Method and apparatus for analyte detection having on-strip standard

A test strip is provided for having liquid applied thereto and for determining the presence or quantity of an analyte in such liquid. Specifically, the test strip comprises a reaction zone which varies in reflectance as a function of the quantity of analtye present in the applied liquid. The strip is to be inserted into an optical reading apparatus. A standard zone is positioned on the strip so as to lead the reaction zone as the strip is inserted into the reading apparatus. The apparatus may then be provided with optical means for sequentially determining the reflectance value of the standard zone as the strip is being inserted into its fully inserted position in the apparatus and the reflectance value of the reaction zone after the strip has been inserted. The apparatus is further provided with means for calculating the presence and/or quantity of analyte in question as a function of the standard zone reflectance and the reaction zone reflectance.

FIELD OF THE INVENTION 
The present invention relates to a test device and method for the optical 
determination of analytes in aqueous fluids, particularly whole blood. In 
one preferred embodiment it concerns a test device and method for 
optically measuring the concentration of glucose in whole blood. 
BACKGROUND OF THE INVENTION 
The quantification of chemical and biochemical components in colored 
aqueous fluids, in particular colored biological fluids such as whole 
blood and urine and biological fluid derivatives such as blood serum and 
blood plasma, is of ever-increasing importance. Important applications 
exist in medical diagnosis and treatment and in the quantification of 
exposure to therapeutic drugs, intoxicants, hazardous chemicals and the 
like. In some instances, the amounts of materials being determined are 
either so minuscule--in the range of a microgram or less per deciliter--or 
so difficult to precisely determine that the apparatus employed is 
complicated and useful only to skilled laboratory personnel. In this case 
the results are generally not available for some hours or days after 
sampling. In other instances, there is often an emphasis on the ability of 
lay operators to perform the test routinely, quickly and reproducibly 
outside a laboratory setting with rapid or immediate information display. 
One common medical test is the measurement of blood glucose levels by 
diabetics. Current teaching counsels diabetic patients to measure their 
blood glucose level from two to seven times a day depending on the nature 
and severity of their individual cases. Based on the observed pattern in 
the measured glucose levels, the patient and physician together make 
adjustments in diet, exercise and insulin intake to better manage the 
disease. Clearly, this information should be available to the patient 
immediately. 
Currently a method widely used in the United States employs a test article 
of the type described in U.S. Pat. No. 3,298,789 issued Jan. 17, 1967 to 
Mast. In this method a sample of fresh, whole blood (typically 20-40 
.mu.l) is placed on an ethylcellulose-coated reagent pad containing an 
enzyme system having glucose oxidase and peroxidase activity. The enzyme 
system reacts with glucose and releases hydrogen peroxide. The pad also 
contains an indicator which reacts with the hydrogen peroxide in the 
presence of peroxidase to give a color proportional in intensity to the 
sample's glucose level. 
Another popular blood glucose test method employs similar chemistry but 
uses, in place of the ethylcellulose-coated pad, a water-resistant film 
through which the enzymes and indicator are dispersed. This type of system 
is disclosed in U.S. Pat. No. 3,630,957 issued Dec. 28, 1971 to Rey et al. 
In both cases the sample is allowed to remain in contact with the reagent 
pad for a specified time (typically one minute). Then, in the first case, 
the blood sample is washed off with a stream of water while in the second 
case, it is wiped off the film. The reagent pad or film is then blotted 
dry and evaluated. The evaluation of the analyte concentration is made 
either by comparing color generated with a color chart or by placing the 
pad or film in a diffuse reflectance instrument to read a color intensity 
value. 
While the above methods have been used in glucose monitoring for years, 
they do have certain limitations. The sample size required is rather large 
for a finger stick test and is difficult to achieve for some people whose 
capillary blood does not express readily. 
In addition, these methods share a limitation with other simple 
lay-operator colorimetric determinations in that their result is based on 
an absolute color reading which is in turn related to the absolute extent 
of reaction between the sample and the test reagents. The fact that the 
sample must be washed, blotted or wiped off the reagent pad after the 
timed reaction interval requires that the user be ready at the end of the 
timed interval and wipe or apply a wash stream at the required time. The 
fact that the reaction is stopped by removing the sample leads to some 
uncertainty in the result, especially in the hands of the home user. 
Overwashing, overblotting, or overwiping can give low results and 
underwashing can give high results. 
Another problem that often exists in simple lay-operator determinations is 
the necessity for initiating a timing sequence when blood is applied to a 
reagent pad. A user will typically have pricked his or her finger to 
obtain a blood sample and will then be required to simultaneously apply 
the blood from the finger to a reagent pad while starting a timer with his 
or her other hand, thereby requiring the use of both hands simultaneously. 
This is particularly difficult since it is often necessary to insure that 
the timer is started only when blood is applied to the reagent pad. All of 
the prior art methods require additional manipulations or additional 
circuitry to achieve this result. Accordingly, simplification of this 
aspect of reflectance reading instruments is desirable. 
Great improvements have been achieved upon the introduction of the systems 
described in U.S. Pat. Nos. 5,179,005, 5,059,394, 5,049,487, and 4,935,346 
wherein an apparatus is provided for accepting a test strip having a test 
pad, one surface of which comprises a reaction zone adapted to be 
optically readable by said apparatus. The test strip is inserted into the 
apparatus, the apparatus is started and then whole blood is applied onto 
the test pad. At least a portion of such blood is allowed to permeate to 
the reaction zone whereby any analyte present therein will react with 
color-producing reagents in the test pad to alter the light reflectance 
characteristics of the reaction zone. The reflectance of the reaction zone 
is then a measure of the presence and/or quantity of analyte present in 
the blood sample. As described in the aforementioned patents, this system 
does not require a large sample of blood nor does it require the user to 
undertake timed manipulations with respect to the beginning or end of the 
reaction. Instead, because the strip is first inserted into the apparatus 
prior to the application of the sample, a standard reflectance reading of 
the reaction zone in the dry state may be obtained. The beginning of the 
reaction can be detected by the first "breakthrough" of the liquid sample 
onto the reaction zone by monitoring the reflectance and comparing the 
reading to the standard reflectance of the dry reaction zone. A 
reflectance reading taken after a predetermined time after the reaction 
has begun, and compared to the standard reflectance, i.e., the dry 
reaction zone reading, will be indicative of the quantity of analyte 
present in the sample. 
While the above described system does indeed solve the problems of the 
prior art and relieves the user of the burden of measurement and timing, 
it does require that the user apply a sample of blood onto the strip while 
the strip is in the apparatus. For the most part this represents no 
problem to the vast majority of users. However, certain users suffer from 
handicaps such as poor vision or impaired motor coordination so that the 
accurate application of blood from such users' pricked fingers to the 
strip, in place on the apparatus, represents a hardship. Further, for 
institutional users, for example, there is the possibility that some 
quantity of blood remains on the device from a prior user since the 
systems necessitate applying one's pricked finger to the device. In such 
instances there is the need to disinfect the device between users. 
Accordingly, for the above reasons, in the case of at least some users, it 
would be preferable to first apply the blood sample to the strip prior to 
inserting the strip into the apparatus. Unfortunately, by doing so the 
apparatus no longer has the capability of reading reflectance of the dry, 
unreacted, reaction zone, i.e., at no time is the dry reaction zone 
presented to the apparatus. This reading was necessary in the prior 
devices to provide a calibration standard for determining the reflectance 
change as a result of the reaction and hence the presence and/or quantity 
of the analyte in the sample. 
Certain prior systems have been devised to provide the apparatus with such 
a calibrated standard so as to allow a strip, with a sample already 
applied, to be introduced into the apparatus. In each such instance, 
however, the prior systems have complicated the user's tasks in obtaining 
a reading and have required the user to employ multiple steps in operating 
such prior systems. 
For example, the system described in U.S. Pat. No. 4,125,372 to Kawai 
discloses a test strip that includes two regions with essentially 
identical optical characteristics wherein one region undergoes a color 
change in the presence of the analyte and the other region does not. In 
this way, color variations of the changing region may be determined 
against the calibrated reading of the unchanging region after the strip is 
inserted. The calibration process, however, requires that the user insert 
the strip in steps. Firstly, the strip is inserted into a first position 
wherein the user manually adjusts a calibration knob to obtain a standard 
reading based on the non-color changing region. Then the user inserts the 
strip into a second position to obtain a reading of the color changing 
region which is then compared to the first reading to obtain a value for 
the quantity of analyte present. Obviously these multiple steps are 
undesirable and particularly so with respect to a handicapped user. In 
U.S. Pat. No. 5,037,614 to Makita again a multi-step process is disclosed 
wherein the user first inserts a clean test strip into an apparatus, then 
obtains a calibrated standard value, then removes the strip, then applies 
the sample and then reinserts the strip, each time activating the 
appropriate mode of operation of the apparatus. 
In the devices described in U.S. Pat. Nos. 5,277,870 and 5,174,963 to 
Fuller, a replaceable calibrated disk element, specific to a lot of test 
strips, is separately employed to provide a calibrated standard. There is, 
however, no means for compensation for the deterioration of such external 
standard disk with time. In addition, there is the inconvenience of the 
multiple steps of inserting the disks and then the strip. 
Accordingly, there is a need for a strip, apparatus, and methodology for 
allowing the user to apply a sample to the strip before inserting it into 
the reading apparatus while also providing a calibrated standard for the 
determination of analyte presence and/or quantity; all without the need 
for excessive manipulation, multiple steps or the danger of the 
deterioration or the misplacing of some separate calibration standard. 
SUMMARY OF THE INVENTION 
In accordance with the teachings of this invention, a test strip for 
determining the presence and/or quantity of an analyte in a liquid sample 
is provided which can be employed by first applying the sample to the 
strip and then inserting the strip into an optical reading apparatus. This 
is accomplished without requiring the user to perform additional 
manipulations to provide the apparatus with a calibrated standard to 
compare against the sample-containing strip. 
Specifically, the test strip comprises a leading edge, a trailing edge, and 
a portion for having the liquid applied thereto, this portion having an 
optically visible surface (i.e., at least with respect to the optics of 
the apparatus to be employed with the strip) defining a reaction zone. The 
reaction zone is such that its reflectance varies as a function of the 
quantity of analyte present in the applied liquid. Preferably, such is 
accomplished by the analyte, if present, reacting with reactants to 
produce a color change of the reaction zone. The test strip further 
comprises an optically visible standard zone having, along its length, a 
substantially constant reflectance. Preferably, the standard zone has a 
substantially constant high reflectance, relative to the reflectance of 
the reaction zone. The standard zone is positioned on the strip so as to 
lead the reaction zone as the strip is inserted into the apparatus. The 
standard zone of choice extends from the reaction zone toward the leading 
edge for a distance of at least 0.3 inches. 
Accordingly, the apparatus may be provided with optical means for 
sequentially determining the reflectance value of the standard zone as the 
strip is being inserted into its fully inserted position in the apparatus 
and the reflectance value of the reaction zone after the strip has been 
inserted. Additionally, the apparatus is provided with means for 
calculating the presence and/or quantity of the analyte in question as a 
function of the standard zone reflectance and the reaction zone 
reflectance. 
Owing to the configuration of the strip of this invention and specifically, 
the provision of a standard zone leading the reaction zone, the 
aforementioned apparatus need be provided with only one set of optics, 
e.g., one light emitting diode and one light detector for reading the 
reflectance at a single position along the path of the strip. Preferably 
for reasons described herein, reflectance at two specific wave lengths is 
desirable and hence two light emitting diodes are provided, albeit both 
focused on the same position along the path of the strip. 
In operation, the user turns on the apparatus, applies the sample to a 
fresh strip and then inserts the strip fully into the apparatus and reads 
the results. Without intervention of the user, the strip, configured in 
accordance with the teachings of this invention, allows the apparatus to 
read the reflectance of light incident upon the standard zone as it passes 
the optics of the apparatus as the strip is inserted. The reading is 
employed to then calibrate the apparatus to account for variations owing 
to changes in the apparatus from the factory condition and to lot-to-lot 
variations in the strip. The fully inserted strip thereafter presents the 
reaction zone to the optics of the apparatus and the reflectance of this 
surface may be read. Means are provided for the apparatus to calculate and 
report the analyte presence or concentration as a function of these 
readings.

DETAILED DESCRIPTION OF THE INVENTION 
Turning now to the drawings, FIG. 1 illustrates an exploded, perspective 
view, a strip 10 for applying a sample thereon and for inserting such 
sample laden strip 10 into an optical reading apparatus 12. This 
embodiment of the strip 10 and apparatus 12 will generally be described 
hereinafter in terms of detection and quantification of glucose but it 
will be understood by those skilled in the art that the teachings herein 
are not limited to glucose determinations, but instead may be applied to 
other analyte determinations. Further, for the purposes of simplification 
and clarity, the strip 10, the apparatus 12 and their respective component 
parts shall all be described as being in the orientation shown in the 
drawings and terms such as "the bottom" and "the top" shall be employed 
consistent with such orientation. It will be appreciated, however, that 
this method of description is merely convenient and that in no way is the 
invention restricted to such orientation and, in fact, the strip and strip 
holder may be rotated through any angle relative to the apparatus and the 
teachings herein still apply. 
As can be seen in FIG. 1, the strip 10 is adapted to be inserted 
longitudinally, into an opening 14 of a strip holder 16 carried on 
apparatus 12. Strip holder 16, shown in more detail in FIGS. 2 and 3, is 
preferably removable from apparatus 12 for cleaning. The apparatus 12 is 
provided on its visible surface with a screen 18 on which messages, 
instructions, error warnings, and most importantly, results may be 
displayed by means such as liquid crystal displays as are well known in 
the art. Such information may be conveyed by letters, words, numbers or 
icons. Additionally, apparatus 12 is provided with a power switch for 
activating the apparatus, preferably with batteries and such power switch 
is shown as push button 20 on the drawings. 
Referring now to FIGS. 2 and 3, illustrated therein in longitudinal and 
transverse cross-sectional views respectively, is the removable strip 
holder 16 with a strip 10 fully inserted therein, together with 
fragmentary views of the adjacent parts of the apparatus 12. The strip 
holder 16 is comprised of an upper guide 22 and a lower guide 24 which 
together form a channel or strip passageway 26 into which the strip is 
inserted via opening 14. The extent of full insertion of the strip is 
determined by strip impeding wall 31. It should be noted that the 
passageway 26 is canted at an angle with respect to the plane of the 
bottom 28 of the apparatus 12, so as to facilitate the insertion of strip 
10 into the apparatus when the apparatus is sitting on a flat surface. 
The lower guide 24 is provided with an aperture 30 through which the bottom 
surface 11 of the strip 10 can be "seen" by the optics located below lower 
guide 24. As will be understood hereinafter, the aperture 30 is positioned 
along the lower guide 24 so as to "see" the bottom surface of a reaction 
zone of strip 10 when the strip 10 is fully inserted into passageway 26. 
The optics for the apparatus are located in optic block 32 affixed to 
apparatus 12. Optic block 32 contains a light emitting diode (LED) 36 
capable of directing light through aperture 30, upon a surface such as the 
lower surface of the strip. The light emitting diode is preferably one 
which emits light of essentially a uniform wavelength in rapid bursts, 
hereinafter referred to as "chops", for a period of time, each time it is 
activated. For the purposes of glucose determination it has been found 
preferable to employ two such LED'S, each emitting light at a different 
wavelength and preferably at 660 and 940 nanometers (LED 660 and LED 940, 
respectively). The optic block 32 also comprises a photodetector 38, a 
device capable of intercepting light reflected from the surface upon which 
the LED's focus and converting such light into a measurable voltage. 
Incorporated into the upper guide 22 is bias means 40 which is adapted to 
be biased toward the upper surface 42 of the lower guide in the area of 
the aperture 30 so as to ensure that the portion of the strip 10 lying 
over the aperture 30 is flat and presents an optically consistent surface 
to the optics. As illustrated in the drawings, bias means 40 comprises an 
elastomeric membrane having, on its surface opposing the aperture, a 
ring-like projecting gasket 44 which is adapted to bear against the strip 
when in place and hold the strip flat to the aperture. Centered within the 
ring-like projection is a colored target, preferably gray, hereinafter 
referred to as the "gray target" 45. As will be described in greater 
detail herein, the gray target 45 presents to the optics a surface for 
assuring the correct calibration of the apparatus before the strip is 
inserted. 
The bias means 40 may take forms other than that of an elastomeric 
membrane. For example, a leaf spring can be used as such bias means. In 
commonly assigned copending U.S. patent application Ser. No. 08/302,282, 
filed Sep. 8, 1994 filed on this same day and bearing (incorporated herein 
by reference), such alternative bias means are described and include a 
particularly useful means in which the passageway 26 is designed in a 
serpentine configuration which in combination with a strip having spring 
properties serves to function as a bias means. Such a passageway is 
illustrated in FIG. 13 wherein upper guide 22 and lower guide 24 are 
shown. 
Table 1, below, recites preferred dimensions for the angles, distances and 
radii; all being based on the X,Y coordinates shown in FIG. 13. 
TABLE 1 
______________________________________ 
DIMENSIONS FOR FIG. 13 
______________________________________ 
ANGLES (Degrees) 
A 26 
B 17 
C 9 
DISTANCES (Inches) 
L.sub.1 
0.562 
L.sub.2 
0.467 
L.sub.3 
0.184 
L.sub.4 
0.013 
CURVATURE 
RADIUS (Inches) 
CENTER (X, Y In) 
R.sub.1 
0.2 0.207, 0.179 
R.sub.2 
0.347 0.391, 0.300 
R.sub.3 
0.100 0.417, 0.006 
R.sub.4 
2.635 0.412, 2.603 
______________________________________ 
Referring now to FIG. 4 illustrated therein is a planar view of the bottom 
surface 43 of a strip 46 embodying the teachings of this invention. FIG. 5 
is a longitudinal, cross-sectional view of strip 46, taken through line 
5--5 of FIG. 4. 
In the embodiment described herein for detecting glucose in whole blood, 
the strip 46 comprises an elongate and generally rectangular support 47 
onto which is attached a test pad 48 containing reactants and provided 
with an overlying transport medium 50. In use the sample is to be applied 
to the top surface of the transport medium 50 overlying the test pad 48. A 
portion of the sample penetrates through the test pad and any glucose 
present reacts with the reactants therein to produce a color change which 
is visible on the bottom surface of the test pad. A support aperture 52 is 
provided through the support for aligning with aperture 30 in the lower 
guide of the apparatus when the strip is fully inserted therein, so that a 
portion of the bottom of the surface of the test pad will be visible to 
the optics of the apparatus (such portion hereinafter, the reaction zone). 
Details of these components of the strip are described in copending U.S. 
Ser. No. 230,447, filed Apr. 20, 1994 and incorporated herein by 
reference. Briefly, the transport medium 50 comprises pores which drain 
the sample therethrough by capillary action. The transport medium may be 
composed of natural materials such as cotton or paper, as well as such 
synthetic materials as polyesters, polyamides, polyethylene and the like. 
The transport medium has pores having an effective diameter in the range of 
about 20 microns to about 350 microns, preferably about 50 to about 150 
microns, e.g., 100 microns. The transport medium is generally hydrophilic 
or may be rendered hydrophilic by treatment with surfactants compatible 
with red blood cells. One such compatible surfactant is MAPHOS.TM. 66 sold 
by Mazer Chemical, a division of PPG Industries Inc. Chemicals of Gurnee, 
Ill. In a preferred embodiment, the transport medium is capable of 
absorbing blood samples of up to about 20 to about 40 microliters, e.g. 30 
microliters. 
The transport medium may be, for example, a filter paper or sintered 
plastic material, such as those porous polyethylene materials commonly 
available from the Porex Corp. of Fairburn, Ga. The transport medium is 
generally fabricated to have a thickness of about 0.022 inch, with about 
0.25 inch width and about 1.0 inch length. The transport medium is treated 
with a red blood cell compatible surfactant solution. Since only about 3 
to about 5 microliters of blood are required to saturate the testing pad, 
the transport medium will preferably possess a small void volume in order 
not to require large volumes of blood. Excess blood applied to the reagent 
strip is absorbed and held in the portion of the transport medium which 
extends beyond the test pad. 
The test pad and its preparation are also set forth in detail in U.S. Pat. 
No. 4,935,346 and need not be described in detail herein. Essentially, the 
test pad is a hydrophilic porous matrix to which reagents may be 
covalently or non-covalently bound. Examples of a suitable material 
include polyamides, which are conveniently condensation polymers of 
monomers of from 4 to 8 carbon atoms, where the monomers are lactams or 
combinations of diamines and dicarboxylic acids, polysulfones, polyesters, 
polyethylene, and cellulose based membranes. Other polymeric compositions 
may also be used. Further, the polymer compositions may be modified to 
introduce other functional groups so as to provide for charged structures, 
so that the surfaces may be neutral, positive, or negative, as well as 
neutral, basic, or acidic. The material of choice is a hydrophilic, 
anisotropic polysulfone membrane having pores varying in size from large 
to small through the thickness of the matrix. The preferred matrix is 
obtained from the Memtec America Corporation of Maryland and has an 
average pore size of from about 125 to about 140 micrometers e.g., 130 
micrometers. The ratio of the average diameter of the large to the small 
pores is about 100. 
The transport medium 50 is attached to the test pad 48 by an adhesive (not 
shown). Suitable adhesives for this purpose include acrylic, rubber, and 
ethylene vinyl acetate (EVA) based formulations. Particularly useful 
adhesives are the hot melt adhesives known in the art. The adhesive may be 
placed in continuous stripes located only near the perimeter of the test 
pad, leaving a central portion of the receiving surface of the test pad 
substantially unobstructed. 
Alternatively, when the transport layer is composed of a material that 
fuses at industrially practical temperatures, the transport layer may be 
attached directly to the test pad by an application of heat and pressure. 
The transport layer is heated until it begins to melt and then pressed 
against the testing pad and cooled. Direct attachment of the transport 
layer to the testing pad by fusion obviates any need for a distinct 
adhesive layer. 
The transport medium is adapted to accept a whole blood sample and 
transport a detectable portion of the sample to the receiving surface by 
capillary action. The transport medium preferably extends past one or more 
ends of the test pad so as to form a reservoir for holding excess amounts 
of blood sample which may be present during actual use. It is usually more 
desirable to retain such excess amounts of the blood sample in the 
transport medium, rather than allowing the excess to drip upon the user or 
upon the viewing means in an uncontrolled fashion. Accordingly, it is 
preferred that the transport medium be capable of holding from about 20 to 
about 40 microliters of blood, preferably about 30 microliters of blood 
and of passing from about 3 to about 5 microliters of blood to the test 
pad. 
The test pad is impregnated with a color forming reagent system specific to 
an analyte. Typical analytes are glucose, cholesterol, urea, and many 
others which will readily occur to those skilled in the art. Preferably, 
the color forming reagent system includes an enzyme which selectively 
catalyzes a primary reaction with the analyte of interest. A product of 
the primary reaction may be a dye which undergoes a change in color that 
is detectable at the reaction zone. Alternatively, the product of the 
primary reaction may be an intermediate which undergoes another reaction, 
preferably also enzyme catalyzed, and participates in a secondary reaction 
which, directly or indirectly, causes a final dye to undergo a change in 
color which is detectable at the reaction zone. 
An exemplary color-forming reagent system is the system which is specific 
to glucose and contains glucose oxidase, a peroxidase, and an oxidizable 
dye. Glucose oxidase is an enzyme, usually obtained from Aspergillus Niger 
or Penicillium, that reacts with glucose and oxygen to produce 
gluconolactone and hydrogen peroxide. The hydrogen peroxide so produced, 
catalyzed by a peroxidase enzyme, such as horseradish peroxidase, oxidizes 
a dye. The resulting chromophore (the oxidized dye) exhibits a color that 
may be observed at the reaction zone. Many suitable oxidizable dyes are 
known in the art including, for example, those set out in U.S. Pat. No. 
5,304,468 incorporated herein by reference. One particularly useful 
oxidizable dye is the 3-methyl-2-benzothiazolinone hydrazone 
hydrochloride/8-anilino 1-naphthalenesulfonate dye couple (MBTH/ANS 
couple) described in copending U.S. patent application Ser. No. 245,940, 
filed May 19, 1994 (LFS-30). Many other suitable color-forming reagent 
systems specific to particular analytes are known in the art. A dye couple 
of choice is a derivative of MBTH, meta3-methyl 2-benzothiazolinone 
hydrazone! N-sulfonyl benzenesulfonate monosodium coupled with ANS. This 
combination is described in detail in U.S. patent application Ser. No. 
08/302,575, filed Sep. 8, 1994 and incorporated herein by reference. 
The support 47 may be of a material having the properties of being 
sufficiently rigid to be inserted into the apparatus without undue bending 
or kinking. Preferably, such support is comprised of materials such as 
polyolefins (e.g., polyethylene or polypropylene), polystyrene or 
polyesters. A preferred material for this support is a polyester material 
sold by the Imperial Chemical Industries Limited of Great Britain under 
the trademark Melinex 329, in thicknesses of about 0.014 inches. 
As viewed in FIG. 4, the bottom surface of the strip (i.e., the surface to 
be inserted in face-to-face relationship with the aperture 30 of the lower 
guide of the apparatus and hence the surface "seen" by the optics of the 
apparatus), can be seen to present a reaction zone 54 comprised of the 
portion of the test pad 48 visible through the support aperture 52. The 
reaction zone 54 is longitudinally placed between the leading edge 56 of 
the strip (leading with respect to insertion into the apparatus) and the 
opposite edge 58. In accordance with the teachings of this invention, a 
standard zone 60 is provided on this bottom surface of the strip, at least 
a portion of which is positioned between the leading edge 56 of the strip 
and the reaction zone 54. As illustrated in FIG. 4, the standard zone 
extends longitudinally from the leading edge to the reaction zone i.e., 
over the dimension L. As will be described in greater detail, the standard 
zone provides a calibrated standard reflectance value against which the 
reflectance of the color-developed reaction zone may be measured so as to 
allow the apparatus to compute and report the presence or quantity of the 
analyte in question in the sample. The standard zone is placed so as to 
lead the reaction zone as the strip is inserted into the apparatus whereby 
the reflectance of the standard zone may be measured as it passes over the 
optics during the process of the insertion. The standard zone should 
exhibit reflectance of a given incident light which is substantially 
constant along its length. Preferably the reflectance of light of 660 
nanometers wavelength should not vary within the length of the standard 
zone by more than from about 70% to about 100% based on the maximum 
reflectance of such light within the standard zone. It is also preferable 
that the reflectance within the standard zone contrast with the 
reflectance of the color-developed reaction zone and more preferably is of 
higher reflectance. For example, when employing a light source having a 
wavelength of 660 nanometers, the standard zone is preferably capable of 
reflecting at least four times as much light as a color-developed reaction 
zone which has had a whole blood sample applied thereto containing 100 
milligrams per deciliter of glucose. Still more preferably, when employing 
a light source having a wavelength of 660 nanometers, the standard zone is 
capable of reflecting from about four to about nine times as much light as 
a color-developed reaction zone which has had a whole blood sample applied 
thereto containing 100 milligrams per deciliter of glucose. The 
reflectance of the material of the standard zone and the material of the 
color-developed reaction zone may be measured with a spectrophotometer 
available from the Macbeth Company, a division of Kollmorgen, Inc., of 
Little Britain, Newburgh, N.Y., model number 545. 
The requisite reflectance for the standard zone may be obtained by any 
number of ways as will occur to one skilled in the art. For example, the 
support may have laminated to it, in the region of the standard zone, a 
layer having the requisite reflectance. Alternatively, the material 
comprising the support may have incorporated therein a coloring material 
imparting the proper reflectance to the region comprising the standard 
zone. As further alternatives, the coloring material may be printed or 
painted on the appropriate region. Preferably, as illustrated in FIGS. 4 
and 5, the entire support strip is comprised of a material colored to meet 
the reflectance requirements of the region of the standard zone. In this 
case there is no clearly visible boundary for the standard zone when 
viewed by the naked eye. Of course, in such case the optics will only read 
as the reflectance of the standard zone that portion of the support 
extending from the leading edge up to the beginning of the reaction zone. 
It will be appreciated that since the apparatus must read the value for 
reflectance of the standard zone as the strip is being inserted into the 
strip passageway 26, the time available for reading such value will be a 
function of the velocity at which the strip is inserted and the length of 
the standard zone, i.e., the dimension L. It has been determined that the 
highest speed that a user is apt to employ when inserting the strip is 
less than about 3.5 inches per second and that an accurate reading may be 
obtained when the standard zone is at least about 0.3 inches and 
preferably at least about 0.4 inches, e.g., 0.55 inches. 
FIG. 4A illustrates an alternative strip 62 wherein a standard zone 64 is 
provided which does not extend to the leading edge 66 but instead extends, 
for a length of dimension L, from somewhat inward of the leading edge to 
the reaction zone 68. In this embodiment, the portion 67 from the leading 
edge 66 to the beginning of standard zone 64 is provided with reflective 
properties in sharp contrast to the standard zone, e.g., a low reflectance 
as contrasted with a high reflectance for the standard zone. Accordingly, 
the apparatus may be programmed to expect first a low reflectance followed 
by a high reflectance if the strip has been properly inserted with the 
bottom surface facing the optics. Should the apparatus fail to detect such 
abrupt change as the strip is being inserted, means may be provided for 
reporting the error, i.e., the strip has been inserted upside down. It 
should be noted, of course, that this means of detecting an upside down 
strip is based on providing a different reflectance pattern on the 
opposite surface of the strip. 
To better understand the strip of this invention and the mode of using the 
same, reference is made to FIG. 6 which illustrates schematically the 
functional features of the apparatus as the strip is inserted therein 
together with FIGS. 7-11 which schematically illustrate the strip in 
various positions during the insertion process. 
As illustrated in FIG. 6, a strip 46 such as that described in connection 
with FIGS. 4 and 5 is being inserted in the direction of the arrow, into 
the strip passageway 26 of apparatus 12. A bias means 40 is provided to 
urge the strip flat against the aperture 30 to ensure consistent optical 
performance. The bottom surface 45 of the bias means 40 presents the gray 
target to the optics of the system when no strip is in place. Within the 
apparatus and focused on the surface presented through aperture 30 is at 
least one LED 36. For the purposes of glucose determination, two such 
LED's are employed emitting beams of light at 660 and 940 nm, 
respectively. A photodetector 38 is positioned to detect light reflected 
from the surface presented to the aperture 30 and communicate such 
detected light to an analog/digital convertor (A/D) 39 whereby reflected 
light is converted from a voltage into a digitized signal which, in turn, 
is communicated to microprocessor 33. The microprocessor also communicates 
to the LED's 36 via a digital/analog convertor (D/A) 35 to control the 
sequence of operations of the LED's in accordance with the programmed 
operation of the apparatus. The microprocessor also controls the operation 
of the output, i.e., the instructions, messages, and results reported on 
the liquid crystal display screen of the apparatus. 
FIGS. 7-11 illustrate, schematically, sequenced positions of the strip with 
respect to the aperture 30 as the strip is inserted into passageway 26. 
Thus, in FIG. 7 the strip 46 has just been inserted into the passageway 
and the leading edge has not yet reached the aperture. Accordingly, the 
surface presented to the optics is solely the gray target at the bottom of 
the bias means (position A). In FIG. 8, the leading edge and the beginning 
of the standard zone have partially occluded the aperture and hence the 
optics see portions of both the gray target and the standard zone 
(positions B through C). In FIG. 9, the strip has totally occluded the 
aperture 30 and the optics see only the standard zone (positions C through 
D). In FIG. 10, the interface between the standard zone and the reaction 
zone, lies over the aperture 30 and the optics see portions of both zones 
(position D through E). Finally, referring to FIG. 11, the strip is fully 
inserted and the optics see only the reaction zone (positions E through 
F). 
The reflectance of the surface presented to the optics is measured by the 
apparatus at each of these positions. Multiple readings are taken at each 
position in spaced periods of time. Each such reading comprises a number 
of bursts of energy imparted to the LED in response to directions from the 
microprocessor. These bursts, referred to as chops, control the amount of 
light energy directed to the surface for each reading, i.e., at a constant 
power level, the greater the number of chops the greater the light energy 
incident upon the surface being measured. The light energy reflected by 
the surface during each reading is captured by the photodetector and 
converted into a voltage. The voltage is allowed to decrease to zero over 
a period of time and the time it takes to decrease to zero is a measure of 
the light energy absorbed by the photodetector, i.e., the light reflected 
from the surface being measured. Such time period is measured in units 
called counts and hence the number of counts represents the light energy 
reflected from the surface. FIG. 12 is a plot of the counts or light 
energy reflected from the surface presented to the aperture as a function 
of the position of the strip as it is inserted into the apparatus. The 
position of the strip corresponding to FIGS. 7 through 11 are noted by the 
corresponding letters A through F. Thus, referring to FIG. 12, when the 
strip is in the position shown in FIG. 7 (positions A through B) only the 
gray target is presented and the light reflectance is at a low constant 
value. When the strip is in position B through C, the gray target is being 
occluded by the highly reflectant standard zone and hence the light 
reflection detected increases as occlusion proceeds. When the strip is in 
position C to D, the standard zone is presented to the optics and the 
light reflection becomes a constant high value. When the strip is in 
position D through E an increasing proportion of the aperture is presented 
with the relatively low reflective surface of the reaction zone and a 
decreasing portion of the relatively high reflective standard zone and 
hence the light reflection detected rapidly decreases. Finally, when the 
strip reaches positions E through F and beyond only the reaction zone is 
visible to the optics and a relatively low constant light reflectance is 
detected. 
With the above described relationship of the strip position and the output 
of the optical reading apparatus in mind, the calibration and operation of 
the system will next be described. 
It will be understood that each apparatus and strip combination will 
qualitatively behave as has been described herein. However, variations 
between specific apparatus, variations in time in a given apparatus and 
variations in lot to lot manufacture of strips must all be accounted for 
before an accurate value for an analyte such as glucose in a sample liquid 
such as blood can be ascertained. To do this, each apparatus must be 
factory adjusted prior to release and each lot of strips must be coded for 
its own reflective characteristics so that when the apparatus is turned on 
and put to use, an internal calibration is made to account for changes in 
the apparatus after it has left the factory and for changes from lot to 
lot for the strips. 
Firstly, each apparatus must be adjusted to provide the proper quantity of 
light energy to be emitted by each LED (for glucose LED 660 and LED 940). 
As has been described, such light energy is a function of the number of 
chops and the power supplied to the LED. These parameters are adjusted in 
the factory so as to produce, in a given apparatus, an arbitrarily chosen 
light reflectance from a white standard zone, such reflectance value 
arbitrarily being selected at 4,000 counts (the time for the photodetector 
to degrade accumulated voltage to 0). The degrees of freedom of the system 
allow the power to be adjusted so as to achieve the goal of about 4,000 
counts while constraining the number of chops to a value which is 
approximately 55 chops per reading. With each of the 660 nm wavelength and 
940 nm wavelength LED's set at its own factory determined value of chops 
(CHP 660 and CHP 940) and power, a reading for the reflectance of the gray 
target of the apparatus for each LED is made and stored in the 
microprocessor as the calibrated gray reading for each LED (RCG 660 and 
RCG 940). 
In operational mode in the hands of the user, when a glucose determination 
is to be made, the user first powers the apparatus. At this point, the 
microprocessor directs certain diagnostic checks to be made. For example, 
the battery voltage is checked to assure that it is sufficient. Further, 
the operating temperature is checked. It will be understood that since the 
determination of the analyte, e.g., glucose, is dependent upon a chemical 
reaction occurring within the test pad of the strip, the rate of such 
reaction going to completion will be a function of temperature. 
Accordingly, if the temperature is too low or too high, e.g., less than 
10.degree. C. or greater than 40.degree. C., the apparatus will report an 
error. If the temperature is low but still operable, the apparatus will 
adjust for such low temperature by extending the reading time of the 
reaction zone. 
Having made these diagnostic tests, the microprocessor will adjust the 
optics (autoscale) to account for any variations occurring after the 
apparatus has left the factory. As has been described above, with a strip 
out of the apparatus, the optics view only the gray target. Accordingly, 
the microprocessor directs a reading of the gray target employing the LED 
940 at 3 chops. If the reflectance reading is less than a predetermined 
value, it is assumed that the gray target is missing or out of position 
and an error is reported. If the reflectance value is higher than a 
predetermined value, it is assumed that a strip has been prematurely 
inserted into the apparatus and again an error message is reported. As is 
generally the case, when the reflectance reading is between the two 
predetermined values, the apparatus begins the adjustment or autoscaling 
as follows. The apparatus views the gray target, reads a value for its 
reflectance at each LED wavelength using the factory determined number of 
chops (calibrated CHP 660 and calibrated CHP 940) and compares these 
values to the factory stored values for the gray reading. If the reading 
differs, an adjustment is made in the number of chops for each LED to 
bring the numbers into closer agreement. Such autoscaling is based on the 
following calculation: 
##EQU1## 
wherein RDG 660 and RDG 940 are the current gray target readings from LED 
660 and LED 940, respectively. 
In the event that the adjustment exceeds a predetermined limit, the 
apparatus will report an error. 
The microprocessor then causes the apparatus to advise the user, via the 
screen, to apply a sample to a strip and then insert the same into the 
apparatus. At this time, the microprocessor initiates the procedure for 
detecting the leading edge of the standard zone. This is accomplished by 
rapid readings of the reflectance of the surface presented to aperture 30 
using LED 940 at a low number of chops per reading, e.g., three chops per 
reading. If a reflectance is read which is greater than a predetermined 
number of counts, chosen to be an indicia of a highly reflective surface 
appearing in the aperture, the leading edge of the standard zone is 
considered detected. 
The apparatus is next programmed to read the reflectance of the standard 
zone. It has been found that calibrating the reflectance of the standard 
zone to the maximum reading obtained, provided at least three valid 
readings are obtained as the strip is inserted, will give accurate 
results. Accordingly, the microprocessor is programmed to cause the 
readings to begin, at detection of the strip, with LED 660 and then 
alternate readings between LED 940 and 660 at a reduced number of chops 
with respect to the autoscaled number of chops. The reduced number of 
chops allows more readings in the time available as the strip is inserted 
and produces adequate resolution for the purposes of calibration. In 
practice, the microprocessor, for each reading at each wavelength, 
replaces the prior value stored as the reflectance with the next read 
value if, and only if, such next read value exceeds the stored value. In 
this way, only the maximum reflectance reading for each wavelength is 
stored, after the entire standard zone is scanned. 
The maximum reflection reading of the standard zone may then be scaled to 
the autoscaled number of chops at each wavelength as follows: 
##EQU2## 
wherein RMX 660 and RMX 940 are the maximum detected readings for the 
reflectance of the standard zone and RW 660 and RW 940 are the now 
calibrated values for the standard zone at the respective autoscaled 
number of chops for each LED. N IS EQUAL TO THE REDUCED NUMBER OF CHOPS! 
For each of the LED 660 readings, the ratio of such reading to the prior 
maximum value is calculated. If such ratio falls below a predetermined 
value, e.g., 0.7, it is assumed that the interface of the standard zone 
and the reaction zone has been reached. Should this occur before three 
valid readings for maximum reflection in the standard zone had been made, 
then the microprocessor shall cause the screen to report an error to the 
user, assuming that the strip has been inserted too fast. Further, should 
the reaction zone not be detected within a predetermined time, e.g., 15 
seconds, it will be assumed that the strip has not been properly inserted 
and an appropriate message will appear on the screen. 
Having determined the calibration value for the standard zone, a further 
internal calculation is made to ensure that the optics are not dirty or 
otherwise impaired by employing the reflectance reading taken on the gray 
area when the apparatus was first started and the now determined 
calibrated standard reflectance. It is assumed that, provided the optics 
are clean and operable, the K/S ratio between the gray target and the 
standard zone are constant over the life of the product, within a 
predetermined tolerance, e.g., .+-.15%. K/S is the calculated value 
employed in the Kubelka-Monk equations derived specifically for 
reflectance spectrometry from employment of Beer's Law and described in 
some detail in U.S. Pat. No. 5,179,005, and in greater detail in Journal 
of Optical Society of America; Vol. 38; No. 5; May, 1948; pp 448-457. In 
accordance with the Kubelka-Monk equations: 
##EQU3## 
wherein R* is the ratio of the reflectance in question to a standard 
reflectance. The K/S for the gray target at each wavelength is determined 
as a function of the initial reading of the gray target and the calibrated 
standard zone reflectance to determine if these correspond with the K/S 
ratios calculated from the factory stored reflectance data for these two, 
within prescribed tolerances. If not, an appropriate error message is 
reported. 
Once this internal check is completed, the apparatus is programmed to 
examine the reflectance of the reaction zone and determine when the 
reaction between the putative analyte (glucose in the described 
embodiment) and the reactants in the test pad has gone to an end point 
(within a prescribed tolerance). The end point is detected by reading the 
reaction zone once every second with the LED 660 at the autoscaled chops 
until completion is detected. The readings are converted into K/S data as 
described above (hence a function of the calibrated standard zone reading 
and the reading taken on the reaction zone) until no change, within 
prescribed limits, is detected in the K/S reading and it can be assumed 
that the end point has been reached. 
In addition to detecting the end point by reading the reaction zone with 
the LED 660, another reading is made by the LED 940 after a predetermined 
interval during the end point detection process, e.g., 30 seconds after 
the process has begun. This is made to assure that a reflectance reading 
is within prescribed ranges indicative of the fact that a proper amount of 
sample has been deposited on the test pad. Should these ranges be 
transgressed, an error will be reported. 
Having determined that the end point has been reached and the proper 
quantity of sample has been applied, calculations are next made to 
determine the analyte (glucose) content of the sample. The last K/S data 
taken from a reading of the reaction zone when the end point has been 
detected, KS660, is employed and is first corrected for the factory 
determined calibration using a linear correlation, e.g.: 
EQU KSMCAL=F(KS660)+G 
wherein F and G are coefficients provided to the microprocessor of the 
particular apparatus in the factory. The KSMCAL value is further corrected 
to provide for idiosyncrasies in an individual manufacturing lot of 
strips. Each lot has been tested in the factory and given a single code 
number. The code number references a set of coefficients, e.g., 21 sets 
stored in the microprocessor of each apparatus and indexed against said 
code number. For example, a linear correlation is believed adequate to 
account for lot to lot variations in the strips in the glucose test and 
hence two coefficients per set are stored against each code number. Upon 
inserting a strip into the apparatus, the user will be asked to enter the 
proper code number found on the package of the strips being employed. The 
microprocessor will then, employing a look-up table, be apprised of the 
proper coefficients. It will be understood that the strip itself may be 
provided with an apparatus readable code thereby obviating the need to 
enter the same. In any event, the apparatus corrected K/S ratio KSMCAL, is 
further corrected with respect to the strip, as: 
EQU KSSCAL=(KSMCAL)M+B 
wherein KSSCAL is now the strip corrected K/S ratio and M and B are the 
looked-up coefficients. Finally, the analyte (glucose) concentration, 
preferably in units of mg/dl of sample, is calculated in accordance with a 
trinomial correlation: 
EQU G=K.sub.1 +K.sub.2 (KSSCAL)+K.sub.3 (KSSCAL).sup.2 +K.sub.4 (KSSCAL).sup.3 
wherein G is the glucose concentration and K.sub.1, K.sub.2, K.sub.3, and 
K.sub.4 are empirically derived constants. 
Alternatively, a look-up table which reflects such correlation may be 
supplied to the microprocessor. 
The invention having now been fully described, it will be apparent to one 
of ordinary skill in the art that modifications and changes can be made 
thereto without departing from the spirit or scope of the invention as 
defined in the following claims.