Note: Descriptions are shown in the official language in which they were submitted.
= =
CA 0221471~ l997-os-o~
WO 96/27795 PCT/IJS96/02828
--1--
ASSAYS FOR COMPOIJNDS IN CONSUMABLE ITEMS
Field of the Inventio~
This invention is in the field of assays for specific compounds in consumable items such
as foods and beverages. In particular, the invention relates to simple assays by which an
individual can deterrnine the presence of caffeine or lactose in consumable items using a simple
non-automated technique.
Bal~k~round of the Invention
The detection of compounds in various foods and beverages is a well-developed
0 technology practiced in analytical chemistry laboratories. Producers of foods and beverages
typically require laboratory services to determine the quality and safety of their products, and
also to insure that their products remain substantially uniform regardless of raw materials,
seasonal variations, etc.
It is relatively easy to ~1Ptermine the presence of a wide variety of compounds using
15 analytical chemistry techniques. However, such methods often are not available, or practical, for
individual consumers seeking to cleterrnine the presence or absence of certain compounds in their
food and beverages. For example, although "decaffeinated" coffees, teas, and soft drinks have
become increasingly popular over the past several years, the average consumer has no way of
verifying the absence (or presence) of caffeine in such beverages when receiving them in
20 re~l~uld~ and other public and private settings. It should be understood that the term
"decaffeinated" as used herein is a relative term referring to beverages having a reduced caffeine
content. For example, a cup of coffee typically contains about 100 mg of caffeine
(approximately 400mg/L) whereas a cup of decaffeinated coffee typically contains about 4 mg of
caffeine (approximately 16mg/L), and a cup of tea typically contains about 35 mg of caffeine
25 (approximately 1 40mg/L) whereas a cup of decaffeinated tea typically contains less than about 2
mg of caffeine (approximately 8mg/L). Additionally, since most people find caffeinated and
decaffeinated beverages to be very similar in taste, a consumer has very limited ability to
o ~1eterrnine whether a beverage received in a re~l~ul~ut or other setting lacks (or contains)
caffeine. Thus, if a group of diners orders a combination of caffeinated and decaffeinated
30 beverages, it becomes very difficult to assure that the server properly distributed the caffeinated
and decaffeinated beverages to the proper persons.
In a related example, a significant percentage of the population lacks the enzymes
necessary to digest lactose. This "lactose-intolerance" increases dramatically with age, starting
CA 02214715 1997-09-05
WO 96/27795 PCT/US96/02828
--2--
in infancy and progressing through adolescence. The prevalence of lactose-intolerance is known
to vary according to ethnicity. For example, virtually all Vietnamese adults arelactose-intolerant, as are approximately 95% of Native American adults, 65% of
African-American adults, 22% of C~llc~ci~n adults, and 7% of Northern European adults.
5 Lactose-intolerant individuals often experience a significant level of gastrointestinal distress if
they consume milk or other products cont~ining lactose. Although milk and other food and
beverage products that are said to have reduced lactose content or to be free of lactose are
available in supermarkets, grocery stores, restaurants and the like, those consuming such
products cannot guarantee that the products consumed actually are free of lactose until a
o significant time period has elapsed following consumption of the products.
Additionally, some foods (such as sorbet) may or may not contain milk or milk products,
and it is often verv difficult to tell if such are present at all, let alone in a lactose-reduced form.
Numerous methods are known to analytical chemists for dete- " ~ i "; l Ig the presence of
caffeine and lactose in various media. For example, in the case of caffeine, the scientific
15 lileldLu~ includes methods such as electrometric determination in which a caffeine-specific
electrode is prepared from a caffeine-picrylsulfonate ion-pair complex dissolved in octanol,
fluorimetric det~ lhldlion in which a buffered solution of caffeine is oxidized with
N-bromosuccinimide and then re~cted with dimethyl o-phenyleneAi~mine followed by a
fluorescence measurement at 480 nm; colormetric cletermin~tion in which an ethenolic solution
20 of caffeine is oxidized by potassium bromate, dried and then redissolved in dimethylformamide
followed by an absorbence measurement at 500 nm; Fourier Transform Infrared
Spectrophotometry (FTIR), thin-layer/gas chromatography; enzyme-linked immunosorbent
caffeine assays in which a caffeine-cont~inin~ sample of plasma or serum is dissolved in a
buffered solution and incubated in a vessel where it competes with peroxidase-labeled caffeine
25 for the binding sites on caffeine antibodies followed by detection of a visible color change with
the addition of o-phenylenç~ mine, imm-m~-assay of theophylline with cross-sensitivity for
caffeine; and immunoliposome assay of theophylline with cross-sensitivity for caffeine.
Likewise, numerous assays exist for determining the presence of lactose in various
media. These include the lactose enzyme electrode in which a lactose specific electrode is
30 prepared using either galactose oxidase or a beta galactosidase/glucose oxidase combination in
conjunction with an H20~ electrode, whereby lactose reacts with the galactose oxidase or
galactosidase/glucose oxidase combination to liberate peroxide which produces an
CA 022147l~ lss7-os-o~
WO 96/27795 PCT/US96/02828
--3--
electrochemical response; lactose detçrmin~tion by micro-calorimetry in which the reaction of
lactose with beta galactosidase (lactase) to produce glucose and galactose is used; Benedict's
reagent in which a cupric citrate z~lk~line solution is used to detect re~ cin~ sugars, and the
method specified in U.S. Patent No. 3,814,668 for the semi-4uaulil~live detçrmin~tion of glucose
s in a sample fluid in which a sample cont~ining glucose reacts with glucose oxidase to liberate
oxygen, the peroxidase accelerates the release of oxygen (peroxide), and the peroxide reacts with
potassium iodide releasing iodine and producing a color change to brown.
~ The problem inherent in most test procedures for lactose is that the majority are not
lactose-specific. Rather, most will also react with glucose. Furthermore, each of the caffeine
o and lactose assays described above requires the use of laboratory instrllment~tion and techniques
in carrying out the assay and, as such, they are not well suited for consumer use.
In view of the above, a need exists for a simple, inexpensive test that can be used by a
consumer to clçtennine the presence (or absence) of compounds such as caffeine and lactose in
con~u.l.able food and beverage products.
Furthermore, a need exists for a simple assay which can be carried out without the need
for instrllment~tion or laboratory techniques.
A need also exists for an assay which can determine the presence (or absence) ofcompounds such as caffeine and lactose in a food or beverage portion without adulterating the
portion (or at least a .~ipnific~nt amount thereof).
A need also exists for a simple assay which is easily portable and may be performed
quickly and discreetly.
Summary of the Invention
The present invention relates to simple assays to detect the presence (or absence) of
materials such as caffeine and lactose in con~-lm~ble items such as foods and beverages. The
assay materials may be easily carried in a pocket or purse, and the assay can be performed in a
simple and discreet manner. Each assay comprises an indicator, in the form of a wick, a capillary
tube, a dipstick or the like having ~I!lol)-iate reagents thereon, which can be contacted with a
sample taken from a food or beverage portion and can provide rapid visual fee~lb~ck indicating
the presence (or absence) of the subject compound, or in sime cases, its concentration.
In one pler~;lled embodiment, caffeine and lactose indicators are enclosed in a
matchbo.ok-type package which includes a plurality of caffeine indicators, lactose indicators, or a
combination thereof. Such a construction offers a simple. disposable, inexpensive package that
CA 02214715 1997-09-05
WO 96/27795 PCTIUS96/02828
--4--
is easily used by a consumer. In that embodiment, a consumer ordering a cup of, for example,
decaffeinated cappuccino or caffe latte, would open the matchbook, tear off indicators for
caffeine and lactose, and briefly contact those indicators with a sample of the beverage. Shortly
thereafter, colored portions on the indicators could be viewed to determine the presence or
5 absence of caffeine and lactose in the beverage.
Brief Description of the Dr~wi-~s
FIG. 1 is a schematic depiction of one embodiment of an indicator for caffeine;
~ FIG. 2 is a sçhem~tic depiction of one embodiment of an indicator for lactose;
FIG. 3 is a schematic depiction of a package cont~ininp: a plurality of indicator strips.
FIG. 4a and 4b depict a housing conts~ininp: an indicator and illustrate its method of use.
Detailed De~e~ tion of the Invention
The present invention provides a simple assay for ~leterrnining the presence (or absence)
of compounds such as caffeine or lactose in a food or beverage sample. In one embodiment, the
subject assays provide a system using a sample "dipstick" which allows a consumer to verify
5 whether the target species are present simply by contacting the dipstick with the food or beverage
sample and observing an indication, typically a colormetric indication, thereon.In the case of caffeine, d~L~ ation is made using an immlmoassay. In particular,caffeine-specific antibodies, raised in various laboratory ~nim~l~, can bond to a specific number
of caffeine molecules at a limited number of binding sites. By ~iml1lt~neously introducing a
20 caffeine sample taken from a beverage and a control standard such as a labeled caffeine sample
to a caffeine-specific antibody, a competitive assay results in which the labeled and unlabeled
caffeine molecules compete for the available binding sites on the antibody. Any unbound,
labeled caffeine is then available to contact an indicator to provide an indication of the presence
of caffeine in the consumable sample. For example, if a control standard of caffeine is labeled
25 with an agent that causes a color change in an indicator and introduced to a caffeine-specific
antibody simultaneously with a beverage sample which does not contain caffeine, all available
binding sites on the antibody would be occupied by the labeled caffeine, thereby leaving few of
the labeled moieties available in an unbound form to induce a color change in the indicator.
Thus, little color change in the indicator would be expected to develop. Likewise, if equal
30 concentrations of labeled and unlabeled caffeine were provided to the antibodies, about half of
the maximum possible color change would be expected, since about half of the antibody binding
sites would be occupied by labeled caffeine with the other half being occupied by unlabeled
CA 022147l~ lss7-os-o~
WO 96l27795 PCT/USg6/02828
-5--
caffeine. The unbound labeled caffeine complexes would remain available to induce a visual
change in the indicator. Thus, as the concentration of caffeine in the sample increases, the
number of binding sites on the antibody occupied by labeled-caffeine decreases, thereby freeing
more labeled caffeine for interaction with the indicator. The result is a greater color change on
5 the indicator. In one preferred embodiment, these effects can be produced by labeling caffeine
with peroxidase and using o-phenylenerli~min~? or other suitable indicating reagents to detect the
presence of unbound, labeled caffeine.
One ~lefelled embodiment of a caffeine indicator is depicted in FIG. 1. In that Figure,
the indicator 10 comprises a test strip 12 formed of a material having the ability to wick fluids.
0 One preferred material is filter paper. The indicator 10 includes five separate portions: a sample
liquid portion 14, a labeled caffeine portion 16, a caffeine antibody portion 18, an indicator
portion 20, and a grip portion 22.
In use, the liquid sample portion 14 is contacted with a liquid, such as coffee, to be
tested. The portion 14 may either be immersed in the coffee or the coffee may be dripped onto
5 the liquid sample portion using a spoon, dropper or the like. It is ~ler~ ~led that only the liquid
sample portion be contacted with the beverage being tested. If the entire indicator is immersed in
the beverage, caffeine present in the sample will be able to bind with the antibody faster than the
labeled caffeine, thereby providing inaccurate results. Additionally, such immersion raises the
possibility of introducing unwanted chemical reagents into the beverage. Each of these effects
20 could be avoided by providing a relatively impermeable sheath or jacket around the indicator
except in the sample liquid portion 14. Suitable jacket materials include any of a wide variety of
transparent polymeric m~teri~l~ approved by regulatory agencies for food and beverage
contacting applications.
As the sample liquid wicks upward along the indicator strip, it first encounters and mixes
2s with a labeled caffeine, such as peroxidase-labeled caffeine, that has been impregnated into the
wickable material in the labeled caffeine portion 16. The labeled caffeine mixes with any
caffeine contained in the liquid sample and is carried upward along the wick as the wicking
process cc-ntinl-~s As the sarnple liquid enters the caffeine antibody portion 18, labeled and
non-labeled caffeine compete for binding sites on the caffeine antibodies. As noted above, if the
30 sample liquid is ~l~b~ lly free of caffeine, substantially all ofthe labeled caffeine will
become bound to the caffeine antibodies. In contrast, as the amount of caffeine in the sample
liquid increases, a greater amount of the labeled caffeine will be unable to attain a binding site on
CA 0221471~ 1997-09-0~
WO 96/27795 PCT/US96/02828
--6--
the antibody and will be free to continue wicking along the indicator strip.
The sample liquid, now cont~ininp a lower percentage of labeled c~ffeine as a result of
interaction with the caffeine antibodies continues to wick along the indicator strip into the
indicator portion 20. Based upon the amount of labeled caffeine interacting with the indicator
5 (o-phenylenerli~mine or the like), the color of the indicator will react. If the sample is relatively
free of caffeine, only a small amount of, for example, peroxidase-labeled caffeine will be
available to interact with the indicator and thus, no significant color change will occur in the
~ indicator. In contrast, if the sample beverage contains a significant amount of caffeine, a greater
amount of unbound peroxidase-labeled caffeine will be available for interaction with the
o indicator and the color change of the indicator will be significant. By ex~minin~ the color
response of the indicator, the consumer thus has an indication as to whether the sample beverage
contains or lacks caffeine. Likewise, by ex~mining the amount of color change, the consumer
can determine not only the presence (or absence) of caffeine, but the relative amount present as
well. The entire indicator strip can be easily manipulated by the consumer via the gripping
15 portion 20.
It should be noted that the present invention is not intçncle~l to be limited to the specific
competitive assay method described above. For example, other approaches may be employed
including apoenzyme reactivated immnno~s~y (ARIS) and "enzyme ch~nnelling". In the ARIS
assay, caffeine is linked to the enzyme prosthetic group flavin adenine dinucleotide (FAD), the
20 co-factor for glucose oxidase. The FAD-caffeine complex competes with free caffeine for
binding to anti-caffeine antibodies. As with the competitive assay described above, the lligher
the content of caffeine in the beverage, the more unbound FAD-caffeine is available for
interaction with apoglucose oxidase, thereby converting it to active enzyme to metabolize
glucose in the presence of peroxidase and tetramethylbenzidine as color reagents. The result is
25 an increasing blue color with increasing caffeine concentration. In this embodiment, the test
device may comprise a plastic strip having filter paper impregnated with an aqueous phase
followed by drying and secondary application of an organic phase. The aqueous dip includes
anti-caffeine antibodies, apoglucose oxidase, anti-glucose oxidase (for enzyme stabilization),
peroxidase and glucose. The organic phase contains FAD-caffeine and tetramethylbenzidine or
30 similar color reagents. Further description of the ARIS procedure may be found in Greenquist,
AACC TDM-T. Vol. 6, No. 6, pp. 1-8, December, 1984, the te~rhing~ of which are incorporated
herein by reference. Of course other enzyme systems may be employed.
CA 02214715 l997-os-05
WO 96/27795 PCTIUS96/02828
--7-
In connection with ARIS arrays, a multilayer immunochemical dipstick employing
multifilm technology may be used. The result is that the reagents are spacially separated in
various parts of the reaction. Such a device is depicted schematically on page 7 of the
Greenquist reference.
In the enzyme channelling reaction, caffeine linked to peroxidase is again used. Free
caffeine in a beverage competes with caffeine-peroxidase for binding to caffeine antibodies
which are linearly spaced along a wicking path. The higher the amount of caffeine in the sample,
the greater the competition for binding sites along the wicking path. As a result, if the sample
has a high caffeine concentration, labeled caffeine is likely to wick further along the path. By
o determining the length of kavel ofthe labeled caffeine, the relative caffeine concentration ofthe
sarnple can be determined. The assay can be achieved in a multilayer dipstick model, sequential
impregn~ted papers on a plastic dipstick, or within a thin plastic "thermometer"-type device
where the height of the color (i.e., distance traveled by free caffeine-peroxidase) is proportionate
to the amount of caffeine in the beverage. Further description of the enzyme ch~nnelinp assay
may be found in Wagman et al., AACC TDM-T, Vol. 7, No. 8, pp. 1-6, February, 1986, the
te~f hin~s of which are incorporated herein by reference.
For each assay, multiple impregn~tt?rl paper strips aligned along a plastic support above a
chemical-free "wick" may be employed. Multifilm layer technology, or a defined volume
capillary tube capable of being immersed in the beverage may be used as well. In the latter case,
the capillary tube can have the a~ ;ate reagents immobilized upon the interior walls of the
lumen. The result is a hollow "thermometer"-type device. The test strips or "thermometers" may
be packaged either in a matchbook like device or in a small container readily carried by a
consumer. Depending upon size and configuration of each device, a small preme~nred plastic
sampling spoon may also be provided or physically incorporated into the assay device. Also, a
strip of a support material such as a semi-rigid plastic may be affixed to each indicator to provide
some level of physical support and ease of manipulation.
In the case of the lactose-indicating devices, the ~lef~ d embodiment includes
indicators for the presence of both glucose and lactose. This is because many of the lactose
indicators are also sensitive to the presence of glucose, but not necess~rily vice-versa. Thus, a
glucose indicator can be compared (as a control) to a lactose indicator with a ~iirr~rellce between
the two being indicative of the presence of lactose. In one ~ler~ d embodiment shown in FIG.
2, the lactose indicator 50 comprises a dipstick 52 having a glucose test portion 54 and a
CA 02214715 1997-09-05
W 0 96t27795 PCTtUS96tO282X
--8--
glucose/lactose test portion 56 mounted at the end of a plastic strip. Each test portion can consist
of an absorbent paper impregnated with an indicator solution, optionally sealed within a
polymeric film. A gripping portion 58 of the indicator is provided as well to allow the consumer
to manipulate the indicator. In the dual indicator embodiment described above, one such
formulation for the test portions is as follows: "
Chemical Name Glucose Indicator Lactose Indicator
% (WM7) % (W/W)
Potassium 1.0 1.0
Iodide
FD&C Blue 10.0 10.0
No. 1 (0.1% aq)
Water 36.4 16.4
Citric Acid, 0.6 0.6
anhyd
Sodium Citrate 5.0 5.0
Methyl vinyl ether 10.0 10.0
solution (thickener,
stabilizer) (10% aq)
Polyvinylpyrollidone 5.0 5.0
(10% aq)
Horseradish peroxidase 2.0 2.0
(5 % aq)
Glucose oxidase 30.0 30.0
(1000 units/ml)
Lactase - 20.0
(3000 LAU/ml)
In use, the consumer will contact an indicator strip with a beverage to be tested. The
40 contacting should be such that each of the glucose and the lactose test squares are each fully
contacted with the sample material. If the lactose test square undergoes a color change that is
different than that of the glucose test square, the presence of lactose in the sample is indicated.
One embodiment of a package for the subject indicator strips is shown in FIG. 3. In the
CA 022l47l~ lgg7-o9-o~
WO 96/27795 PCT/US96/02828
g
Figure, the package 80 is of the matchbox type having a cover 82, a plurality of indicators 84,
each of which is removeably mounted to a retainer 86. The cover is secured to the retainer using
a staple 88, an adhesive or the like. Each of the indicator strips 84 may be torn from the retainer
and used to indicate the presence of the desired compounds in a food or beverage sample. As
depicted in the Figure, the retainer 86 may include indicators for lactose 90 and caffeine 92 in
combination, or the package may include simply a single type of indicator. Additionally, more
than a single retainer/indicator combination can be used in each package, thereby allowing
multiple indicators with each retainer/indicator combination including a single type of indicator.
In each of the embodiments shown in the figures above, an indicator strip is held by the
consumer and contacted with a sample. However, as noted above, it is pler.,lr~d that the entire
indicator not be contacted with the sample, but rather that the sarnple be placed only on a small
portion of the indicator strip. One method for achieving this is by providing a protective jacket
or sleeve around the indicator. In another embodiment, however, the indicator may inserted into
(or provided within) a s~mplin~ enclosure or housing. One such embodiment is depicted in
FIGS. 4a and 4b. In those figures, the sample housing 100 comprises a "U"-shaped tube having
a sample collector 102 positioned at one end of the "U" . The bend 104 of the "U" forms a
sample chamber in which a liquid sample 106 is caused to contact an indicator device 108. If
desired, two indicators, for dirr~ materials, may be positioned back-to-back within the
housing.
In the embodiment depicted in FIGS. 4a and 4b, the indicator 108 is a caffeine indicator
as shown in FIG. 1. However, it should be noted that the specific indicator is not inten~lecl to be
limited to caffeine indicators, but rather, may be any indicator of the type described herein. The
housing may be of a single-use type having a single indicator perm~nently mounted therein, or in
the alternative, the leg ofthe "U" opposite the leg co"li.i..;..~ the sample collector 102 may be
25 open or openable to allow the indicator to be replaced, thereby providing the housing with a
multi-use capability. The housing may be formed of transparent glass; however, a durable,
transparent polymer is ~l~;r~ d, as such materials are less likely to suffer breakage during
transport and use.
In use, the sample collector 102 is dipped into a sample to be analyzed. For example, the
30 collector 102 may be dipped into a cup of coffee to obtain a small liquid sarnple 106 having a
volume of, for example, approximately 1 milliliter. The sample 106 remains within the
collection portion of the device (FIG. 4a) until the device is inverted (FIG. 4b). Once the device
CA 0221471~ 1997-09-0~
WO 96/27795 PCTIUS96/02828
-10-
is inverted, the sample fluid 106 flows down to the bend 104 of the "U" and contacts the
a~pl~,pl;ate portion of the indicator 108. The device is m~intzlin-ocl in the position of FIG. 4b
until the sample has had an opportunity to wick up the indicator and provide the consumer with
the desired assay. Upon completion of the assay, the device may be either discarded (in the
5 single-use embodiment) or emptied and rinsed with fresh water to allow reuse (in the multi-use
embodiment).
Having thus described at least one illustrative embodiment of the invention, various
alterations, modifications and improvements will readily occur to those skilled in the art. Such
alterations, modifications, and improvements are inten~led to be within the spirit and scope of the
o invention. For example, the materials employed, as well as their shapes and llimen~ions may be
modified according to the requirements of the device. Accordingly, the foregoing description is
by way of example only and is not intended as limiting. The invention is limited only as defined
in the following claims and the equivalents thereto.
What is claimed is:
