Patent Publication Number: US-6986869-B2

Title: Test strip for measuring analyte concentration over a broad range of sample volume

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional of U.S. patent application Ser. No. 08/442,035, filed May 16, 1995, now U.S. Pat. No. 6,395,220, which is a continuation-in-part of U.S. patent application Ser. No. 07/960,579, filed Oct. 13, 1992, now U.S. Pat. No. 5,418,142, which is a continuation of U.S. patent application Ser. No. 07/691,192, filed Apr. 25, 1991, abandoned, which is a continuation of U.S. patent application Ser. No. 07/399,055, filed Aug. 28, 1989, abandoned, and is a continuation-in-part of U.S. application Ser. No. 07/736,537, filed Jul. 26, 1991 now U.S. Pat. No. 5,306,623. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a test strip which allows a user to determine the concentration of an analyte in a liquid test sample. The test strip includes an absorbent membrane and a reagent that undergoes a change in color when exposed to the analyte, such as glucose or cholesterol, in a body fluid. 
     2. Description of Related Art 
     Test strips are commonly used for determining the concentration of analytes in liquid test samples. For example, known test strips can detect glucose, cholesterol, proteins, ketones, uric acid, phenylalanine, or enzymes in body fluids, such as blood or urine. The operation of such test strips has become so simple and reliable that the test strips can be used by patients to determine their own analyte concentrations. 
     Many test strips require a user to apply a drop of a body fluid to a reagent pad, or to a transport medium which conducts the fluid to the reagent pad, where an oxidizable dye or other indicator changes color to signal the presence of the analyte. It is sometimes difficult to bring exactly the right amount of body fluid to the test strip. If too little fluid is delivered, the test strip may not function properly. If too much fluid is applied to the test strip, excess fluid may drip from the test strip. Unnecessary contact between the user and the body fluid, which is typically blood, urine, or saliva, is preferably avoided. 
     SUMMARY OF THE INVENTION 
     The invention is directed to a color-indicating test strip which operates with a relatively small volume of body fluid but is capable of absorbing and retaining several times the required volume of body fluid, thereby minimizing any tendency for the body fluid to drip from the test strip. The test strip includes a membrane containing a color-changing reagent in contact with a porous sheet that has a pillow portion adapted to absorb excess body fluid from the membrane. The membrane extends across and protrudes into a window in the test strip. The window serves both to make visible any color change and to allow oxygen access to the membrane. The pillow portion is located in a protective channel defined by a relatively rigid container. The test strip is small, inexpensive, and suitable for mass production by heat-sealable packaging methods. 
     In one aspect, the invention is a test strip that comprises:
         a cover sheet, having an elongated window cut through it;   a lamellar membrane adjacent to the cover sheet, extending across and into the window and containing a reagent that reacts with the analyte to produce a color change;   a porous sheet in fluid communication with the membrane, having a pillow portion and a compressed portion, the pillow portion being substantially aligned with the window; and   a backing sheet, adjoining the porous sheet and having a sample port cut through it:   whereby fluid introduced into the sample port can flow to the membrane, and analyte in the fluid can react with the reagent to produce a color change visible through the window.       

     In another aspect, the invention is a test strip comprising a backing sheet. An opening through the backing sheet leads to an absorbent sheet of a relatively compressible nonwoven material, so that a liquid that passes through the opening is absorbed in the absorbent sheet. The absorbent sheet is sandwiched between the backing sheet and a relatively incompressible planar center plate. Because a central part of the center plate has been removed, as by punching, the center plate extends around an internal relief chamber. One of the faces of the center plate is fastened to the absorbent sheet. The other face of the center plate is attached to one side of a generally planar permeable membrane that has been treated with a reagent capable of changing color on contact with an analyte in a liquid solution. The side of the membrane is held by the face of the center plate, so that a portion of the membrane extends across and protrudes into the internal relief chamber of the center plate. A cover sheet defines a window that is generally parallel to and has a cross section which is approximately the same size as the internal relief chamber. The cover sheet is bound against the other side of the membrane with the portion of the membrane that is visible through the window bulging into the window. 
     The invention also provides a method of making a test strip. The cover sheet with the heat-sealable coating, the membrane, the porous layer, the adhesive, and the backing sheet are stacked as a heat-sealable package on a heatable shaping die. The die is generally flat, but has a groove adapted to accommodate the uncompressed porous layer in a region that is aligned with the window. The die is heated and forced against the package to compress portions of the membrane and porous layer and to bond the cover sheet and backing sheets in place. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is an elevation view of a test strip of the present invention. 
         FIG. 2  is a cross-sectional view taken along the plane  2 — 2  of the test strip of  FIG. 1  before compression by a heated shaping die. 
         FIG. 3  is the cross-sectional view of  FIG. 2  after compression. 
         FIG. 4  is the cross-sectional view of a test strip of the present invention that includes an incompressible center plate between an absorbent sheet and a membrane before compression. 
         FIG. 5  is the cross-sectional view of  FIG. 4  after compression. 
         FIG. 6  is an elevation view of a shaping die that can be used in the method of the present invention. 
         FIG. 7  is a cross-sectional view taken along the plane  7 — 7  of the die of FIG.  6 . 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     A test strip in accordance with the present invention is illustrated in FIG.  1 .  FIGS. 2 and 3  depict a cross section through the strip of  FIG. 1  before and after the strip is compressed, respectively. The test strip  10  comprises a porous membrane  20  having a sample side  22  and a testing side  23 , as can be seen in  FIGS. 2 and 3 . A testing reagent is held within the pores of the membrane. A portion of the membrane is adapted to absorb a body fluid, such as blood, which comes in contact, with the sample side  22 . The membrane transports the body fluid by capillary action from the sample side  22  toward the testing side  23 , where the testing reagent reacts with the analyte to produce a color change. The change in color depends on the concentration of analyte in the sample and enables one to determine the concentration. 
     Depending on the composition of the testing reagent, the strip can be used to measure analytes such as glucose, cholesterol, proteins, ketones, uric acid, phenylalanine, or enzymes in body fluids such as blood or urine. In a preferred embodiment, the testing reagent includes a component, such as glucose oxidase, for converting glucose (present in a sample of blood) to hydrogen peroxide and components for detecting the hydrogen peroxide produced. The components for detecting hydrogen peroxide may be an oxidizable dye and a peroxidase, such as horseradish peroxidase. 
     For use with blood samples, it is preferred that the membrane  20  be an anisotropic porous membrane having pores with effective diameters that decrease in size as the distance from the sample side  22  of the membrane increases. As blood is transported through the membrane by capillary action, red blood cells are removed by progressively smaller pores which the blood encounters as it travels. Separation of red blood cells from the blood sample produces a relatively clear fluid through which the change in color near the testing side  23  may be observed. 
     The thickness of the membrane  20  is usually in the range of about 50 to about 500 micrometers, with a thickness in the range of about 100 micrometers to about 200 micrometers being preferred. For use with blood samples, the membrane  30  preferably has pores with effective diameters in the range of about 5 to about 50 micrometers on the sample side and about 0.1 to about 1.0 micrometers toward the testing side  23 . The membrane  10  may be composed of porous polyamides, polysulfones, polyesters, polyolefins, or cellulosics. Polysulfone is the preferred material for the membrane. The testing reagent may be held within the pores of the membrane covalently or non-covalently. When the binding of the testing reagent is intended to be non-covalent, the testing reagent may be, for example, impregnated in the membrane by applying a solution containing the testing reagent to the membrane and subsequently evaporating the solvent. 
     A porous sheet  30  is in fluid communication with the membrane  20  and is adapted to accept the body fluid sample and transport a detectable portion of the sample to the sample side  22  of the membrane. The sample is absorbed into pores of the porous sheet  30  and passed through the sheet by capillary action. The sheet  30  may be composed of natural fibers, such as cotton or cellulose, as well as polyester, polyamide, polyethylene, polyvinyl alcohol, polyester nylon blends, or other synthetic polymers. Polyester is the preferred material for the porous sheet, with polyester-nylon blends also being favored. The porous sheet  30  is preferably nonwoven, having fibers that are thermally bonded to produce pores having an effective diameter in the range of about 20 to about 200 micrometers, preferably about 50 to 100 micrometers. Its structure provides a venting path for air in the strip that is displaced by the sample, thereby facilitating the passage of sample to the membrane. The sheet is generally intrinsically hydrophilic or rendered hydrophilic by treatment with a surfactant, such as polypropylene glycol. 
     Preferably, the porous sheet  30  is capable of absorbing and retaining in the range of about 2 to about 10, and more preferably, in the range of about 4 to about 6, times the volume of blood that is required to reliably produce a detectable change in color on the testing side of the membrane. Typically, a minimum of about 3 to about 10 microliters of blood is required for determination of the analyte concentration. Therefore, it is preferred that the porous sheet be capable of absorbing from about 6 to about 100 microliters, ideally about 30 microliters. 
     Referring now to  FIG. 3 , the porous sheet  30  includes a pillow portion  32  which retains the major part of body fluids absorbed by the sheet  30 , and a relatively compressed portion  34  intended primarily as a sealing and buffer zone extending laterally in opposite directions from the pillow portion. 
     The strip structure is sandwiched between a cover sheet  41  and a backing sheet  44 , which may be of any suitable plastic, but are preferably polyethylene terephthalate. The cover sheet  41  faces the testing side  23  of the membrane and the backing sheet  44  is adjacent to the porous sheet  30 . The edges, and optionally the ends, of the strip are sealed by the relatively compressed sealing region  24  of the membrane  20  and the relatively compressed portion  34  of the porous sheet  30  which are sandwiched between the cover sheet  41  and the backing sheet  44 . The cover sheet  41  is attached to the membrane  20  by a sealable coating  50  and the backing sheet  44  is fixed to the porous sheet  30  by an adhesive  60 . A relatively thin layer of non-woven polyethylene-coated polyester  80  may optionally be positioned between the membrane  20  and the porous sheet  30  to further improve the seal. 
     An elongated window  48  is cut into the cover sheet, and is in general alignment with the pillow portion  32 , exposing a portion of the absorbent region  21  of the membrane. The window permits the testing side  23  of the membrane to be visible and also provides oxygen access to the color-forming reaction in membrane  20 . Preferably, the cover sheet  41  is formed of a printable material and colors for comparison or markings dividing the exposed membrane into identifiable zones  70  (shown in  FIG. 1 ) are printed on the cover sheet. The membrane  20  extends across the window  48  and the absorbent region  21  of the membrane may extend into the window, thereby increasing visibility of a color change which may take place near the testing side  23 . The pillow portion  32  provides a cushion and support for membrane  20 , which is otherwise unsupported in the region where it spans window  48 . 
     Preferably, the membrane also contains an inhibitor which retards the change in color of the testing reagents until a detectable threshold level of glucose is present at the testing side  23 . Further, the testing side is divided into zones  70 , each having a known concentration of inhibitor and, therefore, a unique but predictable threshold level of glucose. The zones  70  are arranged sequentially along the length of the window  48 . Those of the zones  70  that undergo an observable change in color appear to form a continuous line terminating at a definite point along the length of the window  48 . Markings printed on the cover sheet allow a user to quickly determine which of the zones  70  have undergone the change in color and determine, within incremental ranges, the concentration of glucose present. 
     A sample port  45  provides flow communication from the outside of the strip to the porous sheet  30  and to the membrane  20 . If the sample port  45  is located in the cover sheet  41 , the sample port is in alignment with or adjacent to the absorbent region  21  of the membrane. A body fluid sample that is delivered to the sample port  45  is absorbed by the membrane  20  and any excess fluid is absorbed by the porous sheet  30 , which is in fluid communication with the membrane. Alternatively, if the sample port is located in the backing sheet  44 , the sample delivered to the sample port passes directly into the porous sheet and an amount of sample needed for analyte concentration determination passes to the membrane by capillary action. Locating the sample port  45  in the backing cover  44  is preferred and is especially preferred in small test strips that require less than a single drop of blood for their operation. In any case, when the membrane is anisotropic, the larger membrane pores are nearer the port. When the port is in the backing cover the larger pores may be near one surface of the membrane (the sample side), the smaller pores near the other surface (the testing side). The advantage of an anisotropic membrane is that the highly-colored red blood cells are captured near the sample side and the serum passes through to the testing side. As a result, there is less interference with the observation (from the testing side) of the color change reaction that measures the analyte concentration. Sheet  30  may be treated with polypropylene glycol or similar surfactants to provide some preliminary blood separation even before the sample reaches membrane  20 , which further reduces red cell interference. 
     If coating  50  is a pressure-sensitive adhesive, heat is not required to seal; the coating is preferably a heat-sealable emulsion-based material. Polyisobutylene rubber and ethylene vinyl acetate are suitable, with ethylene vinyl acetate being preferred. The coating  50  is conveniently applied around or along the face  42  of the cover sheet  41 , which is positioned toward the membrane  20 . An opposite face  43  is positioned away from the membrane  20 . 
     Alternatively, the adhesive coating  50  may be a low-density polyethylene film that is co-extruded with the polyethylene terephthalate cover sheet on the face  42  of the cover sheet that is positioned toward the membrane. The coating is in the range from about 5 to about 13 micrometers thick if ethylene vinyl acetate is used, and in the range from about 37 to about 87 micrometers thick if co-extruded low-density polyethylene is used. Similarly, the adhesive  60  may be an emulsion-based material such as ethylene vinyl acetate, acrylic, or polyisobutylene. Alternatively, coating  60  may be a pressure-sensitive adhesive; however, since coatings  50  and  60  are preferably heat-sealed, in the description that follows heat-sealable coatings are emphasized. 
     Another preferred embodiment in accordance with the present invention is shown in cross section in  FIGS. 4 and 5 . Each of the numbered elements of  FIGS. 4 and 5  that corresponds to a numbered element depicted in  FIGS. 1 through 3  is designated by an element number that is one hundred units higher than that of the corresponding element. For example, a membrane  120  in  FIG. 4  corresponds to membrane  20 . Similarly, an opening  145  corresponds to sample port  45 . Elements that have no corresponding element in  FIGS. 1 through 3 , such as an internal relief chamber  199  illustrated in  FIG. 5 , are designated by element numbers whose last two digits are freely assigned. Of course, some corresponding elements are not numbered in any of the figures. 
     Referring now to a test strip  100  illustrated in  FIG. 4 , a sample port  145  passes through backing sheet  144  for admitting blood or other body fluids. Typically, the backing sheet is rectangular or oblong and is white or clear polyethylene terephthalate, with the opening  145  formed by punching. The backing sheet  144  should be relatively rigid to provide support for the test strip  100 . Backing sheet  144  preferably has a thickness in the range from about 25 to about 100 micrometers, with about 50 micrometers preferred. One of the faces of the backing sheet is coated with an adhesive  160 , preferably a heat-sealable adhesive as described above, for adhering the sheet to porous absorbent sheet  130 . 
     Alternatively, the backing sheet  144  may be a thermoplastic material, such as a polyvinyl resin, a polystyrene resin, an acrylic resin, a copolymer of ethylene and vinyl acetate, or a mixture of paraffin wax and polyolefins. In that case, no adhesive coating is required to fasten the backing sheet to adjacent porous sheet  130 . A combination of heat and pressure serves to partially fuse the thermoplastic material, permitting it to penetrate the porous member. On cooling, a portion of the thermoplastic material solidifies within the porous member, affixing and, optionally, sealing the backing strip  144  to the porous member. 
     Porous absorbent sheet  130  is adhered to backing sheet  144  and extends across the opening  145 . Any liquid that passes through the opening  145  contacts the absorbent sheet  130  and is wicked up by capillary action. Preferably, sheet  130  is a polyester non-woven material. Material having a weight in the range from about 10 to about 30 grams per square meter has proven satisfactory, with material of about 20 grams per square meter being preferred. The absorbent sheet  130  may be treated with surfactants, such as polypropylene glycol or polyethylene glycol. Preferably, the surfactants are present in the range of about 0.01 to about 5.0 weight percent, based on the total weight of the absorbent sheet  130 . Sheet  130  usually has about the same size and shape as the backing sheet  144 . 
     A plastic center plate  190  defines and substantially encircles an internal relief chamber  199 . Preferably, the chamber  199  is formed by removing a part of the center plate  190 , preferably by punching it out. Adhesive layers,  185  and  195 , preferably heat-sealable adhesives, are spread upon the faces of the center plate, either before or after part of the center plate  190  is removed. The adhesive layers are not required when the center plate  190  is a thermoplastic material capable of heat-sealing directly to a porous surface. However, it is currently preferred that the adhesive layers  185  and  195  be employed. 
     Preferably, the center plate  190  is a polyethylene terephthalate sheet having a thickness in the range of about 50 to about 125 micrometers. A thickness of about 75 micrometers is preferred. Typically, the internal relief chamber  199  is rectangular or oblong, having a length of about 2.5 cm and a width of about 0.1 cm. It is important that the center plate  190  be of a material that is sufficiently incompressible to maintain the dimensions of the internal relief volume substantially unchanged, despite being subjected to conditions effective to compress the absorbent sheet  130  and the membrane  120 , and to set the heat-sealable adhesives. Typically, the components are laminated using about 480 to about 860 kPa of pressure at a temperature in the range of about 65° C. to about 110° C. for a period of about 0.5 to about 15 seconds. 
     The adhesive layer  185  attaches one face of the center plate  190  to the absorbent sheet  130 . Adhesive layer  195  attaches the center plate  190  to a relatively broad side of the membrane  120 . The membrane  120  is sufficiently permeable to permit liquids, such as body fluids, to pass through relatively freely but solids contained in the liquids such as red blood cells, may be retained at an external surface of the membrane or at some point within the membrane. The membrane  120  may be anisotropic and is, preferably, a polysulfone that has a thickness in the range of about 100 to about 150 micrometers, preferably about 125 micrometers. The membrane  120  is impregnated with a color-change reagent capable of reacting with an analyte in solution. Very preferably, the reagent contains a component for converting glucose to hydrogen peroxide and an inhibitor, as described above. When the reagent changes color, the external appearance of the membrane  120  is altered. 
       FIG. 5  shows a cross-sectional view of the elements depicted in  FIG. 4  after being compressed and heat-sealed by a heated shaping die. The membrane  120  includes a relatively dense portion  124  which is located adjacent to the center plate  190 . The dense portion  124  is formed by the compression and heat-sealing process, and is relatively less permeable to liquids. The membrane  120  also includes an uncompressed portion  121  that is disposed opposite the internal relief chamber  199 . During the compression that takes place in the sealing process, portion  124  of the membrane  120  becomes more dense and portion  121  enters into the internal relief chamber  199 . The uncompressed portion  121  is relatively permeable to liquids, such as body fluids. 
     A cover sheet  141 , preferably of white or clear translucent plastic is adhered to the side of the membrane  120  that is furthest from the center plate  190 . Alternatively, the cover sheet  141  may be a thermoplastic material and attached directly to the membrane  120  by partial fusion and subsequent solidification within the membrane  120 . The cover sheet  141  defines a window  148 , which has a cross section about the same size and shape as that of the internal relief chamber  199 . During compression of the strip, forces that urge the cover sheet  141  in the direction of the center plate  190  tend to cause the uncompressed portion  121  of the membrane  120  to bulge into the internal relief chamber  199  and into the window  148 . 
     Similarly, forces which move the backing plate  144  toward the center plate  190  tend to compress a region  134  of the absorbent sheet  130  which is located between the backing sheet  144  and the center plate  190 . In contrast, region  132  of the absorbent sheet  130 , which is generally adjacent to the internal relief chamber  199 , remains substantially uncompressed and continues to exhibit a relatively high degree of absorbency. During compression, membrane  120  and/or absorbent sheet  130  may buckle, causing the two layers to come into physical contact. 
       FIG. 6  depicts the face of a die  200  that is used to compress the strip of  FIG. 2  (or  FIG. 4 ) to the configuration shown in  FIG. 3  (or FIG.  5 ).  FIG. 7  is a cross-sectional view taken along the plane  7 — 7  on FIG.  6 . The compression step is accomplished by sandwiching strip material between the die of  FIG. 6 and a  flat plate and applying pressure. Preferably, the die is also heated. The die shown forms four strips; however, it is clear that more or fewer strips may be formed at one time by suitably modifying the die. The method for compressing the strip material is described in greater detail as follows. Although described with reference to  FIGS. 2 and 3 , the method is similarly suited for making the embodiment of the test strip depicted in  FIGS. 4 and 5 . The method comprises assembling the cover sheet  41 , sealable coating  50 , membrane  20 , polyethylene-coated polyester layer  80 , porous sheet  30 , adhesive  60 , and backing sheet  44  in their proper orientation (described above) as a sealable package on a shaping die of  FIG. 6  that is preferably heated.  FIG. 2  provides a cross section of the configuration before heat or pressure is applied. As shown in  FIG. 6 , the die has a substantially flat surface which has at least one elongated groove  248  ( 4  grooves in  FIG. 6 ) having a length about equal to or greater than the length of the window  48  (FIG.  1 ). The sealable package is positioned on the die with the groove and window in alignment, so that pressing the package against the die will shape the porous sheet  30  into a pillow portion  32  adjacent the membrane  20  and urge the membrane  20  into the window  48 . 
     The sealable package is pressed between the die and a flat plate. Portions of the membrane  20  and of porous sheet  30 , located on opposite sides of the groove adjacent to flat surfaces of the die, are compressed and become relatively impermeable to body fluids, producing the relatively compressed regions  24  and  34 . Preferably, in addition heat from the die causes the (heat-sealable) coating  50  to bond to the cover sheet  41  and to the membrane  20 . The heat also causes the adhesive  60  to bond to the porous layer  30  and the backing sheet  44 . A pressure in the range of about 340 to about 1400 kPa. preferably about 480 to about 860 kPa, is applied to the materials by the die while the die is preferably heated to a temperature in the range of about 65° C. to about 110° C. 
     Descriptions of the invention and examples of its use have been set forth to communicate the invention fully, not to limit the scope of the invention in any way. It should now be apparent that other forms and embodiments of the invention are possible. The scope of the invention is intended to be as broad as the claims will allow.