BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to a device and a method for separating
plasma from blood and determining one or more analytes. More specifically, the
present invention is directed to plasma separation from blood and analytical testing
of the plasma to measure analytes such as cholesterol, HDL cholesterol, triglycerides,
2. Prior Art Statement
In many applications such as therapeutics, plasma/serum standards,
and particularly for blood analyte determination, there is often a need to separate
cells from whole blood. For rapid determination of analytes in ser um or plasma,
colorimetric methods are quite popular wherein the analyte or a product obtained
by conversion of this analyte, reacts with a chromogen to give color, the intensity
of which is proportional to the concentration of the analyte. When using whole blood
for determination of an analyte, it is essential to remove the red cells from the
blood so that the color of the reaction is not masked by the color of the blood.
Centrifugation of the blood or the clotting of blood are two common prior art methods
used to isolate plasma or serum from whole blood. However, these result in an
additional sample preparation step. It would appear that since red blood cells
are rather large (about 8 microns in diameter), one could conceivably use a microfilter
of a smaller pore size to obtain clear plasma. However, this does not usually
work for the following reasons:
- (a) When one puts a drop of blood on a dry hydrophilic membrane with pore sizes
in the 3 to 8 micron range, the blood gets absorbed into the pores and onto the
microfibrils of the filter. Often due to this rapid absorption, hemolysis of blood
occurs wherein the fragile red blood cells rupture and the broken cells and/or
hemoglobin from the cells leak through. As a result, generally, the plasma coming
through is not clear but reddish and the throughput may also be small because the
filter may clog up rapidly due to the high solids content of the blood (typically
50% of blood volume is cells). To compound the problem, blood may start clotting
in the membrane further promoting the clogging. Due to this clogging, very often
large analytes such as lipoproteins may be held back and whatever passes through
the membrane may be significantly diluted with respect to these large analytes.
The concentration of these analytes in the filtrate may therefore be significantly
lower than in plasma or serum and may vary with both applied sample volume as
well as with blood hematocrit (solid content), hence yielding unreliable results.
- (b) When one places a drop of blood onto a hydrophilic microporous membrane
with pore sizes of less than 3 microns, the clogging on top of the membrane appears
to be especially rapid and usually neglibible plasma gets through. Again the same
problems as mentioned above with clogging are encountered.
- (c) Using a depth filter in addition to or in place of the microfilter may
likewise be problematic. Due to higher hold up and absorptive capacity of the depth
filter, volume requirements of blood become rather excessive and the problems of
hemolysis and clogging still exist. An exception to this is the depth filter made
up of glass fibers which was shown to separate plasma/serum from blood rather effectively.
This method is described in United States Patent No. 4,477,575. However, many of
the problems of other "dry" membrane systems remain, although to a lesser degree.
In order to overcome the problems of dry membrane test systems, numerous
wet or semi-wet microporous membrane test systems have been proposed. Thus, when
wetted with water or with aqueous solutions, the microfibers of these hydrophilic
membranes are hydrated by a thin layer of water. This significantly reduces the
absorptive capacity of the membrane. It is possible to get fairly clean plasma
across this wetted microporous membrane of pore sizes of about 5 microns or less
in this fashion. However, wet and semi-wet systems have three major drawbacks.
First, wet and semi-wet systems may involve wetting the membrane prior to use
and this adds an additional step for a diagnostic test. Second, when dealing with
small volumes of a few microliters, as are encountered in diagnostic tests, this
would dilute the plasma and the extent of dilution would depend upon the volume
of sample. Finally, in a wet membrane, the rate of transport of plasma through
the membrane will be governed by diffusion rather than capillary pull and may take
an excessively long time thereby affecting the overall response time of a diagnostic
This approach of using a wet microporous membrane for separation
of plasma from whole blood is indeed being used very successfully in a process
called plasma phoresis. In this process, the microporous membrane is first wetted
with isotonic saline in a step which is called priming the membrane. Blood is then
pumped through one side of the membrane and a transmembrane pressure differential
is maintained. Because of the externally applied pressure difference, the process
is fairly rapid. Additionally, a relatively large amount of blood is processed
(on the order of a liter) and hence dilution due to isotonic saline in the membrane
is negligible. Therefore, even though plasmaphoresis is an effective way of obtaining
plasma from blood on a process scale, it is not very practical for a simple and
rapid diagnostic test requiring on-line serum/plasma separation for microliter
quantities, without external pressures and preferably without dilution.
Glucose reagent strips were among the first to utilize whole blood
for blood glucose measurements. These colorimetric tests for whole blood based
on dry-strip technology became quite popular due to the possibility to obtain rapid
analysis of glucose from whole blood without sample pre-treatment. These early
tests relied on diffusion of glucose from whole blood through thin, transparent
and tightly structured membranes (U.S. Pats. 3,092,465; 3,298,789; 4,543,338).
A drop of blood is placed on the test-pad and after a pre-determined period of
time the blood is either washed off or wiped off and the color developed underneath
is then compared to a color chart or read in a reflectance meter. These tests do
not require a precise metering of the blood but do require a blood wash-off or
a wipe-off step and precise timing for the same. While this approach is satisfactory
for a relatively small hydrophilic molecule such as glucose which can diffuse rather
easily through a thin tightly structured membrane, it is not feasible to measure
large analytes such as proteins or hydrophobic analytes such as cholesterol or
triglycerides which are complexed with proteins and are carried in the blood stream
as part of lipoproteins ranging in size from a few hundred thousand to several
million in molecular weights. These relatively large molecules cannot penetrate
the dense membranes used in the glucose strips and attempts to separate plasma
or serum from whole blood with more open membranes and without pressure or external
influences were not successful due to reasons mentioned above.
The discovery that certain depth filters made up of glass fibers
could successfully separate red cells from whole blood (U.S. Patent No. 4,477,575)
paved the way for successful commercialization of an instrument called "Reflotron"
by Boehringer Mannheim Diagnostics (Trademark) where the separation of clear serum
or plasma occurs within the dry strip itself without need of an external force
such as pressure or centrifugation. This made analysis of various analytes possible
from whole blood in a simple inexpensive manner. However, although blood analysis
with Reflotron is convenient, a metered amount of blood must be placed onto the
reagent pads and there is a need to crush the pad to bring all the reagents into
intimate contact with the separated serum/plasma. Additionally, it can perform only
one reaction at a time and typically the chemistries take about three minutes
Thus, while many new developments have occurred and have been patented,
the need for a convenient, efficient, easy-to-use, reliable test method and device
for testing one or more analytes from whole blood plasma has not been satisfied
until the development of the present invention.
SUMMARY OF THE PRESENT INVENTION
The present invention involves a method and device for obtaining
plasma from whole blood and determining one or more analytes contained therein.
A sample of blood is first applied to a physical transport medium which moves
at least a portion of the blood from a remote location thereon to a second location
thereon to a first surface of a microporous plasma separation membrane. This tangential
movement is achieved by gravity, absorption and/or capillary action. The physical
transport medium may be a sheet of fabric with weave or otherwise or a channeled
or grooved synthetic material. Once the blood has moved to the first surface of
the microporous plasma separation membrane, the plasma is absorbed and passes through
the membrane, thereby separated from the whole blood (and the red color). The
plasma reaches the second (opposite) surface of the microporous plasma separation
membrane and contacts a plasma collecting test membrane. This test membrane is
capable of reacting with the plasma (i.e. one of its components) to display at
least one analyte characteristic thereof. Preferably, colorimetric reaction takes
place and color intensity indicates concentration level of the analyte in the
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention described herein is more fully appreciated
and understood when the disclosure is taken in conjunction with the appended drawings,
DETAILED DESCRIPTION OF THE PRESENT INVENTION
- Figure 1 illustrates a photographic representation of a 5 micron nitrocellulose
plasma separation membrane used in the method and device of the present invention;
- Figure 2 shows a photographic representation of a 3 micron cellulose plasma
separation membrane used in the present invention;
- Figure 3 shows a side cut view of a present invention device which includes
a support substrate, a lateral physical transport medium, a microporous plasma
separation membrane and a plasma collecting test membrane;
- Figure 4 shows a top diagramatic view of the device shown in Figure 3;
- Figure 5 shows an alternative embodiment present invention device utilizing
a channeled substrate as the physical transport medium;
- Figure 6 shows another alternative embodiment present invention device wherein
the physical transport medium is a combined channeled substrate and capillary action
- Figure 7 illustrates the top view of an alternative present invention device;
- Figure 8 illustrates a side view of a present invention device having multiple
plasma collecting test membranes for determining three different analytes; and,
- Figure 9 illustrates a present invention device which includes radially arranged
plasma collecting test membranes which permit analyte determination for four different
The present invention is directed to obtaining clear plasma from
microliter quantities of whole blood rapidly, efficiently, without causing dilution
of blood analytes and without relying on externally applied forces or without
any sample pretreatment. It is also directed to the process of separation of plasma
in a device for determination of blood analytes. The present invention plasma
separation method can be utilized for colorimetric detection of blood analytes.
The devices of this invention enable the plasma to come into intimate contact
with all the necessary enzymes, proteins or other reagents and chromogens without
external pressure or without denaturing blood or sensitive reagents. The test strip
device of this invention also enables one to do multiple chemistries on the same
strip with a single sample application in a very quick, simple and reliable manner.
Primarily, the present invention relies upon application of blood
at a first (remote) location on a physical transport medium and movement of the
blood to a second location on the medium for separation of plasma and subsequent
testing. Typically, the present invention utilizes tangential flow of blood underneath
a suitable microporous plasma separation membrane so that the red cells are retained
on the underside but clean plasma is obtained on the top surface of the membrane.
Capillary pressure, both within the membrane matrix and by suitable channels and/or
a suitable open mesh underneath the microporous membrane may provide the appropriate
driving force for the tangential flow and no external forces are necessary. For
filtration of highly viscous liquids or for slurries or suspensions with high solids
content, tangential flow across the membrane is much more efficient than the dead-ended
filtration. The problems of clogging of the membrane are substantially reduced
in tangential filtration mode. In this system, a viscous fluid or suspension is
swept across one side of the membrane, and clean filtrate is collected across
the other side.
This invention uses this principle, for a diagnostic strip, on a
miniature scale. For small volumes of blood which can be obtained by finger-pricking,
the driving force for blood flow may be provided entirely by the capillary pull.
To do this efficiently, it relies heavily on both the pore-structure of the separation
membrane and the physical transport medium and support structure underneath it.
Additionally, the choice of the microporous separation membrane becomes even more
important than for other conventional applications. An ideal separation membrane
should wet easily with serum or plasma, should not allow the red cells to migrate
to the top surface, should not cause hemolysis of the red cells, should have a
good capillary pull and a fairly uniform pore-structure near the surfaces. Out
of several dozen commercial microporous membranes which were screened, three were
found to be particularly preferable, a 5 micron microporous membrane made from
nitrocellulose of Schleicher and Schuell (Keene, New Hampshire) performed optimally
in separating plasma from whole blood. In general, suitable pore-sizes of such
membranes should be between about 0.02 to 10 microns, with the preferred range
between 1 to 5 microns. Skinned membranes, wherein one or both surfaces of the
microporous membranes have much smaller and uniform pore-sizes, as compared to
the pore-sizes in the middle region, are found to be particularly suitable. Unlike
most commercial microporous membranes which are generally isotropic in their cross-section,
these membranes show double-skinned structures which may be the reason for their
effectiveness. Figures 1 and 2 show the cross-section of the 5 micron nitrocellulose
and the 3 micron cellulosic membrane respectively.
Other important components of this invention are the physical transport
medium and the substrate onto which this microporous separation membrane is placed.
Typically, this membrane is placed on a plastic substrate and between this substrate
and the membrane is placed a physical transport medium which may be a material
with open structure and hydrophilic surface to achieve tangential flow of the blood.
Examples of materials with such open structures are various types of polymeric
meshes, cloth, tissue papers, gauze etc. Ideally the hold up volume and the absorptive
capacity of these open-structured materials should be very small so as to minimize
the loss of blood due to absorption and the surface on a macroscopic scale should
be fairly smooth so as to provide good contact with the microporous separation
membrane. Woven fabrics from many monofilament yarns such as polyester and nylon
are particularly useful. Even though the pore sizes of the transport media are
not critical, it would be preferable to have them at least an order of magnitude
larger than that of the analyte of interest, so that these materials do not have
any sieving properties and so that their sole function is to assist in smooth
and rapid flow of blood. As a further aid for rapid movement of blood underneath
the microporous separation membrane, channels of well-defined geometry or grooves
may be included on the plastic substrate, underneath the open-structured material.
Channels 1 to 10 mm wide and 1 to 15 mil (25 to 225 micron) deep work quite well,
1 to 4 mm width and 2 to 10 mil depth being the preferred range.
In practice, an open structured material such as a monofilament woven
fabric is placed onto the plastic substrate with or without the channels, and the
microporous separation membrane is placed on the top such that part of the fabric
extends beyond the membrane. A plasma collecting test membrane such as a piece
of filter paper or another hydophilic membrane is placed on top of the microporous
separation membrane. This will absorb the clean plasma as it comes through the
microporous separation membrane and may be loaded with reagents specific for a
given analyte. This whole assembly may then be secured by placing another plastic
cover on the top. Blood applied to the physical transport medium through an orifice
in the plastic cover at a location remote from the separation membrane, migrates
along to a location next to (under) the microporous plasma separation membrane,
thereby creating a thin film of blood along one surface of the microporous plasma
separation membrane. Clean plasma available from the top surface of this membrane
saturates the test membrane and if it is loaded with reagents, the analyte reacts
with these reagents producing color or other analyte indicator.
Referring now to Figures 3 and 4, there is shown a side cut view
and a top view of present invention test strip device 1. The device 1 has an inert
substrate 3 and a physical transport medium 5 attached thereto. In this embodiment,
physical transport medium 5 is a woven cloth material. Atop physical transport
medium 5 is microporous plasma separation membrane 7 and optional hydrophobic barrier
strip 9, adjacent thereto as shown in both figures. Of less length and placed
atop of microporous membrane 7 is plasma collecting test membrane 11. Clear plastic
cover 13 with orifice 15 is adhered over the tops of the other components as shown
by the arrows 2 and 4 in Figure 3. Top view Figure 4 shows all of the components,
at least in part, due to the fact that cover 13 is clear.
As now can be seen, the user simply places a drop of blood in orifice
15 ( a remote location from microporous plasma separation membrane 7 ). The blood
flows down physical transport medium 5 and under microporous plasma separation
membrane 7 to a first (bottom) surface thereof. Plasma separates and flows upwardly
through membrane 7 and is, at this point, essentially clear in color. It migrates
to a second (opposite, i.e. top) surface of membrane 7 to plasma collecting test
membrane 11. Here the analyte(s) react to establish concentration(s). For example,
in this case , test membrane 11 contains reagents specific for the analyte or
analytes of interest, e.g. chromogens which reveal colorimetric measurement of
cholesterol, glucose or the like. Further, the dimensions shown in Figure 4 are
merely exemplory and are not critical.
In general, the essential components of the present invention device
are the physical transport medium, the microporous plasma separation membrane,
and the plasma collecting test strip. Although not preferred, a microporous plasma
separation membrane may be extended and graded to act both as a transport medium
and a separation membrane. However, more leeway as to blood volume, dimensions
and "overflow" control are achieved when the physical transport medium is one
or more components which are elements separate and different from the microporous
plasma separation membrane. Likewise, the transport medium may act as a substrate
or a separate, inert and inactive substrate may be utilized.
The combination of a separate physical transport medium and a separation
membrane is chosen such that when blood is placed at the remote blood application
area, it is rapidly pulled underneath the microporous membrane and clean plasma
seeps across this membrane and saturates the reactant containing test membrane.
Further, the test membrane may be a single layer or may be a compilation e.g.,
a reagent containing layer as well as a chromogen containing layer which is preferably
a porous hydrophilic layer. The enzymes or reagents in these layer(s) dissolve
in the plasma and react with the analyte producing color. This type of system
is particularly appropriate for determining analytes such as glucose, cholesterol,
triglycerides, uric acid, creatinine, alcohol, creatine kinase, cholinesterase,
AST, ALT, etc. wherein an enzyme or a series of enzymes convert an analyte to hydrogen
peroxide which then reacts with chromogen, contained in the test membrane. Many
of the enzymes used in such diagnostic strips are particularly susceptible to
denaturation when contacted by many chromogens for an extended period of time.
By separating the enzymes and the chromogens in two separate layers and yet keeping
them in close proximity, the long term stability of the reagents can be preserved.
For analyzing analytes such as albumin or other proteins, amylases, calcium, inorganic
phosphorus, etc. wherein the reagents used are not biomolecules such as enzymes
or antibodies but are stable chemicals and the color formed is due to selective
complexation of the analyte with the reagents, the porous layer can be eliminated
and all the reagents can be combined in a single layer membrane. It is also possible
to load some of the reagents elsewhere in the system such as in additional layers,
although this is not essential, provided that they don't hemolyze the cells or
damage them in any way. It is best to view the final color at the top of the test
membrane since it also acts as a hiding layer to mask the color of the blood. In
such a case, an additional layer, if necessary for chromogens, could be transparent
or translucent. In order to avoid the bleedthrough of the color developed during
the test downstream from the device onto the test membrane, an optional hydrophobic
material or glue can be applied on the exposed edge of the separation membrane
just downstream of the physical transport medium (not shown in the Figures). Such
a hydrophobic surface barrier prevents the color from diffusing down the separation
membrane, but is not always necessary.
In the alternative, when a test membrane having a first reactant
(reagent) and second reacting component (chromogen) is used, a single test membrane
instead of a plural layer may be used. Thus, in some embodiments, the reagents
and chromogens may be contained in dry or semi-wet form or even wet form in the
same membrane structure, or one or the other may be encapsulated or otherwise isolated
within the same membrane.
Further, the present invention device may be used without any additional
sample preparation for detection of certain analytes which normally requires a
precipitation step for removal of interfering analytes. The precipitants, for example,
can be incorporated within the microporous plasma separation membrane or in the
physical transport medium, so that the precipitation step occurs on-line and the
plasma reaching the test membrane is free of the interferent. If, due to the nature
of the precipitant, it is advisable not to have direct contact of the precipitant
with the blood, then an additional layer containing the precipitant can be placed
between the separation membrane and the physical transport medium. An application
that makes use of the precipitation step is the measurement of high density lipoprotein
cholesterol (HDL cholesterol) which typically needs precipitation steps using precipitants
such as polyethylene glycols, dextran sulfate, or magnesium tungstate to precipitate
out cholesterol in low density and very low density lipoproteins (LDL and VLDL),
followed by centrifugation. The present invention device achieves this on-line
by loading the precipitant into the microporous plasma separation membrane.
These devices are self-monitoring because a microporous membrane
cannot be over-filled or over-saturated by capillary filling. Once the plasma collection
membrane is saturated, the excess blood and plasma simply migrate down the transport
medium. If, for some reason, the sample volume is so large as to exhaust the absorptive
capacity of the transport medium, then the excess blood will merely stay at the
application area without getting absorbed. In case the sample volume is not enough,
then only part of the test membrane will be saturated and colored. The precise
control over the thicknesses (and hence the void volume) of the test membrane is
not critical because even if the saturation capacity of this membrane varies with
the thickness, the concentration of the analyte and hence the intensity of the
final color produced will be independent of the thickness of this membrane. The
minimum volume needed to saturate the plasma collecting test membrane with plasma
will depend on the thickness of this membrane, the geometry of the system and
the hematocrit of the blood: for the system shown in Figures 3 and 4, it is approximately
10-15 microliters. When using plasma or serum in place of whole blood, the volume
requirements are half as much. To further minimize the required minimum volume
the size of the test membrane and/or the thickness may be decreased as long as
the test membrane is sufficiently opaque to mask the color of the blood underneath
it. If the test membrane is not of sufficient opacity, then one could use a geometry
similar to that shown in Figure 7 where the color developed is not directly over
the blood path.
Figure 5 shows a present invention device 21, which is similar to
the one shown in Figures 3 and 4 and like parts are like numbered. Note that physical
transport medium 19 is now channels or grooves established in substrate 17 itself.
Arrows 6 and 8 show assemblage.
Figure 6 likewise shows a device 23 with like parts being identically
numbered, and here, both the cloth physical transport medium 5 of the Figure 3
device and the grooved physical transport medium 19 of the Figure 5 device are
combined. Additionally, absorbant storage medium 25 rests in part on transport
medium 5 and on substrate 17. This optional storage medium may be used to absorb
additional (excess) blood to draw the excess past the separation membrane 7. Any
blood absorbing material may be used, although this is not an essential feature
of the invention.
Figure 7 shows present invention device 33 which has substrate 35,
physical transport medium 37 (e.g. a sheet material such as a gauze or synthetic
having the necessary capillary action), located on substrate 35, plasma separation
membrane 39 over physical transport medium 37, and plasma collecting test membrane
41 which is partly over the separation membrane 39 and partly located directly
on substrate 35, as shown. This arrangement permits the use of a very thin separation
membrane which may show through some of the red color. By offsetting part of the
test membrane, the isolated portions will show true colorimetric readings without
a pinkish background. Top cover 43 with orifice 45 is clear plastic and the user
uses this strip in the same manner as described above.
Advantageously, the present invention method and device may be used
for a plurality of simultaneous testings. Thus, it is possible to combine various
chemistries on the same device. This is particularly advantageous for example
in testing on-site in doctor's office, where a patient needs to be checked for
several analytes to diagnose a condition. In the strips of this invention several
related tests can be combined on the same strip and determination of multiple
analytes can be made within a few minutes from application of a single blood sample
which can be obtained by finger-prick. Thus, Figure 8 shows one geometry combining
a lipid panel (cholesterol, HDL cholesterol and triglycerides) onto a single strip.
There is shown in Figure 8 device 41 having substrate 43. The physical transport
medium in this embodiment includes channels 45 located on substrate 43 as well
as sheet 47 located above channels 45. Three microporous plasma separation membranes
49, 51 and 53 are spaced apart and located on sheet 47 as shown. Membrane 51 additionally
contains a precipitant for LDLs and VLDLs (low and very low density cholesterols).
Atop each of the separation membranes 49, 51 and 53 are multilayer plasma collecting
test membranes 55, 57 and 59 respectively. The plasma collecting test membranes
55, 57 and 59 contain the necessary reactants for three different analytes, in
this case cholesterol, HDL cholesterol and triglycerides respectively. Optional
absorbant storage medium 61 is also included. The top cover in this embodiment
includes a first clear plastic layer 63 with orifice 65 and an opaque layer 67
containing orifice 69 for blood sample transmission and orifices 71, 73 and 75
for visual comparison for colorimetric measurements of the respective analyte
reactions at test membranes 55, 57 and 59.
Figure 9 shows a different geometry for obtaining plural analyte
characteristics and, in this case, contains four different analyte tests. Thus,
device 91 includes substrate 93 and, in a large radially expanding cross, physical
transport medium 95 which is placed on top of substrate 93 and extends radially
from the center to approximately the outer edge of substrate 93 and under all
other components hereafter described. Atop physical transport medium 95 are four
separate microporous plasma separation membranes 97, 99, 101 and 103. Atop a portion
of each of these are plasma collecting test membranes 105, 107, 109 and 111, respectively,
and these in turn contain reactants for colorimetric determination for four different
analytes such as glucose, cholesterol, HDL cholesterol and triglycerides. Optional
absorbant storage media 113, 115, 117 and 119 are placed on top of the outer ends
of physical transport medium 95. Clear plastic cover 121 is placed on top of all
the other components and has a single orifice 123 for blood sample application
in the center as shown. This device 91 permits nearly simultaneous testing of
four different analytes by application of a single blood drop.
The following examples further support the effectiveness of the present
A device such as that shown in Figures 3 and 4 was prepared except
that tissue paper is used as the the physical transport medium to facilitate the
flow of blood underneath a 5 micron pore-size nitro cellulose separation membrane
(Type AE 98, Schleicher and Schuell), a microphotograph of which is shown as Figure
1. The plasma collecting test membrane in this case was a 0.45 micron nylon 6,6
membrane. It is advantageous to use for the test membrane a microporous membrane
of a pore size smaller than the plasma separation membrane for plasma collection;
it will then also act as a secondary membrane in case the primary membrane has
some defects. A drop of blood (20-40 microliters in volume) is placed at the blood
application area and within about 1 to 2 minutes the plasma collection membrane
gets saturated with clear plasma.
A Figure 5 type of device was constructed wherein the bottom support
plastic has a 3 mm wide and 5 mil deep channel built in. No open structured material
is used as a blood transport material. To prevent the blood from going over the
plasma separation membrane, a hydrophobic tape is put on the top surface of this
membrane. The plasma separation membrane is the very same as in Example 1. When
a drop of blood is placed on the blood application area, clear plasma saturates
the plasma collecting test membrane in about 1 minute.
A Figure 6 device with a geometry utilizing both the channels and
an open mesh was made. The channel geometry is the same as in Example 2 but a woven
fabric from monofilament polyester is used to facilitate the transport of blood.
The plasma separation membrane is of the same type as in above examples. When a
drop of blood is placed on the blood application area, clear plasma saturates
the plasma collection membrane in about 10 seconds.
The same geometry as shown in Example 3 is used except that the plasma
separation membrane is 8 micron nitrocellulose (Type AE99, Schleicher and Shuell).
The plasma collecting test membrane is saturated in about 10 seconds.
The same geometry as in Example 4 is used except that cellulosic
membrane (Micron Separations, Inc.) is used for the separation membrane. A microphotograph
of this cellulosic membrane is shown in Figure 2. The plasma collecting membrane
is saturated within 30 seconds.
In the geometry of Example 3, an additional filter paper is introduced
to serve as an absorption pad to soak up excess blood as shown in Figure 7. The
rest of the components are the same as in Example 3. Blood samples from about
15 to 100 microliters are placed in the blood application area and the plasma collection
membrane is saturated in about 10 seconds.
Examples 7 through 15 are illustrative of the geometry, assembly
and functioning of the devices. In these illustrative examples, a 5 micron nitrocellulose
membrane (Schleicher and Schuell) was used as the plasma separation membrane (#1),
0.45 micron pore-size nylon 6.6 membrane (Micron Separation Inc., Westoro, MA)
as the plasma collecting test membrane and a woven polyester mesh as the physical
transport medium to facilitate the transport of blood, lens tissue-paper or a dialysis
membrane of 10,000 dalton cut-off (Spectrum Medical Industries,Inc.,L.A.,CA) was
used to load the chromagen, white PVC or polystyrene plastic (15-25 gauge) was
used as the substrate and a clear PVC or vinyl plastic (8-12 gauge) was used as
the top cover. Glues such as polybutene or silastic or PVC based, or medical grade
double-stick tape, all of which are relatively inert toward the chemistries, were
used in these devices to provide either a hydrophobic barrier or a good seal. The
chromogen used in conjunction with the various chemistries involving enzymes as
the reagents is 3,3&min;, 5,5&min; tetramethylbenzidine dihydrochloride (TMBD) which
in the presence of horseradish peroxidase gives bluish green color when it reacts
with hydrogen peroxide generated from the reactions of the analyte. TMBD can be
substituted by a number of other chromogens such as phenol or its derivatives
and 4 amino antipyrine, or a number of leuco dyes, e.g. from arylmethine family
(e.g. leuco malachite green) or by solubilized reactive dyes in their leuco form.
The porous lower layer of the test membrane was soaked into the solution of the
chromogen and allowed to dry. Typically this solution consisted of 0.3g TMBD, 0.5g
of sodium cholate, and 0.5-2g of polyethylene glycol (PEG) (molecular weight 8000
daltons) dissolved in 20 ml of reagent alcohol and 0.1M pH 3 phosphate buffer (3:1
by vol). The top layer of the test membrane was tissue paper soaked in the chromogen
solution and upon drying stays tacky due to a combination of sodium cholate and
PEG. This tackiness enables it to have an intimate contact with the reagent lower
layer. To load the reagents, reagent solutions in appropriate buffers are made
at the desired concentrations and the membrane is either soaked into this solution
or the solutions pipetted onto the membrane till it is saturated. The membrane
is then allowed to dry at room temperature. Typically the reagent membrane needs
10 microliter of reagent solution per square cm area for saturation. The solutions
used for the various tests illustrated in the examples below are as follows: (a)
cholesterol: 400 units of pancreatic cholesterol esterase, 40 units of cholesterol
oxidase from Nocardia, and 200 units of horseradish peroxidase per milliliter of
0.1M pH 7 phosphate buffer. (b) Triglycerides: 2000 units of lipase from chromobacterium
viscosum, 300 units of glycerol kinase from arthrobacterium sp., 300 units of glycerol
phosphate oxidase (Microbial), and 500 units of horseradish peroxidase per milliliter
of 0.1M phosphate buffer at pH 8. Additionally adenosine triphosphate (ATP) solution
is prepared at a concentration of .625g/ml and is combined with the above solution
in the ratio of 4 parts of enzyme solution to 1 parts of ATP solution. (c) glucose:
80 units of glucose oxidase from aspergillus niger and 300 units of horseradish
peroxidase per milliliter of 0.1M phosphate buffer at pH 7.
Test strips for measurement of total cholesterol were made as per
Figure 6 where the reagent membrane contained the enzymes for cholesterol as described
above. Blood or serum samples ranging in volume from 10 to 40 microliter were
placed at the blood application area. Typically it took about 10 to 15 30 seconds
to saturate the test membrane and in about seconds the color was fully developed
which was then read in a reflectance meter or compared to a color chart.
Test strips of cholesterol were made as per the geometry of Figure
6 and with the reagent composition as described above. Samples of whole blood ranging
in volume from 15 to 50 microliters were applied at the blood application area.
The color development typically was complete in about 30 to 45 seconds.
HDL cholesterol strips were made as per geometry of Figure 6. The
only difference between the cholesterol strips of Example 7 and the HDL strips
is that the plasma separation membrane 1 in this case was first soaked in an aqueous
20% by weight solution of PEG 2000 ( a precipitating agent for LDL and VLDL cholesterol)
and allowed to dry prior to use. 10 to 40 microliter samples (whole blood, serum
or plasma) were applied at the sample application area and color development was
complete in about 1 minute.
Triglyceride strips were made as per the geometry of Figure 6 and
the test membrane was loaded with the enzymes as described above. 10 to 40 microliter
samples (whole blood, serum or plasma) were applied at the sample application
area and color development was complete in about 1 minute.
Glucose test strips were made as per the geometry of Figure 6 wherein
the test membrane was loaded with the enzymes as described above. 10 to 40 microliter
samples (whole blood, serum or plasma) were applied at the sample application
area and color development was complete in about 30 seconds.
Test strips for the lipid measurement were prepared as shown in Figure
8. The three test membranes for individual geometries were as described above
and the separation membrane for HDL cholesterol was treated with PEG as in Example
9. 40 to 100 microliter samples (whole blood, serum or plasma) were applied at
the sample application area and color development was complete in about 3 minutes.
Devices for simultaneous measurement of cholesterol (total and HDL),
glucose and triglycerides was prepared as per the geometry of Figure 9. 50 to 100
microliter samples (whole blood, serum or plasma) were applied at the sample application
area and color development was complete in about 2 minutes.
Devices for the measurement of total cholesterol and HDL cholesterol
were made. 20 to 80 microliter samples were applied at the sample application area
and the color development was complete in about 1 minute.
In the following Examples 15 - 21, the enzymes and chromogens were
loaded in a single layer plasma collecting test membrane. The chromogen was 3,3&min;,
5,5&min; tetramethyl benzidine which was dissolved at a concentration of .03 g/ml
in acetone or at about .02 g/ml in a 3% solution of phenol in toluene. The enzymes
for cholesterol were loaded from their aqueous solutions as described above and
the test membranes were allowed to dry. After drying, the test membranes were
loaded with chromogen solution ( either toluene or acetone based ) and again allowed
to dry. The geometry used in testing was as shown in Figure 7, except that the
separate chromogen layer was eliminated and additionally an aperture was provided
on the top plastic cover over the plasma collecting membrane to allow for oxygen,
since the reactions seemed more sensitive to oxygen requirements than when the
chromogen was loaded separately in another matrix. For aesthetic purposes and
to protect the reagent area from outside contamination, an air-permeable membrane,
such as a thin (∼20 micron thick), transparent polyurethane membrane coated
on one side with porous adhesive (e.g. from Acutek, Inglewood, California) can
be placed over the aperture. Such films were found to allow adequate transport
of oxygen to the reaction zone.
A nylon -6,6 membrane of 0.45 micron pore-size was loaded with cholesterol
enzymes and chromogen from acetone solution. When blood or serum samples in volumes
ranging from 15 to 50 microliters were applied at the blood application zone, in
a Figure 7 type device, color developed in about 30 seconds and colors were accurately
proportional to the cholesterol content.
The same test was done as in Example 16 with a cellulosic test membrane
of 0.2 micron pore-size. The chromogen, however, was loaded from its solution
in toluene. Again similar results were obtained.
The test of Example 15 was repeated. This time, however, after loading
and drying with cholesterol enzymes, the test membranes were saturated with acetone,
and after partial evaporation of acetone, a mixture containing 90:10 parts by volume
of toluene and dodecane was added. After a few minutes of drying the test membrane,
chromogen in toluene solution was applied. When the test membrane dries, the non-volatile
dodecane component is left behind. These membranes are, therefore, not quite dry
but contain the hydrophobic, non-polar dodecane and can be considered pseudo-liquid
membranes. Non-polar liquids such as dodecane create a hydrophobic environment
around the enzymes and give them added stability against denaturation and expecially
under humid environment. When tested, the performance was similar to above.
The test of Example 15 was repeated but this time with a test membrane
from HYPAN® multiblock polymer (Kingston Technology , Dayton, N.J.) and described
in U.S. Patents Nos. 4,331,783; 4,337,327; 4,369,294; 4,379,874; 4,420,589 and
4,370,451. This test membrane was made over a polyester cloth backing and contained
approximately 5-10 mole % of acrylamide groups and the remainder were acrylonitrite
groups. The structure of the test membrane was asymmetric with the pores on the
more open side of being larger than 1 micron. When this membrane was tested as
in Example 15, similar results were obtained.
The same test as that in Example 17 was repeated with HYPAN membrane
of Example 18. HYPAN membrane in this pseudo-liquid state showed similar behavior.
The test of Example 15 was repeated with a microporous HYPAN membrane
which was symmetric in structure. The membrane was impregnated on a polyester
cloth support. The HYPAN membrane in this case had greater than 75 mole % of acrylonitrite
groups, the remainder being glutarimide-derived groups. The pores on this membrane
were in the range of .02-1 micron. The test results were similar to those in Example
The membrane of Example 20 was tried in a pseudo-liquid form as described
in Example 17. Again similar successful results were obtained.
Obviously, numerous modifications and variations of the present invention
are possible in light of the above teachings. It is therefore understood that
within the scope of the appended claims, the invention may be practiced otherwise
than as specifically described herein.