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Description  |
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INTRODUCTION
1. Technical Field
The technical field of this invention is disposable devices for use in
analyte detection assays.
2. Background
Despite the numerous strides that have been made in the last two decades in
the development of diagnostic reagents and instruments, efforts continue
to make diagnoses more accurate, simpler, and more available to
non-technical personnel in a wide variety of environments. There is
continuing interest in being able to carry out individual assays by
non-technical personnel at such sites as doctor's offices, clinics, the
home, rest homes, and the like. In order to ensure that non-technical
individuals may accurately perform these assays, it is essential that the
protocols be simple and that there be few, if any, measurements. Further,
the readings must be relatively automatic.
In designing these types of assays, it is desirable to have a disposable
device which can be used individually for a particular assay determination
and then discarded. The disposable device can provide the various members
of the signal producing system which are necessary for the assay
determination, serve to ensure the appropriate mixing of the reagents of
the signal producing system, and allow for the proper fitting of the
device into an automated instrument which provides the final
determination. Thus, in using the device, the operator need only add
sample and then read the result. In this manner, one can be relatively
assured that assay determinations may be made rapidly and with a minimum
opportunity for error in quantitation.
Clinical laboratories also provide many opportunities for measuring an
analyte in an individual assay determination. Frequently, particular
analytes may be determined only a few times in any one day, so that
individual assays will be the most efficient method of performing a
particular analyte determination. Where one can use a disposable device
which only requires the addition of the sample to the disposable device,
significant savings in labor may be realized, because individuals of high
technical qualification would not be required for operation of the assay
and the accuracy of the analyte determination would be relatively assured.
In response to this need for disposable assay devices, the industry has
responded with devices which allow for the performance of the assay
protocol, with minimal measurement and input from the operator, while
allowing for sensitive and accurate detection of the amount of analyte in
a sample. Despite the development of such disposable devices, there are
problems with currently available designs. Long-standing difficulties have
included problems with efficiently mixing participating reagents with
sample, problems in efficient washing to remove unbound reagents from a
measurement area, problems in controlling the fluid flow through the assay
device, problems in controlling the timing of interaction between members
of the signal producing system in the device, and the like. Further,
problems have been encountered in designing a disposable device which has
a basic design that is sufficiently flexible so that it can be used in a
variety of different assays, thereby limiting the possibility of using the
same structural configuration for a wide variety of assays.
Thus, there is a need for the development of improved disposable assay
devices. The improved devices should provide for increased ease and
simplicity of use, while consistently providing a reliable result. The
improved devices should also provide for improved control and
reproducibility over reagent interaction and fluid flow through the
device. Furthermore, the device should have a structural configuration
that is adaptable for use in a wide variety of diverse assays.
Relevant Literature
Enzyme immunoassays are described in: Tijssen, Practice and Theory of
Enzyme Immunoassays(Elsevier) 1985; Wisdom, "Recent progress in the
Development of Enzyme Immunoassays," Ligand Rev. (1981) 3: 44-49; and
Ishikawa, "Development and Clinical Applications of Sensitive Enzyme
Immunoassays for Macromolecular Antigens-A Review," Clin. Biochem. (1987)
20:375-385. Fluorescence Immunoassays are described in: Smith et al., "A
Review of Fluoroimmunoassay and Immunofluorometric Assay," Ann. Clin.
Biochem. (1981) 18:253-274; Hammila, "Fluoroimmunoassays and
Immunofluorometric Assays," Clin. Chem. (1985)31: 359-370; Diamandis,
"Immunoassays with Time-Resolved Fluorescence Spectroscopy: Principles and
Applications," Clin. Biochem. (1988)21: 139-150. Chemiluminescent
Immunoassays are described in: Kricka & Thorpe, "Luminescent
Immunoassays," Ligand Rev. (1981) 3: 17-24; Seitz, "Immunoassay Labels
Based on Chemiluminescence and Bioluminescence," Clin. Biochem. (1984)
17:120-125; Weeks & Woodhead, "Chemiluminescence Immunoassay," J. Clin.
Immunoassay (1984) 7: 82-89. Fluorescence polarization techniques are
described in: DeGrella, "Fluorescence Polarization: A Review of Laboratory
Applications," Amer. Biotech. Lab. (1988) 6: 29-34; Jolley et al.,
"Fluorescence Polarization Immunoassay. III. An Automated System for
Therapeutic Drug Determination," Clin. Chem. (1981) 27: 1575-1579;
Dandliker et al., "Fluorescence Methods for Measuring Reaction Equilibria
and Kinetics," Meth. Enzymol. (1978) 48: 380-415. Immunoassays are
generally described in: Collins, Alternative Immunoassays (John Wiley
1985); Freytag, "The Future of Immunodiagnostics," J. Clin. Immunoassay
(1991) 14:239-244 and Gosling, "A Decade of Development in Immunoassay
Methodology," Clin. Chem. (1990) 36: 1408-1427.
SUMMARY OF THE INVENTION
A disposable assay device and methods for its use are provided. The device
comprises a sample addition port in fluid communication with at least one
main channel. The main channel comprises, in the direction of fluid flow,
a main reagent area in fluid communication with an incubation area and a
waste area. In fluid communication with the main channel is at least one
side reagent channel. The side reagent channels comprise, in the direction
of fluid flow, a liquid addition port and a side reagent area in fluid
communication with the main channel at a region upstream from the
incubation area. The main and side channels are conveniently located in a
housing comprising a top and bottom plate, where an optically clear window
is present over the incubation area for assay signal detection. Efficient
mixing of reagent in any of the reagent areas and/or in the incubation
area may be accomplished by inclusion of agitation means in these areas.
Control of fluid flow through the device is enhanced through placement of
at least one capillary valve at locations in the main and side channels
upstream from the incubation area.
The device finds use in analyte detection assays where the signal producing
system used in the assay is based on interactions between specific binding
pair members and an optical signal is related to the presence of analyte
in the sample.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an overhead view of an assay path according to the subject
invention.
FIG. 2 is an overhead view of the bottom plate of a device in accordance
with the subject invention.
FIG. 3 is an overhead view of the top plate of the device in accordance
with the subject invention.
FIG. 4 is a three dimensional view of a capillary valve in accordance with
the invention.
FIG. 5 provides three dimensional representations of various impeller
structures in accordance with the subject invention.
FIG. 6 depicts alternative bottom plate designs in accordance with the
subject invention.
FIG. 7 is an overhead view of an alternative embodiment of an assay path of
the subject invention:
FIG. 8 is an overhead view of an alternative embodiment of an assay path of
the subject invention.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
A disposable assay device and methods for its use are provided for
determining the presence of an analyte in a sample. The device comprises a
sample addition port in fluid communication with at least one main
channel. The main channel comprises, in the direction of fluid flow, a
main reagent area in fluid communication with an incubation area and a
waste reservoir. In fluid communication with the main channel is at least
one side reagent channel. The side reagent channels comprise, in the
direction of fluid flow, a liquid addition port and a side reagent area,
where the side reagent channel is in fluid communication with the main
channel at a region upstream from the incubation area. To provide for
homogeneous dispersal of signal producing system members in the sample,
agitation means may be provided in at least one of the main and side
reagent areas and/or the incubation area. Capillary valves located at
various positions along the main and side reagent channels upstream from
the incubation area may be used to control fluid flow through the device.
The device comprises a sample addition port through which sample is
introduced. In fluid communication with the sample addition port is at
least one main channel. The main channel of the subject device originates
beneath the sample addition port in a sample receiving region. The sample
receiving region serves to receive the sample and provide a conduit
between the sample receiving region and the first reagent area.
Preferably, the sample receiving region is sufficiently long to ensure
that air outside the device cannot directly access the main reagent area,
since air in the reagent area may adversely interfere with the assay being
conducted in the device.
In fluid communication with the sample receiving region is the main reagent
area. The main reagent area may serve a variety of purposes. First, a
member or members of the signal producing system, e.g. dry reagent, may be
stored in the main reagent area, so that upon entry of sample into the
main reagent area, dry reagent is rehydrated and available for reaction in
the assay. Further, the main reagent area may serve as a convenient
location where the sample temperature may be modulated, e.g. raised or
lowered, as desired for the particular assay being carried out. Finally,
the main reagent area may comprise agitation means, so that the main
reagent area serves as a mixing chamber where member of the signal
producing system is homogeneously dispersed throughout the sample.
The main reagent area is in fluid communication with at least one
incubation area. In some embodiments, the border between the reagent and
incubation areas may be so indefinite that the two regions may be
considered one region. However, the two regions will typically be
separated by a main transport channel. The main transport channel may
serve as a conduit of sample from the main reagent area to the incubation
area. The main transport channel may further serve a variety of additional
functions. Depending on the nature of the assay, the main transport
channel may serve as a storage area for various reagents which may be
diffusibly or non-diffusibly bound to the walls of the transport channel.
For example, antibodies may be present which serve to remove one or more
components from the sample, e.g. cells, interfering components, etc.
Chemical reagents may also be located in the main transport channel to
change the pH, redox potential or other characteristics of the sample. In
this way, sample introduced in the sample port may be modified as
necessary for the portion of the assay which is conducted in the
incubation area. Finally, the main transport channel may serve as the
receiving area, where fluid from the side reagent channels enters, and
combines with, the main channel.
In fluid communication with, and downstream from, the main transport
channel is the incubation area. The incubation area is where the signal
which is related to the presence of analyte is produced. On one wall of
the incubation area, usually the top wall, will be an optically clear
window which provides for viewing of the signal generated by the
particular assay being performed in the incubation area. Opposite the top
wall is a bottom wall. Diffusibly or non-diffusibly bound to the top
and/or bottom wall, usually the top wall, will be members of the signal
producing system. In one embodiment, an assay platform may be included
which comprises members of the signal producing system. A plurality of
incubation areas may be included in the main channel, where fluid flows
from one incubation area to another sequentially. Where the main channel
comprises a plurality of incubation areas, one may perform different
assays in each of the incubation areas on the same sample.
Located to one side of the incubation area, usually at the far side
opposite the side adjacent the main transport channel, will be the waste
area. The waste area serves to receive the unreacted sample and/or various
wash fluids which flow through the incubation area during a particular
assay.
In addition to the main channel, the subject device also comprises at least
one side reagent channel or flow path, and more usually comprises two side
reagent channels. Side reagent channels provide for the sequential
addition of one or more members of the signal producing system into the
incubation area. Furthermore, the side reagent channels provide
alternative means of introducing liquid, e.g. wash fluid, into the
incubation area, so that one may avoid contamination with reagent
remaining in the main reagent area.
Analogous to the main channel, the side reagent channels of the subject
device originate with a liquid addition port. In fluid communication with,
though not directly below, the liquid addition port will be the side
reagent area. Preferably, the side reagent area is a sufficient distance
from the liquid addition port so as to preclude direct access to air
outside the device to the side reagent area, where air in the side reagent
are may interfere with the assay being conducted in the device. As with
the main reagent area described above, the side reagent area may serve as
an area where members of the signal producing system are stored, as an
area where the temperature of the liquid may be modulated and as an area
for homogeneously dispersing reagent throughout the liquid. Connecting the
side reagent area with the main channel in fluid communication at a region
of the main channel upstream from the waste area, e.g. at the incubation
area or main transport channel, is a side reagent transport channel. The
side reagent transport channel serves as a conduit for liquid, and any
member of the signal producing system present therein, from the side
reagent area to the main channel.
To assist in homogeneous dispersal of the various signal producing system
members in the sample and other liquid mediums flowing through the device,
an agitation means may be provided in at least one of the main and side
reagent areas and/or the incubation area. The agitation means serves to
provide sufficient fluid flow so that dry reagent present in the vicinity
of the agitation means is efficiently hydrated and homogeneously
distributed throughout the fluid. Agitation means includes airflow,
shaking, ultrasonic techniques, suction techniques, e.g. where reagent is
dehydrated onto a porous membrane and fluid in sucked through the membrane
resulting in hydrated reagent, and mechanical means, preferably mechanical
mixing means. Suitable mechanical mixing means include mixing means
fabricated from magnetic and paramagnetic materials, and may take diverse
forms, including propellers, pins, dumbbells, balls, wires, perforated
sheets; discs with fins and the like. In a preferred embodiment, the
agitation means is an impeller device. Where the material from which the
mixing means is magnetic or paramagnetic, agitation is conveniently
accomplished by applying a moving magnetic field above or below the
device, or alternatively, by moving the device through a stationary
magnetic field. The rate and/or timing of mixing may be controlled as
needed to cause the desired level of agitation.
Control of fluid flow through the main and side reagent channels may be
enhanced though use of a variety of means. For example, where one desires
to enhance fluid flow through a device, one may provide for hydrophilic
regions in the channel at the appropriate region, where the hydrophilic
region serves to attract and draw fluid through that region.
Alternatively, where one wishes to slow or impede fluid flow through a
particular region of the channel, one may provide for hydrophobic areas in
that particular region. Another means of enhancing control of fluid flow
through the device is to employ one or a plurality of capillary valves,
which may be located at various positions along the main and/or side
reagent flow paths, usually being positioned at a region of the channel
upstream from the incubation area.
The device may be varied as to size, usually being at least about 1
cm.times.1 cm and not more than about 20 cm.times.20 cm, preferably having
a shorter dimension in the range of about 4-7 cm and in a longer direction
by about 5-10 cm. While for the most part, the device may be any
convenient shape, generally, it will be rectangular where the edges may be
modified by rounding, cutting the corner(s), or other modification which
will allow for easy handling and adapting the device to an instrument with
which it is used. The thickness of the device will generally vary from
about 1 mm 3 cm, more usually from about 1.5-3 mm.
The device may comprise one or more assay paths, where an assay path
comprises the main and side reagent channels. Inclusion of two assay paths
allows for the running of a control with the sample or running two assays
on portions from the same sample.
Also included in the device may be a reference region which serves to
provide an indication of the operability of the members of the signal
producing system being employed. For example, where a signal producing
system comprises enzyme and substrate, where the substrate is converted to
a detectable product, the reference zone may comprise dehydrated enzyme
and substrate. When the device is used, a drop of fluid is placed in the
reference zone. Appearance of enzyme product indicates the enzyme is
active, thus indicating that the enzyme in the device will be operative.
The reference region will be positioned in the device at a region which is
not in fluid communication with the assay path(s) of the device, so as not
to interfere with an assay being conducted with the device.
Applications in which the subject device may find use include assays where
the signal producing system is based on interactions between specific
binding pair members and an optical signal is generated by the system
which is related to the presence of analyte in the assayed sample.
In carrying out such an assay in the device, one may assay any type of
liquid, where the liquid may be assayed directly or may be subjected to
prior treatment, depending upon the nature of the liquid and the analyte
of interest. The liquid may contain a sample or be a sample from any
source, such as a physiological source, e.g. blood, serum, plasma, urine,
saliva, spinal fluid, lysate, nasal pharyngeal aspirates etc.; sample of
ecological interest, e.g. water, soil, waste streams, organisms, etc.;
food, e.g. meat, dairy products, plant products, other organic matter
etc.; drugs or drug contaminants in processing; or the like.
The analyte may be any compound which can be detected and is a member of a
specific binding pair, either ligand or receptor. The term "receptor" is
used arbitrarily, since its origin had to do with surface membrane
proteins, where the compound which bound to the surface membrane protein
was referred to as a ligand. Receptors include naturally occurring
receptors, e.g. enzymes, lectins, surface membrane proteins, antibodies,
recombinant proteins, etc., synthetic receptors, nucleic acids,
c-glycosides, carbohydrates, gangliosides, chelating compounds, etc. For
the purpose of the subject invention, it is sufficient that two molecules
have a significant affinity for each other, where the binding constant
will usually be at least about 10.sup.5 mol.sup.-1 and one may choose to
refer to either member as the receptor. Compounds of interest have to some
degree been indicated by indicating the various sample sources. The
analyte may be any type of compound, e.g. small organic molecules,
peptides and proteins, sugars, nucleic acids, complex carbohydrates,
viruses, bacteria particles, lipids and combinations thereof, naturally
occurring or synthetic or combinations thereof, so long as there is a
complementary binding member. The analytes will frequently include drugs,
both naturally-occurring and synthetic, various components of animals,
including humans, such as blood components, tissue components, and the
like; microorganisms, such as bacteria, fungi, protista, viruses, and the
like; components of waste streams or products or contaminants of Such
products in commercial processing; components in the environment,
particularly contaminants, such as pesticides, microorganisms, and the
like.
Depending upon the nature of the sample, the sample may be subjected to
prior treatment, such as extraction, distillation, chromatography, gel
electrophoresis, dialysis, dissolution, centrifugation, filtration, cell
separation, and the like. For blood, one may wish to remove red blood
cells to provide plasma or serum but their removal is not necessary.
Various media may be employed, which will allow for providing for a sample
solution or dispersion which can be used in the subject device.
After appropriate pretreatment, if any, the sample in liquid form is then
introduced into the sample port. The volume of sample may range from about
1 .mu.l to about 0.5 ml, more usually from about 10 .mu.l to 250 .mu.l,
preferably from about 25 .mu.l to 170 .mu.l. The sample is drawn from the
sample receiving region beneath the sample application port by capillary
action into the main reagent area. In the main reagent area, the sample
may combine with a member of a signal producing system. If the main
reagent area comprises agitation means, the sample may be agitated,
thereby hydrating any dry signal producing system member present in the
main reagent area and dispersing the hydrated member homogeneously
throughout the sample. Depending on the particular signal producing
system, a variety of members may be included in the main reagent area,
including antibodies or fragments thereof, antibody/enzyme conjugates,
antibodies labeled with a dye molecule, specific for analyte or binding
member in the incubation area, and the like.
The sample flows from the main reagent area into the main transport
channel. Where the main reagent area and the main transport channel are
separated by a capillary valve, the capillary valve may be filled to
provide for fluid flow into the main transport channel. Further, the vigor
of agitation in the main reagent area may be increased to provide the
necessary force to move sample into the transport channel. As the sample
proceeds down the main transport channel, it dissolves and/or reacts with
any reagent which may be present in the transport channel.
As the sample flows into the incubation area, it combines with other
members of the signal producing system which are present in the incubation
area. Other members of the signal producing system which may be present in
the incubation area may be members of specific binding pairs, fluorescent
production layers as described in U.S. application Ser. No. 08/089,975,
the disclosure of which is incorporated herein by reference and lipid
layers as described in U.S. Pat. No. 5,156,810 and U.S. application Ser.
No. 08/084,884, the disclosures of which are incorporated herein by
reference.
Signal production systems which find use in the application may involve
competition or cooperation. In the case of competition, the conjugate will
bind to either the analyte or binding sites in the incubation area, e.g.
on the walls of the incubation area, on an assay platform in the
incubation area, on a membrane in the incubation area, and the like. By
having a limited number of conjugate molecules, the number of conjugate
molecules which can bind to the complementary binding member will be
inversely proportional to the number of molecules of analyte in the
sample. Thus, the number of labels which ultimately become bound in the
incubation area will be inversely proportional to the number of analyte
molecules in the sample. This approach will normally be employed with
small analytes, particularly haptenic analytes, where the analyte can only
bind to a single receptor.
By contrast, with larger analytes, which are polyepitopic, one has the
opportunity for two receptors to bind simultaneously. In this way, the
analyte may serve as a bridge between the complementary binding member
bound to the membrane and the complementary binding member which is
labeled. One may also use the competitive protocol, by having the specific
binding pair member of the conjugate capable of competing with the analyte
for binding to the bound binding member.
Where a member of the signal producing system is a membrane, the membrane
in the incubation area may be divided up into a plurality of sections,
where each section may have the same or different specific binding pair
member. In this way, the sample may be assayed simultaneously for a number
of different analytes. Depending upon the nature of the different
analytes, the same or different members of the signal producing system,
e.g. conjugates, would be present in the incubation area. The assay could
be carried out in the same way, except at the time of reading, one would
specifically address different regions of the membrane to identify the
signal coming from each of the individual regions.
After sufficient time for substantially complete reaction of the analyte in
the incubation area, the incubation area may be washed to remove
substantially all of the sample and unreacted members of the signal
producing system. A buffered aqueous solution may be used which is
appropriate for maintaining the binding of the specific binding pair
members. Buffer may be introduced through the main sample addition port or
one of the side reagent ports, where the buffer travels down the main or
side reagent channels respectively and into the incubation area. Usually,
the volume of the wash solution will be at least about equal to the volume
of the sample and may be 40-fold more or greater than the volume of
sample, and preferably at least about 2-fold greater than the volume of
sample. Washing of the incubation area may be enhanced with agitation,
where agitation means are included in the | | |
