fig. 13, a portion of the reagent present in the sample is chemically or biochemically oxidized or reduced, such as with oxidoreductase or similar types. This redox reaction occurs as the sample hydrates the reagents. After oxidation or reduction, electrons can optionally be transferred between the reagent and the mediator in 1330 electron transfer. Accordingly, a measurable ionized type is formed, such as from a reagent or mediator, having a reagent-sensitive sample concentration. It may be advantageous to provide an initial time delay or "incubation period" for the reagents for the reaction with the reagent.

[0110] In the first application of the input signal 1340 of Fig. 13, the system applies the input signal to the sample using a first counter electrode. Input signals are electrical signals, such as current or potential, and can be a sequence of excitation pulses separated by relaxation. The system may apply one or more input signals to the sample, including those used to determine the presence and / or concentration of the reagent and those used to determine other factors such as the hematocrit content of the sample or the state of filling of the test sensor.

[0111] In addition to the first use of the input signal 1340, an initial signaling potential may be introduced before the first use of the input signal 1340 to determine the presence of a sample. Potential can also be used between a pair of electrodes and / or conductors to remove material from the electrode surface and / or conductor, to change the chemical properties of the electrode or to oxidize or reduce parts of the charge transfer system. Such potential can be used before analysis.

[0112] In a second application of the input signal 1350 of Fig. 13, the system uses the second input signal at a different potential relative to the sample, using a second counter electrode. The ability to select the working potential of many working electrodes and / or the ability to choose the potential of multiple counter electrodes provides the biosensor system with the ability to perform many types of analysis. During the analysis, the potential between any pair of multiple working electrodes, counter electrodes and / or reference electrodes can be measured

-32 to provide useful information. By supplying the sample container with multiple sequential filled secondary analysis areas, the progress of sample tank filling can be monitored using two or more input signal applications 1340, 1350.

[0113] When determining sample 1360, the measuring device analyzes the output signals in response to two input signals to determine the presence and / or concentration of at least one measurable type in the sample at each potential. If the oxidoreductase or similar types used for the 1320 redox reaction react with a single reagent, specificity may be provided by part of the generated electrical signal. Since more than one measurable type can be ionized by different parts of the input signal, the presence and / or concentration of many reagents, mediators, interferors and the like can be determined. Additional current, time and / or other values ​​can also be analyzed. For example, the currents specified for one reagent, mediator or interfering agent may be modified with the currents specified for another reagent, mediator or interfering agent to increase the measuring capacity of the system.

[0114] Gated input signals, such as gated amperometric, gated voltometric and / or combinations thereof, can be used to respond to the potential of a specific mediator and solve a set of linear equations. When a test sensor having electrically independent counter electrodes and electrically connected working electrodes is used, for example, the concentrations of three different measurable types can be determined by solving equations (1) by (3):

ilow = A1 * S1 + Int1, (1) imedium = ilow + i2 = k1 * (A1 * S1 + Int1) + k2 * (A2 * S2 + Int2), (2) ihigh = imedium + i3 = k1 * (A1 * S1 + Int1) + k2 * (A2 * S2 + Int2) + k3 * (A3 * S3 + Int3), (3) where ilow, imedium and ihigh are currents from coupling with low, medium and high potential counter electrodes; A1, A2 and A3 are the concentrations of three different measurable types; k1, k2 and k3 are proportionality constants that express the current difference between the two action potentials; and S and Int are the slope and cut-off for each reagent calibration system, respectively.

[0115] Fig. 14A shows an input signal from a sequential gated, amperometric pulse sequence, used in conjunction with a test sensor having independently addressable counter electrodes and working electrodes (WE1-WE4). In this case, one working electrode works at a time, and the input signal is sequentially inserted into each pair of electrodes. In this way, a multi-potential potentiostat is not required to determine the output signal from multiple pairs of electrodes. FIG. 14B shows the input signal from a simultaneous gated amperometric pulse sequence, used in conjunction with a test sensor having independently addressable counter electrodes and working electrodes (WE1-WE4). In this case, all four pairs of electrodes are operated simultaneously with the same potential for each excitation. Although it wasn't

Shown in the figure, the input signal may be applied simultaneously to two or more electrodes, when applied sequentially to other electrodes.

[0116] When connecting multiple independently addressable counter electrodes to a current / voltage converter, the output currents obtained from the analysis can be measured separately. This action can be combined with a gated input signal with one or more electrodes off while the other counter electrode is on. The cascade of measuring currents obtained from independent counter electrodes provides a way of analyzing many reagents and other sample components. Linear combinations of equations can be solved to determine the concentration and / or other parameters of individual reagents.

[0117] The input signals used may have voltages from 0.05 to 1.0 V, preferably from 0.1 to 0.8 V, and more preferably from 0.2 to 0.5 V. The input signals may be provided for a duration of 0.01 seconds to 3 minutes, depending on the reagent or reagents of interest. For example, glucose analysis can be completed in less than 5 seconds, while other reagents may use longer input signals. If the input signal contains multiple excitations and relaxation, the duration of each excitation can be, for example, from 0.01 to 7 seconds, preferably from 0.5 to 3 seconds, and more preferably from 0.1 to 2 seconds for glucose. Other input signal and excitation durations can be used.

[0118] When transmitting 1370 the sample concentration of Figure 13, the measuring device converts at least one concentration of the measurable type to the sample reagent concentration and can display, store for future reference, further process and / or use one or more specified concentrations of measurable type for additional calculations. For example, the value specified for one reagent, mediator or interfering agent may be modified by the value specified for another reagent, mediator or interfering agent to increase the system measurement efficiency.

[0119] A counter electrode having oxidizable types present in the charge transfer system can also be used as a working electrode without oxidoreductases, thereby providing the ability to analyze the hematocrit and determine the background component of the output signal. The reagent concentration can be modified with this or other information to increase accuracy and / or precision. A counter electrode can be used as an open-circuit electrode to measure one or more hematocrit parameters. In another aspect, one or more output signals may be correlated with a calibration curve or lookup table to determine the hematocrit deviation or deviation assigned to the disturbing factor.

[0120] Depending on the nature of the reagent, the concentration of one reagent may be used to change the reading of another reagent. For example, when the concentration of the first reagent positively interferes with the concentration of the second reagent, the concentration of the first reagent may be subtracted from the concentration of the second reagent to increase the accuracy and / or precision of the concentration value determined for the second reagent.

[0121] Fig. 15 shows the results of averaging the results of up to four separate analyzes for the same reagent to determine the reagent concentration in the sample. As shown in the graph, by increasing the number of separate analyzes carried out from one to three, 98% of the obtained concentration values ​​fell within ± 15% of the deviation limit compared to the YSI instrument. Although the underlying data was obtained from separate test sensors, test sensors having two or more secondary analysis regions can be configured to perform the same analysis in more than one secondary region in addition to analyzing for different reagents. Therefore, a plurality of substantially chemically isolated secondary analysis regions can provide the benefits of signal averaging from a single test sensor.

[0122] The ability to perform the same analysis multiple times on a single test sensor can significantly increase the accuracy and / or precision of a particular reagent concentration. Therefore, averaging, enabled by performing the same analysis multiple times on the same test sensor, can provide an improvement in the signal-to-noise ratio of the test sensor by reducing the random signal (as characterized by the standard deviation sd value) at grade 1 / Vn with respect to conventional sensor systems.

[0123] Fig. 16 shows the current attenuations obtained when the gated amperometric input signal is simultaneously applied to eight individually addressable and substantially chemically isolated electrodes. The electrodes were configured in a multi-T design with four electrodes operating relative to four counter electrodes through the primary channel as shown previously in Fig. Each working electrode was formed with a reagent composition containing a HEC connector 0.5% (w / w), 50 mL of Structure I molecule and 2 U / pL of the PQQ-GDH enzyme system in phosphate buffer at pH 7. Each counter electrode was formed with a transfer system charge, containing a HEC linker 0.5% (w / w) and substantially pure 100 mM cyanoferrate in phosphate buffer at pH 7.

[0124] To perform the experiment, a sample containing 100 mg / dL glucose in pH 7 phosphate buffer was introduced into the test sensor and a gated amperometric input signal was simultaneously applied to each of the four opposite electrode pairs. The gated input signal contained two initial excitations having varying pulse widths, followed by seven excitations having a pulse width of 0.375 seconds. The last seven excitations were separated by one second relaxation periods. Approaching the end of the excitation used in the second second, for example, the four current values ​​corresponding to each pair of electrodes (W1-C1, W2-C2, W3-C3, and W4-C4) are averaged. From this average of the four current values, the reagent concentration of the sample can be determined using one or more correlation equations or a similar method. In this way, the previously discussed benefits of accuracy and / or precision, obtained from averaged multiple analyzes, can be obtained from a single test sensor.