US20070017805A1 - Method and device for sampling and analyzing interstitial fluid and whole blood samples - Google Patents

Abstract

The invention disclosed in this application is a method and device for combining the sampling and analyzing of sub-dermal fluid samples, e.g., interstitial fluid or whole blood, in a device suitable for hospital bedside and home use. It is applicable to any analyte that exists in a usefully representative concentration in the fluid, and is especially suited to the monitoring of glucose.

Description

RELATED APPLICATIONS
• This application is a continuation of U.S. application Ser. No. 10/369,120, filed Feb. 13, 2003, which is a continuation of U.S. application Ser. No. 10/166,487, filed Jun. 10, 2002, now U.S. Pat. No. 6,939,312, which is a continuation of application Ser. No. 09/536,235, filed Mar. 27, 2000, now U.S. Pat. No. 6,612,111.
FIELD OF THE INVENTION
• The present invention relates to a method and device for combining the sampling and analyzing of interstitial fluid or whole blood samples which is suitable for hospital bedside and home use.
BACKGROUND OF THE INVENTION
• The management of many medical conditions requires the measurement and monitoring of a variety of analytes in bodily fluid. Historically, the measurement of analytes in blood has required an invasive technique, such as a venipuncture or finger puncture, to obtain blood for sampling purposes. An example of an analyte which is routinely tested by obtaining a blood sample through an invasive technique is glucose. In order to control their condition, diabetics must monitor their glucose levels on a regular basis. Invasive techniques used to obtain a blood sample for analysis have the disadvantage of being painful, which can reduce patient compliance in regular monitoring. Repeated testing, e.g., on a fingertip, can result in scar tissue build-up which makes obtaining a sample in that region more difficult. Moreover, invasive sampling procedures pose a risk of infection or disease transmission.
• An alternative is to sample interstitial fluid rather than whole blood. Interstitial fluid is the fluid that fills the space between the connective tissue and cells of the dermal layer of the skin. An application where interstitial fluid has been shown to be an appropriate sampling substitute for plasma or whole blood is in the measurement of glucose concentration (J. Lab. Clin. Med. 1997, 130,436-41).
• In the patents U.S. Pat. Nos. 5,879,367, 5,879,310, 5,820,570 and 5,582,184 are disclosed methods of sampling using a fine needle in conjunction with a device to limit the penetration depth to obtain small volumes of interstitial fluid for the purpose of glucose monitoring. However, there is no method disclosed for analyzing the drawn samples that is suitable for home use or hospital bedside use.
SUMMARY OF THE INVENTION
• It is desirable to be able to measure the concentration of analytes in humans or other animals without having to draw a blood sample by conventional methods. It is further desirable to be able to do so with an inexpensive disposable device that is simple enough for home or hospital bedside use.
• The invention provides a suitable alternative to conventional sampling devices and methods that is less invasive than traditional whole blood sampling techniques and that requires a considerably smaller sample volume than is required in the conventional venipuncture or finger puncture sampling methods. Because of the smaller sample volume required, a smaller wound is necessary to obtain the sample. In the conventional finger stick method, a drop of blood is formed on the tip of a finger, then the sensor sample entrance is wetted with the drop. Because the sample comes into contact with the skin surface, contamination of the sample by material on the skin surface is possible. The devices and methods disclosed herein do not require forming a blood drop on the surface of the skin, and therefore have less risk of sample contamination.
• In one embodiment of the present invention, a fluid sampling device is provided which includes a body, the body including a dermal layer penetration probe having a penetrating end and a communicating end, and an analysis chamber having a proximal and distal end, the analysis chamber having a volume, wherein the penetration probe is in fluid communication with the analysis chamber such that fluid can flow from the penetration probe toward the analysis chamber. The analysis chamber can have at least one flexible wall which can be compressed to reduce the volume of the analysis chamber. The penetration probe can include, for example, a needle, a lancet, a tube, a channel, or a solid protrusion and can be constructed of a material such as carbon fiber, boron fiber, plastic, metal, glass, ceramic, a composite material, mixtures thereof, and combinations thereof. The penetration probe can include two sheets of material in substantial registration, having a protrusion on each sheet, wherein the sheets are spaced apart such that liquid can be drawn between the sheets by capillary action. The two sheets of material can extend into the device so as to form a pre chamber. The penetration probe can be positioned within a recess in the proximal end of the device, and the recess can be configured to substantially align with a shape of a selected dermal surface.
• In a further embodiment, the device can further include a pre-chamber having a volume and a first and second end, wherein the pre-chamber is interposed between the penetration probe and the analysis chamber such that the first end of the pre-chamber is adjacent the communicating end of the penetration probe and the second end of the pre-chamber is adjacent the proximal end of the analysis chamber. The volume of the pre-chamber can be greater than or equal to the volume of the analysis chamber. The pre-chamber can have at least one flexible wall that can be compressed to reduce the volume of the pre-chamber. The pre-chamber can also include a valve at the first end capable of substantially sealing the pre-chamber from the penetration probe.
• In another embodiment, the device further includes a compressible bladder in communication with the analysis chamber, the compressible bladder being capable of applying a positive or a negative pressure to the analysis chamber.
• In yet another embodiment, the pre-chamber and the analysis chamber can be capable of exerting different capillary forces, The capillary force exerted by the analysis chamber can be greater than the capillary force exerted by the pre-chamber. The differential capillary force can be derived, at least in part, from a difference between the pre-chamber height and the analysis chamber height. In this embodiment, the interior surface of the pre-chamber can include at least first and second pre-chamber walls spaced apart at a first distance to define a pre-chamber height, and the interior surface of the analysis chamber can include at least first and second analysis chamber walls spaced apart at a second distance to define an analysis chamber height, wherein the height of the analysis chamber is less than the height of the pre-chamber.
• In yet another further embodiment, at least one of the chambers can include a substance capable of enhancing or diminishing the capillary force exerted by the chamber. The substance can include, for example, a polymer, a resin, a powder, a mesh, a fibrous material, a crystalline material, or a porous material. Suitable substances include polyethylene glycol, polyvinylpyrrolidone, a surfactant, a hydrophilic block copolymer, and polyvinylacetate.
• In a further embodiment, the device further includes a releasable actuator capable of supplying a force sufficient to cause the penetration probe to penetrate a dermal layer. The actuator can be external to or integral with the body, and upon release propels the body toward the dermal layer.
• In a further embodiment, the analysis chamber can include an electrochemical cell including a working electrode and a counter/reference electrode and an interface for communication with a meter, wherein the interface communicates a voltage or a current.
• In yet another embodiment of the present invention, a method for determining a presence or an absence of an analyte in a fluid sample is provided including the steps of providing a fluid sampling device as described above; penetrating a dermal layer with the penetration probe; substantially filling the analysis chamber with a fluid sample by allowing the sample to flow from the penetration probe toward the analysis chamber; and detecting a presence or an absence of the analyte within the analysis chamber. The sample can include, for example, interstitial fluid and whole blood. A qualitative or quantitative measurement of a characteristic of the sample can be obtained in the detecting step. The characteristic of the sample can include, for example, a reaction product of the analyte, such as a color indicator, an electric current, an electric potential, an acid, a base, a reduced species, a precipitate, and a gas. The analyte can include, for example, an ion such as potassium, an element, a sugar, an alcohol such as ethanol, a hormone, a protein, an enzyme, a cofactor, a nucleic acid sequence, a lipid, a pharmaceutical, and a drug. Cholesterol and lactate are examples of substances that can be analyzed.
• In a further embodiment, the flow of sample toward the analysis chamber can be driven by a driving force, e.g., capillary force or a pressure differential. Where the analysis chamber has a flexible wall, the wall can be compressed to reduce the volume of the analysis chamber prior to penetrating the dermal, then the compression released to form a partial vacuum in the analysis chamber. Where the fluid sampling device further includes a compressible bladder, the bladder can be compressed to reduce its volume, then after penetration of the dermal layer the compression can be released to form a partial vacuum in the compressible bladder and analysis chamber.
BRIEF DESCRIPTION OF THE DRAWINGS
• FIG. 1 shows a top view (not to scale) of one embodiment of a sampling device illustrating an arrangement of the penetration probe, pre-chamber, and analysis chamber.
• FIG. 2 shows a cross section (not to scale) along the line A-A′ ofFIG. 1.
• FIG. 3 shows a top view (not to scale) of one embodiment of a sampling device illustrating an arrangement of the penetration probe, pre-chamber, and analysis chamber wherein the proximal edge of the device forms a recess.
• FIG. 4 shows a top view (not to scale) of one embodiment of a sampling device illustrating an arrangement of the penetration probe, pre-chamber, and analysis chamber.
• FIG. 5 shows a cross section (not to scale) along the line B-B′ ofFIG. 4.
• FIGS. 6aand6b(not to scale) depict an embodiment of the invention wherein the device is loaded in a releasable actuator to facilitate penetration of a dermal layer by the penetration probe.FIG. 6adepicts the device loaded in the actuator, wherein the actuator is in the cocked position, ready to be triggered.FIG. 6bdepicts the device and actuator after triggering.
• FIG. 7 is a schematic drawing (not to scale) of a first embodiment according to the invention shown in side elevation.
• FIG. 8 shows the embodiment ofFIG. 7 in plan, viewed from above.
• FIG. 9 shows the embodiment ofFIG. 7 in plan, viewed from below.
• FIG. 10 shows the embodiment ofFIG. 7 viewed in end elevation.
• FIG. 11 is a schematic drawing (not to scale) of a second embodiment according to the invention in side elevation.
• FIG. 12 shows the embodiment ofFIG. 11 in plan, viewed from above.
• FIG. 13 is a schematic drawing (not to scale) of a third embodiment according to the invention, in side elevation.
• FIG. 14 shows the embodiment ofFIG. 13 in plan, viewed from above.
• FIG. 15 is a schematic drawing (not to scale) according to the invention in plan view, viewed from above.
• FIG. 16 shows the embodiment ofFIG. 15 in end elevation.
• FIG. 17 shows the embodiment ofFIG. 15 in side elevation.
• FIG. 18 shows a schematic drawing (not to scale) of a hollow cell embodiment according to the invention, viewed in cross section.
• FIG. 19 is a graph showing a plot of current (ordinate axis) versus time (co-ordinate axis) during conduct of a method according to the invention.
• FIG. 20 is a further graph of use in explaining the method of the invention.
• In FIGS.11 to12, components corresponding in function to components of the embodiment of FIGS.7 to10 are identified by identical numerals or indicia.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
• Introduction
• The following description and examples illustrate various embodiments of the present invention in detail. Those of skill in the art will recognize that there are numerous variations and modifications of this invention that are encompassed by its scope. Accordingly, the description of a preferred embodiment should not be deemed to limit the scope of the present invention. Methods and devices for optimizing sampling of fluid samples are discussed further in copending U.S. patent application Ser. No. 09/536,234, filed on Mar. 27, 2000, entitled “METHOD OF PREVENTING SHORT SAMPLING OF A CAPILLARY OR WICKING FILL DEVICE,” which is incorporated herein by reference in its entirety.
• The invention disclosed in this application is a method and device for combining the sampling and analyzing of a fluid sample from sub-dermal tissue in a device suitable for hospital bedside and home use. The fluid sample can comprise, but is not limited to, interstitial fluid or whole blood samples obtained from an animal. Any fluid sample obtained from sub-dermal tissue of a plant or an animal can sampled and analyzed, thus the invention has broad application in the fields of human medicine, veterinary medicine, and horticultural science. The device and method are applicable to any analyte that exists in a usefully representative concentration in the fluid sample. For clarity, the present disclosure will discuss the application to glucose monitoring. However, it is to be understood that the invention is not limited to the monitoring of glucose, and that other analytes, as discussed below, can also be measured.
• The method utilizes an integrated sampling and analyzingdevice10 incorporating apenetration probe12 capable of penetrating a patient's dermal layers to extract an interstitial fluid or whole blood sample, and a method for transferring the sample from thepenetration probe12 to theanalysis chamber20. In one embodiment, thedevice12 can be a one-shot disposable device which can be inserted into a meter which communicates with theanalysis chamber20 to perform the analysis of the sample and present and optionally store the result.
• In thedevice10, apenetration probe12 for penetrating the subject's dermal layers to collect an interstitial fluid or whole blood sample is integrated with ananalysis chamber20. A property of sampling interstitial fluid is that it can take from several to tens of seconds to collect sufficient sample to analyze. This is often not desirable for ananalysis chamber20 wherein the analyte undergoes a reaction as part of the analysis process, as it can be difficult to obtain an accurate start time for the test as well as achieve an even reacting reagent distribution in the sample. In a second aspect of the current invention a method is disclosed for collecting the sample in a pre-chamber14 and, when full, transferring the sample quickly to ananalysis chamber20.
• In this disclosure, unless a different meaning is clear from the context of its usage, “proximal” refers to a region or structure of the device situated toward or adjacent to the dermal surface to be penetrated, and “distal” refers a region or structure of the device situated toward the opposite (non-proximal) end of the device. For example, thepenetration probe12 is at the proximal end of the device.
• The Penetration Probe
• Thepenetration probe12 can be any device capable of penetrating the patient's dermal layers to the desired extent and capable of transporting a sample to a pre-chamber14 oranalysis chamber20. Thepenetration probe12 comprises two ends, as illustrated inFIG. 1. The—penetratingend11 of thepenetration probe12 is the end inserted into the dermal layer. The communicatingend13 of thepenetration probe12 is the end which is in communication with either the pre-chamber14 or theanalysis chamber20.
• One ormore protrusions12 with at least one sharp edge or point are suitable as thepenetration probe12. Thepenetration probe12 can be fabricated from materials including plastic, metal, glass, ceramic, a composite material (e.g., a composite of ceramic and metal particles), or mixtures and combinations of these materials. Thepenetration probe12 can be in the form of a solid protrusion, a needle, a lancet, a tube or a channel. The channel can optionally be open along one or more of its elongated sides. As illustrated inFIG. 2, a preferred embodiment of thepenetration probe12 is twosheets30 of material formed so as to have a sharply pointedprotrusion12 on eachsheet30 in substantial registration, with thesheets30 spaced apart such that liquid can be drawn between thesheets30 by capillary action. In a particularly preferred embodiment, the twosheets30 of material extend to and overlap with theanalysis chamber20 to form a pre-chamber14 for sample collection.
• When interstitial fluid is sampled, the penetration depth can be controlled by limiting the length thepenetration probe12 protrudes from theproximal surface34 of thesampling device10 to less than the thickness of the dermal layer. In a preferred embodiment, the length of theprotrusion12 will be less than 2 to 3 mm, more preferably about 1.5 mm. After penetration to a suitable depth corresponding to the length of theprotrusion12, contact between the surface of the dermal layer and thesurface34 of the analyzing device prevents further penetration. For other uses, such as in sampling interstitial fluid from regions having a thick dermal layer, or for veterinary uses, it can be desirable for the length of theprotrusion12 to be greater than 3 mm. Accordingly, the invention contemplatesprotrusions12 of any length, wherein the length is sufficient to sample interstitial fluid. When whole blood is sampled, a slightlylonger penetration probe12 should be used, i.e., one having a length greater than 2 to 3 mm.
• The diameter or width of thepenetration probe12 depends upon the design of thepenetration probe12. Suitable diameters or widths are those which provide sufficient sample flow. In the case of aprotrusion12 forming a sharp edge or point, or a tube or channel, the minimum diameter or width is typically greater than about 10 μm. When the penetratingmeans12 comprises twosheets30 in substantial registration, each having a sharply pointedprotrusion12, the twoprotrusions12 are typically spaced from 1 mm to 10 μm apart.
• Thepenetration probe12 can be located on any suitable part of thetest strip10, i.e., anedge34, acorner42, or one of the flat surfaces44. Protection can be provided to thepenetration probe12 by locating it within a recess formed in thedistal edge34 of thetest strip10, as shown inFIG. 3, or in a depression on thesurface44 of thetest strip10. In a preferred embodiment, the recess in thedistal edge34 of thetest strip10 can be configured to substantially align with the shape of a selected dermal surface, e.g., a fingertip. However, the recess can be configured in other suitable shapes, e.g., a square recess, a V-shaped recess, a curved recess, a polygonal recess, and the like. In a preferred embodiment, thepenetration probe12 does not protrude past the proximal-most portion of theproximal edge34 orsurface44 of thedevice10, but when pressed against the skin, the skin deforms into the recess and is punctured by thepenetration probe12. Such an arrangement aids sampling by compressing the area of the skin around the sampling point. Thepenetration probe12 can form an integral part of another component of thetest strip10, e.g., a side of the pre-chamber54, as shown inFIG. 2. Alternatively, thepenetration probe12 can comprise a separate part which is attached to or incorporated into thetest strip10 by any suitable means, e.g., adhesive, thermal bonding, interlocking parts, pressure, and the like. Thepenetration probe12 can be retractable or non-retractable.
• Penetration itself can be accomplished by any suitable means, including inserting thepenetration device12 manually or by means of areleasable actuator84 such as, for example, a spring-loadedmechanism84 as depicted inFIGS. 6aand6b. Such aspringloaded mechanism84 incorporates aspring86 which is compressed and held in place by atrigger88 which can release the force compressing thespring86 when the triggering mechanism is activated. Thetrigger88 can be activated manually, or thedevice84 can incorporate a pressure sensor which indicates that sufficient pressure has been applied to obtain the sample, thereby activating thetrigger88. In one embodiment, the distal end of thedevice10 is placed in the spring-loadedmechanism84 such that when the force compressing thespring86 is released by activating thetrigger88, force is transferred to thedevice10, which is ejected from themechanism84, thereby inserting the penetratingprobe12 into the dermal layer.
• Any suitable body part can be used for sampling. In a preferred embodiment, the sampling area is one which does not have a high density of nerve endings, e.g., the forearm. Typically, 5 to 15 seconds is required to obtain sufficient sample. Application of pressure to the sampling area can be needed to extract interstitial fluid or whole blood. To facilitate the appropriate amount of pressure being applied, a pressure sensor can be incorporated into thedevice10 which indicates when sufficient pressure has been applied. Sample acquisition time can be improved by applying increased pressure to the area surrounding the direct sampling area. Some of the factors that can affect interstitial fluid or whole blood sample acquisition include the patient's age, skin thickness, temperature, and hydration. The amount of interstitial or whole blood sample collected for testing can preferably be about 0.02 μl or greater, more preferably 0.1 μl or greater, and most preferably about 0.5 μl or greater.
• In one preferred embodiment, thedevice10 can be inserted into a meter prior to sample acquisition. In such an embodiment, the meter serves multiple functions, including supporting thedevice10, providing an automated means of initiating sample acquisition, and indicating when sample acquisition is complete.
• Transfer of Sample from Penetration Probe to Analysis Chamber
• In a preferred embodiment of thesampling device10, the device comprises two parts—thepenetration probe12 and ananalysis chamber20. In another preferred embodiment, illustrated inFIGS. 1 and 2, thedevice10 comprises thepenetration probe12 and a pre-chamber14. The pre-chamber14 can then be integrated with or can be interfaced to theanalysis chamber20.
• In a further embodiment, theanalysis chamber20 is integrated with or can be interfaced to a means for facilitating filling of theanalysis chamber20. This means can comprise a collapsible orcompressible bladder22, as shown inFIGS. 3 and 4, which can be used to apply a positive or negative pressure (i.e., partial vacuum) to theanalysis chamber20. Thecompressible bladder22 can comprise any chamber with flexible walls that can be compressed to reduce the volume of the chamber. When the force compressing thecompressible bladder22 is released, a partial vacuum is formed which draws sample into theanalysis chamber20. In a preferred embodiment, the volume of thecompressible bladder22 is sufficiently large so that when thebladder22 is substantially fully compressed, the reduction in volume of thebladder22 is larger than or equal to the total volume of theanalysis chamber20, thereby ensuring that theanalysis chamber20 is substantially filled. However, acompressible bladder22 with a smaller volume than theanalysis chamber20 can also be effective in assisting the filling of theanalysis chamber20.
• Alternatively, theanalysis chamber20 itself can be collapsible or compressible. In such an embodiment, a piston or other compressing agent, such as a patient's or clinician's fingers, can first compress then release theanalysis chamber20, thereby forming a partial vacuum. When the compressing force is released, the partial vacuum causes the sample to flow from the penetration probe toward the analysis chamber.
• Pre-Chamber
• In a preferred embodiment, as illustrated inFIGS. 1 and 2, a pre-chamber14 is provided in the integrated sampling andtesting device10 for accumulation and storage of the collected sample prior to its being transferred to theanalysis chamber20. A pre-chamber14 is useful when using an analysis method which requires that the sample fill theanalysis chamber20 in a short period of time to return accurate results, i.e., a time shorter than that required to draw sufficient sample from the dermal layer. In a preferred embodiment, the volume of the pre-chamber14 is larger than that of theanalysis chamber20, thus ensuring that once the pre-chamber14 is filled, sufficient sample has been collected to completely fill theanalysis chamber20.
• In a preferred embodiment, as illustrated inFIGS. 1 and 2, thepenetration probe12 opens into the pre-chamber14 at a first end, and at the second end the pre-chamber14 opens to theanalysis chamber20. The pre-chamber14 can be free of reagents or other substances, or can optionally contain one or more substances to enhance or diminish the capillary force exerted by the walls of the pre-chamber14 or to pre-treat the sample prior to analysis. These substances can include, for example, polymers, resins, powders, meshes, fibrous materials, crystalline materials, porous materials, or a mixture or combination thereof. To facilitate effective filling of theanalysis chamber20, a preferred embodiment utilizes a pre-chamber14 andanalysis chamber20 of different heights, as shown inFIG. 2. Where theanalysis chamber20 is formed so that its height (typically referring to the smallest chamber dimension) is smaller than the height of the pre-chamber14, a capillary force is generated that is capable of drawing fluid out of the pre-chamber14 and into theanalysis chamber20. Afirst air vent64 can be formed at theend70 of theanalysis chamber20 opposite theopening62 to the pre-chamber14, facilitating the filling of theanalysis chamber20 by allowing air to be displaced from theanalysis chamber20 as sample enters. Optionally, asecond vent74 can be formed opening into the pre-chamber14 at the substantiallyopposite end60 of the pre-chamber14 to where thepenetration probe12 opens into the pre-chamber14. Thisvent74 provides air to the pre-chamber14 to replace the sample as it is transferred from the pre-chamber14 to theanalysis chamber20. Thevent74 can be placed in any suitable position on thetest strip10. In a preferred embodiment, thevent74 incorporates a sharp corner, e.g., at a 90° angle, which functions as a “capillary stop” to prevent sample from exiting thedevice10 through thevent74.
• In another embodiment, the pre-chamber14 consists of a tube, or other shaped chamber, with flexible walls, attached to thepenetration probe12. In this embodiment, the pre-chamber14 is either permanently fixed to theanalysis chamber20 or is placed next to and aligned with a port to theanalysis chamber20. Such alignment can occur during use by suitable placement in an external device such as the measurement meter.
• In one aspect of this embodiment, the pre-chamber14 further comprises a valve, defined as a device to control the flow of fluid sample between thepenetration probe12 and the pre-chamber14. The valve can comprise one or more rollers, pistons, or squeezing devices capable of simultaneously closing off thefirst end60 of the pre-chamber14, and compressing the pre-chamber14 such that the fluid in the pre-chamber14 is forced towards thesecond end62 of the pre-chamber14 and subsequently into theanalysis chamber20.
• Alternatively, theanalysis chamber20 consists of a tube, or other shaped chamber, with flexible walls, attached to thepenetration probe12. In one aspect of this embodiment, theanalysis chamber20, prior to penetration, is compressed by one or more rollers, pistons, or other squeezing devices. After thepenetration probe12 is inserted, the compression is released, forming a vacuum which pulls sample into theanalysis chamber20. In such an embodiment, the pre-chamber14 can not be necessary if sufficient vacuum is generated for rapid sample acquisition. In such an embodiment, thedevice10 can not require avent64,74 if such would interfere with forming a vacuum.
• In another embodiment, illustrated inFIGS. 3 and 4, apre-chamber14 of suitable size is formed which opens to thepenetration probe12 on oneend60 and to theanalysis chamber20 on theother end62. Theend70 of theanalysis chamber20 opposite to that opening to the pre-chamber14 opens to acompressible bladder22. Thebladder22 can be formed separately and attached to theend70 of theanalysis chamber20. Alternatively, it can be formed by removing a section on the middle laminate82 in thetest strip10, similar to those described in W097/00441 (incorporated herein by reference in its entirety), as illustrated inFIGS. 3 and 4.
• In use, thebladder22 in thestrip10 is compressed by suitable means prior to thepenetration probe12 being inserted into the patient. Insertion of thepenetration probe12 can be confirmed by use of a sensor, such as a pressure sensor, or the patient can confirm that thepenetration probe12 is inserted either visually or by touch. In the latter case, the patient sensing can signal the meter, such as by pushing a button. At this point, the means compressing thebladder22 is withdrawn to a halfway position to draw sample into the pre-chamber14. When the pre-chamber14 is full, as indicated by a suitable sensor, the meter indicates to the patient to withdraw thepenetration probe12. The compressing means then moves to its fully withdrawn position and so draws the sample from the pre-chamber14 into theanalysis chamber20. In the case where the initial suction from thebladder22 causes the sample to be accumulated with sufficient speed, the pre-chamber14 can be dispensed with and thebladder22 used to draw sample through thepenetration probe12 directly into theanalysis chamber20. Avent64,74 which would interfere with forming a vacuum need not be incorporated into the device in some embodiments.
• Analysis Chamber
• In a preferred embodiment, theanalysis chamber20 is contained in an analyzingdevice10 comprising a disposable analysis strip similar to that disclosed in W097/00441. The analysis strip of W097/00441 contains a biosensor for determining the concentration of an analyte in a carrier, e.g., the concentration of glucose in a fluid sample. Theelectrochemical analysis cell20 in this strip has an effective volume of 1.5 μl or less, and can comprise a porous membrane, a working electrode on one side of the membrane, and a counter/reference electrode on the other side. In a preferred embodiment, ananalysis cell20 having an effective volume of about 0.02 μl or greater is used. More preferably, thecell20 has a volume ranging from about 0.1 μl to about 0.5 μl.
• In one aspect of this embodiment, thepenetration probe12 is a small needle integrated into theanalysis strip10 by being inserted through a wall of theanalysis chamber20 such that one end of theneedle12 opens into thestrip analysis chamber20. In using adevice10 having this arrangement to obtain and analyze a sample of interstitial fluid, theneedle12 is inserted into the patient's dermal layer and sample is drawn into theneedle12 via capillary action. The sample is then transferred from theneedle12 into theanalysis chamber20 by capillary action whereupon the sample is analyzed. Anopening64 in theanalysis chamber20 to atmosphere, remote from the point where theneedle12 opens into the chamber, acts as avent64 to allow the escape of displaced air as theanalysis chamber20 fills with sample. Analysis devices of the type disclosed in W097/00441 are particularly suited for use with this arrangement because of their ability to utilize the very small volumes of sample typically available with interstitial fluid sampling.
• Theanalysis chamber20 can contain one or more substances to enhance or diminish the capillary force exerted by the walls ofanalysis chamber20. Such materials can include polymers, resins, powders, meshes, fibrous materials, crystalline materials, porous materials, or a mixture or combination thereof, as can also be used in the pre-chamber, discussed above. For example, thewalls24 of theanalysis chamber20 can be coated with a hydrophilic material to encourage the flow of fluid sample into the analysis chamber. Suitable hydrophilic materials include polyethylene glycol, polyvinylpyrrolidone, a surfactant, a hydrophilic block copolymer, and polyacylicacid. Theanalysis chamber20 can also contain reagents capable of reacting with the analyte or other substances present in the sample. Such other substances can include substances which interfere in determining the presence or absence of the analyte. In such cases, the reagent will react with the substance so that it no longer interferes with the analysis.
• Any analyte present in a fluid sample in a detectable amount can be analyzed using thedevice10. A typical analytes can include, but is not limited to, an ion, an element, a sugar, an alcohol, a hormone, a protein, an enzyme, a cofactor, a nucleic acid sequence, a lipid, and a drug. In a preferred embodiment, glucose is the analyte to be tested. Typical analytes could include, but are not limited to, ethanol, potassium ion, pharmaceuticals, drugs, cholesterol, and lactate.
• The presence or absence of the analyte can be determined directly. Alternatively, the analyte can be determined by reacting the analyte with one or more reagents present in the analysis chamber. The product of that reaction, indicative of the presence or absence of the analyte, would then be detected. Suitable reaction products include, but are not limited to, a color indicator, an electric current, an electric potential, an acid, a base, a precipitate, or a gas.
• Any suitable analytical method can be used for determining the presence or absence of the analyte or a reaction product of the analyte. Suitable analytical methods include, but are not limited to, electrochemical methods, photoabsorption detection methods, photoemission detection methods, and the measurement of magnetic susceptibility. In the case of a reaction product having a different color than the analyte, or the formation of a precipitate or a gas, a visual determination can be a suitable method for determining the presence or absence of the analyte.
• With reference to FIGS.7 to10 there is shown a first embodiment of apparatus of the invention, in this case a biosensor for determining glucose in blood. The embodiment comprises athin strip membrane1 having upper andlower surfaces2,3 and having acell zone4 defined between a workingelectrode5 disposed onupper surface2 and acounter electrode6 disposed onlower surface3. The membrane thickness is selected so that the electrodes are separated by a distance “1” which is sufficiently close that the products of electrochemical reaction at the counter electrode migrate to the working electrode during the time of the test and a steady state diffusion profile is substantially achieved. Typically, “1” will be less than 500 μm. A sample deposition or “target”area7 defined onupper surface2 ofmembrane1 is spaced at a distance greater than the membrane thickness fromcell zone4.Membrane1 has adiffusion zone8 extending betweentarget area7 andcell zone4. A suitable reagent including a redox mediator “M”, an enzyme “E” and a pH buffer “B” are contained withincell zone4 of the membrane and/or betweencell zone4 andtarget area7. The reagent may also include stabilizers and the like.
• In some cases it is preferable to locate the enzyme and mediator and/or the buffer in different zones of the membrane. For example the mediator may be initially located withinelectrochemical cell zone4 while the enzyme may be situated belowtarget area7 or indiffusion zone8.
• Haemoglobin releases oxygen at low pH's, but at higher pH's it binds oxygen very firmly. Oxygen acts as a redox mediator for glucose oxidase dehydrogenase (GOD). In a glucose sensor this competes with the redox mediator leading to low estimates of glucose concentration. Therefore if desired a first pH buffer can be contained in the vicinity oftarget area7 to raise the pH to such a level that all the oxygen is bound to haemoglobin. Such a pH would be non-optimal for GOD/glucose kinetics and would consequently be detrimental to the speed and sensitivity of the test.
• In a preferred embodiment of the invention a second pH buffer is contained as a reagent in the vicinity of the working electrode to restore the pH to kinetically optimal levels.
• The use of a second buffer does not cause oxygen to be released from the haemoglobin as the haemoglobin is contained within the blood cells which are retained nearblood target area7 or are retarded in diffusion in comparison with the plasma and therefore not influenced by the second buffer. In this manner oxygen interference may be greatly reduced or eliminated.
• In use of the sensor a drop of blood containing a concentration of glucose to be determined is placed ontarget zone7. The blood components wick towardscell zone4, the plasma component diffusing more rapidly than red blood cells so that a plasma front reachescell zone4 in advance of blood cells.
• When the plasma wicks into contact with the reagent, the reagent is dissolved and a reaction occurs that oxidises the analyte and reduces the mediator. After allowing a predetermined time to complete this reaction an electric potential difference is applied between the working electrode and the counter electrode. The potential of the working electrode is kept sufficiently anodic such that the rate of electrooxidation of the reduced form of the mediator at the working electrode is determined by the rate of diffusion of the reduced form of the mediator to the working electrode, and not by the rate of electron transfer across the electrode/solution interface.
• In addition the concentration of the oxidised form of the mediator at the counter electrode is maintained at a level sufficient to ensure that when a current flows in the electrochemical cell the potential of the counter electrode, and thus also the potential of the working electrode, is not shifted so far in the cathodic direction that the potential of the working electrode is no longer in the diffusion controlled region. That is to say, the concentration of the oxidized form at the counter electrode must be sufficient to maintain diffusion controlled electrooxidation of the reduced form of the mediator at the working electrode.
• The behavior of a thin layer cell is such that if both oxidised and reduced forms of the redox couple are present, eventually a steady state concentration profile is established across the cell. This results in a steady state current. It has been found that by comparing a measure of the steady state current with the rate at which the current varies in the current transient before the steady state is achieved, the diffusion coefficient of the redox mediator can be measured as well as its concentration.
• More specifically, by solving the diffusion equations for this situation it can be shown that over a restricted time range a plot of ln (i/i−1) vs. time (measured in seconds) is linear and has a slope (denoted by S) which is equal to −4π²D/1², here “i” is the current at time “t”, “V” is the steady state current, “D” is the diffusion coefficient in cm²/sec, “1” is the distance between the electrodes in cm and “π” is approximately 3.14159 The concentration of reduced mediator present when the potential was applied between the electrodes is given by 2π²i/FA1S, where “T” is Faraday's constant, “A” is the working electrode area and the other symbols are as given above. As this later formula uses S it includes the measured value of the diffusion coefficient.
• Since 1 is a constant for a given cell, measurement of i as a function of time and i enable the value of the diffusion coefficient of the redox mediator to be calculated and the concentration of the analyte to be determined.
• Moreover the determination of analyte concentration compensates for any variation to the diffusion coefficient of the species which is electrooxidised or electroreduced at the working electrode. Changes in the value of the diffusion coefficient may occur as a result of changes in the temperature and viscosity of the solution or variation of the membrane permeability. Other adjustments to the measured value of the concentration may be necessary to account for other factors such as changes to the cell geometry, changes to the enzyme chemistry or other factors which may effect the measured concentration. If the measurement is made on plasma substantially free of haematocrit (which if present causes variation in the diffusion coefficient of the redox mediator) the accuracy of the method is further improved.
• Each ofelectrodes5,6 has a predefined area. In the embodiments of FIGS.7 to10cell zone4 is defined byedges9,10,11 of the membrane which correspond with edges ofelectrodes5,6 and by leading (with respect to target area7) edges12,13 of the electrodes. In the present example the electrodes are about 600 angstrom thick and are from 1 to 5 mm wide.
• Optionally, both sides of the membrane are covered with the exception of thetarget area7 by laminating layers14 (omitted from plan views) which serves to prevent evaporation of water from the sample and to provide mechanical robustness to the apparatus. Evaporation of water is undesirable as it concentrates the sample, allows the electrodes to dry out, and allows the solution to cool, affecting the diffusion coefficient and slowing the enzyme kinetics, although diffusion coefficient can be estimated as above.
• A second embodiment according to the invention, shown inFIGS. 11 and 12, differs from the first embodiment by inclusion of a second workingelectrode25 and counter/reference electrode26 defining asecond cell zone24 therebetween. These electrodes are also spaced apart by less than 500 μm in the present example.Second electrodes25,26 are situatedintermediate cell zone4 andtarget area7. In this embodiment the redox mediator is contained in the membrane below or adjacent to targetarea7 orintermediate target area7 andfirst cell zone4. The enzyme is contained in the membrane in thefirst cell zone4 andsecond cell zone24. The enzyme does not extend intosecond cell24. In this case when blood is added to the target area, it dissolves the redox mediator. This wicks along the membrane so that secondelectrochemical cell24 contains redox mediator analyte and serum including electrochemically interfering substances. First electrochemical cell receives mediator, analyte, serum containing electrochemically interfering substances, and enzyme.
• Potential is now applied between both working electrodes and the counter electrode or electrodes but the change in current with time is measured separately for each pair. This allows the determination of the concentration of reduced mediator in the absence of analyte plus the concentration of electrochemically interfering substances in the second electrochemical cell and the concentration of these plus analyte in the first electrochemical cell. Subtraction of the one value from the other gives the absolute concentration of analyte.
• The same benefit is achieved by a different geometry in the embodiment ofFIGS. 13 and 14 in which the second working electrode and second counter/reference electrode define thesecond cell24 on the side oftarget area7 remote from firstelectrochemical cell4. In this case the enzyme may be contained in the membrane strip between the target area andcell1. The redox mediator may be in the vicinity of the target area or between the target area and each cell. The diffusion coefficient of mediator is lowered by undissolved enzyme and the arrangement ofFIGS. 13 and 14 has the advantage of keeping enzyme out of the thin layer cells and allowing a faster test (as the steady state current is reached more quickly). Furthermore the diffusion constant of redox mediator is then the same in both thin layer cells allowing more accurate subtraction of interference.
• Although the embodiments of FIGS.7 to14 are unitary sensors, it will be understood that a plurality of sensors may be formed on a single membrane as shown in the embodiment of FIGS.15 to17. In this case the electrodes of one sensor are conductively connected to those of an adjacent sensor. Sensors may be used successively and severed from the strip after use.
• In the embodiment of FIGS.15 to17 electrode dimensions are defined in the diffusion direction (indicated by arrow) by the width of the electrode in that direction.
• The effective dimension of the electrode in a direction transverse to diffusion direction is defined betweencompressed volumes16 of the membrane in a manner more fully described in co-pending Application PCT/AU96/00210. For clarity optionallaminated layer14 ofFIG. 7 has been omitted from FIGS.15 to17.
• In the embodiment ofFIG. 18 there is shown a hollow cell according to the invention wherein theelectrodes5,6 are supported by spaced apartpolymer walls30 to define a hollow cell. Anopening31 is provided on one side of the cell whereby a sample can be admitted intocavity32. In this embodiment a membrane is not used. As in previous embodiments, the electrodes are spaced apart by less than 500 μm, preferably 20-400 μm and more preferably 20-200 μm. Desirably the effective cell volume is 1.5 microlitres or less.
• It will be understood that the method of the invention may be performed with a cell constructed in accord with co-pending application PCT/AU95/00207 or cells of other known design, provided these are modified to provide a sufficiently small distance between electrode faces.
• The method of the invention will now be further exemplified with reference toFIGS. 19 and 20.
• A membrane 130 microns thick was coated on both sides with a layer ofPlatinum 60 nanometers thick. An area of 12.6 sq. mm was defined by compressing the membrane. 1.5 microlitres of a solution containing 0.2 Molar potassium ferricyanide and 1% by weight glucose oxidase dehydrogenase was added to the defined area of the membrane and the water allowed to evaporate.
• The platinum layers were then connected to a potentiostat to be used as the working and counter/reference electrodes. 3 microlitres of an aqueous solution containing 5 millimolar D-glucose and 0.9 wt % NaCl was dropped on to the defined area of the membrane. After an elapse of 20 seconds a voltage of 300 millivolts was applied between the working and counter/reference electrodes and the current recorded for a further 30 seconds at intervals of 0.1 seconds.
• FIG. 19 is a graph of current versus time based on the above measurements.
• Using a value of the steady state current of 26.9 microamps the function ln(il26.9−1) was computed and plotted versus time. The slope of the graph (FIG. 20) is −0.342 which corresponds to a diffusion coefficient of 1.5×10⁻⁶cm²per second and a corrected glucose concentration (subtracting background ferrocyanide) of 5.0 millimolar.
• The steady state current is one in which no further significant current change occurs during the test. As will be understood by those skilled in the art, a minimum current may be reached after which there may be a drift due to factors such as lateral diffusion, evaporation, interfering electrochemical reactions or the like. However, in practice it is not difficult to estimate the “steady state” current (i). One method for doing so involves approximating an initial value for i. Using the fit of the i versus t data to the theoretical curve a better estimate of i is then obtained. This is repeatedly reiteratively until the measured value and approximated value converge to within an acceptable difference, thus yielding an estimated i.
• In practice, the measurements of current i at time t are made between a minimum time t min and a maximum time t max after the potential is applied. The minimum and maximum time are determined by the applicability of the equations and can readily be determined by experiment of a routine nature. If desired the test may be repeated by switching off the voltage and allowing the concentration profiles of the redox species to return towards their initial states.
• It is to be understood that the analysis of the current v. time curve to obtain values of the Diffusion Coefficient and/or concentration is not limited to the method given above but could also be achieved by other methods.
• For instance, the early part of the current v. time curve could be analysed by the Cottrell equation to obtain a value of D^1/2×Co (Co=Concentration of analyte) and the steady state current analysed to obtain a value of D×Co. These two values can then be compared to obtain D and C separately.
• It will be understood that in practice of the invention an electrical signal is issued by the apparatus which is indicative of change in current with time. The signal may be an analogue or digital signal or may be a series of signals issued at predetermined time intervals. These signals may be processed by means of a microprocessor or other conventional circuit to perform the required calculations in accordance with stored algorithms to yield an output signal indicative of the diffusion coefficient, analyte concentration, haemocrit concentration or the like respectively. One or more such output signals may be displayed by means of an analogue or digital display.
• It is also possible by suitable cell design to operate the cell as a depletion cell measuring the current required to deplete the mediator. For example in the embodiment ofFIG. 5 the method of the invention may be performed usingelectrodes5,6, which are spaced apart by less than 500 μm. An amperometric or voltametric depletion measurement may be made usingelectrodes5 and26 which are spaced apart more than 500 μm and such that there is no interference between the redox species being amperometrically determined atelectrodes5,26.
• The depletion measurement may be made prior to, during or subsequent to, the measurement of diffusion coefficient by the method of the invention. This enables a substantial improvement in accuracy and reproducibility to be obtained.
• In the embodiments described the membrane is preferably an asymmetric porous membrane of the kind described in U.S. Patent Nos. 4,629,563 and 4,774,039. However symmetrical porous membranes may be employed. The membrane may be in the form of a sheet, tube, hollow fibre or other suitable form.
• If the membrane is asymmetric the target area is preferably on the more open side of the asymmetric membrane. The uncompressed membrane desirably has a thickness of from 20 to 500 μm. The minimum thickness is selected having regard to speed, sensitivity, accuracy and cost. If desired a gel may be employed to separate haematocrit from GOD. The gel may be present between the electrodes and/or in the space between the sample application area and the electrodes.
• The working electrode is of any suitable metal for example gold, silver, platinum, palladium, iridium, lead, a suitable alloy. The working electrode may be preformed or formed in situ by any suitable method for example sputtering, evaporation under partial vacuum, by electrodeless plating, electroplating, or the like. Suitable non-metal conductors may also be used for electrode construction. For example, conducting polymers such as poly(pyrrole), poly(aniline), porphyrin “wires”, poly(isoprene) and poly (cis-butadiene) doped with iodine and “ladder polymers”. Other non-metal electrodes may be graphite or carbon mixed with a binder, or a carbon filled plastic.
• Inorganic electrodes such as In₂0₃or SnO₂may also be used. The counter/reference electrode may for example be of similar construction to the working electrode. Nickel hydroxide or a silver halide may also be used to form the counter/reference electrode.
• Silver chloride may be employed but it will be understood that chloridisation may not be necessary and silver may be used if sufficient chloride ions are present in the blood sample. Although in the embodiments described the working electrode is shown on the upper surface of the biosensor and the counter/reference electrode is on the lower surface, these may be reversed.
• It is preferable that the working electrode and counter (or counter/reference) electrodes are of substantially the same effective geometric area.
• If a separate reference and counter electrode are employed, they may be of similar construction. The reference electrode can be in any suitable location.
• It will be understood that the features of one embodiment herein described may be combined with those of another. The invention is not limited to use with any particular combination of enzyme and mediator and combinations such as are described in EP0351892 or elsewhere may be employed. The system may be used to determine analytes other than glucose (for example, cholesterol) by suitable adaptation of reagents and by appropriate membrane selection. The system may also be adapted for use with media other than blood. For example the method may be employed to determine the concentration of contaminants such as chlorine, iron, lead, cadmium, copper, etc., in water.
• Although the cells herein described have generally planar and parallel electrodes it will be understood that other configurations may be employed, for example one electrode could be a rod or needle and the other a concentric sleeve.
• Display/Storage of Measurement Data
• In a preferred embodiment, an analysis strip as described above or another embodiment of thesampling device10 is integrated with a measuring device, e.g., a meter, which can display, store or record test data, optionally in computer-readable format. In such an embodiment, thetest strip10 comprises an interface for communicating with the meter, e.g., conductive leads from the electrodes of theelectrochemical cell20. In the case of obtaining an electrochemical measurement, the interface communicates a voltage or a current to theelectrochemical cell20.
• The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention as embodied in the attached claims.

Claims (19)

1. A fluid sampling device, the device comprising a test strip, wherein the test strip comprises:

(a) an upper surface;

(b) a lower surface;

(c) an analysis chamber defined between the upper surface and the lower surface, wherein the analysis chamber comprises a proximal end and a distal end, a first analysis chamber wall, a second analysis chamber wall, a volume, a working electrode and a counter and/or reference electrode;

(d) an opening for admitting a sample comprising an analyte to the analysis chamber;

(e) an opening to the atmosphere on the distal end of the analysis chamber; and

(f) an interface for communication with a meter, wherein the interface comprises conductive leads from the electrodes for obtaining an electrochemical measurement.

2. The device ofclaim 1, wherein an electrode of the analysis chamber is disposed on the upper surface.

3. The device ofclaim 2, wherein the electrode is the working electrode.

4. The device ofclaim 1, wherein an electrode of the analysis chamber is disposed on the lower surface.

5. The device ofclaim 4, wherein the electrode is the counter and/or reference electrode.

6. The device ofclaim 1, wherein the upper and lower surfaces are covered by laminating layers.

7. The device ofclaim 1, wherein the electrodes are comprised of a metal selected from a group consisting of palladium, gold, platinum, iridium and silver.

8. The device ofclaim 1, wherein a flow of sample to the analysis chamber is driven by a driving force, wherein the driving force comprises a capillary force.

9. The device ofclaim 1, wherein the analysis chamber comprises a substance capable of enhancing or diminishing a capillary force.

10. The device ofclaim 9, wherein the substance is selected from a group consisting of a polymer, a resin, a powder, a mesh, a fibrous material, a crystalline material, a porous material, and a combination thereof.

11. The device ofclaim 10, wherein the substance is selected form the group consisting of polyethylene glycol, polyvinyl pyrrolidone, a surfactant, a hydrophilic block copolymer, and polyvinylacetate.

12. The device ofclaim 1, wherein the analysis chamber is integrated with a means for facilitating filing of the analysis chamber.

13. The device ofclaim 12, the means is a penetration probe.

14. The device ofclaim 1, wherein the analysis chamber comprises a porous membrane.

15. The device ofclaim 1, wherein the volume of the analysis chamber is less than 1.5 μl.

16. The device ofclaim 1, wherein the analysis chamber contains one or more reagents capable of reacting with the analyte or a substance in the sample.

17. The device ofclaim 1, wherein the sample is selected from the group consisting of interstitial fluid and whole blood.

18. The device ofclaim 1, wherein the analyte is selected from the group consisting of an ion, an element, a sugar, an alcohol, a hormone, a protein, an enzyme, a cofactor, a nucleic acid sequence, a lipid, a pharmaceutical, and a drug.

19. The device ofclaim 1, wherein the analyte is selected from the group consisting of a potassium ion, ethanol, cholesterol, glucose, and lactate.

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