BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to a device and a method for separating
fluid components from one another and especially undesirable components from desirable
ones. More specifically, the present invention is directed to separation of one
or more undesirable components from a liquid mixture to obtain one or more desirable
components for further use, e.g. identification of the component, testing or otherwise
performing operations thereon or therewith.
2. Prior Art Statement
In many applications such as therapeutics, plasma/ serum standards,
pollution or contaminant testing, pH testing, color testing, ion concentration
testing, isomer testing, quality control evaluation, etc. there is often a need
to separate one or more undesirable components from a liquid mixture to obtain
desirable component(s) in furtherance of these applications. Other applications
are within the purview of the artisan whereby such separations may be necessary
or useful. Many separation techniques are available in the prior art, including
filtering, reacting, ionizing, centrifuging, etc. However, simple techniques for
fast yet reliable separation are lacking. While not limited to the testing of
blood, the relative benefits of the present invention over prior art techniques
are described as exemplary.
When using whole blood analyte determination techniques, 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 a drop of blood is placed on a dry hydrophilic membrane with 3 to
8 micron pore sizes, rapid absorption results in hemolysis of blood 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 may not
be clear but reddish. Also, blood may start clotting in the membrane further promoting
the clogging, hence yielding unreliable results.
- (b) When a drop of blood is placed on 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 negligible plasma gets through. 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
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 components
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, this 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 component and the
extent of dilution would depend upon the volume of sample. Last, in a wet membrane,
the rate of transport of material 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 test.
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.
Many other separation requirements in liquid mixture operations have
similar drawbacks which are unique to the particular constituents, yet can be
improved by the device and method of the present invention.
SUMMARY OF THE PRESENT INVENTION
The present invention involves a method and device for separating
fluids from one another in a liquid mixture, and especially for separating undesirable
components from the liquid mixture to obtain one or more desirable components for
subsequent use. A sample of the liquid mixture is first applied to a physical
transport medium which moves at least a portion of the liquid mixture along the
physical transport medium from a remote location thereon to a second location
thereon and in contact with a first surface of a microporous separation membrane.
The primarily tangential movement is achieved by gravity, absorption or capillary
action and the physical transport medium may be a sheet of fabric with weave or
otherwise or a channeled or grooved synthetic material. Once a portion of the liquid
mixture has moved to the first surface of the microporous separation membrane,
one or more undesirable components are separated from one or more desirable components
and the desirable components are absorbed and pass through the membrane. The desirable
component(s) reach the second (opposite) surface of the microporous separation
membrane and contact a collecting membrane. This collecting membrane collects
the desirable components for further use, e.g. it may be capable of reacting with
at least one of the desirable components to display at least one chemical reaction
characteristic thereof. For example, colorimetric reaction may take place and
color intensity may indicate concentration level or other quantitative or qualitative
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 shows a side cut view of a present invention device which includes
a support substrate, a lateral physical transport medium, a microporous separation
membrane and a collecting membrane;
- Figure 2 shows a top diagramatic view of the device shown in Figure 1;
- Figure 3 shows an alternative embodiment present invention device utilizing
a channeled substrate as the physical transport medium;
- Figure 4 shows another alternative embodiment present invention device wherein
the physical trans port medium is a combined channeled substrate and capillary
action transport membrane;
- Figure 5 illustrates the top view of an alternative present invention device;
- Figure 6 illustrates a side view of a present invention device having multiple
collecting membranes for determining three different component characteristics;
- Figure 7 illustrates a present invention device which includes radially arranged
collecting membranes which permit determination of four different component characteristics.
The present invention is directed to obtaining desirable components
of liquid mixtures in microliter quantities rapidly, efficiently, and without causing
dilution, destruction, or contamination of the desirable components. Separation
and movement of components are achieved without relying on externally applied
forces or without any sample pretreatment. It is also directed to the process of
and device for separation of one or more components from a liquid mixture for
determination of qualitative and/or quantitative characteristics or for other subsequent
use of the desirable components. Thus, the separation method can be utilized for
therapeutics, plasma/serum standards, pollution identification and/or testing,
pH testing, isomer testing, water hardness, water mineral content or water contaminant
testings, laboratory analysis, separation and chemical reaction, quality control
of liquid mixtures, pH testing of liquids, ion separation and determination, shelf
life determination, urine testing, other bodily fluid testing, etc. In fact, the
present invention method and device may be used for the separation of components
from any liquid mixture in which components are subject to separation by membrane.
Thus, "liquid mixture" as used herein means a mixture which is flowable and has
one or more components which are separable by membrane separation and in which
at least one of the components is liquid phase. Included would be aqueous solutions,
slurries, multicomponent suspensions, impure water, bodily fluids, liquid-liquid
mixtures, non-aqueous solutions, flowable monomer and/or polymer mixtures, etc.
Further "components" as used herein may themselves be solid(s), liquid(s) or liquid(s)
with ions and/or solids suspended or dissolved therein.
The devices of this invention enable desirable component(s) to come
into intimate contact with a collecting membrane for subsequent use such as testing
or analyzing such as suggested above. The collecting membrane may contain active
chemicals, ions, enzymes, proteins, chromogens or other reagents and movement
and subsequent reaction is achieved without external pressure. The separation 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 a liquid
mixture at a first (remote) location on a physical transport medium and movement
of at least a portion of the mixture to a second location on the medium to contact
a separation membrane for separation of undesirable and desirable components.
Typically, the present invention utilizes tangential flow of the liquid mixture
underneath a suitable microporous separation membrane so that the undesirable
component(s) are retained on the underside but desirable component(s) are obtained
on the top surface of the separation 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 separation strip, on a miniature
scale. For small volumes of liquid mixture which can be obtained by eyedropper
or the like, the driving force for the liquid mixture 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, where applicable,
on the support structure underneath it. Additionally, the choice of the microporous
separation membrane is important. An ideal separation membrane should wet easily
with the liquid mixture, should not allow the undesirable component(s) to migrate
to the top surface, should not cause destruction or breakdown of the component(s),
should have a good capillary pull and have a fairly uniform pore-structure near
the surfaces. There are several dozen commercial microporous separation membranes
which are available and the actual selection of a particular separation membrane
for a particular liquid mixture application is within the purview of the artisan.
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, in cases involving aqueous mixtures, a hydrophilic surface.
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 liquid 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 support structures
are not critical, it would be preferable to have them at least an order of magnitude
larger than that of the microporous separation membranes, so that these substrate
materials do not have any sieving properties and so that their sole function is
to assist in smooth and rapid flow of liquid. 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 may vary in size, depending upon the application. For example,
channels 1 to 10 mm wide and 1 to 15 mil (25 to 225 micron) deep work quite well
for bodily fluids, with 1 to 4 mm width and 2 to 10 mil depth being the preferred
range for blood serum, for example. However, as mentioned, size selection is within
the skill of the artisan.
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 collecting membrane such as a piece of filter
paper, non-woven or woven sheet or other membrane is placed on top of the microporous
separation membrane. This collecting membrane will absorb the desirable liquid(s)
as it comes through the microporous separation membrane and may be loaded with
reagents specific for a given test or reaction, if desired. This whole assembly
may optionally be secured by placing another plastic cover on the top. A liquid
mixture sample 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 separation membrane, thereby creating
a thin film of liquid mixture along one surface of the microporous separation
membrane. Desirable liquid(s) available from the top surface of this membrane
saturate the collecting membrane and if it is loaded with reagents, the desirable
liquid reacts with these reagents producing color or other characterization.
Referring now to Figures 1 and 2, 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 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 collecting membrane 11. Clear plastic cover 13
with orifice 15 is adhered over the tops of the other components as shown by the
arrows 17 and 19 in Figure 1. Top view Figure 2 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 a liquid mixture
in orifice 15 ( a remote location from microporous separation membrane 7 ). The
liquid mixture flows down physical transport medium 5 and under microporous separation
membrane 7 to a first (bottom) surface thereof. Desirable liquid(s) separate and
flow upwardly through membrane 7 and, at this point, are essentially free of any
undesirable liquids which have been separated out. Then it migrates to a second
(opposite, i.e. top) surface of membrane 7 to collecting membrane 11. Here the
desirable liquid(s) may be stored, examined, aged, tested, reacted or otherwise
used as desired. For example, in this case, test membrane 11 may contain reagents
specific for the acidity or other characteristic(s) of the liquid(s), of interest.
In the case of blood serum testing, chromogens which reveal colorimetric measurement
of cholesterol, glucose or the like may be included.
In general, the essential components of the present invention device
are the physical transport medium, the microporous separation membrane, and the
collecting "membrane". Although not preferred, a microporous separation membrane
may be extended and graded to act both as a transport medium and a separation
membrane. However, more leeway as to liquid 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 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 liquid mixture is placed at the remote liquid
application area, it is rapidly pulled underneath the microporous membrane and
desirable liquid(s) seep across this membrane and saturate the collector (collecting
membrane). Further, the collecting membrane itself 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 desirables and react with them producing color.
This type of system is particularly appropriate for determining blood 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 collecting membrane. Many of the enzymes used in such diagnostic s rips
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 damage the liquid(s) e.g. in the case of blood, 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 original
liquid mixture. In order to avoid the bleedthrough of color developed during the
test downstream from the device onto the collecting 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 as shown in Figures 1 and 2. Such
a hydrophobic surface barrier prevents the color from diffusing down the separation
membrane, but is not always necessary.
In the alternative, when a collecting membrane having a first reactant
(reagent) and second reacting component (chromogen) is used, a single collecting
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 blood or other analytes which normally
requires a precipitation step for removal of interfering analytes. The precipitants,
for example, can be incorporated within the microporous separation membrane or
in the physical transport medium, so that the precipitation step occurs on-line
and the plasma reaching the collecting 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 separation membrane.
These devices are self-monitoring because a microporous membrane
cannot be over-filled or over-saturated by capillary filling. Once the collecting
membrane is saturated, the excess liquid mixture ( with desirable liquid(s) )
simply migrates 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 liquid mixture will merely stay at the application area without getting
absorbed. In case the sample volume is not enough, then only part of the collecting
membrane will be saturated and used as desired. The precise control over the thicknessess
(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 any reactants in the desirables, and hence the intensity of the final results
produced may intentionally be independent of the thickness of this membrane. The
minimum volume needed to saturate the collecting membrane with desirables will
depend on the thickness of this membrane, the geometry of the system, the nature
of the desirables and its ultimate use.
Figure 3 shows a present invention device 21, which is similar to
the one shown in Figures 1 and 2 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 4 likewise shows a device 23 with like parts being identically
numbered, and here, both the cloth physical transport medium 5 of the Figure 1
device and the grooved physical transport medium 19 of the Figure 3 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) liquid mixture to draw the excess past the separation membrane
7. Any liquid absorbing material may be used, although this is not an essential
feature of the invention.
Figure 5 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, separation membrane
39 over physical transport medium 37, and collecting 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 original liquid mixture color. By offsetting part
of the test membrane, the isolated portions will show true colorimetric readings
without a shaded background. Top cover 43 with orifice 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 purposes, e.g. multiple 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 or at a pollution
site, chemical pond, spill or the like where on site broad classification information
preliminary to determining procedures for further testing is used.
In the strips of this invention, several related tests can be combined
on the same strip and determination of multiple results can be made within a few
minutes from application of a liquid mixture sample. Thus, Figure 6 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, 52 and 53 are spaced apart and located on sheet
47 as shown. Atop each of the separation membranes 49, 51 and 53 are multilayer
plasma collecting test membranes 53, 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 7 shows a different geometry for obtaining plural test result
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
The type of device shown in Figures 1 and 2 is made for testing bacteria
in murky water. The collecting membrane is seeded with colorimetric bacteria indicating
reagent. The separation membrane is of a size chosen to filter out the murky color
contaminents. A water sample is applied to the application area, some of which
moves along the physical transport medium. At the separation membrane, the murkiness
is removed and clear water surfaces at the top of the collecting membrane, showing
a colorimetric bacteria count.
A device such as is shown in Figure 3 is made for pH testing of colored
liquids. The purpose is to remove the color pigment before pH testing. The separation
membrane is chosen to remove pigments and permit only clear liquid to the collecting
membrane so that accurate litmus type testing is achieved.
A device such as that shown in Figures 1 and 2 is prepared for testing
blood, except that tissue paper is used as the physical transport medium to facilitate
the flow of blood underneath a 5 micron pore-size nitrocellulose separation membrane
(Type AE98, Schleicher and Schuell). The plasma collecting test membrane in this
case is 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 3 type of device is 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 3. 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 4 device with a geometry utilizing both the channels and
an open mesh is made. The channel geometry is the same as in Example 4 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 Example 3. 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 5 is used except that the plasma
separation membrane is 8 micron nitrocellulose (Type AE97, Schleicher and Shuell).
The plasma collecting test membrane is saturated in about 10 seconds.
The same geometry as in Example 6 is used except that cellulosic
membrane (Micron Separations, Inc.) is used for the separation membrane. The plasma
collecting membrane is saturated within 30 seconds.
In the geometry of Example 5, an additional filter paper is introduced
to serve an an absorption pad to soak up excess blood as shown in Figure 5. 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.
A filter paper (collecting membrane) is used as a collection membrane
and placed on a component separation membrane of the type of Figure 1. A drop of
blood (approximately 40 microliters) is placed at the sample application area.
The device separates out the blood cells and the filter paper is saturated with
clean plasma. The filter paper is then removed from the device and the pH of blood
plasma is tested by pressing the wet filter paper against a pH paper which changes
color at increments of 1 pH unit. The pH of the plasma, as would be expected, is
found to be between 7 and 8 from comparisons with the color chart.
Example 9 is repeated, except that pH paper is used as the collecting
membrane instead of the filter paper. The testing is repeated and plasma is collected
at the pH paper collecting membrane. In a very short time, the collecting membrane
changes color to show pH as between 7 and 8. In contrast, when blood is applied
directly on the filter paper it becomes red, making it impossible to determine
the pH level of blood.
A suspension of blue dye Intrapel Blue 12 (Crompton and Knowles)
is made in an aqueous buffer at pH 3. On the device of Figure 3, a filter paper
is used as a sample collecting membrane and a drop of aqueous suspension is placed
at the sample application area. Filter paper collects clean colorless buffer which
when tested for pH by pressing it against a pH paper shows a pH of 3. This device
is useful for separating out particles which may contribute to turbidity/color
to the sample and interfere with a test such as acidity.
Examples 12 - 20
Examples 12 through 20 are illustrative of the geometry, assembly
and functioning of the devices for blood testing. In these illustrative examples,
a 5 micron nitrocellulose membrane is used as the plasma separation membrane,
0.45 micron pore-size nylon 6.6 membrane 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 (Spectra-por)
is used to load the chromagen, white PVC or polystyrene plastic (15-25 gauge)
is used as the substrate and a clear PVC or vinyl plastic (8-12 gauge) is 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,
are used in these devices to provide either a hydrophobic barrier or a good seal.
The chromogen which may be 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 is soaked into the solution
of the chromogen and allowed to dry. Typically this solution consists 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 is 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.
Test strips for measurement of total cholesterol are made as per
Figure 4 where the reagent membrane contains the enzymes for cholesterol as described
above. Blood or serum samples ranging in volume from 10 to 40 microliter are placed
at the blood application area. Typically it takes about 10 to 15 seconds to saturate
the test membrane and in about 30 seconds the color is fully developed which is
then read in a reflectance meter or compared to a color chart.
Test strips of cholesterol are made as per the geometry of Figure
4 and with the reagent composition as described above. Samples of whole blood ranging
in volume from 15 to 50 microliters are applied at the blood application area.
The color development typically is complete in about 30 to 45 seconds.
HDL cholesterol strips are made as per geometry of Figure 4. The
only difference between the cholesterol strips of Example 12 and the HDL strips
is that the plasma separation membrane 1 in this case is 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 ) are applied at the sample application area and color development
is complete in about 1 minute.
Triglyceride strips are made as per the geometry of Figure 4 and
the test membrane is loaded with the enzymes as described above. 10 to 40 microliter
samples ( whole blood, serum or plasma ) are applied at the sample application
area and color development is complete in about 1 minute.
Glucose test strips are made as per the geometry of Figure 4 wherein
the test membrane is loaded with the enzymes as described above. 10 to 40 microliter
samples ( whole blood, serum or plasma ) are applied at the sample application
area and color development is complete in about 30 seconds.
Test strips for the lipid measurement are prepared as shown in Figure
6. The three test membranes for individual geometries are as described above and
the separation membrane for HDL cholesterol is treated with PEG as in Example 9.
40 to 100 micro liter samples ( whole blood, serum or plasma are applied at the
sample application area and color development is complete in about 3 minutes.
Test strips for the lipid measurement are made as per the geometry
of Figure 6. 40 to 100 microliter samples are applied at the sample application
area and the color development typically takes about 1 minute.
Devices for simultaneous measurement of cholesterol ( total and HDL
), glucose and triglycerides are prepared as per the geometry of Figure 7. 50 to
100 microliter samples ( whole blood, serum or plasma ) are applied at the sample
application area and color development is complete in about 2 minutes.
Devices for the measurement of total cholesterol and HDL cholesterol
are made. 20 to 80 microliter samples are applied at the sample application area
and the color development is complete in about 1 minute.
Examples 21 - 28
In the following Examples 21 - 28, the enzymes and chromogens are
loaded in a single layer plasma collecting test membrane. The chromogen is 3,3&min;,
5,5&min; tetramethyl benzidine which is dissolved at a concentration of .03g/ml
in acetone or at about .02 g/ml in a 3% solution of phenol in toluene. The enzymes
for cholesterol are loaded from their aqueous solutions as described above and
the test membranes are allowed to dry. After drying, the test membranes are loaded
with chromogen solution ( either toluene or acetone based ) and again allowed to
dry. The geometry used in testing is as shown in Figure 5, except that the separate
chromogen layer is eliminated and additionally an aperture is provided on the top
plastic cover over the plasma collecting membrane to allow for oxygen, since the
reactions seem more sensitive to oxygen requirements than when the chromogen is
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 are found to allow adequate transport of oxygen
to the reaction zone.
A nylon -6,6 membrane of 0.45 micron pore-size is loaded with cholesterol
enzymes and chromogen from acetone solution. When blood or serum samples in volumes
ranging from 15 to 50 microliters are applied at the blood application zone, in
a Figure 5 type device, color develops in about 30 seconds and colors are accurately
proportional to the cholesterol content.
The same test is done as in Example 21 with a cellulosic test membrane
of 0.2 micron pore-size. The chromogen, however, is loaded from its solution in
toluene. Again similar results are obtained.
The test of Example 21 is repeated. This time, however, after loading
and drying with cholesterol enzymes, the test membranes are saturated with acetone,
and after partial evaporation of acetone, a mixture containing 90:10 parts by volume
of toluene and dodecane is added. After a few minutes of drying the test membrane,
chromogen in toluene solution is 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 especially
under humid environment. When tested, the performance is similar to above.
The test of Example 21 is repeated but this time with a test membrane
from HYPAN® multiblock polymer ( Kingston Technologics, 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 is made over a polyester cloth backing and contains
approximately 5-10 mole % of acrylamide groups and the remainder are acrylonitrile
groups. The structure of the test membrane is asymmetric with the pores on the
more open side of being larger than 1 micron. When this membrane is tested as in
Example 21, similar results are obtained.
The same test as that in Example 23 is repeated with HYPAN membrane
of Example 24. HYPAN membrane in this pseudo-liquid state shows similar behavior.
The test of Example 21 is repeated with a microporous HYPAN membrane
which is symmetric in structure. The membrane is impregnated on a polyester cloth
support. The HYPAN membrane in this case has greater than 75 mole % of acrylonitrite
groups, the remainder being glutorimide-derived groups. The pores on this membrane
are in the range of .02-1 micron. The test results are similar to those in Example
The membrane of Example 26 is tried in a pseudo-liquid form as described
in Example 23. Again similar successful results are obtained.
A device of the type shown in Figure 3 is prepared for testing phenol
type compounds from murky, oil/water mixtures. A filter paper soaked in a solution
of horseradish peroxidase, 4-aminoantipyrine and hydrogen peroxide serves as the
reagent membrane. The presence of phenol is indicated by the pink color (quinoimine
dye) created by the oxidative coupling of phenol and 4-aminoantipyrine in the presence
of hydrogen peroxide and peroxidase enzyme. Alternatively, reagents such as strontium
peroxide, tartaric acid and calcium acetate can be used in place of hydrogen peroxide
solution to generate hydrogen peroxide in-situ in presence of water, thereby enabling
one to make a "dry" reagent pad. Hydrolyzable phenol moeities are present in many
pesticide residues and such a test could be useful to check for toxic pesticide
residues in a water supply.
The device of Figure 6 is prepared with separate reagent membranes
for testing free chlorine/bromine, total chlorine/bromine and pH of a liquid sample.
Free chlorine/bromine and total chlorine/bromine reagent pads were utilized from
Environmental Test Systems (Elkhart, Indiana) and a laboratory pH paper was used
for pH pad. Such a device can be used for simultaneous testing of multiple analytes.
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.