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Description  |
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FIELD OF THE INVENTION
The present invention relates to a technique which allows the user to
comparatively determine analyte levels in whole blood. More particularly,
the present invention relates to a comparative reagent strip which allows
the user to determine levels of analytes in whole blood. Most
specifically, the present invention relates to a comparative reagent strip
which separates whole blood into cells and a fluid and from which is
determined analyte levels through means of a visual test or various
instrumental means.
BACKGROUND OF THE INVENTION
Numerous simple visual test devices have been developed for the analysis of
body fluids in order to determine component analyte amounts. These tests
include such devices as means for detecting glucose or other sugars in
urine or in blood as well as protein in urine, ketones, uric acid,
phenylalanine or enzymes, only to mention a few. All of these tests detect
various soluble analytes.
Yet, it has been particularly difficult to perform visual tests of these
constituents in whole blood. This difficulty lies in the problems
associated with visual responses to the presence of red blood cells in
whole blood. The dense red coloration of red blood cells and hemoglobin
seriously interferes with such analysis.
Means have been proposed for separating and removing highly colored red
cell components from whole blood prior to analysis. Some of the simpler
methods involve the use of a carrier member impregnated with a test
reagent composition and coated with a semipermeable membrane which
effectively acts as a means for screening out large molecules such as
hemoglobin. This semipermeable membrane permits the passage of smaller
molecules or ions in the solution. A substantially clear fluid containing
the constituent diffuses into the test reagent in the carrier to cause a
chromogenic reaction with the reagent.
Other methods provide for the drawing of whole blood, then allowing the
blood to clot. Once clotted, the blood is centrifuged to separate cell
components.
These methods are cumbersome and generally laborious and require at least
one extra manipulative step such as wiping, blotting or rinsing with
water. This amounts to considerable loss in time and more importantly,
accuracy and efficiency. Moreover, the membrane screens out larger
molecules in solution, which precludes these molecules from reaching the
test reagent. This sometimes renders these methods inoperative for
particularly needed determinations. These methods are also
technique-dependent and difficult for untrained operators to perform.
Other methods have included taking whole blood samples and placing such
samples on a bicomponent reagent strip. After a predetermined time period
lapses, the blood sample is blotted to remove excess blood. At that point,
constituents of the whole blood sample react with molecules in the reagent
strip, and a visual comparison test is performed.
Other test systems may comprise a single matrix which contains both a
separating reagent and a test reagent in such a way that the whole blood
first contacts the separating reagent to form a substantially colorless
fluid which then contacts the test reagent. In employing such a single
matrix test system the separating reagent must be compatible with the test
reagent for both reaction and stability during storage. The matrix must be
designed so that the blood sample reaches the area of the device where the
response is read substantially free of any blood coloration. In such an
embodiment, a porous support is first coated or impregnated with the test
reagent and subsequently the surface of the matrix is coated or
impregnated with the separating reagent. In such a test device, the whole
blood is contacted with the separating reagent and the test response is
observed in an area not initially contacted with the blood and to which
the substantially colorless fluid or serum has migrated.
Examples of such single matrix test strips included separating reagents
which have been found to be, among other things, water-soluble salts,
amino acids and carbohydrates such as mannitol. Some of these chemicals
cause hemolysis which releases cellular constituents, including
hemoglobin. The salts found effective as separating reagents are
non-volatile and do not decompose to any extent under the conditions of
preparing and utilizing the test device. The salts have been defined as
having solubility in distilled water of at least about 1 gram per liter at
20.degree. C.
In many instances, red blood cells or hemoglobin continue to seep through
the separating reagent so that the test reagent encounters colored blood
components. When this occurs, accuracy levels are destroyed, and visual
comparison is difficult.
It is thus an object of the present invention to provide a unitary test
device, wherein during one step the user can apply an unmeasured sample of
whole blood and determine analyte levels in the whole blood sample.
It is therefore another object of the present invention to provide a
unitary test device wherein the test device, whether single or
multi-layer, contains separating means as well as test reagent.
It is a further object of the present invention to form a test device
consisting of a single matrix wherein whole blood samples can be applied
to one side and visual comparisons of analyte levels can be made at the
opposite side of the test strip, or alternatively in a longitudinal
transport device, such readings made on a second portion of test strip
after wicking.
It is yet a further object of the present invention to determine glucose
levels in whole blood samples where a wholly unmeasured sample of whole
blood is applied to a single side of a reagent strip. The separating
reagent and test reagent are coated on or trapped within the reagent strip
and both work effectively and simultaneously to separate and react with
the separated clear fluid sample in order to determine, visually, glucose
levels of the wholly unmeasured whole blood sample.
It is finally an object of the present invention to provide a test device
such that whole blood is analyzed in a single manipulative step for
selected molecular constituents such as glucose by a combination of
separation means and detection means.
SUMMARY OF THE INVENTION
These and other objects of the present invention are accomplished in a
single membrane test strip which is attached to a support member. This
test strip is treated with both a separating reagent and a test reagent.
Both the separating reagent and test reagent may be found throughout the
entire test strip matrix or may be found predominately on one side of the
matrix. In the method of the present invention, whole blood is applied to
one side of the matrix. As the whole blood passes through the matrix,
separation and reaction occur. Ultimately, the whole blood is separated
into red blood cells and a substantially colorless fluid. Because the
matrix is configured with such a thickness to cause the red blood cells to
become separated within a first portion of the strip of the matrix, the
lower portion of the matrix containing a substantially colorless
constituent reacts with the test reagent alone. The test reagent is, of
course, configured to accurately determine (visually) the predetermined
levels of analytes. Therefore, the resulting configuration on the test
side of the matrix will be a test reagent which has reacted to the clear
constituent and enables visual or instrumental determination of analyte
levels.
In an alternate embodiment of the present invention, whole blood is placed
on a testing surface comprising a disc of porous material, which is then
contacted with a matrix treated with both separating reagent and test
reagent. This closed strip then allows the blood to separate and react in
the same fashion. A final visual comparison is then made on the test side
of the matrix.
In addition, the present invention may be configured so that a separating
membrane and reagent membrane are incorporated within the same test strip.
The same separation techniques are applied to the whole blood sample.
After separation, the same reaction takes place between the separated
sample and the reagents in a reagent membrane. Alternately, the device may
be configured so that varying degrees of separation or reaction may take
place simultaneously in the matrix on the same layer. Therefore, visual
comparison can be made on the test side of the reagent matrix.
The present invention will be more accurately understood in conjunction
with the following detailed description of the invention as well as the
present detailed description of the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective view of a preferred embodiment test strip of the
present invention illustrating a matrix fastened to a plastic holder which
defines a hole;
FIGS. 2a, 2b and 2c are top, bottom and side views respectively of an
alternate preferred embodiment of the present invention, respectively,
depicting a plastic support upon which are fastened two separate matrices;
FIG. 3 is a perspective view of a second alternate preferred embodiment of
a test strip of the present invention in a closed position showing a clear
support which defines a well;
FIG. 4 is a perspective view of a second alternate embodiment of the
present invention in an open position; and FIG. 5 is a top view of the
second alternate preferred embodiment of the present invention in the
closed position, which displays the manner in which the support may be
folded.
DETAILED DESCRIPTION OF THE INVENTION
The subject invention provides an improved rapid, simple methodology
implying reliable and easy to operate apparatus for determination of
analytes such as glucose, particularly involving a substrate which results
in the change in color in order to determine analyte levels in whole
blood. The method involves applying to a porous matrix a small volume of
whole blood, sufficient to saturate the matrix. The matrix may be either
one single layer or a combination separation matrix and reagent matrix.
Bound to or contained in the matrix are one or more reagents of a signal
producing system, which results in production of a change in the color of
the matrix when combined with analytes in blood. The liquid sample
penetrates the matrix and an observation is made on the opposite side of
the matrix from where the sample is placed, as a result of the separation
of whole blood into clear and colored constituent components and reaction
of the clear component with a testing reagent.
For measurements of blood, particularly glucose measurements, whole blood
is typically used as the assay sample. The matrix will contain both a
separating agent and a reaction agent. The reaction agent produces a light
absorbing product which changes either color or intensity dependent upon
concentration of the analyte in the whole blood sample. The time span
within which the blood is able to be separated and reacted typically
varies from about 15 seconds to about 5 minutes.
The first component of the present invention comprising the reagent test
strip 10 to be considered is a reagent element 11, as seen in FIG. 1. This
reagent element 11 comprises an inert porous matrix 16 and the component
or components of a signal producing system, which is capable of reaction
with an analyte to produce a color variable reaction product on the
non-sampling side of the porous matrix 16. As previously noted, the porous
matrix 16 may be a single or multi-layer element. The signal producing
system allows flow of liquid through the matrix. In order to assist in
reading the color-produced scheme, it is preferred that the matrix 16 have
at least one test side 18 which is substantially smooth and flat.
Typically the matrix will be formed into a thin sheet with at least one
smooth, flat side.
In use, the liquid sample being analyzed is applied to one side of the
matrix 16 sheet whereby the desired analyte passes through the reagent
element by means of capillary action, wicking, gravity flow and/or
diffusion. The components of the signal producing system present in the
matrix will react to give a light absorbing reaction product, whose color
will be dependent upon the analyte concentration in the liquid sample.
The first component of the reagent element 11 is the matrix 16. The matrix
will be a matrix to which reagents may be covalently or noncovalently
bound or impregnated. The matrix 16 will allow for the flow of an aqueous
medium through the matrix 16. The matrix 16 will also retard passage of
whole blood cells through the matrix without substantial hemolysis and
without significantly adversely affecting the identity or concentration of
the analyte in the blood sample. Composition of the matrix 16 will be of
sufficient thickness, preferably 50 to 3000 microns, to permit the
formation of a colored reaction product on the test side 18 of the matrix,
opposite a side where the sample is applied, so that essentially clear
constituent reacts with the test reagent embedded in the matrix 16. The
matrix 16 also should not deform substantially upon wetting so as not to
interfere with subsequent quantitation. The matrix 16 thus substantially
retains its original size and flatness.
As exemplary of matrix surfaces are porous polyethylenes, especially
matrices having a porosity of between 0.5 and 150 microns. Especially
useful are matrices which are coated with polyethylene glycol, polystyrene
sulfonic acid or polyvinyl sulfonic acid at a pH between 4.0 and 8.0.
However, it has been observed that sufficiently opaque thicknesses of
paper will also be effective as a matrix, as well as woven or non-woven
polyesters and polyamides and other absorptive surfaces, such as
nitrocellulose.
Most particularly however, it has been found that a composite polyester
membrane is most effective when treated with reagents such as polyethylene
glycol. Yet, also effective is the porous polyamide reagent membrane used
in the One-Touch.TM. device made by the present assignee.
One manner of preparing the porous material is to cast the polymer onto a
core of nonwoven fibers. The core fibers can be any fibrous material with
requisite integrity and strength, such as the aforementioned polyesters or
polyamides. The reagent that will form the separating and reacting
material is present within the pores of the matrix 16 but does not block
liquid flow through the matrix 16. Thus, the separated clear constituent
can pass through the pores of the matrix 16, while red blood cells and
hemoglobin are retarded at or near the matrix surface.
A matrix of less than about 3000 microns thickness is usually employed with
about 100 microns to about 1000 microns being preferred. Typically, the
matrix 16 will be attached to a holder in order to give it physical form
and rigidity, although this is not essential. FIG. 1 shows an embodiment
of the invention in which a thin reagent element comprising reagent
element 11 is positioned at one end of a plastic holder 12 by means of an
adhesive 13 which directly and firmly attaches the reagent pad. A hole 14
is present in the plastic holder 12 in the area to which reagent element
11 is attached so that sample can be applied to one side of the reagent
element 11 and reaction product observed on the opposite side 18.
A liquid sample to be tested is applied to reagent element 11. Generally,
with blood being exemplary of a sample being tested, the matrix 16 will be
on the order of about 10 mm sq. to about 100 mm sq. in surface area,
especially 10 mm sq. to 50 mm sq. in area, which normally a volume of 5 to
20 microliters of sample will more than saturate. As can be seen in FIG.
1, the plastic holder or support 12 holds reagent element 11 so that the
sample can be applied to one side of the reagent element 11 while color
can be observed on the opposite side of the reagent element 11.
FIGS. 3, 4 and 5 show a system in which reagent is applied to a porous disc
22 on one side of a folding reagent test strip 20. This folding strip
contains a reagent matrix well 24 opposite disc 22 which fits into well 24
when strip 20 is folded. As seen in FIGS. 3 and 4, the strip 20 is folded
so that the reagent matrix 25 can react with a whole blood sample. As seen
in FIG. 5, what is observed will be the reaction product which can be
colorimetrically compared to typical colors formed by reaction product
placed on scale 28 alongside the pad.
The matrix 11, embodied in FIG. 1, and matrix 24 embodied in FIGS. 3, 4 and
5 may be attached to the plastic holder 12, embodied in FIG. 1, and the;
after "support", delete support "30" and insert therefor 20, embodied in
FIGS. 3, 4 and 5, by any convenient means, e.g. a holder, clamp or
adhesives; however, the preferred method is bonding. The bonding can be
done with any nonreactive adhesive, by a thermal method in which the
backing surface is melted enough to entrap some of the material used for
the matrix, or by microwave or ultrasonic bonding methods which likewise
fuse the matrix to the backing. It is important that the bonding be such
as to not itself interfere substantially with the reaction between reagent
element and whole blood sample as well as the separation process in the
matrix. For example, referring now to FIG. 1, adhesive 13 can be applied
to the backing of plastic roller 12, followed first by punching hole 14
into the combined plastic holder and reagent element 11 and then applying
matrix 11 to the adhesive 13 in the vicinity of hole 14 so that the
peripheral portion of the reagent pad element 11 attaches to plastic
holder 12.
Among other things, the separating agent should be capable of producing a
relatively clear colorless fluid by removing the red cells from whole
blood. Separating reagents must be contained within the matrix in
cooperation with reaction reagents, which will later be explained. In
varying degrees, water soluble salts effect such separation. Among salts
operable as separating reagents in the present test device are citrates,
formates and sulfates as well as certain acids such as amino acids, citric
acid, phytic acid and malic acid.
In addition to such salts or acids, polymeric separating agents have also
been effective, such as polyethylene glycol, polystyrene sulfonic acid,
polyvinyl sulfonic acid and polyvinyl alcohol in conjunction with
membranes such as the Pall BioSupport.TM. membrane. It is necessary to
treat a portion of the matrix with such a separating agent.
Signal producing systems typically employed in light reflectance
measurements can also be used for colorimetric readings. As previously
described, the separating reagents cause whole blood to be separated from
red blood cells producing a substantially clear constituent. At that
point, signal producing systems such as those embodied in the previously
referenced One-Touch.TM. test strip may be employed with the analyte in
the sample to produce compounds characteristically visually observable on
the opposite side of the matrix bound to the reagent strip. Alternately,
the strip may be optically tested in conjunction with a meter, such as
that employed using the previously referenced One-Touch.TM. system.
The preferred analysis method is to apply an unmeasured drop of whole blood
on one side of the reagent pad. As the whole blood sample moves across the
reagent pad it reacts with the separating agent to become separated from
red blood cells. At that point, a substantially clear colorless component
is separated from the red blood cells and the analyte in the component
reacts with the embedded reacting agent in order to produce a colorimetric
change.
In an additional preferred embodiment as seen in FIGS. 2a, 2b and 2c, there
is made available a reagent test strip 400 comprising plastic support 200,
which is adhesively connected to a coated-reaction matrix 250 and a coated
separation matrix 300. Each of these components of the test strip 400 will
be explained in sequence.
The first component is the coated separation matrix 300. This separation
matrix will generally be between 50 and 3000 microns in thickness. The
matrix is formed from among the families of polyesters, polyamides,
polyolefins or cellulosics. Among the available materials useable to coat
the separation matrix 300 are polyvinyl sulfonic acid, (PVS 19),
polyethylene glycol (PEG), polystyrene sulfonic acid (PSSA), hydroxypropyl
cellulose (commercially available as Klucel.TM.), polyvinyl alcohol (PVA),
polyvinylpyrrolidone (PVP), polyacrylic acid (PAA) or any such materials
with particulate additives such as silica or clay including a type of clay
commonly identified as bentonite.
This separation matrix layer 300 is combined with a reagent coated reaction
matrix 250 placed below or within the separation matrix 300. The reagent
matrix 250 may be chosen from among polyamides, polyesters, polyolefins or
cellulosics. Reaction matrix 250 is embedded with solution. All indicator
solutions described are provided in a 0.1M, pH 5.0 citrate buffer
containing 1% Klucel.TM.-EF with glucose oxidase at 6 mg/ml and
horseradish peroxidase at 2 mg/ml. The indicator solutions useful as
reagents for coating the reaction matrix 300 may be chosen from among (a)
3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) combined with
3,3-dimethylaminobenzoic acid (DMAB); (b) MBTH combined with
3,5-dichloro-2-hydroxybenzenesulfonic acid (DCHBS); (c)
4-aminoantipyrene(4-AAP) (at 4 mg/ml) and
5-oxo-1-(p-sulfophenyl)-2-pyrazoline-3-carboxylic acid (OPSP); (d) 4-AAP
(at 4mg/ml) and N-(m-tolyl)-diethanolamine (NDA); (e)
2,2'-azino-di(3-ethylbenzthiazoline) sulfonic acid (ABTS); or (f) 4AAP (at
4 mg/ml) and 4-methoxynaphthol.
Further regarding the indicator solutions described above, the MBTH
concentrations are found most effective at 2 mg/ml. In addition, when MBTH
is combined with DMAB or DCHBS, each of these other components are used
within the matrix at concentrations of 2 mg/ml. The 4-AAP/OPSP
concentration is generally used 1 mg/ml. On the other hand, NDA
concentrations can be used most effectively at 0.2 mg per ml. As well, the
ABTS combination is most useful at 5 mg per ml. In addition, these
reagents can be combined with substances such as polyethylene glycol or
Klucel.TM. in order to be better bound to the reaction matrix 250.
It has been found that the polymer coated separation matrix 300 may use a
reagent as a surface tension modifier or analyte releaser and then be
combined with a reagent coated reaction matrix 250. In fact, it has been
found that tetraethyleneglycol dimethyl ether is quite useful in
performance of the present invention.
In addition, the separation matrix 300 has been found effective when a
reagent component is coated within the separation matrix 300 itself. Of
course, additional reagent components are then used within the reaction
matrix 250 of the same test strip 400. It has been found quite useful to
use a separation matrix 300 comprised of polyethylene with a polyethylene
glycol separation coating and including within the separation matrix 300 a
glucose oxidase and an appropriate citrate buffer. As well, in the
reaction matrix 250 it is useful to provide any of the listed indicator
solutions combined with a coating of horseradish peroxidase and the
MBTH-DMAB combination.
Certain separation matrix 300 coatings have been found quite useful to
adequately separate whole blood samples. Any of the above matrix materials
can be used wherein the separation matrix coatings and solvents and
combinations are chosen from the following:
1. 35% weight per volume (W/V) PEG 3500 in methylene chloride on fine
polyethylene
2. 10% (W/V) PVSA and 1% (W/W) Bentonite in water at pH 5.0 fine
polyethylene
3 13% (W/V) Monostearate of PEG in methylene chloride on fine polyethylene
4. 20% (W/V) PEG 1000 and 2% (W/W) Bentonite in methylene chloride on
nonwoven rayon
5 4% (W/V) Tetraethyleglycol dimethyl ether and 30% (W/V) PEG 1000 in
methylene chloride on
nonwoven polyester
6. 15% (W/V) PVSA and 0.2% (W/V) PVA 10000 in water at pH 4.5 on a
polyethylene or a woven nylon membrane
7. 7% (W/V) PVSA in water at pH 4.5 on Pall L/4 polyester
Finally, the preferred reagent matrixes 250 have been found to be useful:
1. "One Touch".RTM. reagent membrane,
2. MBTH plus DCHBS on a polyamide membrane,
3. 4-AAP plus NDA on a polyamide membrane
Finally, it should be noted that the plastic support 200 should be between
50-1000 microns in thickness and be comprised of a transparent, clear
plastic. This plastic support provides support for the entire mechanism,
and provides the base for the testing apparatus.
Thus, the separation matrix 300 is placed above the reaction matrix 250 and
is adhered to the clear plastic support 200. When a whole blood sample is
placed upon the separation matrix 300 layer, the blood sample is separated
in the separation matrix 300 and then enters the reaction matrix 250. In
the reaction matrix 250, the separated blood reacts with the reagent,
which is coated in the reaction matrix 250, and a color change is visible
through the clear plastic support 200. At the surface of the clear plastic
support 200 a comparison can be made with a standardized color chart to
determine levels of analyte, in this particular case, glucose.
Generally, in all the preferred embodiments it is preferred for the color
to vary in intensity or hue dependent on analyte concentration. It has
been found that the particular configurations of reagents is particularly
suited to vary intensity of reaction product color from a light to a dark
color with glucose measurements.
Of course, two factors must be present. First, the reagent test strip must
have a matrix or matrices of the above specified thicknesses in order to
appropriately separate the blood and create a large enough barrier to
maintain the originally clear surface on the opposite side of the matrix.
Second, the varying color change must suitably reflect analyte level
concentrations to the human eye or any other measuring device.
It is well recognized that once the separating agent has separated the red
blood cells or hemoglobin from the substantially clear colorless
constituent, one is capable of performing any desired test for analyte
present in such separated constituent. Specifically, with appropriate
reagents, one can measure cholesterol or alcohol levels in whole blood.
Such is an intended use of the present device in conjunction with the
appropriate known reagents embedded within the matrix.
It is therefore intended that the previous examples not limit the scope of
the present invention which is to be determined from the following claims
and their equivalents.
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