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WO1999046582A1 - Dispositif, composition de dosage, et procede d'optimisation de la resolution de dosage - Google Patents

Dispositif, composition de dosage, et procede d'optimisation de la resolution de dosage Download PDF

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Publication number
WO1999046582A1
WO1999046582A1 PCT/US1998/004607 US9804607W WO9946582A1 WO 1999046582 A1 WO1999046582 A1 WO 1999046582A1 US 9804607 W US9804607 W US 9804607W WO 9946582 A1 WO9946582 A1 WO 9946582A1
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WO
WIPO (PCT)
Prior art keywords
opacifier
matrix
detection zone
reflectance
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1998/004607
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English (en)
Inventor
Joel M. Blatt
Wilma M. Mangan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer Healthcare LLC
Original Assignee
Metrika Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Metrika Inc filed Critical Metrika Inc
Priority to PCT/US1998/004607 priority Critical patent/WO1999046582A1/fr
Priority to AU64552/98A priority patent/AU6455298A/en
Publication of WO1999046582A1 publication Critical patent/WO1999046582A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G01N21/8483—Investigating reagent band
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G01N33/521—Single-layer analytical elements

Definitions

  • the present invention relates to a device, composition, and method which optimizes the resolution of detecting a physical change on the surface of a sample-exposed analytical chemistry strip in a diagnostic device which displays medical information.
  • the reflected optical radiation is detected visually or with the assistance of a reflectometer, problems often arise obtaining accurate and precise measurements of the diffusely reflected optical radiation from the surface of the assay strip within the sampling area or detection zone.
  • One of the causes is that the physically detectable change which is being measured in the test area does not occur within the most sensitive or optimal range of the analytical assay. Assay results detected outside of the optimal range are usually not as accurate by comparison.
  • the optimization of assay resolution should be sufficiently inexpensive, timely, efficient, durable, and reliable for use in a diagnostic device which permits point-of-care use by untrained individuals in locations such as the home, sites of medical emergencies, or locations other than a clinic.
  • the present invention provides an assay composition for producing a physically detectable change upon contact with a sample which correlates with the amount of selected analyte in the sample.
  • the composition includes a support matrix pervious to optical radiation and a chemical reagent yielding a physically detectable change which correlates with the amount of selected analyte in the sample.
  • the support matrix has at least one detection zone for detecting the physical change.
  • the detection zone having a cross-sectional area and a depth profile extending into the support matrix.
  • the composition includes an opacifier present in an amount sufficient to increase the resolution of the physically detectable change.
  • the opacifier is distributed uniformly across the cross-sectional area of the detection zone and is distributed through at least a portion of the depth profile of the support matrix within the detection zone.
  • the chemical reagent is substantially immobilized relative to the opacifier when detecting the physical change.
  • the matrix includes a detection zone having a chemical reagent yielding a physically detectable change which correlates with the amount of selected analyte in the sample and an opacifier present in an amount sufficient to increase the resolution of detecting the physical change.
  • the detection zone has a cross-sectional area and a depth profile extending into the matrix.
  • the opacifier is distributed uniformly across the cross-sectional area of the detection zone and being distributed through at least a portion of the depth profile of the matrix within the detection zone.
  • the opacifier is substantially immobilized relative to the chemical reagent in the detection zone when detecting the physical change.
  • the present invention also provides a diagnostic device for determining the presence of a selected analyte in a sample.
  • the device includes a housing having an exterior surface and sealing an interior area.
  • a receptor is configured to receive the sample containing an analyte selected for determining its presence.
  • the receptor is located on the exterior surface of the housing.
  • At least one transport matrix of the type described above is provided for reacting the sample with a chemical reagent to yield a physically detectable change in a detection zone which correlates with the amount of selected analyte in the sample.
  • the present invention also provides a method for determining the level of a selected analyte in a sample.
  • the method includes optimizing a physically detectable change in an assay composition which correlates with the amount of selected analyte when contacted with the sample.
  • Another method provided by the present invention optimizes the assay results of a selected analyte in a sample-exposed assay composition.
  • the method includes increasing the reflection of optical radiation detecting a physical change of the sample-exposed assay composition to be within the range of optimal assay resolution.
  • Fig. 1 is a partial top plan view of a diagnostic device having a portion cut-away to view the illumination and detection optics of the present invention
  • Fig. 2 is a partial cross-sectional view of the diagnostic device illustrated in Fig. 1 along the lines 2-2;
  • Fig. 3 is a top plan view of a non-instrumented diagnostic device using the present invention.
  • Fig. 4 is an isolated view of a transport matrix having two detection zones utilizing the present invention
  • Fig. 5 is a graph of error in K/S (%) versus percentage change in reflectance (%R) generally illustrating measurement error as a function of reflectance and analog/digital resolution of an instrument detecting the physical change;
  • Fig. 6 is a side view of one embodiment of an assay strip suitable for use in a general chemistry assay such as analyzing samples containing sarcosine;
  • Fig. 7 is a top plan view of the assay strip in Fig. 6;
  • Fig. 8 is a graph of the K/S value for four levels of titanium dioxide (5%, 10%, 15%, and 20% w/v TiO 2 ) versus different concentrations of sarcosine (mM) in a sample; and Fig. 9 is a graph of the K/S value for three levels of titanium dioxide (10%, 15%, and 20% w/v TiO 2 ) versus different concentrations of sarcosine (mM) in a sample.
  • the present invention may be utilized in either instrumented or non-instrumented assay devices.
  • instrumented devices include the disposable single- and multiple- use digital electronic instruments and assay devices described in detail in the above-identified related applications previously incorporated by reference.
  • the present invention provides for more accurate measurement, by either visual observation or instrumentation, of a physically detectable change corresponding to the amount of the selected analyte in one or more detection zones of an assay.
  • the present invention optimizes the resolution of an assay composition which produces a physically detectable change upon contact with a sample which correlates with the amount of selected analyte in the sample.
  • the assay resolution is optimized by increasing the amount of optical radiation reflected from the assay composition to fall within the most sensitive range of reflectance for the concentration range of clinical interest of the selected analyte.
  • the present invention positions elements within the assay composition which scatter the optical radiation directed into assay composition through its surface. The scattering elements increase the amount of optical radiation which is reflected out of the assay composition back through the surface the optical radiation entered. The reflected optical radiation is then detected either visually or with the assistance of an optically sensitive instrument like a reflectometer.
  • opacifier is defined as an element added to a material to make it opaque which makes the material less penetrable by light, but still reflects light.
  • the opacifier is present in an amount sufficient to increase the resolution of detecting the physical change by scattering the optical radiation directed into the assay composition and increasing the reflectance of the optical radiation to where it is detected. Since the opacifier scatters light, it is preferably distributed within at least a portion of the depth profile of the assay composition. Distributing the opacifier on a surface of the assay composition, either closest or furthest from the point which the optical radiation enters the assay composition, produces a negligible increase in assay resolution.
  • the opacifier In order to promote the scattering effect of the opacifier, it is distributed through at least a portion of the depth profile of the assay composition within a detection zone for detecting the physical change. Increasing the number of scattering centers shortens the mean path length of the optical radiation between striking different scattering centers within the assay composition and shortens the path length between the points of entry and exit for the optical radiation.
  • the opacifier is distributed uniformly through the entire depth profile of the assay composition in the detection zone when the reflected optical radiation is detected.
  • the opacifier is also distributed uniformly across the cross-sectional area of the detection zone to promote reliable and reproducible detection of the physical change.
  • the assay composition includes a chemical reagent or an indicator which are defined herein to yield a physically detectable change which correlates with the amount of selected analyte in the sample.
  • the indicator generally scatters a negligible amount of the optical radiation directed into the assay composition.
  • the opacifier modifies the physical change, such as color intensity, yielded by the indicator by increasing the ratio of scattered light over absorbed light.
  • the assay composition also includes a support matrix pervious to the optical radiation.
  • the support matrix includes at least one detection zone for detecting the physical change.
  • the detection zone has a cross-sectional area and a depth profile extending into the matrix.
  • the support matrix defines the physical distance and relation of the chemical reagent and the opacifier to one another within the detection zone. It is preferred that the physical relationship between the opacifier and the chemical reagent is substantially immobilized at the time the reflected optical radiation from the physical change is detected.
  • a material suitable for use as the support matrix in the assay composition includes, but is not limited to, conventional water-based gels like polyvinyl alcohol, polyethylene glycol, or gelatin and translucent materials which include porous materials of various opacities such as microporous membranes.
  • An opacifier suitable for use in the present invention should be inert and stable in contact with biologically active molecules such as the reaction of the reagent and sample analyte.
  • the opacifier should also be finely dispersible with a small particle size. Ease of uniformly dispersing the opacifier is desirable.
  • the opacifier should also have a refractive index greater than that of water.
  • the opacifier has an index of refraction in the range of about 2.5 to about 3.
  • the most preferred particle size of the opacifier is in the range of about 0.2 ⁇ m to about 0.4 ⁇ m with a preferred transport matrix pore size of about 1 ⁇ m to about 8 ⁇ m, although a pore size of about 0.45 ⁇ m to lO ⁇ m is suitable for light in the visible portion of the electromagnetic spectrum.
  • the optimal particle size is dependent on the scattering efficiency of the centers and the wavelight of light. The optimal particle size will increase for light in the near infrared region, and decrease for light in the far ultraviolet region of the spectrum.
  • the opacifier has a surface area in the amount greater than about 10 m 2 /g.
  • a preferred material for use as the opacifier in the present invention is titanium dioxide, particularly in the rutile form.
  • suitable materials include barium sulfate, zinc oxide, silica, lead oxide, diatomaceous earth materials, colloidal materials, and microcrystalline synthetic polymers.
  • An assay device for the present invention can have many configurations, some of which are specifically illustrated herein.
  • One embodiment of an instrumented diagnostic device 10 having an analytical chemistry strip or transport matrix of the present invention is illustrated in Figs. 1 and 2.
  • the device 10 includes a housing 12 having a receptor such as an inlet port 14 which extends from the surface 16 of the housing to its interior 18 for receiving a sample 20 containing the one or more analytes to be determined.
  • the inlet port 14 allows the sample 20 to be introduced to a first 22 and second transport matrix 24 containing chemical reagents for determining the presence of one or more selected analytes in the sample 20.
  • the sample 20 is chemically reacted with at least one reagent on each of the transport matrices 22, 24 to produce a reaction product mixture corresponding to the reagent.
  • a portion of the reaction product mixture is transported to at least one detection zone on each of the transport matrices 22, 24 and produces a physically detectable change which correlates with the amount of the corresponding selected analyte in the sample 20.
  • each of the first 22 and second 24 transport matrices contains two detection zones 26, 28 and 30, 32 respectively.
  • Detectors 34 are positioned to measure optical radiation reflected from the detection zones 26, 28 on the first transport matrix.
  • Detectors 36 are positioned to measure optical radiation reflected from the detection zones 30, 32 on the second transport matrix.
  • the quality control zone 42 does not exhibit the physically detectable change measured in each of the detection zones.
  • Each of the detection zones and the quality control zone are examples of different types of sampling areas on the transport matrices where reflected optical radiation is sampled and measured by one of the detectors.
  • a light-emitting diode (LED) 44 provides a source of optical radiation which is directed to each detection zone 26, 28 and 30, 32 and the quality control zone 42 by a plurality of totally internal-reflecting elements (TIR) 46 which act as mirrors and as a consequence of the refractive index of the transparent material from which they are formed, require no reflective coating.
  • TIR totally internal-reflecting elements
  • the illumination from the LED 44 is split four ways. A part of the illumination is directed to the reference detector 40 from the reflecting element 48. Another part of the illumination is directed to detection zones 26, 28 from a series of reflecting elements 50, 52. The illumination is also directed to detection zones 30, 32 from a series of reflecting elements 54, 56. The reflecting element 46 illuminates another sampling area on the second assay strip 24 for a quality control detector 38.
  • Fig. 2 specifically illustrates another view of the device with an optics assembly 58 and printed circuit board (PCB) 60 disposed within the interior 18 of the housing.
  • the inlet port 14 leads to the first 22 and second 24 assay strips which are supported on the optics assembly 58.
  • Each of the detectors 34, 36, 38, 40 and the LED 44 are mounted directly to the PCB 60.
  • a liquid crystal display (LCD) 62 is also located on the PCB 60 and is positioned to direct its display through a window 64 or opening in the exterior of the housing 12.
  • the LED 44, each of the detectors 36, and the LCD 62 are connected through the PCB 60.
  • a pocket of desiccant 66 can be provided to prevent moisture from affecting the shelf life stability or the operation of the device 10.
  • the device 70 includes a housing 72 having a receptor such as an inlet port 74 which extends from the surface 76 of the housing to its interior for receiving the sample 20 containing one or more analytes to be determined.
  • the inlet port 74 allows the sample 20 to be introduced to an assay strip 78 containing chemical reagents for determining the presence of one or more selected analytes in the sample 20.
  • the sample 20 is chemically reacted with at least one reagent on the assay strip 78 to produce a reaction product mixture corresponding to the reagent.
  • a portion of the reaction product mixture is transported to at least one detection zone 80 on the assay strip and produces a physically detectable change which correlates with the amount of the corresponding selected analyte in the sample 20.
  • the resulting color in the detection zone 80 can then be compared to a color bar 82 or other reference to visually determine the presence and concentration of the selected analyte.
  • detection zone shall mean the area measured, by either visual observation or by instruments, for the physically detectable change.
  • the chemistry and configurations of the present invention may be used in an integrated assay device, the present invention can be used in any other instrumented reflectance or transmission meter as a replaceable reagent.
  • the present invention also encompasses integrated assay instruments and analytical assay instruments comprising the present assay device.
  • assays can be carried out with the present invention for a wide variety of analytes.
  • Assays that can be performed include, but are not limited to, general chemistry assays and immunoassays. Both endpoint and reaction rate type assays can be accomplished with the present invention.
  • Single or multiple assays can be done at one time. For example, a single assay can be performed measuring cholesterol or one device can be set up to measure both total and HDL cholesterol from a single sample. One test device can be set up to measure one, two, three, or more analytes at one time.
  • Analyte is the substance to be detected which may be present in the test sample.
  • general chemistry assays can be performed for analytes such as, but not limited to, glucose, cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, and BUN.
  • the analyte can be any substance for which there exists a naturally occurring specific binding member (such as, an antibody), or for which a specific binding member can be prepared.
  • an analyte is a substance that can bind to one or more specific binding members in an assay.
  • Analyte also includes any antigenic substances, haptens, antibodies, macromolecules, and combinations thereof.
  • the analyte can be detected by means of naturally occurring specific binding partners (pairs) such as the use of intrinsic factor protein as a member of a specific binding pair for the determination of Vitamin B12, or the use of lectin as a member of a specific binding pair for the determination of a carbohydrate.
  • the analyte can include a protein, a
  • such analytes include, but are not intended to be limited to, ferritin; creatinine kinase MB (CK- MB); digoxin; phenytoin; phenobarbital; carbamazepine; vancomycin; gentamicin, theophylline; valproic acid; quinidine; luteinizing hormone (LH); follicle stimulating hormone (FSH); estradiol, progesterone; IgE antibodies; vitamin B2 micro-globulin; glycated hemoglobin (Gly Hb); cortisol; digitoxin; N-acetylprocainamide (NAP A); procainamide; antibodies to rubella, such as rubella-IgG and rubella-IgM; antibodies to toxoplasma, such as toxoplasmosis IgG (Toxo-IgG) and toxoplasmosis IgM (Toxo-IgM); testosterone; salicylates; ace
  • Drugs of abuse and controlled substances include, but are not intended to be limited to, amphetamine; methamphetamine; barbiturates such as amobarbital, secobarbital, pentobarbital, phenobarbital, and barbital; benzodiazepines such as librium and valium; cannabinoids such as hashish and marijuana; cocaine; fentanyl; LSD; methaqualone; opiates such as heroin, morphine, codeine, hydromorphone, hydrocodone, methadone, oxycodone, oxymorphone, and opium; phencyclidine; and propoxyphene.
  • amphetamine an amphetamine
  • methamphetamine barbiturates
  • amobarbital such as amobarbital, secobarbital, pentobarbital, phenobarbital, and barbital
  • benzodiazepines such as librium and valium
  • cannabinoids such as hashish
  • the sample to be tested by the present invention for the presence of an analyte can be derived from any biological source, such as a physiological fluid, including whole blood or whole blood components including red blood cells, white blood cells, platelets, serum and plasma; ascites; urine; sweat; milk; synovial fluid; peritoneal fluid; amniotic fluid; cerebrospinal fluid; and other constituents of the body which may contain the analyte of interest.
  • the test sample can be pre-treated prior to use, such as preparing plasma from
  • the analyte can be any compound or composition to be detected or measured and which has at least one epitope or binding site.
  • the present invention preferably uses particle detection for a physically detectable change or detectable response in each test zone related to the level of analyte in the sample.
  • Other means for providing a physically detectable change in the test zones are suitable for use in the present invention.
  • the analyte may be labeled with an indicator to measure electrical conductance or the reflectance or absorption of a characteristic light wavelength.
  • the analyte may also be reacted with other chemicals to convert a dye, chromogenic compound or the like into a colored form detectable by means of transmission or reflectance photometry.
  • signal producing reagent and indicator are meant to include all compounds capable of labeling the analyte or conjugate thereof and generating a detectable response or signal indicative of the level of analyte in the sample.
  • Another embodiment of the present invention non-diffusively immobilizes a chemical reagent on a solid phase support or a transport matrix which provides a zone in the path through which the sample flows.
  • the transport matrix can be any solid material to which a chemical reagent can be immobilized and includes, but is not intended to be limited to, beads, magnetic particles, paramagnetic particles, microparticles or macroparticles, slides made of glass or other transparent material, capillary and test tubes, fabric or mesh that is woven or cast, and microtiter plates.
  • the transport matrix can be made from synthetic materials, naturally occurring materials, or naturally occurring materials which have been synthetically modified, and includes, but is not intended to be limited to, cellulose materials, such as paper, cellulose and cellulose derivatives such as cellulose acetate and nitrocellulose; fiberglass;
  • porous such as cotton
  • synthetic cloth such as nylon
  • porous gels such as silica, agarose dextran, and gelatin
  • porous fibrous matrices such as starch based materials, such as cross-linked dextran chains
  • ceramic materials olefin or thermoplastic materials including polyvinyl chloride, polyethylene, polyvinyl acetate, polyamide, polycarbonate, polystyrene, coploymers of vinyl acetate and vinyl chloride, combinations of polyvinyl chloride-silica; and the like.
  • the transport matrix is a porous material or wicking member.
  • porous is meant that the material is one through which the test sample can easily pass and which supports the chemical reagent for exposure to the test sample.
  • the transport matrix is used to transport the sample across an assay test zone for non-instrumented or instrumented assays to produce qualitative or quantitative results.
  • a preferred embodiment of the present invention provides the lateral flow assay strip 22 from Fig. 1 which includes three zones of which two detection zones 26 and 28 are test zones and one of the test zones is a reference zone.
  • a first zone 84 treats the sample with a chemical reagent.
  • the first detection zone 26 produces a signal with intensity inversely proportional to analyte concentration and the second detection zone 28 acts as a reference and produces a signal that is directly proportional to analyte concentration.
  • the sum of the signals from the first and second detection zones 26, 28 is substantially equal at all analyte concentrations.
  • Quantitative or qualitative results are achieved by instrumental reading of color intensity on the first detection zone 26, the second detection zone 28 or both the first and second detection zones 26, 28.
  • the results expressed by any one detection zone can also be determined as a proportion of the sum of the actual results expressed by both detection zones.
  • Quality reference is achieved by instrumental reading of both detection zones, the sum of which should be substantially constant within a specified range.
  • Both detection zones 26 and 28 contain an opacifier 86 which is preferably uniformly distributed both across the cross sectional area 88 and the depth profile 90 of the matrix 22 within the detection zones 26, 28.
  • the opacifier 86 can be distributed in one or more of the detection zones depending on whether the resolution of the particular zone requires optimization beyond its initial reflectance.
  • the term baseline reflectance signal defines the reflectance of the assay composition alone, or in a system, without the opacifier.
  • 13 can include the assay composition in combination with the components of the transport matrix and device described herein.
  • the transport matrix configuration may be of any dimension which provide the desired number of zones and which permit (a) the desired binding reactions to be completed in a reproducible manner and (b) detection of the physically detectable change or the reaction indicator to occur.
  • the present transport matrix is a total of no more than about 100 mm in length and about 6 mm wide, and more preferably, from about 10 mm to about 40 mm in length and about 1 mm to about 5 mm wide.
  • the transport matrix is advantageously integrated into any reflectance based instrument, and more preferably, into a disposable electronic assay device, such as that described in above related applications, previously incorporated by reference.
  • the transport matrix can comprise a plurality of zones along its length.
  • the zones can contain diffusively or non-diffusively bound reagents.
  • Each zone can be from about 0.1 mm to about 10 mm wide, more preferably from about 0.25 mm to about 5 mm wide. There will be a minimum of two zones and a maximum of about 10 or more zones, depending on the number of assays to be conducted on one transport matrix.
  • the transport matrix can be one continuous section of bibulous material or can be composed of one, two, three or more sections.
  • Each zone may be a separate bibulous material where each zone is in fluid communication with adjacent zones, or two or more adjacent zones may share a common material, with the other zones being different materials.
  • the transport matrix including each of the zones can be composed of the same or different bibulous materials.
  • the bibulous material permits fluid communication between the various zones, spacers (if present) and sample application site by wicking or capillary action upon application of a fluid sample.
  • the transport matrix includes a bibulous substrate to which the chemical reagent, which may be labeled, is diffusively or non-diffusively
  • Non-diffusive immobilization can be conducted by adsorbing, absorbing, crosslinking or covalently attaching the capture reagent to the bibulous substrate.
  • Diffusive immobilization can be conducted by formulating the assay reagent(s) to be immobilized (e.g., by dissolving in a suitable solvent such as water, a C,-C 4 alcohol or mixture thereof, along with any desired additives), applying the resulting formulation to the bibulous material of the membrane, filter or transport layer in the desired location(s), and drying the material.
  • suitable additives may include detergents, proteins, blocking agents, polymers, sugars or the like.
  • the additive(s) and assay reagent(s) may be applied to the membrane, filter or transport layer by precoating with a "blocking agent", water soluble polymer, sugar or detergent, followed by depositing the conjugate or conjugate formulation and drying the material.
  • Diffusive immobilization allows rapid reconstitution and movement of reagents, whether reacted or unreacted, through the bibulous substrate.
  • Non- diffusive immobilization can be accomplished by covalently attaching, adsorbing or absorbing the capture reagent to the transport matrix.
  • the zones can contain reagents diffusively or non-diffusively bound including, but not limited to, antibodies, antigens, enzymes, substrates, small molecules, proteins, recombinant proteins, viral or bacterial lysate, receptors, sugars, carbohydrates, polymers like PVA and detergents.
  • Adjusting the concentration of the opacifier is to place the analyte range in the region of most intense clinical interest or greatest resolution, requiring the greatest precision or lowest standard deviation of the error curve as illustrated in Fig. 5.
  • This usually means between 20% and 80% reflectance, but can mean as low as 10% reflectance if the electronics of the reflectometer are of high quality and do not contribute a large amount of noise.
  • S/N signal-to-noise ratio
  • the signal range or "curve separation” has only a linear affect on S/N.
  • noise has a quadratic affect because the noise component is squared. The lower the signal as represented by a lower reflectance percentage (%R) in Fig.
  • the reflectance percentages are the percentages of relative reflectance. Whereas, absolute reflectance is defined as intensity of the reflected light over the intensity of incident light.
  • the method of determining a desired range of concentration for the opacifier to optimize the resolution of the assay includes formulating a series of membranes with varying opacifier concentrations and with the desired chemical reagent for the analysis of interest.
  • a dose response experiment yields reflectance values over the desired range of analyte. If the reflectance results are at either the high (>80%) or low ( ⁇ 20%) end of the reflectance scale, then the optimal concentration of titanium dioxide is one that restores the balanced distribution of reflectance values.
  • the present invention provides for decreasing the baseline reflectance signal for the assay composition.
  • the baseline reflectance signal is optimized for the assay composition to produce at least the minimal level of reflectance decrease for the lowest analyte concentration expected to assay in the sample. This step is done prior to increasing the reflectance of optical radiation detecting a physical change of the sample-exposed assay composition to be within the range of optimal assay resolution.
  • the baseline reflectance signal is decreased for the assay composition to produce no more than the minimal level of reflectance increase for the lowest analyte concentration expected to assay in the sample.
  • Another method of optimizing the baseline reflectance signal includes increasing the intensity of the detectable color change for a given concentration of analyte expected to assay
  • Another method of optimizing the baseline reflectance signal includes adjusting the performance of the electronic components of a reflectometer, if an instrument is used to detect the physical change.
  • a decrease of the baseline reflectance signal will result in a decrease in the signal to noise ratio.
  • the signal to noise ratio of a reflectometer is determined prior to the step of increasing the reflectance.
  • the signal to noise ratio of the reflectometer is adjusted over a predetermined range of reflectance.
  • the opacifier is then impregnated in the assay composition in an amount sufficient to increase the reflectance of optical radiation detecting a physical change of the sample exposed assay composition to be within the range of optimal assay resolution.
  • the present invention provides a method for determining the level of a selected analyte in a sample.
  • the method includes optimizing a physically detectable change in an assay composition which correlates with the amount of selected analyte when contacted with the sample.
  • the assay composition is supported in a detection zone on a transport matrix and the opacifier is uniformly distributed across the two dimensional plane forming the cross-sectional area of the detection zone.
  • the opacifier is also distributed at least partially across the depth profile of the matrix in the detection zone.
  • the opacifier is distributed by contacting the assay composition or matrix with a solution containing the opacifier to impregnate the opacifier into at least a portion of the depth profile of the assay composition or matrix.
  • Another example of distributing the opacifier is to spray the assay composition or matrix with a solution containing the opacifier to achieve the desired impregnation.
  • the transport matrices shown and described herein can be configured by several assembly methods. Conventional methods of immobilizing a reagent, by dipping a transport matrix in a solution containing the reagent and subsequently drying are suitable for use in portions of the present invention. In a hybrid method, an approximately uniform layer or coating of a reagent is first deposited on the transport matrix. The uniform deposit is then
  • Example 1 The following strip assembly and chemical reagent immobilization methods were used in the construction of the inventive examples.
  • a dipping mixture of 400 mL of 15% (w/v) titanium dioxide was prepared for the creatinine reagent strip by mixing about 60 g of titanium dioxide (K-ronos 2020 - Titanium Dioxide, Rutile, purchased from Kronos, Inc., Houston, TX) and about 0.3 g. silicon dioxide (Aerosil 200 - Silicon Dioxide, amorphous fumed silica, obtained from the Degussa Corporation, Ridgefield Park, NJ) in a container.
  • About 0.3 g of sodium tripolyphosphate obtained from Sigma Chemical Co., St. Louis, MO was weighed and added along with the other dry agents to the same mixing container. Using a metal spatula, the dry agents were carefully stirred until evenly mixed.
  • the titanium dioxide mixture was transferred into a 500 mL graduated cylinder. The volume was increased to about 400 mL with 1% PVA 186K. The titanium dioxide mixture was transferred into a 1 L glass Erlenmeyer flask containing a magnetic stir bar and stirred for about an hour prior to use. This mixture was kept at room temperature, but was stirred for at least 1 hour or until homogenous before use.
  • mixtures were prepared in accordance with this procedure by varying the concentration of the titanium dioxide.
  • additional mixtures containing 5%, 10%, and 20% (w/v) titanium dioxide were also prepared.
  • Figs. 6 and 7 illustrate a laminated strip layout 130 for a sarcosine, creatinine or other general chemistry assay that is suitable for use in the preferred embodiment of the diagnostic device described above.
  • the strip layout 130 includes a sample pad 132 for receiving the sample through the inlet port (not shown) on the topside 134 of the pad 132 at the proximal end 136 of the strip 138.
  • the sample pad 132 is made of either CytoSep No. 1660 or GF/QA from Whatman.
  • the GF/QA material from Whatman, Inc. of Fairfield, NJ which is an quaternary ammonium cellulose matrix having a basis weight of about 68 g/m 2 , a thickness of about 373 ⁇ m, and a mean pore size of 4.0 ⁇ m.
  • the GF/QA material has a protein binding capacity for bovine serum albumin of 0.296 g/dg with a linear wicking (Klemm) of 2 min for a 7.5 cm rise and a derivative content of 2.0 mg/cm 2 .
  • the GF/QA material includes trimethylhydroxy propyl quaternary ammonium (QA) as a high performance strong base quaternary ammonium exchanger with fast kinetics, high protein capacity, and is effective over a wide pH range.
  • the material had approximately square dimensions of about 7 mm with a thickness of about 0.023 inches.
  • the sample flows from the sample pad 132 to a sample treatment pad 140 that is made of a material from Pall Biosupport Accuwik No. 14-20, is about 7 mm long and 3 mm wide with a thickness of about 0.00945 inches.
  • the sample treatment pad 140 is in fluid communication with a transport matrix 142 made of polyester substrate from Tetko P/N 7- 2F777 BM having a size of about 11 mm long and about 3 mm wide with a thickness of about 0.00846 inches.
  • the transport matrix 142 allows the treated sample to flow quickly towards the distal end 144 of the strip.
  • Substantially overlapping the transport matrix 142 is a spreading layer 146 that assists in spreading the treated sample across the length of the strip.
  • a reagent layer 148 substantially overlaps the spreading layer 146 and contains the chemical reagents for performing the assay to produce a physically detectable change on the top surface 150 of the reagent layer that is measured by the detector previously described.
  • the reagent layer contains the dried chemical components needed to measure sarcosine in the sample: the solution for dipping the indicator included 0.5% w/v sucrose, 1.0% w/v polyvinyl- pyrrolidone (avg. mw.
  • 5% v/v surfactant 10G p- isononylphenoxypoly(glycidol)
  • the enzyme solution used for dipping the reagent layer included 1000 u/ml horse radish peroxidase (EC 1.11.17), 500 u/ml sarcosine oxidase (EC 1.5.3.1), (all from the Toyobo Company), 1% w/v poly( vinyl alcohol) (avg. mw.
  • Triton X-l 00 t-octylphenoxypolyethoxyethanol
  • 1 % w/v sucrose 5 mg/ml Bovine Serum Albumin
  • the transport matrix 142 was dipped in the titanium oxide solution described above for several minutes and allowed to air dry.
  • the sample treatment pad 140 and the transport matrix 142 were supported and attached to a backing material 152 made of poly(ethylene terephthalate) plastic from Adhesives Research with an adhesive P N 8565.
  • the backing material was about 22.5 mm long and about 3 mm wide with a thickness of about 0.01 mm.
  • Fig. 8 presents assay results for various concentrations of sarcosine, namely about 2mM, 3mM, 5mM, 15mM, and 30mM, using transport matrices impregnated with different concentrations of titanium dioxide, namely 5%, 10%, 15%, and 20% (w/v) titanium dioxide.
  • the reflectance densities presented in Fig. 8 was measured with a hand-held reflectance densitometer made by Gretag which uses about a one mm diameter measurement area.
  • the reflectance percentage was increased by increasing the concentration of titanium dioxide, particularly with increasingly higher concentrations of
  • the increase in reflectance percentage demonstrates a means of modulating the color response of the assay by impregnating scattering centers into the transport matrix.
  • Example 2 Additional assays were prepared in a manner similar to that described in Example 1.
  • Fig. 9 presents assay results for various concentrations of sarcosine, namely about 2mM, 3mM, 5mM, 15mM, and 30mM, using transport matrices impregnated with different concentrations of titanium dioxide, namely 10%, 15%, and 20% (w/v) titanium dioxide.
  • the assay results in Fig. 9 were measured and calculated in the same manner as in Fig. 9.

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Abstract

L'invention concerne une composition de dosage pour la production d'un changement physiquement détectable lors de la mise en contact avec un échantillon, changement qui dépend de la quantité d'analyte sélectionné dans l'échantillon. La composition comporte une matrice de support (22) perméable au rayonnement optique et un réactif chimique (84) produisant un changement physiquement détectable, fonction de la quantité d'analyte sélectionné dans l'échantillon. La matrice de support présente au moins une zone de détection (26, 28) pour la détection du changement physique ainsi qu'une section et un profil de profondeur. Ladite composition comprend un opacifiant (86) présent en quantité suffisante pour que la résolution du changement physiquement détectable soit accrue.
PCT/US1998/004607 1998-03-10 1998-03-10 Dispositif, composition de dosage, et procede d'optimisation de la resolution de dosage Ceased WO1999046582A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US1998/004607 WO1999046582A1 (fr) 1998-03-10 1998-03-10 Dispositif, composition de dosage, et procede d'optimisation de la resolution de dosage
AU64552/98A AU6455298A (en) 1998-03-10 1998-03-10 Assay device, composition, and method of optimizing assay resolution

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PCT/US1998/004607 WO1999046582A1 (fr) 1998-03-10 1998-03-10 Dispositif, composition de dosage, et procede d'optimisation de la resolution de dosage

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4645744A (en) * 1983-05-12 1987-02-24 Miles Laboratories, Inc. Unified test means for ion determination
US4895704A (en) * 1985-04-23 1990-01-23 Fuji Photo Film Co., Ltd. Integral multilayer analytical element

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4645744A (en) * 1983-05-12 1987-02-24 Miles Laboratories, Inc. Unified test means for ion determination
US4895704A (en) * 1985-04-23 1990-01-23 Fuji Photo Film Co., Ltd. Integral multilayer analytical element

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