EXHIBIT 1015
`
`U.S. PATENT NO. 5,597,532 TO CONNOLLY
`
`
`
`Infopia Ex. 1015 pg. 1
`
`

`
`lllllllllllllllllIllllIlllllllllIllllllllllllllllllllllllllllllllllllllllll
`US005597532A
`
`[11] Patent Number:
`p
`
`5 597 532
`5
`9
`
`[45] Date of Patent:
`
`Jan. 28, 1997
`
`1/1989 Tarsio ...................................... 530/387
`4,797,473
`6/1989 Llotta
`.... .. 437/7
`4,837,145
`
`4/1990 Evers
`422/58
`4,913,881
`8/1990 Sugarmanetal.
`422/61
`4,952,373
`1/1991 Allen et al.
`......
`422/61
`4,987,085
`9/1991 Phillips
`...... 435/4
`5,049,437
`5,059,394 10/1991 Phillips
`422/68.1
`5,104,619
`4/1992 de Castro .
`422/56
`5,135,716
`8/1992 Thakore .......
`422/56
`5,139,685
`8/1992 de Castro .
`210/767
`5,166,051
`11/1992 Killeen
`435/71
`2:133:88
`3/1333, §1’§33%:':::.............................:::'§§é??
`
`
`
`_
`Primary Examiner—I5yle A. Alexander
`‘Wmey’ Agem’ or F’"”+1<=H°1d 1 Winger
`[57]
`ABSTRACT
`
`An apparatus for the optoelectronic evaluation of test paper
`strips for use in the detection of certain analytes in blood or
`other body fluids. The test strip comprises an elongated
`plastic part including a hinged portion to allow a first portion
`to be folded over a second portion. A series of layers of test
`strips are disposed between the folded over portions of the
`test strip. The test strip is configured such that the chemistry
`layers are placed in contacting engagement with one
`another, but not compressing one another. A reflectance
`photometer is provided and includes various features,
`including a lot number reader wherein if the test strip does
`not match the memory module, a test is not performed, and
`the user is instructed to insert a correct memory module.
`
`20 Claims, 5 Drawing Sheets
`
`Infopia Ex. 1015 pg. 2
`
`United States Patent
`
`[19]
`
`Connolly
`
`[54] APPARATUS FOR DETERMINING
`SUBSTANCES CONTAINED {N ABODY
`FLUID
`
`,
`I“"e“‘°"
`
`[76]
`
`-
`.
`1J)“m§Sd(i:°““°}1y’f1d8142§3f;“§‘§$d°
`"1 “, a“aP°‘S’
`’‘
`'
`‘
`
`[21] Appl. No.2 326,788
`_
`[22] Filed:
`Oct. 20, 1994
`159
`Int 01-“ --------------------------- GOIN 33/48:G°1N 33/49
`[52] U.S. Cl.
`................................. 422/58; 422/56; 42%61;
`435/55; 435/154
`[521 Field of Search .................................. 422/56, 58, 61,
`422/68.1; 436/66-68, 164
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`.
`
`422/58
`
`..... 435/7
`422/58
`. 436/170
`. 356/446
`. 356/446
`422/78
`422/61
`.. 422/58
`
`3/1973 Liotta ........................................ 23/230
`3,723,064
`4/1980 Tidd ..............
`. 356/448
`4,199,261
`4/1980 Wielinger et al.
`4,199,550
`5/1984 Liolta ........ .. ..
`4,446,232
`9/1984 Moore
`4,472,353
`4,477,575 10/1984 Vogel
`4,509,859
`4/1985 Markart
`4,523,853
`6/1985 Rosenbladt
`4,604,264
`8/1986 Rothe ........
`4,645,743
`2/1987 Baker et al.
`4,668,472
`5/1987 Sakamoto et al.
`4,676,653
`6/1987 Strohrneier
`4,729,657
`3/1988 Cooper .................................
`
`
`
`.
`
`356/319
`
`

`
`U.S. Patent
`
`Jan. 28, 1997
`
`Sheet 1 of 5
`
`5,597,532
`
`lnfopia Ex; 1015 pg. 3
`
`

`
`U.S. Patent
`
`Jan. 28, 1997
`
`Sheet 2 of 5
`
`5,597,532
`
`FiG. 2
`
`lnfopia Ex. 1015 pg. 4
`
`
`

`
`U.S. Patent
`
`Jan. 23, 1997
`
`Sheet 3 of 5
`
`5,597,532
`
`4 F
`
`IG.
`
`lnfopia Ex. 1015 pg. 5
`
`

`
`U.S. Patent
`
`Jan. 28, 1997
`
`Sheet 4 of 5
`
`5,597,532
`
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`Infopia Ex. 1015 pg. 6.
`
`
`
`
`

`
`
`
`5,597,5325,597,532
`
`

`
`5,597,532
`
`2
`
`1
`APPARATUS FOR DETERMINING
`SUBSTANCES CONTAINED IN A BOD
`FLUID
`V
`
`BACKGROUND OF THE INVENTION
`
`in a central area of the fiat body. A reagent chemistry pad is
`disposed in the pocket and a snap fit cover is received in the
`pocket and arranged over the pad to retain the pad in
`position. The device size and configuration allows for bar
`code graphics to "be printed on the underneath side of the
`device. The bar code may contain lot specified data about the
`reagent chemistry, and is read by the meter during device
`insertion. This data may further contain critical parameters
`for the software algorithm within the meter electronics. U.S.
`Pat. No. 5,139,685 also discloses a separation filter assembly
`having a snap fit lid. In this patent, glass fibers are utilized
`and maintained in a compressed state under pressure.
`Accordingly, a need exists for an integrated system for
`assaying analytes and whole blood samples which are not
`affected by the chemical or physical interferences normally
`caused by red blood cells and other portions of whole blood.
`
`SUMMARY OF THE INVENTION
`
`The present invention, in one form thereof, comprises a
`dry solid phase diagnostic test strip and system for the
`chemical, enzymatic, and/or immunological analysis of
`. whole blood analytes, comprising a refiectance photometer,
`a solid support strip, a porous detection zone member, a
`permeable spreading layer, an overlay sample receiving
`membrane containing an agent for the exclusion of intact red
`blood cells and a strip-receiving platform for positioning the
`strip inside the retiectance photometer. The detection area
`membrane may contain chemical, enzymatic, and/or immu-
`nological reagents that generate specific signals in the pres-
`ence of a target analyte. The agent, in contact with the
`overlay membrane, prevents passage and hemolysis of red
`blood cells while facilitating rapid transport and reaction of
`the plasma or serum portion of introduced whole blood
`samples.
`In addition, the present invention, in one form thereof,
`comprises a reflectance photometer which utilizes test strips
`that are color coded for test diiferentiation. For example, a
`blue strip may indicate a glucose test, whereas a red strip
`may indicate a cholesterol test. These colors are then divided
`into shades such as 64 shades of blue equal to 64 lot numbers
`of glucose strips. The photometer includes a separate optical
`read head that determines the color and shade of the base of
`the test strip device as the strip is inserted into the photo-
`metric instrument. The shade is converted into a lot number
`ranging from I to 64. The instrument also has a memory
`module (preferably an electrically erasable programmable
`read-only memory) that has a corresponding lot number to
`the shade of the strip to ensure lot number verification. The
`instrument
`then compares the inserted memory module
`programmed lot number to ensure that it is the same lot
`number as the test strip. If the strip lot number does not
`match the memory module lot number,
`the test is not
`performed, and the user is instructed to insert the correct
`memory module.
`-
`The lot number verification allows for the automated
`coding of lot numbers so that the user does not need to enter
`a lot code for each vial of strips. This prevents the running
`of the incorrect, old, or expired lot number tests in the
`instrument.
`
`The “plug-in memory” of the module includes the lot
`number of the test strip, the expiration date, and the perfor-
`mance criteria for the actual strip measurement. The perfor-
`mance criteria include the wavelength, measurement algo-
`rithm, and unreacted density qualifications necessary for a
`valid test result.
`'
`
`lnfopia Ex. 1015 pg. 8
`
`The present invention relates generally to an assay system
`for biological and nonbiological fluids. More particularly,
`the present invention relates to an apparatus for separating -
`10
`serum or plasma from particulate matter and then optoelec-
`tronically evaluating the serum or plasma in order to mea-
`sure analytes within the serum.
`It has long been desirable to utilize devices that can be
`used for on-site testing of blood products. Particularly
`important is the analysis of body fluids from humans and
`animals to diagnose disease, monitor the course of therapy,
`or determine the presence of illicit drugs. Commonly, the
`analytical methods used to carry out
`these objects are
`performed on blood samples.
`Clinical chemists have a preference for working with
`serum over plasma and plasma over whole blood because of
`the clarity of the sample matrix and the lack of interfering
`substances from the solid portion of the blood. In order to
`facilitate analysis, a separation step must be carried out since
`the presence of red blood cells, either intact or hemolyzed
`interferes with the signal generated by the chemical reaction
`performed by the test.
`Conventionally, the separation of blood components has
`been carried out by placing a blood sample in a centrifuge
`and centrifuging the sample for ten minutes at approxi-
`mately 3,000 rpms. The serum obtained from this centrifug-
`ing step is then used to carry out the test, thus avoiding
`interferences from blood solids such as red blood cells.
`
`25
`
`30
`
`15
`
`20
`
`An embodiment for chemical tests called dry reagent
`strips was developed first for urinalysis. Thereafter, various
`elforts to combine dry reagent strip technology in blood
`testing were started in the early l950’s. Notably, U.S. Pat.
`No. 3,092,465 discloses areagent in a bibulous carrier with
`a superimposed semipermeable coating to exclude the
`chemical and ‘nonchernical
`interferences from red blood
`cells. The device, while performing analysis on whole blood,
`still required additional manipulations by the user, in the
`form of washing of excess blood after a specified time
`interval. Additionally, U.S. Pat. Nos. 3,552,925 and 3,552,
`928 disclose the use of salts and amino acids to perform
`in-situ separation. U.S. Pat. No. 4,477,575 discloses the use
`of a glass fiber matrix.
`More recently, membranes have been employed in a
`variety of devices. These include devices disclosed in the
`following United States and foreign patents and publica-
`tions: U.S. Pat. Nos. 4,774,192 and 5,166,051; European
`Published Applications EP 0408222 A1, EP 0408223 A1, EP
`0407800 A2 and EP 0388782; and PCT Published Applica-
`tions Nos. WO 93/22453 and W0 90/10869. The use of the
`various membranes disclosed in the above patent documents
`operate on size exclusion principles, and several of these are .
`limited by rates of capillary flow and do not completely
`eliminate interference from intact or hemolyzed red blood
`cells. Fresh red blood cells are elastic in nature and may pass
`through pores smaller than their nominal diameter. Hemoly-
`sis may occur on contact with some of the architectural or
`chemical components of the strips. Consequently, errors
`may be introduced into the measurement system.
`U.S. Pat. No. 5,104,619 discloses a disposable diagnostic
`system comprising a test card having a substantially flat
`body and a generally cylindrical reagent pad pocket formed
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
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`

`
`3
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`4
`
`5,597,532
`
`The optoelectronic measurements of the chemistry test
`reaction on and in a surface enhances the dynamic range of
`the dry phase test. Algorithms that read at diiferent wave-
`lengths at difl’erent
`times in the chemistry reaction can
`extend the dynamic range of the test system. This is par-
`ticularly applicable when using multiple chromophores in a
`single measurement system. The early portion of a chemistry
`could be read at the peak wavelength of a reaction, while the
`later portion or darker or more dense portion of color
`development could be read at a wavelength not near the peak
`of the color development.
`In addition, different chro-
`mophores may respond in a more linear manner in dilferent
`portions of the dynamic range of the chemistry. Manipula-
`tion of these two data points can significantly increase the
`dynamic range (in mg/dl) of a chemistry reaction.
`The optoelectronic measurement of the chemistry test
`reaction on and in a surface reduces error due to orientation
`of the surface to be read to the instrument. Multiple wave-
`lengths and different angles are used to correct possible
`problems in positioning the strip in the instrument. If the
`detector is at “0” angle and the emitters of the same or
`diiferent wavelengths are at different angles (e.g., one at 40°
`and one at 50°), the tilting of a surface will positively
`contribute to one reading while it will contribute in a
`negative manner to the other reading thus it is able to cancel
`the error presented by the angle presentation of the surface.
`These same measurement methods can be used to eliminate
`interferences from substances such as bilirubin and others.
`
`The optoelectronic measurements of the chemistry test
`reaction on and in the surface enhance the stability of timed
`and untimed dry phase chemistry reaction. Algorithms are
`used to determine the “end point” of a chemistry. In other
`words, measurements can be done at similar or dissimilar
`wavelengths to predict the stable portion or end point of a
`chemistry. If kinetic measurements are made, the kinetic
`readings can be subjected to an algorithm to determine that
`the rate is slow enough to declare the extrapolate chemistry
`is at an end or completion. When known standards are run
`and predicted by this pseudo-endpoint, the same measuring
`criteria can be applied to unknowns to determine the “end-
`poin ” of the test reaction.
`The use of colored or shaded visual indicators in the
`instrument enhance the interpretation of test results. A
`colored bar graph is used to aid the user in knowing when
`the user test results are in a normal or safe range. Out of
`range colors such as orange for caution and red for danger
`are used when results are outside the green “safe” range.
`This is particularly useful to new testers who are not familiar
`with the number scale of the diiferent test results. A voice
`module can also be used to warn the user of unsafe results
`or operation of the instrument system to make the system
`usable by the visually impaired by providing, for example,
`a sound beep for each unit of glucose during a glucose test.
`
`20
`
`30
`
`35
`
`45
`
`50
`
`BRIEF DESCRIPTION
`
`THE DRAWINGS
`
`FIG. 1 is a perspective view of the reflectance photometer
`in accordance with an embodiment of the present invention;
`FIG. 2 is an exploded perspective view of the plastic test
`strip of present invention in its unlocked position;
`FIG. 3 is a perspective view of the plastic strip of FIG. 2
`in its locked position; and
`FIG. 4 is a sectional view of the plastic strip;
`FIG. 5 is a block diagram schematic of one embodiment
`of the reflectance photometer of the present invention;
`
`60
`
`65
`
`FIG. 6 is a graph plotting sample size, elapsed test time
`and percentage of reflectance illustrating how endpoint
`determinations may be utilized to speed chemistry measure-
`ment.
`’
`
`DESCRIPTION OF THE PREFERRED
`EMBODIMENT
`
`In accordance with the embodiment of the present inven-
`tion, the diagnostic chemistry measurement device 10 for
`dry solid phase chemical, enzymatic, immunological assay
`of whole blood or sera analytes is made up of. an injection
`molded carrier test strip 20 in which several porous and
`nonporous materials containing chemicals and reactants are
`contained for the purpose of generating a detectable signal
`in the presence of certain analytes. The test strip 12 is
`inserted into a reflectance photometer. The reaction material
`layer on the test strip 12 is held in intimate noncompressed
`contact with a whole blood separation layer in the absence
`of adhesives for the purpose of providing a liquid sample
`free of red blood cells to the reaction layer or layers.
`
`HOLDER
`
`p
`
`The holder test strip 12 of this invention acts as holder for
`the diiferent layers of the test reaction system. It provides a
`convenient handle as well as a mechanism for placing test
`strip 12 into an instrument 10 for the reading of the density I
`changes of the reaction layers. As shown in FIG. 2 test strip
`12 includes an elongate body 16 preferably formed by
`injection molding. Elongated body 16 includes a first end
`portion 18 and a second end portion 20. A hinged portion 22
`is located between first and second and end portions 18 and
`20 so that first end 18 is foldable over elongated body 16 into
`contact with second end 20.
`
`As shown in FIG. 2 first end portion 18 includes a opening
`24 while second end portion 20 includes a complementary
`spaced opening 26. When first end portion 18 is folded over
`body 16, each opening 24 and 26 are aligned. In its folded
`position as shown in FIG. 3 opening 24 in test strip 12
`defines an area for depositing a body fluid sample while
`opening 26 defines an area in which optoelectronic mea-
`surements of chemistry test reactions are conducted.
`Test strip 12 further includes an adhesiveless carrier layer
`14 formed from, for example, three particular layers. In a
`standard diagnostic test strip, carrier layer 14 may include a
`disbursement layer 28, formed of for example woven mate-
`rials such as polyester or cotton, for rapidand even dis-
`bursement of body fluid along carrier layer 14. Beneath that
`may be included a separating layer 30 constructed of known
`materials such as shown in Table IX infra, that, when
`exposed to a sample liquid, may separate analyte and analyte
`disrupting elements such as red blood cells from whole
`blood. This action would permit the serum analytes to pass
`through separating layer 30 while preventing red blood cells
`or other analyte disrupting elements from passing through.
`The last layer shown in FIG. 2 is that of the test reaction
`membrane 32 on which the dry chemicals and reactants are
`contained for generating a visible signal in the presence of
`serum analytes. Molded carrier body 16 serves as a support
`for the reacting and nonreacting layers 28,'_ 30 and 32 which
`may be formed from papers, membranes and deles materials.
`The test strip holder 12 positions the different layer
`materials 28, 31, 32 within the holder the correct X, Y, and
`Z axis positions. Carrier layer 14 made up, for example, the
`disbursement separating and test reaction layers 28, 30 and
`32 are held in noncompressed adhesiveless locations by first
`
`Infopia Ex. 1015 pg. 9
`
`

`
`5
`
`end portion 18 folding over to second end portion 20. This
`may be accomplished in a number of diiferent ways. The
`preferred way of noncompressingly holding carrier layer is
`of an upstanding annular rim 34 may help locate the carrier
`layer 14 within test strip 12. Additionally, small upstanding
`protuberances 36 along second end portion 20, radially
`located away from opening 26 prevent movement of carrier
`layer 14. The purpose of both annular rim 34 and small
`upstanding protuberances 36 is to hold the layers of carrier
`layer 14 without compression between opening 24 and
`opening 26,
`thereby preventing pooling of any sample
`within carrier layer 14. This consideration of noncompres-
`sion of the carrier layer 14 is of greater importance when
`larger numbers of layers are utilized. The positioning of a
`carrier layer 14 without adhesives or compression allows for
`efficient transport of sample and reactants contained in the
`system and test strip 12. Annular rim 34 or alternatively
`other areas of test strip 12 may include sawtooth protrusions
`to increase flow rate through carrier layer 14.
`Test strip 12. includes a locking mechanism to prevent any
`unlocking of front end portion 18 from its folded position
`over elongated body 16. As shown in FIG. 2, one type of
`locking mechanism may include a plurality of upwardly
`extending tabs or projections 38 that interfit or lock into
`corresponding openings 40 in first end portion 18. When flrst
`end portion 18 is folded to second end portion 20, lock
`projections 38 will interfit and snap lock within openings 40.
`Other types of one way locking mechanisms may also be
`used, such as snap rivets.
`More than one test reaction system can be housed in a test
`strip 12. A second set of holes 24, and 26 may be included
`in test strip 12 so that two tests may be l.'l1Il at once.
`The described holding mechanism allows for the rapid
`separation of whole blood into its liquid and solid compo—
`nents. It also allows sample volumes as low 2.0 rnicroliters
`to be used in dry phase chemistry reactions. Test strip 12
`allows the use of several reaction and non-reaction layers. A
`typical holder could contain from 1 to 8 layers of material
`with thicknesses from approximately 0.002 inches to 0.007
`inches, for example.
`Chemicals and materials are employed to allow for the
`treatment of samples such as whole blood, which will allow
`the whole blood sample to be separated without disrupting
`the red blood cells while rapidly moving the liquid portion
`of the whole blood sample to one or more reaction sites in
`the holder, norrually on a test reaction membrane 32. These
`chemicals can be composed of p_olymeric and nonpolymeric
`substances that are dried onto one or more surfaces of the
`materials contained in the device holder. Additionally, light
`metal salts of elements such as Potassium, Lithium, Sodium,
`and Calcium may be utilized to treat red blood cells before
`and during the separation process. The materials which may
`be used in the holder for treatment by or containment of
`these chemicals can be composed of woven, nonwoven,
`napped, or flocked materials.
`
`ANALYTES
`
`5,597,532
`
`6
`training. This advantage allows use at home, or by mobile
`health care delivery people. Examples of this are diabetics
`that must monitor themselves for glucose and ketone bodies,
`patients on home dialysis who would benefit by monitoring
`of urea nitrogen and people Kendeavoring to lower their
`cholesterol levels.
`
`Further, by combining several different reagents on a
`single support, a panel of tests may be done. Examples of
`this would be a liver panel consisting of ALT, AST, Alkaline
`Phosphates. A diabetic panel might consist of glucose, beta
`hydroxybutryrate and glycated hemoglobin. A coagulation
`panel might consist of Prothrombin time, AC'IT, and ACT.
`
`FAMILIES OF ANALYTES BY STRUCTURE
`
`Family
`
`TABLE I
`
`Examples
`
`Carbohydrate
`Nitrogen Moiety
`Lipid
`Enzyme
`
`glucose, lactose, galactose
`urea nitrogen, creatinine, uric acid
`cholesterol, triglycerides, LDL, HDL
`ALT, AST, Alkaline Phosphatase.
`CPK, CK-MB
`HCG, LH
`theophylline
`cocaine. marijuana, barbiturates,
`salicylates
`NIP‘. K+, Cl‘, Li’, CO2
`Electrolyte
`infectious disease, forensic, applications,
`Nucleic Acids
`genetic disorders
`
`Hormone
`Therapeutic Drugs
`Drugs of abuse
`
`20
`
`25
`
`30
`
`FAMILIES OF ANALYTES BY DISEASE
`
` TABLE II
`
`Disease
`
`35
`
`Examples
`
`Diabetes
`
`glucose, beta hydroxybutyrate, hemoglobin
`All:
`ALT, AST, bilirubin
`Liver problems
`p0,, pCO2, pH
`Acidosis/Alkalosi
`Na*', K*
`Hypertension
`
`Nutritional status Ca“, Mg“, Zn”, trace minerals
`
`EXAMPLES
`
`The following illustrative examples teach various corn»
`binations of bullets, dyes, stabilizers and other reactive and
`functional components which may be combined by a person
`having ordinary skill in the art into the system test reaction
`areas.
`
`Table IX gives various types of dyes and indicators used
`in diagnostic reagents.
`
`EXAMPLE 1
`
`Glucose measuring system
`
`40
`
`45
`
`50
`
`55
`
`TABLE III
`
`
`
`Ingredient
`
`Function
`
`Amount
`
`Available from
`
`A wide variety of analytes can be determined by using the
`disclosed apparatus. Examples are given in tables I and 11,
`infra.
`
`Further, given the small size and robust nature of the
`~ reagent strips and instrumentation, analyses need not be
`limited to traditional clinical laboratory settings. The device
`of the present invention is also simple enough to be used by
`people with minimal or no chemical or medical technology
`
`60
`
`Glucose
`Oxidase
`Peroxidase
`
`reactant
`
`reactant
`
`Silwet 7500
`
`surfactant
`
`65
`
`PVP K 30
`
`Citric Acid
`
`enzyme
`stabilizer
`Buffer
`
`25.000
`
`75,000
`
`0.10 ml
`
`0.50 gms
`
`1.25 gms
`
`Sigma Chemicals,
`St. Louis, MO
`Sigma Chemicals,
`St. Louis, MO
`Dow-Corning,
`Midland, MI
`ISP, Linden, NJ
`‘
`Aldrich Chemical,
`
`lnfopia Ex. 1015 pg. 10
`
`

`
`7
`
`8
`
`‘ 5,597,532
`
`TABLE HI-continued
`
`TABLE V-continued
`
`Ingredient
`
`Function
`
`Amount
`
`Available from
`
`Ingredient
`
`V
`
`Function
`
`Amount
`
`Available from
`
`Sodium citrate
`
`DOW I520
`
`Milwaukee, WI
`Dow-Coming,
`Midland, MI
`Aldrich Chemical,
`Milwaukee, WI
`Aldrich Chemical,
`Milwaukee, WI
`3,5 DCHBS
`chromophore
`0.25 mgs
`Boehringer
`Mannheim
`solventDistilled H20 QS to 100 ml
`
`Preparation: same as example 1
`Alternatively,
`the chromogen may be prepared in an
`organic solvent matrix and treated as avfirst or 2nd applica-
`tion to the membrane or paper.»
`
`TABLE VI
`
`
`
`Ingedient
`
`Function
`
`Amount
`
`Available from
`
`Solvent
`
`Acetone/methanol
`1:1
`Tetrarnethyl
`solvent
`1.00 gm
`Biosynth Inc.,
`
` benezicline chromogen Chicago, IL
`
`
`
`100 ml
`
`Aldrich
`
`EXAMPLE 4
`
`Blood Urea Nitrogen Measuring System
`
`TABLE VH
`
`
`
`Rrnction
`Ingredient
`reactant
`Urease
`solvent
`I-I20
`chroruogen
`Bcornthymol blue
`PVP K90
`film former
`Fructose
`filler
`
`
`4 AAP
`
`chrornophore
`
`0.25 gms
`
`
`
`
`
`Preparation: Approximately 50 ml of distilled H20 was
`placed in a" beaker on a stirring plate. A magnetic bar was
`added and the ingredients added sequentially after the pre-
`vious gradient was dissolved and dispersed. After all ingre-
`dients were added the volume was adjusted to 100 ml of
`distilled H20.
`
`20
`
`EXAMPLE 2
`
`Triglycerides measuring system
`TRIGLYCERIDES+H2O CHOLESTEROL ESTERASE GLYC- -
`EROL-l~FREE FATTY ACIDS GLYCBROL+ATPG“/CEROL
`KINASE L—ALPHA~GLYCEROPHOSPHATE+H202 H2O2+
`4-AMINOANTIPYRIN'E+DCHBS PEROXIDASE QUlNON-
`EIMINE CHROMOPHORE
`
`TABLE IV
`
`
`
`Ingredient
`
`Function
`
`Amount
`
`Available from
`
`reactant
`
`15,000 units
`
`5,000 units
`
`35
`
`Preparation: Same as experiment #1.
`
`
`
`
`system
`Butfer
`system
`antifoam
`
`0.10 ml
`
`1.00 gms
`
`5
`
`TMB
`Distilled H20
`
`chromogen
`solvent
`
`10.0 gms
`QS to 100 ml
`
`Aldrich Chemical.
`
`
`
`
`
`TABLE VHI
`
`Types of Indicators
`
`Chromogenic substrate
`Redox
`
`'Leuco dyes
`Oxidative couplers
`Benzidene Derivatives
`Fluorescent labels
`Dye releasing system
`
`TABLE IX
`
`Separation mechanisms used in dry reagents
`
`
`
`Chemical
`
`‘
`
`Physical
`
`Mechanical
`
`Dextran
`sugars
`lectin
`amino acids
`
`hydrophilic polymers
`porous latex films
`polymer & swelling agent
`membranes
`
`centrifuge
`filters
`filters & pressure
`membranes &.
`diiferenfial
`pressure
`wedge shape
`
`microfiber cloth
`napped cloth
`sintered porous matrix
`density gradient
`glass fibers
`
`PEG/polyacrylate
`thromhin
`gels
`coagulants
`agglutinating
`agents
`hollow fibers
`amine polymers
`
`trivalent cations membrane
`
`lnfopia Ex. 1015 pg. 11
`
`Cholesterol
`esterase
`glycerol ldnase
`
`glycerophosphate
`oxidase
`peroxidase
`
`4 AAP
`3, 5 DCHBS
`
`MES
`
`reactant
`
`reactant
`
`reactant
`
`5,000 units
`
`5,000 units
`
`chrornogen
`chromogen
`
`buffer
`
`1.00 gm
`0.25 gm
`
`2.50 gm
`
`Shinko-American,
`N.Y., N.Y.
`Shinko-American,
`N.Y.. N.Y.
`Shinko—American,
`N.Y., N.Y.
`Shinko-American,
`N.Y., N.Y.
`Aldrich
`Boehringer
`Mannheim
`Research
`Organics
`PVP K30
`stabilizer
`0.50 gm
`ISP
`glucose
`filler
`2.50 gm
`Sigma
`triton X-100
`surfactant
`0.10 gm
`Boehringer
`Mannheim
`solventDistilled H10 -QS to 100 ml
`
`
`
`
`
`Preparation: Same as example 1
`
`EXAMPLE 3
`
`Cholesterol measuring system (all amounts approximate)
`
`TABLE V
`
`
`
`Ingredient
`
`Function
`
`Amount
`
`Available from
`
`Cholesterol
`Oxidase
`cholesterol
`
`sodium phosphate
`0.5M pH 7.0
`B.S.A.
`peroxidase
`DOSS
`
`.
`
`sucrose
`
`reactant
`
`reactant
`
`hulfer
`
`surfactant
`reactant
`surfactant
`
`stabilizer
`
`10,000
`
`7,000
`
`750 ml
`
`15 gm
`170,000
`7.0 gms
`
`10 gms
`
`Shinl<o—American,
`N.Y., N.Y.
`. Shinko-American,
`N.Y., NLY.
`Do\v—Corning,
`
`Aldrich‘ Chemical,
`Shinko-American,
`Boehringer
`Mannheim
`Sigma Chemicals,
`
`60
`
`

`
`5,597,532
`
`9
`SPECTROPHOTOMETER
`
`The present invention also includes use of a spectropho-
`tometric device 10 for determining the density of the color
`reaction on and in the membrane surface of the test reaction
`layer 32 within test strip 12. Photometric device 10 as shown
`in FIG. 1 includes a hand-held housing 50 for containing
`electronic control circuitry for operating the aforementioned
`tests. ln the embodiment shown in FIG. 1, a test strip holding
`region 52 is located above three light detectors or sensors 54
`each disposed within a port 56. During test operation, a test
`strip 12 is inserted into holding region 52 so that test strip
`openings 26 are located adjacent ports 56. Light sensors may
`take a reading from light reflected from the exposed test
`reaction membrane layer 32 or from test strip 12 itself to
`determine its color.
`
`Housing 50 further includes a specialized display device,
`such as a liquid crystal display 58. Display 58 is utilized for
`relating test results and other information to the user. In
`particular, a color scale 60 is used to facilitate interpretation
`of test results operating concurrently with digital display
`segments 62. Additional display segments on display 58
`include a test wait indicator segment 64 to inform the user
`to wait while device 10 is performing the selected tests, and
`a test name segment 66 which the unit determined from the
`type of test strip 12 inserted.
`Color scale 60 may easily by constructed by a plurality of
`shaded or colored segments arranged adjacent each other to
`form a bar graph like indicator. Electrically controllable
`segments 68 are oriented over the color or shaded segments
`so that when segments 68 are activated segments 68 become
`dark, preventing certain colored or shaded segments 60 from
`being visualized or viewed. Segments 68 that are not acti-
`vated permit the underlying colored or shaded segments of
`color scale 60 to be visualized. In this way it is possible for
`an electronic control to permit only a single colored or
`shaded segment to be viewed thereby communicating test
`results.
`’
`
`A possible result range spectrum for color scale indication
`segments may include particular colors with particular test
`result meanings such. as:
`Very high result danger, RED
`high result danger, RED
`high result caution, YELLOW
`high result caution, YELLOW
`high normal result, GREEN
`normal result, GREEN
`normal result, GREEN
`low normal result, GREEN
`low result caution, YELLOW
`low result caution, YELLOW
`very low result danger, RED
`Color scale 60 permits an unsophisticated user to instantly
`visually determine, in one embodiment, if a test result is
`normal (a green segment visualized), slightly abnormal (a
`yellow segment visualized) or dangerous high or low result
`(a red segment visualized). Alternatively, if a color liquid
`crystal display is utilized, the electronic control for test unit
`10 may directly indicate a colored segment, rather than
`covering all but one colored segment.
`A suitable instrument, such as a ditfuse refiectance spec-
`trophotometer 10 with appropriate software, can be made to
`automatically read reflectance at certain points in time,~
`calculate the rate of reflectance change, and by using cali-
`bration factors and software, output the level of analyte in
`
`l0
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`10
`the fluid tested. The electronic control mechanism of pho-
`tometric unit 10 is shown in schematic form in FIG. 5. One
`
`or more light sources 70, for example high intensity light
`emitting diodes (LED) are disposed in housing 50 to illu-
`minate test strip 12 as shown by arrows 72. A light detector
`or sensor 54, for example a photo transistor, is able to take
`a reading of light reflected either from the surface of test
`strip 12 or from its associated test reaction membrane 32.
`Light source 70 and light sensor 54 can be adapted to
`generate or respond to particular wavelengths of light.
`Sensor 70 transmits a signal to an amplifier 74 as is known
`in the art. One type of amplifier available for use is, for
`example, a linear integrated circuit which converts the
`phototransistor current to a voltage signal.
`Appropriate electronic Circuitry is utilized to take the
`output of amplifier 74, normally a sample and hold unit 76,
`and transfer the signal to an analog-to-digital converter 78.
`Analog-to-digital converter takes the analog voltage output
`from the sample and hold unit 76 and converts it to, for
`example a 16 bit binary digital number upon command of a
`microprocessor/rnicrocontroller unit 80.
`‘
`Preferably an electronic microprocessor/microcontroller
`80 utilizing digital
`integrated circuitry is used to time
`selected tests, read signals, and together with associated
`programs and data memory 82, calculate and store reflec-
`tivity valves and calculate analyte levels from the stored
`data.
`Additional information for particular tests may be stored’
`in a removable EEPROM unit 84 operably connected to
`microprocessorlmicrocontroller 80. EEPROM unit 84 is an
`interchangeable plug-in memory module containing mea-
`surement parameters, software, calibration data, and reagent
`recognition data for particular test strips 12. Additionally,
`EEPROM unit 84 contains the shelf life data and identity
`verification information for particular production runs or lots
`of test strips 12_.
`Automated lot coding is done by the color coding the
`plastic material used to make the test strip holder 12. The
`color used in test strip holder 12 preferably has 16 different
`densities that can be distinguished by at least one of the
`wavelengths used in the optical sensor head 54 of instrument
`10. For instance the dynamic range of the % reflectances of
`the strip holder color could be as follows to determine the
`diiferent shades of color density:
`
`% Reflectauce
`Green LED
`
`% Reflectance
`Red LED
`
`Lot #
`
`