HK1096151B - Devices and methods relating to electrochemical biosensors - Google Patents

Description

Apparatus and methods relating to electrochemical biosensors

Reference to related applications

The present application claims priority from U.S. provisional application No.60/480,243 entitled "DEVICES AND METHODS RELATING TO electronic components", and also relates TO applications entitled SYSTEM AND METHOD for USING a catalyst system AC implementation (here U.S. application No.10/688,343, "AC implementation application"), METHOD OF making a reactor (here, application No. RDID-9958-CIP-US, "biosensing"), and DEVICES AND METHODS RELATING TO electronic components (here, U.S. provisional application No.60/480,397, "analog sensing"), which were filed on day 20 OF the year 6, and TO U.S. patent application No.10/264,891 (filed on day 5 OF the year 2002, incorporated by reference as part 10 OF the entire date OF this application), all OF which are filed on day 20 OF the year 6.

Background

The present invention relates to devices, systems, and methods for measuring analytes from biological samples, such as bodily fluid samples. More particularly, the present invention relates to biosensors and methods for testing analytes using certain electrical response characteristics.

Measuring the concentration of substances, especially in the presence of other confounding substances ("interferents"), is important in many fields, particularly in medical diagnostics and disease control. For example, the measurement of glucose in body fluids such as blood is crucial for the effective treatment of diabetes.

Various methods are known for measuring the concentration of an analyte, such as glucose, in a blood sample. These methods generally fall into two categories: optical methods and electrochemical methods. Optical methods typically involve absorbance, reflectance, or laser spectroscopy to observe spectral shifts in the fluid caused by the concentration of the analyte (typically in combination with a reagent that produces a known color when combined with the analyte). Electrochemical methods typically rely on a correlation between the charge transfer or charge movement characteristics (e.g., current, interfacial potential, impedance, conductance, etc.) of the blood sample and the concentration of the analyte (typically bound to a reagent that generates or alters charge carriers when combined with the analyte). See, for example, U.S. patent No.4,919,770 to Preidel et al, and No.6,054,039 to Shieh, which are incorporated herein by reference in their entirety.

An important limitation of electrochemical methods for measuring the concentration of chemical substances in blood is the effect of confounding variables on the impedance of the blood sample. For example, the geometry of the blood sample must closely correspond to the geometry on which the impedance versus concentration mapping function is based.

The geometry of the blood sample is generally controlled by the sample receiving portion of the testing device. In the case of a blood glucose meter, for example, a blood sample is typically placed on a disposable test strip that is inserted into the meter. The test strip may have a sample chamber to define the geometry of the sample. Alternatively, the effect of the sample geometry can be limited by ensuring a virtually infinite sample size. For example, the electrodes used to measure the analyte may be spaced sufficiently close together that a drop of blood on the test strip extends sufficiently beyond the electrodes in all directions. Regardless of the strategy used to control the sample geometry, typically one or more dose sufficiency electrodes are used to ensure that a sufficient amount of sample is present to ensure accurate test results.

Other examples of limitations on the accuracy of blood glucose measurements include changes in blood chemistry (rather than the analyte of interest being measured). For example, changes in the hematocrit (red blood cell concentration) or concentration of other chemicals, components or formed components in the blood can affect the measurement. In measuring blood chemistry, changes in the temperature of a blood sample are another example of confounding variables.

Therefore, there is a need for systems and methods for accurately measuring blood glucose, even in the presence of confounding variables, including changes in temperature, hematocrit, and concentrations of other chemicals in the blood. There is also a need for systems and methods for accurately measuring analytes in fluids. It is an object of the present invention to provide such a system and method.

Many approaches have been taken to attenuate or mitigate the effects of one or more sources of interference, or otherwise compensate or correct the measurements. Multiple design solutions are typically employed to adequately compensate for the sensitivity associated with the selected measurement method.

Well-known design solutions involve permeability-selective and/or size-selective membranes, filters or coatings. This design solution has the disadvantage of increased cost of the goods, and the additional manufacturing process steps further aggravate manufacturing costs, complexity, and manufacturing speed. Systems employing these methods (disposable test strips and instruments) take a general approach to solving problems within the scope of test strip design.

Another general approach involves the use of advanced excitation (sophisticatedexlocation) and signal processing methods coupled with co-optimization algorithms. Simpler, less complex test strip structures and manufacturing processes can be achieved; however, manufacturing techniques are required that require the expense of instrument use, memory and processor requirements, associated complex coding, and calibration. Systems employing this technique take a general approach to solving problems within the scope of use of the instrument.

More recent methods do not involve test strips nor instrumentation per se, but rather employ measurement methodologies. An example of this is the use of coulometry to attenuate the effects of hematocrit and temperature.

In addition, all of the above methods are further supported by the initial design of the reagent system, as is well known to those skilled in the art. In the detection of glucose, for example, this may involve the use of selective redox mediators and enzymes to overcome the deleterious effects of the presence of redox active substances or other sugars.

The object of the present invention is to provide a simpler, less expensive method for attenuating the effects of interferents in a manner that does not suffer from the disadvantages associated with the general methods that are widely used today.

Disclosure of Invention

Roughly two aspects

In one aspect, the invention relates to providing two pairs of electrodes, which allows for interferent correction or compensation for analyte measurements using two measurements. For example, in one embodiment, one pair of electrodes defines a first measurement zone, while a second pair defines a second measurement zone. The pairs are approximately coplanar and in a pair of electrodes, each has a length that is substantially parallel to the length of the other. At least one electrode of the first pair of electrodes comprises at least two elongated, rectangular conductive elements that are interdigitated with the conductive element(s) of the other electrode of the pair. Each element of the electrode is conductively connected to the same contact for electrical communication with the driver and/or meter. The sample establishes electrical contact with the two pairs after dosing.

Several variations of the above are contemplated. For example, in one method, an agent or agents may be selectively deployed on at least one of the at least two pairs of electrodes present in the sample chamber. Both electrode pairs are coated with a first reagent. Optionally, one of the two pairs is coated with a first agent and the second pair is coated with the same agent but lacking an enzyme or a mediator. Alternatively, one of the two pairs is coated with a first reagent and the other pair is coated with a second reagent. In another embodiment, one of the at least two pairs is coated with a reagent and the other pair lacks a reagent coating, and the downstream pair preferably has a reagent coating. In variations of this embodiment, the other of these pairs is covered with a permeability selective, size selective coating, or otherwise affects the electrode response in the presence of one or more analytes and/or interferents.

In further aspects, dose detection and dose sufficiency electrodes are included. For example, a third electrode system may be included which is located further from the edge than the first two electrode pair, i.e. downstream into the sample fluid, and is operable to detect when sufficient sample fluid is present to perform an accurate test. The third electrode system may comprise a single electrode element or a plurality of elements. In a single element embodiment, the element functions in combination with one or more other electrodes to test sample sufficiency. Alternatively, the dose sufficiency electrode system may include a pair of electrode elements that cooperate to indicate sample sufficiency. Comparable electrode systems can be similarly employed to detect when sample fluid is applied to the biosensor.

Drawings

FIG. 1 is a perspective view of a test strip according to one embodiment of the present invention.

Fig. 2 is an exploded view of selected layers of the test strip of fig. 1.

Fig. 3 is a cross-sectional view of the electrode portion of the test strip of fig. 1.

Fig. 4-15 are exploded views of alternative test strips according to the present invention.

Detailed Description

For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended; any alterations and further modifications in the described and illustrated embodiments, and any further applications of the principles of the invention as illustrated therein are contemplated as would normally occur to one skilled in the art to which the invention relates.

Introduction to the design reside in

In general, the test strips of the present invention provide for the testing of analytes in bodily fluids or other fluids using multiple electrode arrays that perform different functions or have different responsive functions to the sample. One specific embodiment involves a combination of macroscopic electrodes and microelectrodes operating in corresponding pairs, but the final determination of the analyte concentration contributing information, for example by using the information obtained from one electrode pair to compensate and correct the results obtained from the other electrode pair, or by combining the responses of these electrode pairs in a predetermined manner.

These electrode arrays can also be combined in a number of other ways to accomplish a number of related functions, including analyte concentration, hematocrit detection, correction factor determination, and sample sufficiency and dose detection, all on a single test strip and in a minimal space. Alternatively, by using multiple arrays with different sensitivities to interferents, two measurements may be taken to provide more accurate results, as would be generally appreciated by those skilled in the art.

In various embodiments, different electrochemical excitation techniques (e.g., DC, AC phase, AC amplitude, or combined DC/AC) are applied to these different electrode arrays to achieve the intended goals. Examples of such techniques are well known in the art and are further exemplified in AC authentication, which is incorporated by reference above.

Another exemplary technique compensates for changes in the diffusion coefficient of the electrochemically active material being tested. A faraday current is generated in the soluble reagent at the electrode surface due to physical diffusion of these species, and the value of the diffusion coefficient affects the measured response. Commercial systems are typically calibrated and set up such that the nominal sensor response (faraday current) for a given amount of glucose is repeatable if the diffusion coefficient remains constant. Unfortunately, the temperature and Hematocrit (HCT) of each individual sample change the effective diffusion coefficient of the electroactive species being measured. If these factors are not considered, the glucose measurement is erroneous for any temperature or hematocrit value that is different from that used in the calibration process of the system.

In this exemplary technique, the system determines the faradaic response of the electrochemical sensor caused by the analyte of interest and provides an estimate of the actual effective diffusion coefficient of the species undergoing redox reactions at the electrode surface. In particular, the system compensates for changes in diffusion coefficient by using two electrode systems (preferably of different types) exposed to the same reagent sample mixture. Soluble electroactive species commonly used in glucose biosensors, such as redox mediators, diffuse onto a planar macroscopic electrode, producing a current response to a potential step according to Cottrell equation (1).

Thus (1a)

(1b)

Where n is the number of electrons involved in electron transfer, F is the Faraday constant (96, 485.3C/equivalent), A_pIs the area of the macroscopic electrode in contact with the solution, C is the analyte concentration in the sample, D is the effective diffusion coefficient of the substance, and i_pIs the current response at the macroscopic electrode.

Similarly, those skilled in the art will appreciate that the response of these same species to the same level of potential at the microelectrodes will produce a current response characterized by equation (2).

Thus (2a)

(2b)

Wherein A is_sIs the area of the micro-electrode, v is the accessibility factor depending on the shape of the electrode, and i_sIs the current response at the microelectrodes. In equations (1b) and (2b), t (∞) means a time long enough to establish a "quasi-infinite" or "steady state" diffusion condition at the electrode, respectively.

One embodiment would apply the same potential between (a) the planar, macroscopic electrode and the counter/reference electrode, and (b) the microelectrode(s) and the counter/reference electrode. At the macro and micro electrodesAfter all the potentials were applied, the time-dependent current response was measured at several time points.Will yield the slope in equation (3)_pTo do soWill produce an intercept_sAs shown in equation (4).

Suppose that in the present invention, i_pAnd i_sAll derived from the same reaction and sample, it is possible to calculate the apparent diffusion coefficient of the electrochemically reactive species in the device, independently of the species concentration according to equation (5), where the area a of both electrode types_sAnd A_pAnd the radius r of the microelectrode(s)_oAre known. For example, spherical microelectrodes produce:

once D is estimated, it can be applied in a number of different ways to provide a correction for the measured concentration C of the electrochemical species. Some embodiments simply use the estimate of D in equation (3) to calculate C. This determination of C is less affected by uncompensated changes in D, which is common in amperometric sensors, whose current response is primarily described by equation (1). It is also worth noting that the correction is independent of the cause of D variation (e.g., temperature, hematocrit, viscosity change, etc.) -the correction is provided by the different functional relationship of the two electrode pairs to the chemical properties of the sample.

In each of the test strips shown herein, an array of electrodes is used to measure an analyte, such as glucose, in a sample. When a sample reaches the array, it combines with a reagent placed adjacent to the array to provide some property of electrical impedance in the presence of some electrical signal, as is well known in the art, which impedance serves as the first data. Another array, either upstream or downstream of the first array, but preferably not covered by the reagent, is used to provide another electrical stimulus to the sample, and the electrical response at that array is used as second data which is affected in a known manner by interferents such as hematocrit, temperature, etc. The two data are combined to produce a corrected analyte concentration value. The two arrays can be used simultaneously to analyze a single sample in a common volume of small size.

General information

System for controlling a power supply

The present invention relates to a system for assessing an analyte in a sample fluid. The system includes an apparatus and method for assessing a target analyte in a sample fluid. The assessing includes detecting the presence of the analyte to determine a concentration of the analyte. The analyte and sample fluid may be any analyte and sample fluid for which the test system is suitable. For illustrative purposes only, a preferred embodiment is described wherein the analyte is glucose and the sample fluid is blood or interstitial fluid. It will be clear, however, that the invention is not limited thereto.

Sensor with a sensor element

One component of the system is an electrochemical sensor that includes a sample-receiving chamber for a sample fluid, and a reagent for generating an electrochemical signal in the presence of a test analyte. The sensor preferably comprises a disposable test strip, particularly a test strip having a layered structure providing an edge opening for the sample-receiving chamber. Reagents are placed in appropriate locations within the sample receiving chamber to provide an electrochemical signal to a working electrode also placed within the receiving chamber. Where appropriate, for example for glucose detection, the reagent may comprise an enzyme and optionally a mediator.

Measuring meter

The sensor is used in conjunction with a meter to determine the presence and/or concentration of an analyte in a sample fluid. The meter typically includes connections to the electrodes of the sensor and circuitry for evaluating the electrochemical signal corresponding to the analyte concentration. The meter may also include means for determining that the sample fluid has been received by the sensor and that the amount of sample fluid is sufficient for testing. The meter will typically store and display the results of the analysis, or may provide data to another device.

Analyte-property

The system can provide a qualitative or quantitative representation of the analyte. In one embodiment, the system simply indicates the presence of an analyte in the sample fluid. The system may also provide a reading of the amount or concentration of analyte in the sample fluid. In a preferred embodiment, the invention is characterized by obtaining highly accurate and precise readings of analyte concentration.

Analyte-type

The system can be used to determine a wide variety of analytes. For example, the test strip is readily adapted for use with any suitable chemical that can be used to assess the presence of an analyte. Most preferably, the system is configured and used for testing an analyte in a biological fluid. Such analytes may include, for example, glucose, lactate, urate, ketones, and the like. Equivalent modifications to the system will be apparent to those skilled in the art. For illustrative purposes, and in a particularly preferred embodiment, the system is described with respect to the detection of glucose in a biological fluid.

Interfering substance

The test method may be affected differently by the presence of interferents in the sample fluid. For example, testing for glucose in a blood sample can be affected by factors such as bilirubin, hematocrit, uric acid, ascorbic acid, acetaminophen, galactose, maltose, and lipids. The present system is adapted to minimize or eliminate the adverse effects of interferents that may also be present in the sample fluid. These effects can be addressed by appropriate selection of test materials and parameters, for example by selection of chemicals which are known to be less or not at all affected by possible interferents. They may also be addressed by selecting two or more reagents that have different sensitivities to interferents, but substantially the same sensitivity to the analyte of interest. Other steps may also be taken to address possible interferent effects, such as using a coating or film that prevents interferents from entering the test zone, as is known in the art. In addition, modifications of the electrode structure or interrogation method may also be used to minimize the effects of interferents.

Type of fluid

The system can be used for a wide variety of sample fluids and is preferably used for detecting analytes in biological fluids. In this context, the term "biological fluid" includes any bodily fluid in which an analyte can be measured, for example, interstitial fluid, skin fluid, sweat, tears, urine, amniotic fluid, spinal fluid and blood. In the context of the present invention, the term "blood" includes whole blood and its cell-free components, i.e. plasma and serum. In addition, the system may be used in conjunction with a reference fluid in a conventional manner to verify the integrity of the system for testing.

In a preferred embodiment, the system is used for testing glucose. In this case, the sample fluid may specifically include, for example, fresh capillary blood, fresh venous blood obtained from a fingertip or approved replacement site (e.g., forearm, palm, upper arm, lower leg, and thigh), and control fluid provided to or used in the system.

The fluid may be obtained and transferred to the test strip in any manner. For example, a blood sample can be obtained in a conventional manner by lancing the skin, for example, with a lancet, and thereafter contacting the test strip with a fluid present at the skin surface. One aspect of the present invention is that the test strip can be used with very small fluid samples. It is therefore a desirable feature of the present invention that only a slight incision in the skin is necessary to generate the amount of fluid required for testing, and pain and other concerns associated with this method can be minimized or eliminated.

Electrode for electrochemical cell

Electrode type

The present invention relates to "electrochemical sensors" which are devices configured to detect the presence of an analyte and/or measure the concentration of the analyte by electrochemical oxidation and reduction reactions within the sensor, and/or the progression of movement of a charged layer within a solution. These reactions are converted into an electrical signal that is related to the amount or concentration of the analyte. Thus, the test strip includes an electrode system that includes at least a working electrode and a counter electrode within a sample-receiving chamber. The sample receiving chamber is configured such that sample fluid entering the chamber is in electrolytic contact with the working electrode and the counter electrode. This allows current to flow between the electrodes to effect electro-oxidation or electro-reduction of the analyte or its products.

In the context of the present invention, a "working electrode" is an electrode at which an analyte or product is oxidized or reduced electrically, with or without the aid of a redox mediator. Here, the term "counter electrode" refers to an electrode paired with a working electrode, and an electrochemical current equal in magnitude and opposite in sign to a current flowing through the working electrode flows through the counter electrode. The term "counter electrode" includes a counter electrode that also functions as a reference electrode (i.e., a counter/reference or auxiliary electrode).

Electrode material

The working and counter electrodes, as well as the remainder of the electrode system, may be formed from a variety of materials as are known in the art. These electrodes should have a relatively low resistance and should be electrochemically inert over the operating range of the test strip. Suitable conductors for the working electrode include gold, palladium, platinum, carbon, titanium, ruthenium dioxide, iridium, and indium tin oxide, as well as others, such as those disclosed in the analysis Sensors application, which is incorporated by reference above. The counter electrodes may be made of the same or different materials. In a preferred embodiment, both electrodes are gold electrodes.

Electrode application

The electrode system employed in the present invention may be applied to the substrate in any manner that results in an electrode having suitable conductivity and integrity. Exemplary processes are well known in the art and include, for example, sputtering, printing, and the like. In a preferred embodiment, the electrodes and other electrically conductive components are provided by coating the substrate, followed by removal of selected portions of the coating to produce the components. The preferred Method of removal is Laser ablation, more preferably broad area Laser ablation, as disclosed in the "Method of making a Biosensor" application, which is incorporated by reference above, and further relevant discussion is found in U.S. patent application nos. 09/866,030 (entitled "Biosensors with Laser absorption Electrodes with a Continuous coating Coyerlay Channel", filed 2001, 5/25), and 09/411,940 (entitled "Laser definedFeatures for Patterned coatings and Electrodes", filed 1999, 10/4). Various other methods of manufacture and application are well known in the art for providing electrical components, and in particular the electrode systems described herein.

Reagent composition

The test strip includes a chemical reagent within the sample-receiving chamber for reacting with a test analyte to generate an electrochemical signal that is representative of the presence of the analyte in the sample fluid. The test chemical is selected relative to the analyte to be assessed. It is well known in the art that there are a large number of chemicals that can be used with each of a variety of analytes, including, but not limited to, the preferred chemicals described in the patent application entitled "Reagent Strip for test Strip" (attorney docket No. 7404-. Accordingly, the selection of appropriate chemicals is within the skill of the art, and need not be further described herein in order for anyone to make and use the present invention.

However, to this end, a preferred embodiment is described wherein the analyte is glucose, but it is to be understood that the scope of the invention and claims is not so limited, unless specifically indicated. In the case of glucose, the active ingredients of the test chemical will typically include a glucolase and a redox mediator. The enzyme oxidizes the glucose in the sample, and the mediator reacts with the reduced enzyme. The mediator then transports the redox equivalents of the analyte products to the electrode surface by diffusion. Where the mediator is quantitatively oxidized at a defined anodic potential and the resulting current is related to the apparent glucose concentration. There are a variety of reagent systems suitable for detecting glucose, and examples of these are contained in AC Excitation, analytical Sensors, and Biosensor applications, U.S. Pat. Nos. 5,385,846 and 5,997,817, and U.S. (reissued) patent application No.10/008,788 ("electrochemical Biosensor Test Strip"), which are incorporated herein by reference.

Glucose chemistry utilizes a redox mediator to carry the current between the working electrode and the glucose analyte, which is otherwise not well suited for direct electrochemical reactions at the electrodes. The mediator acts as an electron transfer agent to transport electrons between the analyte and the electrode. A wide variety of redox species are known and can be used as redox mediators. Generally, the preferred redox mediators are molecules that can be rapidly reduced and oxidized. Examples include ferricyanide, nitrosoaniline and its derivatives, and ferrocene and its derivatives.

Measurement scheme

In one aspect of the invention, the first electrode pair provides a first measurement that is combined with a second measurement obtained by the second electrode pair. As described above, conventional test strips employ at least two electrode pairs (e.g., a working electrode and a counter electrode, respectively) to determine an analyte concentration based on a reaction of the analyte with a reagent located on or adjacent to one of the electrode pairs. Thereby obtaining a basic measurement of the analyte concentration. However, it is often desirable to correct or compensate for other factors of the measurement, such as hematocrit, temperature, presence of other substances in the sample fluid, and so forth. In one embodiment of the invention, biosensors and methods are provided that employ two electrode pairs, one for making a primary measurement of an analyte and the other providing such correction or compensation for the primary measurement, in some cases producing a final measurement map.

The use of two electrode pairs may involve the use of disparate groups of electrodes, where one pair includes macroscopic electrodes and the other pair includes microelectrodes. As used herein, the term macroscopic electrode refers to an electrode whose principal effective diffusion properties are perpendicular to the electrode surface. The macroscopic electrodes are dimensioned and arranged such that the main diffusion characteristic is a linear diffusion characteristic. The term microelectrode refers to an electrode that exhibits a convergent, steady-state, or quasi-steady-state diffusion on a characteristic time scale of measurement. Microelectrodes are electrodes to which radial diffusion provides a significant change in the response function. Microelectrodes may, for example, be sized and positioned such that their primary impedance characteristics are, for example, characteristics of edge-to-edge dynamics between the nearest edges of the fingers. This functionality will be discussed more with respect to the embodiments shown in the figures.

One advantage of using microelectrodes is that these devices can be configured and used to achieve quasi-steady state of current flow at the electrodes very quickly, e.g. only in 0.50-3.25 seconds, or even in less than half a second. This rapid achievement of quasi-steady state allows for faster and more accurate determination of analyte concentration. This is in contrast to prior art methods which estimate or predict results, for example from readings taken before quasi-steady state is reached.

Another advantage derived from some embodiments of the present invention is that the quasi-steady state response to an applied DC signal is at a higher magnitude than quasi-steady state in many prior art systems. This improves the signal-to-noise ratio of the signal, thereby enabling the system to provide more accurate results.

Another advantage derived from the interdigitated array of electrode fingers used in some forms of the invention is the significantly increased electrode edge length that can be achieved in a given space. Depending on the design, results can be obtained with smaller samples in those systems, while achieving the same quality of results as systems requiring larger samples.

Note that equations can be derived and used for a variety of microelectrode configurations, as would occur to one of ordinary skill in the art in view of this disclosure and the ACException application, the latter of which is incorporated above by reference. Empirical measurements can also be used to directly determine the response function of the proposed electrochemical structure in each sensor design. Note that neither the analytical description of the response function nor the achievement of the steady state current is necessary to improve system performance.

General description-Structure

The present invention provides electrode structures and systems for use in various biosensing devices. Described herein are exemplary test strip configurations that demonstrate the utility of the present invention. However, it should be understood that the principles of the present invention are equally applicable to a variety of other biosensor designs. The particular composition, dimensions and other characteristics of the basic biosensor components are not critical and thus are not limiting.

Referring to FIG. 1, generally, strip 210 has a first end 211 for communicating with a drive circuit and a metering circuit (not shown), while end 218 is adapted to receive body fluid in contact with the electrodes, as will be discussed herein. The driver circuit provides a known current and/or potential through the contacts 216 and monitors the current and/or voltage response over a period of time. The respective signals are carried between the contact 216 and the electrode (shown in fig. 2-14) via conductors 270, 272, 274, and 276. These conductors are made of any one or combination of a variety of conductive materials, including, for example, gold or carbon, as will be appreciated by those skilled in the art.

At end 218, notched fluid directing device 214 is generally rectangular, and rectangular notch 148 is cut away therefrom, as can be seen in FIG. 2. Fluid directing means 214 is located on substrate layer 212 (polyimide or other material, as disclosed in the "Method of Making a biosensor" application, which is incorporated by reference above, or other material known in the art) and provides an opening 251 (see fig. 2) for fluid to be drawn by capillary action from edge 224 to outlet 262. The cover layer 250 is positioned atop the guide layer 236 and provides an upper closure for the fluid path defined in part by the recess 248. These structures will be described in more detail below.

Turning now to fig. 2, with continued reference to some of the structures shown in fig. 1, strip 210 includes a substrate layer 212, a reagent strip 264, a fluid directing device 214, and a cover layer 218. When assembled, the channel 248 is horizontally defined by the inner recess surface 249, above by the bottom surface 258 of the cover layer 218, and below by the reagent strip 264 (which is located above the electrode pair 284, but not above the electrode pair 280) and the electrode regions 266 on the upper substrate surface 232. During testing operations, fluid to be tested enters the channel 248 through the end 240 of the fluid directing device 214, the edges 254 and 224 of the cover layer 218 and substrate 212, respectively. The fluid is drawn into the channel 248 by capillary action, follows a path extending away from the edges 224 and 254, and flows out 262 (see fig. 1).

The capillary channel provides a sample-receiving chamber in which the measurement electrodes and associated reagents are contained, and the fluid sample containing the analyte contacts these components of the biosensor. The invention is characterized in that the dimensions of the capillary channel can be varied considerably. In one embodiment, the channel is a volume 1000 μm wide, 100 μm high, and 2000 μm long. Other embodiments, and measurements of channels are generally discussed in the analytical Sensors application referenced above. As the fluid travels along this path, it comes into contact with the reagent and the electrodes, as will be described in further detail below.

On substrate 212, contacts 278 are connected to electrodes 280 via traces 279. These electrodes 280 extend perpendicular to the length of the substrate 212, parallel to the edge 224, and parallel to each other. In a preferred embodiment, the electrode 280 is rectangular, having a length sufficient to extend beyond the width of the recess 248, a width of at least 50 μm, and a spacing between its closest points of greater than about 50 μm. In another preferred embodiment, the electrodes 280 are about 100 μm wide with a 100 μm gap. In another preferred embodiment, the electrodes 280 are about 250 μm wide with a gap of 250 μm. Other configurations and dimensions will occur to those skilled in the art and may be used in the present invention as needed or desired in view of the design considerations of the particular bar and system.

Contact 282 is connected to electrode pair 284 via trace 277. The electrodes 284 each comprise a plurality of parallel, elongated rectangles ("fingers"), each extending approximately parallel to the edge 224 and perpendicular to the centerline of the recess 248, beyond the width of the recess 248 at both ends. These rectangles are connected to traces 274 or 276 at one end or the other in an alternating pattern to form an interdigitated finger, which will be discussed in further detail below. In various embodiments, each rectangular finger of microelectrode pair 284 is about 5 μm to about 75 μm wide and the gap between adjacent fingers is about 5 μm to about 75 μm. The finger width and the gap between adjacent fingers preferably each remain constant over the width of the notch 248.

Turning now to fig. 3, with continued reference to fig. 2, a larger, enlarged view of the electrode portion of the strip 210 of fig. 2 is shown. As discussed above, electrodes 280 extend parallel to edges 224 of strips 210 and are connected at opposite ends to their conductive traces 270 and 272, forming electrode pairs 266. Their nearest edges 281 are separated by a distance ("gap") indicated by reference numeral 286 that is substantially constant throughout their length. Similarly, the interdigitated fingers 284 form the electrode pair 268, and the interleaved fingers are connected to the conductive traces 274 and 276.

Turning to fig. 4, strip 310 shows substrate layer 212, reagent strip 364, notched fluid direction device 214, and cover layer 218. In this embodiment, fluid entering the capillary recess 348 defined by the fluid directing arrangement 214 first encounters the macro electrode 280. The macro electrodes 280 are connected to contacts 378 at the ends 368 of the strips 310 by conductors 379. The electrodes 280 are each, for example, about 250 μm wide and the gap between them is also about 250 μm. Slightly further from the strip end 366 is an electrode pair 284, which are two electrodes for each five fingers, with each finger on one side connected by a conductor 377 to a contact 382 at the strip end 368. Each finger in the electrode pair 284 is rectangular with a width of about 20 μm and each adjacent finger is separated from the next by a gap of about 20 μm. Reagent strip 364 covers electrode pair 280 but not electrode pair 284.

During testing, an AC signal is applied to the contact 378 for a period of time while the sample covers the electrode pair 280. Similarly, for an overlapping time period after the sample covers the electrode pair 284, a DC signal is applied to the contact 382 and the electrical response between the electrodes in the pair 284 is used to estimate the glucose concentration in the sample. The sample response between the fingers of electrode pair 280 is sensitive to the hematocrit of the sample, which, along with the temperature value provided by the thermistor-based circuit, provides a correction factor for the estimate obtained with electrode 284. Note that this "correction factor" need not be a multiplicative or additive factor, but may instead be used in a formula, in a look-up table, and/or otherwise to correct an estimate based on temperature and the presence or absence, or characteristics, of other materials in the sample, as will be appreciated by those skilled in the art. See, for example, AC specification, which is incorporated by reference above. In this embodiment, the volume of blood within the capillary recess 348 sufficient to cover the measurement electrode is about 130 nL.

An alternative embodiment is shown in fig. 5 as bar 410. The substrate layer 212 is depicted with two contacts 478 and partially covers the reagent strip 464 (above the electrodes 480), the notched fluid direction device 214, and the cover layer 218. Contact 478 is electrically connected to a first electrode pair 466 and a second electrode pair 468 by conductive trace 477, one electrode of each pair being connected to one of contacts 478 on each side. Note that in this embodiment, a single contact pair 478 is used by a drive and measurement circuit (not shown) to drive and measure the response from both electrode pairs. Note also that the relative arrangement of the microelectrodes 484 and macroscopic electrodes 480 is reversed with respect to the embodiment shown in FIG. 4. The macroscopic electrodes 480 are also, for example, about 250 μm in width and the gap between them is about 250 μm. Also, each electrode of microelectrode pair 466 is comprised of five fingers that are interdigitated with fingers in the other electrode of the pair. Each finger is also about 20 μm wide and the gap between adjacent fingers is about 20 μm.

In this embodiment, the reagent strip 464 covers the electrode pair 468, but not the electrode pair 466. When the sample covers the electrode pairs 466, the system uses the AC signal through the pair to determine a correction factor for the analyte measurement. When the sample has covered the electrode pairs 468, an estimate of the analyte concentration is obtained using DC excitation methods known in the art, for example, U.S. patent application nos. 09/530,171 and 10/264,891, PCT application No. (WO) US 98/27203, U.S. patent No.5,997,817, and Electrochemical Biosensor Test Strip (reissue) application. With the above exemplary dimensions, the volume of the capillary cavity is about 130 nL.

Turning now to fig. 6, it can be seen that strip 510 also includes substrate layer 212, reagent strip 564, notched fluid direction 214, and cover layer 218. In this embodiment, the working electrode 581 is located between two opposing electrode fingers 580, which are connected to the same contact by one of the conductors 216. These electrodes 580 and 581 form a first electrode pair 480, and each of the three macroscopic electrode fingers in the electrode pair 480 is about 250 μm wide, and the gap on each side of the working electrode 581 is about 250 μm.

The second electrode pair 284 includes two electrodes each having six and seven fingers, respectively, that are interdigitated in a staggered pattern. Each finger is also about 20 μm wide and the gap between adjacent fingers is about 20 μm. In this embodiment, reagent layer 564 covers both electrode pairs 480 and 284. The macroscopic electrode pair 480 provides a Cottrell-like response where the current is proportional to the square root of the diffusion coefficient, while the microelectrode pair 284 provides a current that is directly proportional to the diffusion coefficient. Together, these two responses correct for environmental factors to produce an improved response. In this example, the sample volume required for the measurement is about 200 nL.

Another alternative embodiment is shown in fig. 7. Strip 610 includes substrate layer 212, reagent strip 664, notched fluid direction 214, and cover layer 218. As in fig. 6, the first electrode pair 572 comprises opposing and working macroscopic electrodes 581, respectively, each about 250 μm wide with a gap of about 250 μm between them. However, in this embodiment, the electrode pair 661 includes two electrodes each having three fingers. Each finger is about 50 μm wide and the gap between adjacent fingers is about 50 μm.

The first electrode pair (macro electrode pair 572) reached by the sample was used to obtain hematocrit based measurements using AC excitation techniques. The second electrode pair (microelectrode 661) was used to obtain measurements dependent on glucose and hematocrit in the sample using DC excitation. Reagent strip 664 covers only electrode pairs 661, and requires a sample volume of about 200nL to fill the capillary volume in the relevant zone. These measurements are combined as parameters of a formula, depending on electrode structure, reagent system, and other factors, as will occur to those skilled in the art.

Fig. 8 provides another embodiment of the present invention. The strip 710 includes a substrate layer 212, a reagent strip 364, a notched fluid direction device 214, and a cover layer 218. In this embodiment, the first electrode pair 366 includes two macroscopic electrodes, each having a single rectangular finger, while the second electrode pair 770 includes two microelectrodes, each having five fingers in an interdigitated pattern. In this embodiment, the fingers are about 50 μm wide and the gap between them is about 30 μm, and the reagent strip 364 covers the second pair 770. The volume required to cover the electrodes in the relevant part of the capillary path is about 170 nL.

Turning now to fig. 9, the strip 810 includes the substrate layer 212, the reagent strip 364, the notched fluid direction device 214, and the cover layer 218. A single contact pair 878 is connected to both the first electrode pair 866 and the second electrode pair 868 by conductors 877. The first electrode pair 866 includes two single finger macroscopic electrodes 884, while the second electrode pair 868 includes two microelectrodes 880, each having five fingers in an interdigitated pattern. Each of the first electrode pair 866 is also approximately 250 μm wide and the gap between them is approximately 250 μm. First electrode pair 866 is used to obtain a first measurement based on the hematocrit of the sample. Each finger of the second pair 868 is about 50 μm wide and the gap between adjacent fingers is about 30 μm. When the sample covers the second electrode pair 868, a DC signal is applied to contact 878. The resulting impedance between electrodes 868 is used to obtain a second measurement based on the concentration of glucose and hematocrit in the sample. This measurement is combined in an equation with the measurement obtained by the first electrode pair 866 and the temperature signal from the thermistor (not shown) to obtain a corrected glucose concentration value. Reagent strip 364 covers second electrode pair 868 and the desired sample volume is also about 170 nL.

Fig. 10 shows another alternative embodiment, a strip 1010, comprising a substrate layer 212, a reagent layer 1064, a notched fluid-directing device 214, and a cover layer 218. In this embodiment, the first electrode pair 1081 encountered by the sample includes a working electrode 1071, which is a single finger electrode. The first electrode pair 1081 also includes a counter electrode pair 1072, i.e., a two-finger electrode, with one finger on each side of the working electrode 1071. Each finger in the first electrode pair 1081 is about 250 μm wide and each opposing electrode finger is separated from the working electrode finger by a gap of about 250 μm. Each electrode in the first electrode pair 1081 (i.e., working electrode 1071 and counter electrode 1072) is electrically connected to contact 1067 by conductive trace 216. The system driver is connected to the contact 1067 to obtain an estimated concentration of the analyte in the sample using the first electrode pair.

The second electrode pair 1082 includes two electrodes each having five fingers. These fingers are each about 50 μm wide and the spacing between them is about 30 μm. Each electrode of the second pair is connected to conductive trace 216 to be electrically connected to contact 1068, which is used for driving and measuring a correction factor, such as hematocrit, based on the interaction of the analyte with the second electrode pair.

The third electrode pair 1083 is also a microelectrode structure, and each of the two electrodes in the third pair 1083 has five fingers interdigitated with the five fingers in the other electrode. Each finger is also about 50 a wide and the gap between them is about 30 μm. Each electrode in the third pair 1083 is connected to contacts 1069 by conductive traces 216 and is driven by these contacts to detect the sufficiency of the sample volume based on the electrical response between these electrodes when the sample has passed through the sample chamber 1048 to a sufficient degree. Note that in this embodiment, the reagent layer 1064 covers the upstream electrode pair 1081. In this example, the sample chamber requires about 220nL of sample fluid to cover all three electrode pairs.

Turning now to FIG. 11, the strip 1110 includes a substrate layer 212, a reagent strip 1164, a notched fluid conducting layer 1114 and notch 1148, and a cover layer 1118. The first electrode pair 1170 at the sample end 1166 of the strip 1110 includes two electrodes each having five fingers, with each finger being about 20 μm wide and the gap separating each adjacent finger being about 20 μm. The pair of electrodes is used to determine the concentration of interferents, such as hematocrit, by using AC excitation and impedance measurement techniques. For examples of these techniques, see AC authentication, which is incorporated by reference above.

The second electrode pair 1171 at the sample end 1166 of the strip 1110 includes two electrodes each having three fingers. Each finger is about 20 μm wide and the gap separating adjacent fingers is about 20 μm. The system derives a temperature compensated estimate of the glucose concentration by applying AC or DC excitation techniques to the second electrode pair 1171. In this example, the sample volume required to fill the capillary channel and cover the electrodes is about 69 nL.

Turning now to FIG. 12, the strip 1210 includes the base plate 212, reagent strips 1264, notched fluid guides 1114, and a cover layer 1118. A first electrode pair 1266 at the sample end 1260 of the strip 1210 includes two electrodes each having five fingers. The system uses a first electrode pair 1266 in the strip 1210 to obtain one measurement that is mostly based on interferent detection for combination with another measurement using a second electrode pair 1268. The second electrode pair at the sample end of the strip 1210 is an electrode pair 1268 that includes two electrodes, each having three fingers, and the pair 1268 is covered by a reagent layer 1264. The fingers in the second electrode pair 1268 are also about 20 μm wide and separated by a gap of about 20 μm. The second electrode pair 1268 is used by the system to estimate the analyte concentration in the sample. When the first electrode pair 1266 implements AC technology, the second electrode pair 1268 is driven by an AC or DC signal. Further downstream (beyond the second electrode pair 1268) from the sample end is a third electrode 1270, which is a single electrode finger, approximately 20 μm wide, connected to a contact 1272 by a conductor 1274. The AC signal response between the third electrode 1270 and the first electrode pair 1166 or the second electrode pair 1168 provides a sample sufficiency signal for the system. In a variation of this embodiment, the third electrode 1270 is used as an electrode in a circuit with the second electrode pair 1168 for applying various detection and measurement techniques known in the art.

FIG. 13 shows strip 1410, which includes substrate 212, reagent strip 1464, fluid direction device 1414 having notch 1448, and cover 1418. The first set of electrodes 1170 at the sample end 1166 of the strip 1410 includes two electrodes, each having five fingers. The fingers in electrode 1170 are each about 20 μm wide and the gap separating adjacent interdigitated fingers is about 10 μm.

The second electrode set 1171 includes two electrodes each having three fingers. The fingers of electrode 1171 are each about 20 μm wide and the gap between adjacent, interdigitated fingers is about 10 μm. In the presence of sample and reagent, an estimate of the glucose concentration is derived from the response of the second electrode set 1171 when the first electrode pair 1170 is used by the system to determine the hematocrit of the sample and calculate a correction factor. The third electrode pair 1471 is two electrodes each having two fingers. In this embodiment, a potential is applied across the third pair 1471 until the sample reaches the pair, thereby changing the impedance existing between the electrodes. The system can then conclude that the sample has sufficiently covered the first set 1170 and the second set 1171 of electrodes for accurate analysis. In this exemplary embodiment, a sample volume of about 63nL is required to cover the three electrode sets.

Fig. 14 shows strip 1410 having substrate layer 212, reagent strip 1464, notched fluid direction device 1414 (having notch 1448), and cover layer 1418. First electrode pair 1466 defines a first sensing region 1476 and includes two electrodes each having five fingers. These fingers are about 20 μm wide and include a gap of about 20 μm between the interdigitated fingers. The pair 1466 is used to provide a response reflecting the hematocrit of the sample, allowing the system to correct the estimated concentration of glucose in the sample as determined by using the second electrode pair 1468. Second electrode pair 1468 defines a second sensing region 1478 and includes two electrodes each having three fingers. The finger size and gap of the second electrode pair 1468 is the same as the finger size and gap of the first electrode pair 1466. The second electrode pair 1468 is used to obtain a correction factor for the concentration estimate obtained by the first electrode pair 1166 and uses an AC/impedance measurement technique.

Fig. 15 shows a variation of the strip 1510, i.e. the strip in fig. 11, where the electrode pairs 1570 and 1571 and the layers covering them are slightly modified. Specifically, electrode pair 1570 includes working electrodes with four fingers, each 50 μm wide and a gap width of 20 μm. The corresponding opposing electrode in electrode pair 1570 has three fingers, also 50 μm wide. The second electrode pair 1571 includes a working electrode with two fingers, each 100 μm wide, and an opposing electrode with a single finger, which is also 100 μm wide, and a gap width of 20 μm. In this embodiment, reagent 1564 will cover only electrode pair 1571, while coating 1565 will cover electrode pair 1570. Coating 1565 is a perm-selective, size-selective, ion-selective, or other coating that limits the fraction or composition of the sample that affects the measurement at electrode pair 1570, as is well known in the art. In a variation of this embodiment, there will be three or more electrode pairs, and each electrode pair will be covered with a different reagent or other coating, or combination of coatings, to provide a corresponding number of measurements with different sensitivities, which will be combined to determine the final measurement output. Among other considerations, the measurements are performed as described with respect to fig. 11, except for constants and functions derived from the cell geometry and the selection of coating 1565 and agent 1564.

Aspects of the described embodiments may be combined as desired or needed depending on the design parameters and preferences of a given system. For example, there may be a one-to-one correspondence between electrodes and contacts on a strip, as shown in FIG. 4. Alternatively, it means that all electrodes combined on the same side of the strip may be electrically connected to the same contact, as shown in fig. 5, which provides a many-to-one relationship.

Furthermore, any of the designs discussed herein may accommodate one or more "dose sufficiency" electrodes downstream of those used to analyze the sample, as shown in fig. 11 and 14. Such dose sufficiency electrodes may include two or more electrodes, and the associated circuitry may determine whether the sample has reached those electrodes based on the impedance existing between them. An alternative embodiment includes a single dose sufficiency electrode and the measurement and drive circuitry uses the impedance between it and the measurement electrode (working or counter electrode, i.e. evaluation or calibration pair) to detect the presence of sample fluid in the space between these electrodes.

As mentioned above, the biosensor may similarly include a dose detection electrode system that is comparable to the dose sufficiency electrode system, except that it is closer to the edge of the test strip, upstream of the measurement electrodes, as the sample enters the test strip. Such a dose detection electrode system may comprise a single electrode which works in combination with a separately provided measurement or other electrode. Alternatively, the dose detection electrode system may comprise a pair of electrodes which cooperate to indicate when sample fluid has bridged the gap between the dose detection electrodes. The dose detection electrodes may thus be seen as similar to dose sufficiency electrodes in terms of operation, but are different in terms of the position of the electrodes relative to the measurement electrodes in their upstream position.

In other variations, a thermistor in the system is used to determine the temperature, which is used along with the hematocrit reading to correct the glucose estimate. In other variations, the second electrode pair provides a temperature compensated glucose estimate using techniques known to those skilled in the art.

In other variants, the electrode pair that the sample first encounters is a macroscopic electrode pair, while in other variants, it is a microscopic electrode pair. In each case, each electrode comprises 1, 2, 3, 4, 5, or more appropriately sized fingers, all electrically connected to each other and to contacts for communication with measurement/drive electronics.

Other variations use other combinations of measurements to achieve the desired result. Typically, these variations apply electrical signals to two or more electrodes to obtain a corresponding number of response signals. The response signal is sensitive to different combinations of analyte concentration and interferents due to differences in signal (AC vs DC, spectrum, amplitude, etc.), electrode shape or size, reagents applied to the sample (or reagents may not be present at one or more electrodes), and/or other differences. In one such example, the first response is related to the hematocrit of the sample, and the second response is related to a combination of the hematocrit and the concentration of glucose in the sample. In another such example, the first response is related to temperature, the second response is related to a combination of temperature and hematocrit, and the third response is related to a combination of temperature, hematocrit, and glucose. The resulting function(s) may be different for each design, but they may be determined empirically by one skilled in the art without undue experimentation.

It will be appreciated by those skilled in the art that although embodiments have been described herein with respect to combining measurements, or taking measurements and determining correction factors, systems in accordance with the present invention may use any suitable geometry and any suitable technique to obtain and combine multiple measurements to obtain a final detection or measurement. That is, more or fewer electrodes may be used in practicing the invention, and any formula for combining readings that is appropriate in terms of geometry, reagents, and other system design choices made in connection with the design.

As discussed in the analysis Sensor application, which is incorporated above by reference, accurate detection of analytes can be achieved in a strip-based system having a smaller volume than prior art systems without detrimental effects on the connector, according to the present invention. This allows a smaller sample to be sufficient for the measurement, saving time and trouble for the user of the system.

All publications, prior applications, and other documents cited herein are hereby incorporated by reference in their entirety as if each had been individually incorporated by reference and fully set forth.

While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the inventions are desired to be protected.

Claims (20)

1. A test strip defining a capillary channel sized to cause a bodily fluid sample to move through the channel along a predetermined path by capillary action, characterized in that:

a first set of electrodes in electrical communication with the channel to obtain a first measurement related to an analyte concentration in the sample; and

a second set of electrodes in electrical communication with the channel to obtain a second measurement related to one or more characteristics of the analyte and sample;

one of the electrode sets includes a pair of electrodes having substantially parallel, interdigitated fingers, each electrode having at least two fingers, and each finger having a first distance from the nearest other finger in the one of the electrode sets of less than 50 μm, and

the other set of electrodes comprises a pair of macroscopic electrodes each having one finger separated by a second distance of at least 50 μm,

where two sets of electrodes contribute information to the final determination of the analyte concentration.

2. The test strip of claim 1, wherein the first set of electrodes includes an electrode pair having substantially parallel, interdigitated fingers.

3. The test strip of claim 1, wherein the second set of electrodes includes pairs of electrodes having substantially parallel, interdigitated fingers.

4. The test strip of any one of the preceding claims, wherein each of the interdigitated fingers is at most 50 μm wide.

5. The test strip of any one of the preceding claims, wherein each of the macro electrodes is at least 50 μm wide.

6. The test strip of any one of the preceding claims, wherein the first distance is a distance of at most 30 μm.

7. The test strip of any one of the preceding claims, wherein the second distance is a distance of at least 100 microns.

8. The test strip of any one of the preceding claims, further comprising a third set of electrodes in electrical communication with the path to determine when the sample has entered the channel a predetermined distance.

9. The test strip of claim 8, wherein the third set of electrodes includes at least one dose detection electrode that is closer to the entrance of the channel than the first set of electrodes and the second set of electrodes.

10. The test strip of claim 9, wherein the third set of electrodes includes a first dose detection electrode and a second dose detection electrode, both electrodes being closer to the entrance of the channel than the first set of electrodes and the second set of electrodes.

11. The test strip of claim 8, wherein the third set of electrodes includes a pair of sample sufficiency electrodes, both of which are farther from the entrance of the channel than the first set of electrodes and the second set of electrodes.

12. The test strip of claim 11, further comprising a fourth set of electrodes including at least one electrode closer to the entrance of the channel than the first set of electrodes and the second set of electrodes.

13. The test strip of any one of the preceding claims, wherein the first set of electrodes includes a first electrode and a second electrode, and the second set of electrodes includes a third electrode and a fourth electrode, the first electrode and the third electrode being electrically connected to each other, and the second electrode and the fourth electrode being in electrical communication with each other.

14. The test strip of any of the preceding claims, further comprising a reagent material over the first set of electrodes, wherein the reagent material combines with bodily fluids to produce a redox reaction.

15. The test strip of any of the preceding claims, further comprising a reagent material over the second set of electrodes, wherein the reagent material combines with bodily fluids to produce a redox reaction.

16. The test strip of any one of the preceding claims, wherein the channel has a volume of at most 240 nL.

17. The test strip of any one of the preceding claims, wherein the first and second sets of electrodes are positioned within the channel such that fluid moving along the channel by capillary action will successively encounter the first and second sets.

18. A method of measuring an analyte concentration in a bodily fluid sample, comprising:

providing a test strip according to any one of the preceding claims;

obtaining a first response to applying a first electrical signal to the first set of electrodes;

obtaining a second response to applying a second electrical signal to the second set of electrodes; and

the first response and the second response are used to derive a measurement of the analyte concentration in the sample.

19. The method of claim 18, further comprising, prior to the obtaining step, detecting sample application to the test strip.

20. The method of any one of claims 18 or 19, further comprising, prior to the obtaining step, detecting sufficiency of the sample volume.