US20110097755A1

US20110097755A1 - Apparatus and Method for Optically Sensing Analyte Concentration in an Erythrocyte-Containing Fluid

Info

Publication number: US20110097755A1
Application number: US12/992,337
Authority: US
Inventors: Hiroshi Nomura
Original assignee: Light Pointe Medical Inc
Current assignee: Light Pointe Medical Inc
Priority date: 2008-02-29
Filing date: 2009-05-13
Publication date: 2011-04-28
Also published as: WO2009139889A3; US8008068B2; WO2009139889A4; WO2009139889A2; US20090219510A1; EP2281186A2; EP2281186A4

Abstract

A device for determination of analytes has optical material with a deposit on a surface thereof. The deposit includes an analyte-reactive reagent and a particulate material for enhancing reflectance of a light beam emitted through the optical material into the analyte-reactive reagent. The enhanced reflectance provides a return of light through the optical material at a level which is not only analytically useful, but involves sufficient returned light intensity so as to enable markedly improved sensitivity and accuracy in analytical determinations. A suitable particulate material is one of hollow spherical particles. Moreover, the particulate material may be selected for a property such as a neutralizing charge that suppresses the membrane-disruptive effects of certain types of optical materials and hemolytic reagents on blood cells such as erythrocytes.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and is a continuation-in-part of U.S. patent application Ser. No. 12/152,173 filed May 13, 2008, which claims priority to and is a continuation-in-part of U.S. patent application Ser. No. 12/072,940 filed Feb. 29, 2008, all of which hereby are incorporated herein in their entirety by reference thereto.

BACKGROUND OF THE INVENTION

1. Field of the Invention
The present invention relates to sensing analyte concentration in an erythrocyte-containing fluid, and more particularly to sensing analyte concentration in an erythrocyte-containing fluid using optical materials.
2. Description of Related Art
One method of analyzing a fluid for specific properties such as the presence and concentration of specific moieties is by analyzing changes in light directed through a surface-borne reagent to which a sample of the fluid is brought into contact. This is achieved by utilizing a reagent which produces an optically detectable physical or chemical change upon contact with one or more targeted analytes in the fluid. This method of analyzing fluids is especially effective when a colorimetric reaction is effected in the sample or on a surface in contact with the sample, wherein a color change relates to one or more specific analytes in the fluid. Such a means includes generally a specific source of light of a desired frequency tuned to the spectral range of the colorimetric reaction, and also a detector for such a light. One approach in common use is to impregnate a microporous sheet-like member with a color-producing reagent. Typically, the microporous sheet-like member is a membrane whose composition comprises a “nylon” or polyamide polymer. A fluid sample, such as of blood, is deposited on one surface of the membrane, resulting in a color change reaction with the resident reagent, and the color shade and intensity are read by means of a light beam reflected from the opposite face of the membrane. Another, increasingly contemplated, method of conducting these types of analyses is by use of optic fibers as waveguides for a light beam, both to introduce a light beam from a light source into the fluid sample and as a conduit for returning light from the fluid sample to a detector. Changes in the light beam's spectral pattern, or more often in the intensity of the beam at a specific frequency, can be correlated in some manner to the concentration of a specific analyte present in the fluid sample. Application of this concept in the field of glucose measurement in body fluids is found, for example, in U.S. Pat. No. 6,157,442 issued Dec. 5, 2000 (Raskas, Micro Optical Fiber Sensor Device), which is incorporated herein in its entirety by reference thereto. This approach customarily uses an analyte-reactive reagent or combination of reagents that undergoes a color change or develops one or more colored reaction products. Selective absorption of light at one or more frequencies associated with the spectral range of the colored reaction products occurs, resulting in measurable, (i.e., quantifiable) alterations in the light beam being accessed and evaluated by a suitable detection system. In some cases, such analyses may be based on light frequencies that occur outside the normal human range of visible light, such as ultraviolet and infrared, and may entail other than “colored” reaction products. The analyte-reactive reagent is advantageously bound or otherwise located on a surface of an optic fiber, in effect making the optic fiber into a sensor probe. For instance, an analyte-reactive reagent can be positioned on the distal tip of an optical fiber waveguide, the other end of the optic fiber being attached to an apparatus or assembly that includes a suitable detector. A suitable light source may be located extraneous to the optic fiber sensor, or brought to the fluid sample by means of the optic fiber sensor itself, or brought to the fluid sample through another optic fiber waveguide bundled with the optic fiber sensor.
Some difficulties are inherent in the use of optic fiber sensors for fluid analyses. For instance, the distal tip of an optic fiber probe presents very little surface area for attachment of an analyte-reactive reagent, resulting in a very minimal effect when assayed by means of changes in a light beam shone therethrough. Accuracy and reliability of analyses can be severely compromised by the lack of enough reagent to effect significant changes in a sensor's light beam. Another difficulty resides in designing a suitable arrangement whereby a light beam can be directed through a colored reaction product, captured by an optic fiber probe, and directed to a light detector in a reliable manner. When operating a single optic fiber probe as both the conduit for a light beam from a source into the fluid sample and the conduit for light returning from the fluid sample into the optic fiber and from thence to a suitable detection system, for example, the proportion of returned light from the fluid medium adjacent the distal tip of a fiber optic waveguide is normally very poor. When the tip is textured such as disclosed in U.S. Pat. No. 5,859,937 issued Jan. 12, 1999 (Nomura, Minimally Invasive Sensor), which hereby is incorporated herein in its entirety by reference thereto, the surface area for attachment of analyte-reactive reagents is greatly increased and light return is mildly enhanced by the texturing, such that a few percent of the light is recaptured by the optical fiber. Nevertheless, most of the light is lost by emanation from the surface area of the tip of even a textured tip optic fiber waveguide. The option of directing a beam of light down an optic fiber probe to a distal tip having a high surface area for disposition of a diagnostic reagent is still compromised by the loss of light signal and the minimal return of light from the distal tip through the same optic fiber to a detector.
In this regard, returned light may occur in two ways, by reflection and by reflectance. Reflected light is that portion of the light beam that, in traveling to the tip, encounters an interface between the fiber matrix and the environment (fluid sample, surface-deposited diagnostic chemistry, etc.), and which bounces back from the interface rather than crosses it. Cladding along the periphery of most optic fiber strands is designed to cause reflection of internal light beams, but no such cladding is present on a distal tip of a fiber optic probe, and a very high proportion of any light being beamed down the length of the optic fiber strand will customarily escape the confines of the optic fiber strand at that tip. Reflectance refers to that portion of light that enters into a sample medium such as powder or paper, and re-emerges in the general direction of the source, where the spectral nature of the light has likely been altered by contact with the sample medium. In the aforementioned Nomura U.S. Pat. No. 5,859,937, for example (which is incorporated herein in its entirety by reference thereto), the zone of analyte-reactive reagent in contact with the fluid sample represents the sample medium rather than paper or powder. The phenomenon of reflectance is treated at some length in the publication by E. L. Simmons entitled “Reflectance Spectroscopy: Applications of the Kubelka-Munk Theory to the Rates of Photoprocesses of Powders,” in Applied Optics, Vol. 15, No. 4, April 1976, pages 951-954 (reference made to the original publication by Kubelka and Munk in a German periodical in 1931), which is incorporated herein in its entirety by reference thereto. Such light will commonly be changed by absorption of some frequencies to varying degrees by the medium. Some patent publications loosely refer to reflectance as “reflected” light, and vice versa some patent publications refer to reflected light as “reflectance.”
In the analysis of analytes by a reagent combination disposed on the tip of an optic fiber probe, the light of interest for analytical determinations is mostly lost from the tip of the optic fiber. Reflectance light re-transmitted through the optic fiber from the tip is normally of such weak intensity as to greatly lower the sensitivity of the optic fiber probe in potential analytical applications. Collecting the light by means external to the optic fiber tip may overcome this difficulty, but necessarily results in more complexity of design and higher cost for analytical applications, as well as introducing possible artifacts.
The need exists for improved sensors wherein reflectance can be captured effectively by the fiber optic probe itself.
Similar difficulties are encountered even with sensors employing sheet-like members such as polyamides. When such sensors are wetted by aqueous fluid samples to be analyzed, the wetting in itself tends to make the microporous member significantly more transparent optically, and needing enhanced reflectance for optimum performance.
The need exists for improved sensors having microporous members, wherein reflectance can be captured effectively even when wetting in itself tends to make the microporous member significantly more transparent optically.
An additional problem encountered with various materials, and in particular these sheet-like members, is the fact that the microporous polyamide membranes used for sensor construction, particularly positively charged nylon membranes such as disclosed in U.S. Pat. No. 6,420,128 issued Jul. 16, 2002 (Ouyang et al., Test Strips for Detecting the Presence of a Reduced Cofactor in a Sample and Method for Using the Same), which is incorporated herein in its entirety by reference thereto, are themselves hemolytic toward erythrocytes in samples of whole blood. This releases hemoglobin, which migrates far more freely through the sample matrix than erythrocytes, and generates interferences in the subsequent colorimetric analyses. In the case of some color-producing reagents—and particularly those based on tetrazolium salts—the hemoglobin can actually react with the dye system to produce color bodies, just as would blood glucose. Since the primary use of such sensors is the determination of glucose concentrations in whole blood of diabetic patients, this drawback is of major concern. One approach has been to add a porous transport layer atop the membrane to filter out erythrocytes, such as the “double-layer” embodiment disclosed in U.S. Pat. No. 5,789,255 issued Aug. 4, 1998 (Yu, Blood Glucose Strip Having Reduced Sensitivity to Hematocrit), which is incorporated herein in its entirety by reference thereto. Another approach has been to modify the color change reagent with a soluble acrylic acid polymer, also disclosed in this same reference. Nitrite salts have also been employed in tetrazolium-salt-based diagnostic strips, wherein the nitrite binds to the released hemoglobin, thus suppressing the interfering dye formation caused non-enzymatically by the hemoglobin, as disclosed in U.S. Pat. No. 6,200,773 issued Mar. 13, 2001 (Ouyang et al., Diagnostics Based on Tetrazolium Compounds), which is incorporated herein in its entirety by reference thereto.
These difficulties and subsequent approaches illustrate an ongoing need for optical sensors wherein not only enhanced reflectance is valuable, but also wherein hemolysis of erythrocytes in body fluid samples does not occur or can be reduced to such a low background level as not to develop any significant interference in the enzymatic analysis of, say, blood glucose.

BRIEF SUMMARY OF THE INVENTION

One or more of these or other problems in the prior art are solved in various ways by various embodiments of the present invention.
One or more of the embodiments may provide a means for effectively directing an analytically useful beam of light through an optical conduit from a source coupled to the conduit, directing it to a sampling zone of a surface-borne analyte-reactive reagent, then capturing and directing an enhanced portion of reflectance from the sampling zone back through the same probe toward a suitable detector.
One or more of the embodiments may provide a means within a reagent combination incorporating an analyte-reactive reagent for enhancing reflectance of light from a sampling zone adjacent the surface of an optical guide back through the same optic guide.
One or more of the embodiments may provide a means for enhancing reflectance within a reagent combination incorporating an analyte-reactive reagent, wherein the reagent combination is disposed within a textured zone on a surface of an optical guide.
One or more of the embodiments may provide a means for effectively directing an analytically useful beam of light from a light source to a sampling zone of a surface-borne analyte-reactive reagent, then capturing and directing an enhanced portion of reflectance from the sampling zone back through the same sensor toward a suitable detector, while additionally providing on the sampling surface of the sensor a means to simultaneously suppress hemolysis of any erythrocytes in contact with the sensor materials.
One embodiment of the present invention is a sensor for determination of an analyte, comprising a body of optical material; an analyte-reactive reagent for the analyte, disposed on the optical material body in optical communication therewith; and a plurality of particulate bodies dispersed through the analyte-reactive reagent. The particulate bodies are adapted to contribute to reflectance of light from the optical material body back into the optical material body through at least a portion of the analyte-reactive reagent, when the analyte-reactive reagent is in reaction with the analyte.
Another embodiment of the present invention is a sensor for determination of at least one analyte in a fluid comprising an optic fiber having proximal and distal ends, and further having a textured surface on a distal end; a coating comprising at least one analyte-reactive reagent disposed on the textured surface, said reagent producing an optically detectable change upon reaction with at least one analyte in a fluid to be presented to the distal end; and reflectance enhancement means for enhancing reflectance into the fiber from a sampling zone associated with said coating.
Another embodiment of the present invention is a device for measurement of an analyte in a fluid comprising an optical material having a deposit on a surface thereof. The deposit comprises an analyte-reactive reagent and a plurality of hollow polymeric particles. A sampling zone is associated with the analyte-reactive reagent, a portion of the reagent being within the sampling zone and disposed between the surface of the optical material and the hollow polymeric particles, and the hollow polymeric particles providing for reflectance of a light beam emitted from the optical material into the sampling zone.
Another embodiment of the present invention is a device for measurement of an analyte in a fluid comprising an optical material having a deposit on a surface thereof. The deposit comprises an analyte-reactive reagent and a plurality of hollow polymeric particles. A sampling zone is associated with the analyte-reactive reagent, a portion of the reagent being within the sampling zone and disposed between the surface of the optical material and the hollow polymeric particles, and the hollow polymeric particles providing for reflectance of a light beam emitted from the optical material into the sampling zone.
Another embodiment of the present invention is a method of analyzing a fluid sample for an analyte comprising presenting a fluid sample containing the analyte to a site on an optical material, the site having a deposit comprising an analyte-reactive reagent and a particulate means for enhancing reflectance; developing an optically detectable change in a reagent sampling zone associated with the analyte-reactive reagent; emitting a beam of light through the optical material into the reagent sampling zone; returning reflectance from the reagent sampling zone through the optical material to a detector; and measuring the reflectance for spectral qualities correlative to the analyte.
Another embodiment of the present invention is a sensor for measuring concentration of an analyte in an erythrocyte-containing biological fluid comprising an optical material having a region of analyte-reactive reagent for contact with a biological fluid to be analyzed; and a deposit comprising a plurality of negatively-charged particles disposed in the region. The particles suppressing hemolysis of erythrocytes come into contact with the region.
Another embodiment of the present invention is a method of manufacturing a sensor for determining an analyte, comprising co-depositing an analyte-reactive reagent for the analyte and a plurality of particulate bodies into a sampling zone on an optical material body; and maintaining the analyte in optical communication with the optical material body.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a schematic diagram of a surface coating on an optical guide material illustrating a coating combination with enhanced reflectance capability.

FIG. 2 is a schematic diagram of another surface coating on an optical guide material illustrating a coating combination with enhanced reflectance capability.

FIG. 3 is a schematic diagram of a textured surface of an optical guide material illustrating a coating combination with enhanced reflectance capability.

FIG. 4 is a schematic diagram of one type of reagent dip-coated sheet-like microporous “nylon” member.

FIG. 5 is a graph of percent reflectance of 635 nm light as a function of time at various blood glucose concentrations with reflectance enhancement.

FIG. 6 is a graph of K/S calculated as a function of time for various blood glucose concentrations with reflectance enhancement.

FIG. 7 is a graph of percent reflectance as a function of blood glucose concentrations with and without enhanced reflectance capability for a textured optical fiber.

FIG. 8 is a graph of percent reflectance as a function of blood glucose concentrations with and without enhanced reflectance capability for a nylon microporous membrane having an asymmetrical structure.

FIG. 9 is a representation of a photograph of hemolysis per Example 4.

The figures are provided to be fully descriptive, even while providing for ease of explanation and understanding. Deviation from the figures with respect to number, position, order, relationship and dimensions are explained herein or are within the ordinary skill of the art after study of this patent document. Furthermore, the apparatus, materials and other operational parameters to conform to specific size, force, weight, strength, velocity, temperatures, flow, and similar requirements are likewise within the ordinary skill of the art after study of this patent document. If used in reference to the figures, the terms “top,” “bottom,” “right,” “left,” “forward,” “rear,” “first,” “second,” “inside,” “outside,” and similar terms should be understood to reference the structure and methods described in the specification and illustrated in the drawings, and are utilized for purposes of explanation.

DETAILED DESCRIPTION OF THE INVENTION, INCLUDING THE BEST MODE

The present detailed description relates to devices and methods for analysis of aqueous samples, most notably biological fluid samples such as blood or other body fluids, particularly using optical sensor technology, including particularly optical fibers as sensor probes, but also dry test strips. While reference will be made at times to blood and body fluids in this detailed description, the fluid sample can include not only samples of biological origin, such as blood, saliva, and urine, but also fluid samples derived from nonbiological sources. Some of the devices and methods described herein optimally utilize textured surface sites, wherein multiple advantages are achieved through increased surface area for deposition and subsequent reaction of analyte-reactive reagents and through spatial separation of cellular elements present in some types of body fluids, most particularly blood where such cellular elements include erythrocytes, leucocytes and such. Some of the devices and methods described herein enhance the intensity of light being transmitted from a sampling zone back through the optical sensor material to a suitable detector, wherein a means for enhanced reflectance is disposed in the sampling zone, preferably in association with an analyte-reactive reagent deposited on a surface site of the optical sensor. Some of the devices and methods described herein are particularly useful with reference to whole blood, with respect to which hemolysis of erythrocytes therein causes significant interference in fluid analyses. Some of the devices and methods described herein are useful for reducing hemolysis of erythrocytes, such as may occur through the action, for example, of tetrazolium dyes.
The term “analyte” is used to refer to the substance to be detected in the fluid sample.
“Detected” refers to identification of the presence of a targeted analyte in the fluid sample, but most often also extends to the quantification of the targeted analyte.
The phrase “analyte-reactive reagent” is defined as any reagent that can interact physically or chemically with an analyte such as to produce an optically discernible change, including changes correlative with analyte concentration in a fluid within the sampling zone.
A sample of blood or other body fluid is presented to a surface of an optical material of a sensor whereon an analyte-reactive reagent is present as a coating. The surface may be smooth, or the surface may be advantageously textured so that it presents the morphology of a field of elongated projections. The projections may be suitably spaced apart to exclude certain cellular components such as blood cells in a body fluid sample from entering into the spaces between the projections, while permitting the remaining part of the body fluid sample, which contains the analyte, to enter into those spaces.
The targeted analyte contacts an analyte-specific chemistry on the surface of the sensor, whereupon the analyte and a specific reagent interact in a manner that is optically detectable. Suitable analyte-specific chemistries may include receptor molecules as well as reactive molecules. Commonly, analyte-specific chemistries include components that generate colored species, and optical detection is based on the density and spectral nature of the colored species. In the case of blood containing cellular elements such as erythrocytes, spatial exclusion of the erythrocytes from the zone of the reagent is an important advantage of suitably textured analytical sites, when certain color development chemistries are used where the erythrocytes would interfere in such color-based assays. In commonly applied chemistries for analyzing blood sugar levels, for instance, erythrocytes often absorb light in the same general range of light frequencies in optical determinations, and must be excluded in some manner so as not to negatively influence the analytical results. The nature and arrangement of the analyte-specific chemistry varies depending on the application. For example, the analyte-specific chemistry may be a layer of one type of chemistry or an ordered array or a finely mixed composite of different types of analyte-specific chemistries. For convenience, these various options are grouped together during the remainder of this disclosure by using the term “analyte-reactive reagent”. The optically detectable change may occur specifically in the coating of the analyte-reactive reagent, or in a deposit developed on the coating (such as by binding of targeted analytes), or by development of reaction products in the fluid immediately in contact with the reagent. For purposes of this disclosure, these various possibilities which each involve slightly different spatial regions are included under the term “reagent sampling zone”.
A light beam of a suitable frequency or range of frequency is transmitted from the optical material into the reagent sampling zone. Changes in the spectral nature of the light beam advantageously occur as a function of optically detectable changes in the reagent sampling zone due to interaction of the analyte-reactive reagent with an analyte in the fluid to be analyzed. When detecting the spectral changes using reflectance spectroscopy, any part of this light beam that radiates in a direction away from the optical material through which the light beam is brought to the reagent sampling zone does not generally reenter the optical material, and does not contribute to the measurement. The techniques described herein enhance the amount of this light beam that is returned to and captured within the optical material for subsequent analysis. This technique for enhanced reflectance is optimally in the form of particles that scatter light. These particles may be composed of inorganic or organic materials. Inorganic particles useful as reflectance enhancing agents include silicates and related glasses, and may be in the shape of beads or similarly spheroidal shapes. They may be solid or hollow. Organic particles useful as reflectance enhancing agents include natural and synthetic polymers of various compositions, and may also be solid or hollow. Hollow beads are particularly effective. So that the analyte-reactive reagent may be accessed by the fluid sample, these reflectance enhancing particles are desirably not film-forming, meaning that a coating or array of these particles does not form a film impenetrable to fluid transport. These particles are associated with an analyte-reactive reagent coating, preferably by co-deposition as a mixed coating on the surface of an optical material, so as to be present in the reagent sampling zone. In practice, the analyte-reactive reagent will be present as a coating intimately in contact with a surface of the optical material, and the suitable reflectance enhancing particles will be in contact with the reagent layer but optimally will extend beyond the reagent layer spatially. Thus, in a preferred configuration, a majority of the analyte-reactive reagent is advantageously sandwiched between the optical material and the reflectance enhancing particles. A particularly effective arrangement is a textured surface on an optic material wherein both the analyte-reactive reagent and the reflectance enhancing particles are deposited within valleys or crevices of the surface.
The reflectance enhancing particles are normally to be applied to the surface of an optical material from an aqueous dispersion. The particles may be co-deposited on the surface along with the analyte-reactive reagent in a single step. Alternatively, the particles may be deposited in a separate step, preferably after first depositing a coating of the reagent. Drying of the coating or coatings at some point in the process is accomplished so as to present a dry sensor for handling and storage.
FIG. 1 shows a schematic representation of a surface coating on an optical guide material illustrating a coating combination with enhanced reflectance capability. The optical material 1 has on a surface thereof a coating 2 containing an analyte-reactive reagent. Embedded in this coating and extending outward therefrom are a plurality of reflectance enhancing particles 3. A beam of light, whose direction is indicated by the arrow 4 is transmitted through the optical material 1 into the coating 2. A portion of this light, having traveled into the reagent sampling zone (indicated by the bracket 6, and is the thickness of the deposit) is redirected back into the optical material, as indicated by the arrows 5. Other beam directions may be used as desired, including parallel to the reagent sampling zone 14 as described in the aforementioned Nomura U.S. Pat. No. 5,859,937, which is incorporated herein in its entirety by reference thereto. In practice, a fluid sample to be analyzed will be presented to and will bathe the reagent sampling zone, including immersion of the reflectance enhancing particles. The reagent sampling zone 6 may be made relatively thin, illustratively about 1 μm or 2 μm for particles having a diameter of about 0.4 μm, for example, to facilitate a quick response time.
FIG. 2 shows a schematic representation of a surface coating on an optical guide material illustrating a coating combination with enhanced reflectance capability. The optical material 10 has on a surface thereof a coating 11 containing an analyte-reactive reagent. Embedded in this coating 11 and extending outward therefrom are a plurality of reflectance enhancing particles 12. A beam of light, whose direction is indicated by the arrow 13, is transmitted through the optical material 10 into the coating 11. A portion of this light, having traveled into the reagent sampling zone (indicated by the bracket 14, and is the thickness of the deposit) is redirected back into the optical material, as indicated by the arrows 15. Other beam directions may be used as desired, including parallel to the reagent sampling zone 14 as described in the aforementioned Nomura U.S. Pat. No. 5,859,937, which is incorporated herein in its entirety by reference thereto. In practice, a fluid sample to be analyzed will be presented to and will bathe the reagent sampling zone 14, including immersion of the reflectance enhancing particles. The relatively thick reagent sampling zone 14, illustratively in the range of about 2 μm to 10 μm for particles having a diameter of about 0.4 μm, for example, and the many reflectance enhancing particles 12 contained therein facilitate high sensitivity.
Particles with optimal reflectance enhancing characteristics have a refractive index significantly different from water (refractive index=1.333) or aqueous fluid samples (refractive index typically slightly above 1.333). Divergences in refractive index from 1.000 will generally result in greater light scattering by the particles at greater divergences, and thereby greater reflectances. Polytetrafluoroethylene (PTFE) has a refractive index if 1.35, which is inconveniently close to the index of refraction of water and provides minimal reflectance enhancement. Silicate glass particles typically have a refractive index of about 1.47, which is sufficiently different from 1.333 as to provide some enhancement of light reflectance. Diatomaceous earth such as Celatom (trademark of Eagle Picher Industries) has been used in blood glucose test strips, but tends to “go clear”, i.e., become optically transparent to visible light, when immersed in aqueous media, such as to not provide good reflectance enhancement, due to the closeness of its index of refraction to that of water. Most polymeric materials have refractive indices in the range of 1.49 to 1.65, and serve well in enhancing reflectance, with optimal results being obtained at the higher index values. Hollow particles having air or other gas in the hollow interior possess an additional advantage, in that the refractive index of air, for example, is 1.000, which augments the scattering of any light entering into the hollow particles. Thus, hollow particles composed even of glass or PTFE may suitably enhance light reflectance in aqueous media in a reagent sampling zone. However, hollow particles of synthetic polymer materials having air within the hollow cavities are most preferred, being generally the most effective in light scattering characteristics and reflectance enhancements. Coatings comprising analyte-reactive reagents combined with reflectance enhancing particles may be as thick as desired for sensitivity, but for faster reaction rates, are preferably no more than 10 microns (μm) in thickness, more preferably no more than 5 μm in thickness, and most preferably are in the range of 2 μm in thickness. Wherein textured surfaces are employed, coatings in the range of 2 microns or less in thickness are particularly suitable for deposition within the crevices or valleys of the textured surface morphology.
Various optical materials are suitable for the sensors described herein. The optical material may be in sheet form, fiber form, or in other suitably shaped forms. A particularly suitable optical material is the type of light wave guide commonly referred to as an optical fiber. The optical fiber may be a single optical fiber, or may be a bundle of optical fibers. A minimally invasive sensing device that uses a light conducting fiber having a localized textured site thereon and methods for its manufacture and use are described in the aforementioned Nomura U.S. Pat. No. 5,859,937, which is incorporated herein in its entirety by reference thereto. Optical fibers may be fabricated from a variety of polymers or plastics such as polymethylmethacrylate (PMMA), polycarbonate, polysulfones, polyamide, polystyrene, polyimide, polyvinyl chloride (PVC), and from other types of optical materials such as glass, plastic, glass/glass composite and glass/plastic composite fiber waveguides. Optical fibers typically although not necessarily are provided with a cladding to support the fiber and assist in guiding light along the fiber. Prior to texturing, the fiber tip is given a desired geometric shape, which is dependent on the application and performance requirements, and which includes planar surfaces either normal with respect to or otherwise angled with respect to the fiber axis, convex and concave conical surfaces, and convex and concave semi-spherical surfaces.
A textured surface may be provided on a variety of optical materials other than fibers. Another type of sensor element is made from a sheet of transparent optical material such as, for example, a polymer or plastic (including polycarbonate and polyimide), glass, and quartz. If sample receiving areas are desired in the sheet, they may be formed by any of various processes depending on the type of optical material. Where the material is quartz, for example, the sample areas may be etched using dry or wet etch processes. Where the material is a molded plastic, the mold may contain certain surface recesses and protrusions for forming the sample areas. The sheets may include other optical components such as lenses. Multiple sensor elements may be made from each sheet by dicing, laser cutting, stamping, or otherwise dividing the sheet. Individual sensor elements or entire sheets or parts of sheets may be incorporated into a variety of sensing instruments having a diversity of different applications.
While various surface texturing processes are available, plastic optical materials preferably are textured by etching with atomic oxygen. Generation of atomic oxygen can be accomplished by several known methods, including radiofrequency, microwave, and direct current discharges through oxygen or mixtures of oxygen with other gases. Directed beams of oxygen, such as by an electron resonance plasma beam source, may also be utilized, as set forth in U.S. Pat. No. 5,560,781 issued Oct. 1, 1996 (Banks et al., Process for Non-Contact Removal of Organic Coatings from the Surface of Paintings), which hereby is incorporated herein in its entirety by reference thereto. Techniques for surface texturing are described in the aforementioned Nomura U.S. Pat. No. 5,859,937, which is incorporated herein in its entirety by reference thereto.
Atomic oxygen can be used to microscopically alter the surface morphology of polymeric or plastic materials in space or in ground laboratory facilities. For polymeric or plastic materials whose sole oxidation products are volatile species, directed atomic oxygen reactions produce surfaces of microscopic cones. However, isotropic atomic oxygen exposure results in polymer surfaces covered with lower aspect ratio sharp-edged craters. Isotropic atomic oxygen plasma exposure of polymers typically causes a significant decrease in water contact angle as well as altered coefficient of static friction. Atomic oxygen texturing of polymers is further disclosed and the results of atomic oxygen plasma exposure of thirty-three (33) different polymers, including typical morphology changes, effects on water contact angle, and coefficient of static friction, are presented in Banks et al., “Atomic Oxygen Textured Polymers,” NASA Technical Memorandum 106769, Prepared for the 1995 Spring Meeting of the Materials Research Society, San Francisco, Calif., Apr. 17-21, 1995, which hereby is incorporated herein in its entirety by reference thereto.
The general shape of the projections in any particular field is dependent upon the particulars of the method used to form them and on subsequent treatments applied to them. Suitable projection shapes include, for instance, conical, ridge-like, pillared, box-like, and spike-like. While the projections may be arrayed in a uniform or ordered manner or may be randomly distributed, the distribution of the spacings between the projections preferably is fairly narrow with the average spacing being such as to exclude certain cellular components of blood, such as the red and white blood cells, from moving into the space between the projections. The projections function to separate blood components so that the analyte that reacts with the surface-resident agent on the biosensor substrate is free of certain undesirable body fluid components. Most particularly preferred texturing is as disclosed in U.S. Patent Application Publication No. 2006/0211126 published Sep. 21, 2006 (Banks, Method for Using Texturing Surfaces of Optical Fiber Sensors for Blood Glucose Monitoring), which hereby is herein incorporated in its entirety by reference thereto, wherein polymer surfaces are exposed to a directed beam of energetic oxygen atoms or ions, these atoms or ions having isotropic energy levels greater than 1 eV, preferably in the range of 50 to 100 eV, and were referred to therein as being hyperthermal. Atomic oxygen texturing is discussed also in U.S. Patent Application Publication No. 2006/0257558 published Nov. 16, 2006 (Nomura, Plasma Polymerization of Atomically Modified Surfaces), and in U.S. Patent Application Publication No. 2005/0123451 published Jun. 9, 2005 (Nomura, System and Apparatus for Body Fluid Analysis Using Surface-Textured Optical Materials), which hereby are incorporated herein in their entirety by reference thereto. As a result of atomic oxygen texturing of the optical fiber or other optical material, the surface of the optical fiber/material includes a plurality of elongated projections. The optical material may include one, two, or more surface textured areas. The tip of the fiber may be textured, as well as the end of the optical fiber. The atomic surface texturing of optical materials is believed to improve sensitivity and provide an increased effective sensing area and limit background noise by supporting multiple ray reflections responsive to the light-influencing property of the analyte-specific chemistry.
The projections are suitably spaced apart to exclude certain cellular components, such as red and white blood cells, of the body fluid sample, such as blood, from entering into the micro-wells or valleys between the projections, while permitting the remaining part of the body fluid sample, such as plasma, which contains the analyte, to enter into those wells or valleys. This is particularly preferred in blood glucose determinations, although utility is not limited to blood glucose determinations. Analytes/biomarkers in the blood plasma, which are indicative of cellular and/or soluble platelet activation and coagulation activation, contacts or associates with the analyte specific chemistries on the surface of the elongated projections, whereupon the analyte and the analyte specific chemistry interact in a manner that is optically detectable. This permits almost instantaneous analysis of the available plasma component of blood.
For some applications, the atomic oxygen textured surface on the optical guide material may be advantageously modified by plasma polymerization to allow for the adherence of the analyte specific chemistries specific for the desired analyte to be assayed. If there is more than one textured surface, one or more of the textured surfaces may be modified by plasma polymerization. Plasma polymerization and treatment are processes to modify the surface of substrate materials to achieve specific functionality. Such surfaces may be modified to become wettable, non-fouling, slippery, crosslinked, reactive, reactable and/or catalytic. The plasma polymerization process is a chemical bonding technology in which a gaseous plasma is created at or near ambient temperatures in a modest vacuum, causing a gaseous monomer to chemically modify the surface of a substrate material. The modification of surfaces by gaseous plasmas in this manner is treated at length in the book by H. Yasuda, “Plasma Polymerization,” Academic Press, New York, 1985, ISBN 10:127687602, which hereby is incorporated herein in its entirety by reference thereto. The plasma polymerization of textured surfaces is described in the aforementioned Nomura U.S. Patent Application Publication No. 2006/0257558, which is incorporated herein in its entirety by reference thereto.
FIG. 3 is a schematic diagram of a textured surface of an optical guide material illustrating a coating combination with enhanced reflectance capability. A series of peaks and valleys achieved by means of atomic oxygen texturing are displayed, wherein an array of projections 21 (peaks) and micro-wells 22 (valleys and crevices) are present on the surface of an optical material 20. A deposit is present within the micro-wells which includes a coating 23 of an analyte-reactive reagent in admixture with light scattering particles 24. In operation, a light beam travels through the optical material 20 in the direction indicated by arrow 25, and exits the optical material into the reagent sampling zone 26 present within the micro-wells 22. Illustratively, the thickness of the sampling zone 26 which includes the texturing is from 5 μm to 7 μm. Reflectance from the reagent sampling zone 26 reenters the optical material 20 and is transmitted in a direction through the optical material 20 generally opposite to the arrow 25. Reflectance is enhanced due to the presence of the light scattering particles 24, which serve to re-direct the path of light incident upon them. Much of this light would otherwise continue in a direction away from the matrix of the optical guide material 20, much of it generally continuing in the same direction indicated previously by the arrow 25. The light scattering particles 24 are optimally small enough to fit within the micro-wells 22, and further are advantageously held in place by one or more binder materials used in preparing a coating of the analyte-reactive reagent although, as will be seen later with Example 1 and associated Table 1, a binder is not always needed. Appropriate examples of such binders are water-soluble or water-dispersible polymers such as polyacrylic acid and polyacrylamide. Preferred binders will be polymeric in nature and, in most cases, readily wetted by aqueous media. The optimal binder will be usually selected on the basis of its compatibility with the specific chemistry of a chosen analyte-reactive reagent, and this is well within the capability of one of normal skill in the art, since the immobilization of the light scattering particles along the surfaces of the micro-wells is rather easy to accomplish.
Suitable light scattering particles preferably are of such dimensions as to fit within the wells between the elongated projections, but larger dimensions may be accommodated such that the light scattering particles are excluded from the wells in the same manner as blood cellular components. It is preferable, however, that the dimensions of the light scattering particles allow such particles to fit into and reside within the wells of the textured surface. Suitable light scattering particles may be polymeric in nature or may be fashioned from inorganic matter. One example of a particularly effective light scattering material is an opaqueness-generating polymer, in emulsion form, consisting of spherical styrene-acrylic beads, available under the tradename Ropaque™ from Rohm and Haas, a wholly owned subsidiary of The Dow Chemical Company, of Philadelphia, Pa., USA. The intended purpose of these beads is as an opacifying agent in paint films, in that they provide high hiding power. These beads are hollow, and are customarily supplied as aqueous emulsions wherein the cavities within the beads are water-filled. During drying of these beads, water permanently diffuses from the hollow interiors, resulting in encapsulated air voids. The hollow beads, in the dry state, exhibit highly effective light scattering characteristics, which results in their utility as opacifying agents. In addition, these particles preferably do not effectively form impenetrable or impermeable films under conditions of usage herein, so that fluid from a body fluid sample can still enter and bathe the surfaces of the textured site. In practice, the textured site is impregnated with an analyte-reactive reagent plus the light scattering particles in such a manner as to replace the water inside the hollow particles with a gaseous medium such as air, such as by means of a drying step.
Hemolysis is a complicating factor when the sensor is in the form of a dry test strip employing a sheet-like microporous “nylon” member, or when the sensor in either format (dry test strip or optical fiber) employs tetrazolium dyes for assay of blood components such as glucose. Both the microporous “nylon” members and the tetrazolium dyes have been implicated with hemolysis. Advantageously, certain types of light reflectance enhancement particles simultaneously suppress hemolysis. Particularly preferred are particles that have a weak negative surface charge. While the invention as set forth in the claims is not to be bound by the following theory, it is believed that positively charged surfaces and/or positively charged analyte-reactive reagents interact destructively with the cell membranes of erythrocytes, causing cell membrane disruption and leakage of otherwise contained hemoglobin into the sampling zone. A non-film-forming surface array of these negatively charged particles effectively shields the erythrocytes from the membrane-disrupting effects of the aforementioned hemolytic reagents. Of particular effectiveness are Ropaque™ hollow particles, which are known to have a plurality of surface-borne carboxylate groups. Other negatively charged groups may be pendant on the particle surfaces in place of carboxylate groups, including for example sulfonate, sulfinate, phosphate, and phosphate groups. Combinations of such groups may be present on the surface of such particles. But the weakly anionic nature of carboxylate groups are sufficient and effective.
FIG. 4 is a schematic representation of one type of reagent dip-coated sheet-like microporous “nylon” member, illustratively a multizone membrane which has a more open pore side 36 and a tighter pore side 37. When dip solution is applied to the more open pore side 36, reflectance enhancing particles 38 remain in the more open pore side 36 along with some of the analyte-reactive reagent (not shown), while a substantial amount of the analyte-reactive reagent 39 migrates to the tighter side of the membrane. The reflectance enhancing particles 38 in the larger pores and the analyte-reactive reagent in the smaller pores together form a sampling zone 32. Illustratively, the thickness of the sampling zone 32 which includes the pores of the membrane, is essentially the thickness of the membrane, which is variable but may be about 125 μm. To use the multizone membrane of FIG. 4, one applies a droplet of whole blood 30 to the more open pore side 36, and applies a beam of light, whose illustrative direction is indicated by the arrow 31, to the tighter pore side 37.
The following examples illustrate the salutary effects of negatively charged particles, such as Ropaque™, in both enhanced reflectance and in suppressing hemolysis in whole blood deposited on “nylon” membranes such as used in dry test strips.

Example 1

The following example illustrates the applicability of this techniques described herein in enhancing reflectance. Optic fibers of 2 mm in diameter, having tips textured with atomic oxygen in accordance with the method described in the aforementioned Banks U.S. Published Patent Application 2006/0211126, which is incorporated herein by reference thereto, were treated with 2 microliters of an aqueous reagent containing 3.7 mg/ml WST-5[2, 2′-Dibenzothiazolyl-5, 5′-bis{4-di (2-sulfoethyl) carbamoylphenyl}-3,3′-(3,3′-dimethoxy-4,4′-biphenylene) ditetrazolium, disodium salt], 2 mM methoxyPMS [1-Methoxy-5-methylphenazinum methylsulfate, obtained from Dojindo Co, Japan], 900 U/ml PQQ-dependent GDH [pyrroloquinolin-quinone 1-oxidoreductase—dependent glucose dehydrogenase, obtained from Amano Enzyme Inc., Japan], 4 mM calcium chloride and Ropaque™ Ultra Emulsion (30 solid weight %) diluted by volume to varied concentrations from zero to 10 volume percent, followed by drying for 5 min at 50° C. No binder needed to be added to the diluted emulsion, in that the Ropaque™ beads showed sufficient natural adhesion to the textured surface so as to remain in place. A beam of light was directed through the optic fibers toward the textured tips, and light reflectances at 635 nm were measured and compared both with the tips dry and the tips immersed in whole blood containing 100 mg/dl glucose. Results are shown in Table 1, wherein data are expressed in Relative Light Units (RLU). The data in Table 1 show greatly increased light reflectance due to the presence of Ropaque™ opacifying particles disposed on the textured optic fiber tips. It can be seen from the data in Table 1 that reflectance was enhanced by as much as 450% with whole blood, and to as high as 650% in a dry state.

TABLE 1

Reflectance Enhancement with Ropaque ™ at Varied Concentrations

Ropaque ™
Concentration	Light Reflectance	Light Reflectance (635 nm)
(vol %)	(635 nm) Dry (RLU)	with Whole Blood (RLU)

0	200	142
1	900	440
5	1215	667
10	1317	634

Example 2

Optic fibers 2 mm in diameter having tips textured with atomic oxygen were treated with 1 microliter of a reagent blend for blood sugar analysis containing 16 mM MES (2-morpholinoethanesulfonic acid, monohydrate), 6 mM calcium chloride, 4 mM methoxy PMS (1-Methoxy-5-methylphenazinum methylsulfate, obtained from Dojindo Co, Japan), 1800 U/ml PQQ-dependent GDH (pyrroloquinolin-quinone 1-oxidoreductase—dependent glucose dehydrogenase, obtained from Amano Enzyme Inc., Japan), 14.8 mg/dl WST-5 (2,2′-Dibenzothiazolyl-5,5′-bis(4-di (2-sulfoethyl)carbamoylphenyl)-3,3′-(3,3′-dimethoxy-4,4′-biphenylene)ditetrazolium, disodium salt), 0.125 wt % poly(acrylic acid) (partial sodium salt solution, MW ca. 5,000, obtained from Sigma-Aldrich Chemcal Co.) and 2 vol % Ropaque™ Ultra Emulsion. The treated fibers were then dried for 5 minutes at 50° C. Dried, coated fibers were stored covered at room temperature until testing. In testing, whole blood standards were prepared having one of four different glucose levels. Fibers were mounted in an optical detector and light reflectance testing was initiated. After an initialization period of approximately 3 seconds, each optic fiber tip was contacted with an amount of the whole blood (hematocrit 40%) equaling 0.5 μl, one for each glucose concentration level at 0, 49, 212, and 437 mg/dl. FIG. 5 shows a graph of the tracings of percent reflection of a 635 nm light beam obtained at each of the four glucose concentration levels: trace 42 was obtained at 0 mg/dl, trace 44 was obtained at 49 mg/dl, trace 46 was obtained at 212 mg/dl, and trace 48 was obtained at 437 mg/dl. The tracings showed stable development of light reflectance signals easily readable by the light sensor apparatus. K/S (Kebulka-Munk absorption parameter) values were also calculated and plotted versus time for these four whole blood glucose concentrations, and the results are displayed in the graph in FIG. 6: trace 52 was obtained at 0 mg/dl, trace 54 was obtained at 49 mg/dl, trace 56 was obtained at 212 mg/dl, and trace 58 was obtained at 437 mg/dl. This example demonstrates utility.

Comparative Example

Optic fibers of 2 mm in diameter having tips textured with atomic oxygen were treated with 1 microliter of a reagent blend for blood sugar analysis containing 16 mM MES, 6 mM calcium chloride, 4 mM methoxy PMS, 1800 U/ml PQQ-dependent GDH, and 14.8 mg/dl WST-5. No reflectance enhancing particles were provided in the recipe. Fibers were dried for 5 minutes at 50C. Dried, coated fibers were stored covered at room temperature until testing. In this example, 0.5 microliter of whole blood (HCT 40%) was added after an initial waiting period of approximately 3 seconds. FIG. 7 shows the percent reflectance at 10 seconds and at different glucose concentrations (lower curve 64), along with comparison data taken from Example 2 at 10 seconds (upper curve 62). Reflectances were in the range of 3% to 4%, compared to reflectances of about 15% to 25% in the presence of Ropaque™ particles, the latter representing at least a fourfold increase in light reflectance compared to the former. The result showed that there was a rather small percent reflectance change depending on glucose concentration (lower curve 64), with only a small amount of differentiation to determine glucose concentration in blood.

Example 3

A single-layer reinforced nylon microporous membrane, available commercially from Cuno Corporation, USA, under the designation BLA080, was used in this and the following example. The nylon membrane had an asymmetric structure, the more-open side having a 2.5 μm nominal pore size and the tighter side having a 0.8 μm nominal pore size. A tetrazolium reagent composition was applied to the more open side of the nylon membrane in this example, wherein the reagent contained Ropaque™ particles (i.e., 30 solid wt % Ropaque™ Ultra Emulsion) added at 10% by volume to the reagent. The reagent also contained 100 mM MES, 4.0 mM calcium chloride, 2 mM methoxy PMS, 900 U/ml PQQ-dependent GDH, 10 mM WST-5, 0.25 wt % PAA, 10 wt % Trehalose, 0.2 wt % Triton X-100, and 16 mg/ml PSSA (polystyrenesulfonic acid, sodium salt, MW 70,000). In testing, whole blood (hematocrit 40%) samples equaling 2 μl with several glucose concentrations were applied to the more open pore side of membranes. The reflectance values at 635 nm were obtained at 10 seconds exposure time, and are plotted in FIG. 8. A curve 72 fitted to the plotted values shows very good sensitivity.

Comparative Example

Samples of the same membrane substrate as in Example 3 were treated with the same reagent, but no reflectance enhancing particles were provided in the recipe. In this example, as before, 2 μl samples of whole blood (HCT 40%) with varying glucose concentrations were applied. FIG. 8 shows the percent reflectance at 10 seconds and at different glucose concentrations (lower curve 74) for the treated membrane without reflectance enhancing particles, alongside the 10 second data from Example 3 (upper curve 72) for the treated membrane without reflectance enhancing particles. Reflectance values were in the range 3 to 7% without reflectance enhancing particles, compared to the enhanced reflectances of about 14 to 35% in the presence of Ropaque™ particles, the latter representing at least a fourfold increase in light reflectance compared to the former. The reflectance change also improved substantially. In trace 72, the change in reflectance between 50 and 300 mg/dl glucose was approximately 16.8%, or about 5.6% per 100 mg/dl. In trace 74, the change was approximately 4.8% over the same range of glucose concentrations, or about 1.6% per 100 mg/dl.

Example 4

A single-layer reinforced nylon microporous membrane, available commercially from Cuno Corporation of Meriden, Conn., USA, under the designation BLA080, was used. The nylon membrane had an asymmetric structure, the more-open side having a 2.5 μm nominal pore size and the tighter side having a 0.8 μm nominal pore size. The reagent composition as used above in Example 3 was applied to the more open side of the nylon membrane in this example, along with varying amounts of Ropaque™ particles. Specifically, 30 solid wt % Ropaque™ Ultra Emulsion was added at 0%, 2.5%, 5%, 7.5% and 10% by volume to the tetrazolium reagent composition of Example 3. The treated membranes were then dried. In this application, the suspended particles remain adsorbed on the original surface of application, and a substantial portion of the analyte-reactive reagent chemicals migrated to the tighter side of the membrane. Whole droplets of whole blood without glucose (0 mg/dl) were applied to the more open side of the membranes. Hemoglobin permeation through the membrane, indicative of hemolysis, was easily observed with the 0% Ropaque™ condition, and to a minor extent for the 2.5% condition, looking at the tighter side (the underside) of the membranes. At 0% Ropaque™, the color of the underside changed from white to red. At 2.5% Ropaque™, color changed from white to pink. Color change was nearly absent at higher Ropaque™ concentrations. This color development was readily within less than 10 seconds after blood was applied. After 10 seconds, a photograph was taken to visually compare the color change (white to red/pink) on the underside. FIG. 9 represents the progression in color change, from a deep red (81) through decreasing shades of pink (82, 83, 84, 85). The intensity of the color at 635 nm, reflectance mode, was measured by spectroscopy using an instrument manufactured by Ocean Optic Inc., USA, and results are listed in Table 2.

TABLE 2

Inverse Relationship: Hemoglobin Vs. Ropaque ™ Concentration

	Ropaque ™	Level of Unabsorbed
	Concentration (vol %)	Light at 635 nm (%)

	0	52.7
	2.5	55.2
	5	71.9
	7.5	80.2
	10	81.0

In this example, concentrations of Ropaque™ at higher than 5% applied to the nylon membrane surface achieved essentially full suppression of hemolysis that would otherwise have occurred by the combined effects of the nylon membrane surface and the tetrazolium dye. Later, at approximately 5 minutes after application of the blood droplets, tetrazolium dye reaction with the hemoglobin was observable, being decidedly pronounced in the 0% Ropaque™ condition, was present to a minor extent in the 2.5% Ropaque™ condition, and at generally baseline values in the other conditions, as observed approximately 5 minutes after blood application. The baseline values represent returned light showing minimal or no absorption of the 635 nm frequency due to any hemoglobin interference. Thus, the utility of these weakly negatively-charged particles in suppressing erythrocyte hemolysis, when dispersed on the surface of an optical sensor, has been discovered and is herein disclosed. Applicability of the hemolysis-suppressing particles includes both sheet-like and optical-fiber-type sensors, as well as both textured and nontextured surfaces.
Improvements in sensitivity are achieved over a broad range of particle densities. As to Ropaque™ hollow sphere pigments, for instance, Example 1 shows significant improvement in light reflectance for concentrations as low as one percent. Assuming the spheres in the undiluted Ropaque emulsion are essentially tightly packed, a one percent Ropaque concentration is in effect a one percent average particle density, meaning that significant improvement in light reflectance and therefore in sensitivity are achievable at average particle densities of as low as one percent. Ropaque sphere densities were varied in the experiments (2% concentration of Ropaque emulsion in Example 2, 10% concentration of Ropaque emulsion in Example 3, and 2.5%, 5.0% and 7.5% and 10.0% concentration of Ropaque emulsion in Example 4), all with improved reflectance over measurements without the Ropaque material.
The description of the invention including its applications and advantages as set forth herein is illustrative and is not intended to limit the scope of the invention, which is set forth in the claims. Variations and modifications of the embodiments disclosed herein may be made, and practical alternatives to and equivalents of the various elements of the embodiments may be substituted. These and other variations and modifications of the embodiments disclosed herein, including of the alternatives and equivalents of the various elements of the embodiments, are contemplated, and may be made without departing from the scope and spirit of the invention.

Claims

1. A sensor for determination of an analyte, comprising:

a body of optical material;

an analyte-reactive reagent for the analyte, disposed on the optical material body in optical communication therewith; and

a plurality of particulate bodies dispersed through the analyte-reactive reagent, the particulate bodies being adapted to contribute to reflectance of light from the optical material body back into the optical material body through at least a portion of the analyte-reactive reagent, when the analyte-reactive reagent is in reaction with the analyte.

2. The sensor of claim 1 wherein the analyte-reactive reagent produces an optically detectable change upon reaction with the analyte.

3. The sensor of claim 2 wherein the analyte is for blood glucose.

4. The sensor of claim 2 wherein the particulate bodies are adapted to scatter the light from the optical material body.

5. The sensor of claim 2 wherein the particulate bodies comprise hollow polymeric beads.

6. The sensor of claim 1 wherein a surface region of the optical material presents a textured field of elongated projections.

7. The sensor of claim 6 wherein:

the body of optical material comprises an optical fiber; and

the surface region is upon a distal tip of the optical fiber.

8. The sensor of claim 6 wherein:

the body of optical material comprises an optical fiber; and

the surface region is upon a sidewall of the optical fiber.

9. The sensor of claim 6 wherein:

the body of optical material comprises a sheet of optical material; and

the surface region is upon a major surface of the sheet.

10. The sensor of claim 1 wherein:

the body of optical material comprises a sheet of optical material; and

the analyte-reactive reagent is disposed upon a major surface of the sheet.

11. The sensor of claim 1 wherein:

the body of optical material comprises a microporous nylon membrane having a first major surface and a second major surface, the membrane having first pores of a first pore size along the first major surface, and second pores of a second pore size less than the first pore size along the second major surface; and

the analyte-reactive reagent is disposed at least in part within the first pores.

12. The sensor of claim 1 wherein:

the optical material comprises a sheet of optical material; and

the analyte-reactive reagent and the particulate bodies form a coating having an average thickness of less than 5 μm.

13. The sensor of claim 1 wherein:

the optical material comprises a textured surface; and

the analyte-reactive reagent and the particulate bodies form a coating within valleys and crevices of the textured surface having an average thickness of less than 2 μm.

14. The sensor of claim 1 further comprising:

a light source optically coupled to the body of optical material for furnishing light to the analyte-reactive reagent; and

a light sensor optically coupled to the body of optical material for receiving reflectance from the analyte-reactive reagent.

15. The sensor of claim 1 wherein the particulate bodies simultaneously enhance light reflectance and suppress hemolysis.

16. The sensor of claim 1 wherein the particulate bodies are dispersed through the analyte-reactive reagent in a wet state at an average particle density greater than about one percent.

17. The sensor of claim 1 wherein the particulate bodies are dispersed through the analyte-reactive reagent in a wet state at an average particle density greater than about five percent.

18. A sensor for determination of at least one analyte in a fluid comprising:

an optic fiber having proximal and distal ends, and further having a textured surface on a distal end;

a coating comprising at least one analyte-reactive reagent disposed on the textured surface, said reagent producing an optically detectable change upon reaction with at least one analyte in a fluid to be presented to the distal end; and

reflectance enhancement means for enhancing reflectance into the fiber from a sampling zone associated with said coating.

19. The sensor of claim 18 wherein the proximal end of said fiber receives light from a source and emanates reflectance light to a detector.

20. The sensor of claim 18 wherein the reflectance enhancement means comprises light scattering particles co-disposed with the analyte-reactive reagent on the textured surface.

21. The sensor of claim 20 wherein the light scattering particles comprise inanimate particulate matter deposited within valleys and crevices within the textured surface.

22. The sensor of claim 21 wherein the inanimate particulate matter comprises hollow polymeric particles.

23. A device for measurement of an analyte in a fluid comprising:

an optical material having a deposit on a surface thereof;

wherein the deposit comprises an analyte-reactive reagent and a plurality of hollow polymeric particles; and

wherein a sampling zone is associated with the'analyte-reactive reagent, a portion of the reagent being within the sampling zone and disposed between the surface of the optical material and the hollow polymeric particles, and the hollow polymeric particles providing for reflectance of a light beam emitted from the optical material into the sampling zone.

24. The device of claim 23 wherein the hollow polymeric particles comprise beads having a light scattering characteristic.

25. The device of claim 24 wherein the hollow polymeric particles comprise beads of a composition transparent to visible light.

26. The device of claim 23 wherein the deposit is wettable by water and body fluids.

27. A method of analyzing a fluid sample for an analyte comprising:

presenting a fluid sample containing the analyte to a site on an optical material, the site having a deposit comprising an analyte-reactive reagent and a particulate means for enhancing reflectance;

developing an optically detectable change in a reagent sampling zone associated with the analyte-reactive reagent;

emitting a beam of light through the optical material into the reagent sampling zone;

returning reflectance from the reagent sampling zone through the optical material to a detector; and

measuring the reflectance for spectral qualities correlative to the analyte.

28. The method of claim 27 wherein the site on the optical material comprises a surface textured by means of atomic oxygen texturing.

29. The method of claim 28 wherein the particulate means comprises hollow polymeric particles.

30. The method of claim 27 wherein the particulate means comprises hollow polymeric particles.

31. A sensor for measuring concentration of an analyte in an erythrocyte-containing biological fluid comprising:

an optical material having a region of analyte-reactive reagent for contact with a biological fluid to be analyzed; and

a deposit comprising a plurality of negatively-charged particles disposed in the region;

wherein said particles suppressing hemolysis of erythrocytes coming into contact with the region.

32. The sensor of claim 31 wherein:

the optical material comprises a microporous membrane; and

the deposit comprises a porous array of the negatively-charged particles.

33. The sensor of claim 31 wherein:

the optical material comprises a textured surface; and

the negatively-charged particles are disposed along the textured surface.

34. The sensor of claim 31 herein the particles comprise hollow polymeric beads.

35. The sensor of claim 34 wherein the particles have a plurality of carboxylate groups on their surfaces.

36. The sensor of claim 31 wherein the particles have a plurality of carboxylate groups on their surfaces.

37. The sensor of claim 36 wherein the region contains a tetrazolium dye reagent for blood glucose determination.

38. The sensor of claim 31 wherein the region contains a tetrazolium dye reagent for blood glucose determination.

39. The sensor of claim 37 wherein the negatively-charged particles simultaneously enhance light reflectance and suppress hemolysis.

40. The sensor of claim 31 wherein said negatively-charged particles simultaneously enhance light reflectance and suppress hemolysis.

41. A method of manufacturing a sensor for determining an analyte, comprising:

co-depositing an analyte-reactive reagent for the analyte and a plurality of particulate bodies into a sampling zone on an optical material body; and

maintaining the analyte in optical communication with the optical material body.

42. The method of claim 41 further comprising;

forming a textured field of elongated projections on a surface of the optical material body that presents into the sampling zone;

wherein the co-depositing step comprises depositing the particulate bodies into valleys and crevices of the textured field.

43. The method of claim 42 wherein the forming step comprises exposing the optical material body to atomic oxygen texturing.

44. The method of claim 41 wherein the particulate bodies comprise light scattering particles.