 |
Description  |
|
|
TECHNICAL FIELD
The present invention relates to a diagnostic device useful in chemical and
particularly biological and biochemical assays. The invention is
particularly directed to multiple well filtration devices, such as
microtitration plates, able to retain fluids for extended periods of time
and, under specified conditions, to remove liquid quickly and completely.
BACKGROUND ART
Diagnostic devices, including test plates, and, in particular, multiple
well or microtitration plates, have been used for both quantitative and,
especially, qualitative chemical and biological tests for decades. Various
designs and configurations have proliferated as the area of enzyme
immunoassays has expanded. Test devices and, particularly, plates having a
plurality of wells which include microporous membrane filters, have also
become routinely used in clinicallaboratories in recent times. This has
resulted, at least in part, from development of cell and tissue culture
techniques and assays in fields such as virology and immunology.
It is common in the clinical assay to simultaneously run a number of
different tests on the same liquid sample, to run duplicate tests, or to
perform the same test procedure on a number of different samples. In such
instances, it is preferred to employ a multiple well filtration plate,
such as a ninety-six well plate. Such test devices have advantages in that
they provide a single test apparatus rather than multiple test apparatus
and also provide side-by-side comparison of test results within a single
device. Such plates, however, have several significant shortcomings. Many
of the materials used to form at least the bottom portion of such test
devices are porous in nature and permit liquid in the wells to pass
through the bottom either by gravity flow or capillary action. Although
such liquid loss may be permissible and even desirable in many instances,
uncontrolled loss of fluid in many assays leads to inaccurate or
unreliable results. This is particularly true in treating or conducting
tests on living cells or tissues. In such applications, the biological
material is frequently grown or maintained in media of specified
composition for periods of from several hours to several days. Losses of
even small volumes of liquid can in some instances alter the results
drastically.
A second common problem encountered with the use of such multiple well test
plates involves a phenomenon known by some as "cross-talk". Such
occurrence involves the migration of liquid, sometimes in the form of a
pendant drop suspended from the bottom of one well, to an adjacent well.
Two causes of the type of migration known as cross-talk are (1) wicking of
fluid or diffusion of solutes laterally through the membrane between
adjacent wells, and (2) coalescing of pendant drops suspended below the
wells. Such migration may lead to spurious results, both when the liquid
removed from the wells is to be analyzed or, possibly, when liquid flows
back into an adjacent well.
DISCLOSURE OF THE INVENTION
The present invention is directed to a diagnostic test device which
includes a plate having at least one well, and preferably a plurality of
wells, each well having an open bottom. At the bottom of the well and
forming a hydrophobic, liquid-tight seal at the periphery thereof is
placed a composite membrane comprising three layers which are, preferably,
in intimate contact with one another. Proceeding from the top or upstream
side to the bottom or downstream side of the composite membrane, in
sequence, the first layer is a reaction or filtration layer formed from a
thin, liquophilic microporous membrane, such as a membrane of filtration
material. After transfer of a test sample to a well and removal of liquid,
in those situations when subsequent reactions are performed on substances
retained by the composite membrane, it is the reaction layer which
generally forms the site at which reaction occurs. Placed below the
reaction layer is a second or sealing layer. This second, preferably
porous layer functions as both a means of securing or adhering the
reaction layer to a liquophobic barrier layer as well as forming a
liquophobic seal at the periphery of the well where the side walls of the
well contact the composite membrane. Because of the liquophobic seal,
cross-talk by lateral diffusion or wicking is eliminated. Preferred is a
hydrophobic material for the second layer. The third or downstream layer
is a liquophobic barrier layer. This layer includes a small aperture
located substantially at the center of the well. This barrier layer
substantially eliminates dripping, enhances isolation of each well in
multiple well devices, and inhibits lateral migration of a pendant liquid
drop from one well to another well.
In addition to the aforementioned advantages, the multiple well diagnostic
plate device of the present invention permits greater or enhanced
sensitivity in tests performed with the device as a result of the
increased surface area afforded by the porous nature of the reaction
layer. In some instances, the substantially increased sensitivity
resulting from the substantially higher surface area of the microporous
membrane provides results making the difference between operability and
inoperability of the particular protocol. Furthermore, the rapid and
efficient removal of sample and reagent solutions only after application
of a pressure differential permits the solutions to be removed rapidly and
completely without the use of a pipette.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a preferred embodiment of the present invention in
combination with a manifold.
FIG. 2 is a sectional view of FIG. 1 taken along line II--II.
FIG. 3 is a sectional view of an embodiment of the present invention which
includes an upper well-containing plate portion having a composite
membrane at the bottom opening of each well and a lower receptacle
portion.
BEST MODE FOR CARRYING OUT THE INVENTION
The Diagnostic Plate
The diagnostic plate employed in the present application may have a variety
of forms and be made from any suitable material. Preferred materials are
thermoplastic resins including polyolefins, such as polypropylene, and
polystyrene. The plate may contain as few as one well or as many wells as
can be arranged within the plate and effectively serviced by the source of
differential pressure, i.e., reduced or positive pressure, employed.
Filtration plates currently being used typically have ninety-six wells and
this number of wells is not incompatible with the present invention.
Typically, the wells in such devices are closely spaced for efficiency and
economics. As a result, the potential for cross-talk in such devices is
enhanced.
The wells themselves may be formed in the shape of cylinders projecting
upwardly from a base of the plate or, as preferred, may be formed as
cylinders projecting downwardly from an upper surface or plane of the
plate. Each of the wells may have vertical or downwardly tapered side
walls, the latter forming conical or tapered wells.
The dimensions and shape of each well may be varied depending upon the
application of the device. Thus, the well will typically have a
cylindrical configuration but other configurations, such as a square or
rectangle, may also be used. Likewise, the diameter of the well may vary
considerably depending upon such factors as the size of the sample to be
used in each well and whether the device is to be used for diagnostic
purposes, its most widespread anticipated application, or for separation
and isolation of substances.
The size of the liquid sample used and, therefore, the dimensions of the
well may also depend on the sensitivity of the reaction performed in the
well when the device is used for diagnostic purposes. Typically, the
diameter of a well will be from about 1 mm to about 100 mm. Preferably,
the diameter of a well will be about 2 mm to about 25 mm. Typically, the
height of a well is about 1 to about 75 mm, preferably about 5 to about 10
mm.
The bottom of each well has an opening formed therein. For example, the
well may have the configuration of an open cylinder or of a hollow
truncated or frustoconical cone. The periphery or brim of the opening to
which the composite membrane is adhered generally forms a horizontal
surface.
Composite Membrane
Across the bottom of the well and sealed in liquid-tight relationship to
the periphery of the well is a composite membrane. The composite membrane
comprises at least three layers. The first or uppermost layer constitutes
a "reaction layer" which, when the device of the present invention is used
for diagnostic purposes, is the layer in or upon which reagents are added
and tests are performed. This layer may also be used as a filtration
medium to separate liquid and solid components when the material retained
by the composite membrane is not intended for further characterization.
The material from which this layer is formed, like the other layers, must
not react adversely with substances found in either the samples, reagents
or solvents employed in the analyses. In addition, the reaction layer must
be formed from a liquophilic, microporous membrane, typically having an
absolute pore rating of about 0.001 to about 20 microns, preferably about
0.02 to about 8 microns, and most preferaly about 0.2 to about 3 microns.
The reaction layer preferably is also skinless. Materials which are
suitable for use as the reaction layer also have voids volumes in the
range of about 60 to about 90 percent, preferably in the range of about 75
to about 90 percent. Preferred materials are hydrophilic in nature and
are, therefore, easily water-wettable and tend to freely pass aqueous
solutions. Examples of liquophilic materials which may be used in the
present invention include, but are not limited to, polyamides, such as
nylon 66, polyvinylidene difluoride, cellulose esters, and nitrocellulose.
Liquophilicity, as used herein, refers to the wettability of the membrane
by the liquid(s) with it is contacted. The wettability or liquophilicity
of a solid structure, e.g., a membrane, is a function of that structure's
critical surface energy and the surface tension of the applied liquid. If
the critical surface energy is at least as high as the surface tension of
the liquid, the liquid will spontaneously wet the solid structure. For
example, a microporous membrane having a critical surface energy of 72
dynes/cm or higher will be wetted by water which has a surface tension of
72 dynes/cm, i.e., it is hydrophilic.
The capability of a porous structure (membrane) to be wetted by a liquid
can be determined by placing a drop of liquid on the porous structure. The
angle of contact provides a quantitative measure of wetting. A very high
angle of contact indicates poor wetting, while a zero angle of contact
defines complete or perfect wetting. Materials used in the subject
invention as the wettable or liquophilic porous layer are characterized by
being readily or spontaneously wetted by the applied liquid and have a low
angle of contact with the applied liquid. Indeed, when a drop of a test
liquid(s) is placed on a spontaneously wettable or liquophilic microporous
membrane layer, the drop of liquid penetrates the layer amd wets the
membrane, effectively providing a zero angle of contact therewith.
Suitable materials should also be capable of being treated with and
retaining or immobilizing a substance being analyzed and/or a reactant
which may be used to perform a specified test or reaction with the
substance being analyzed for in a sample. The reactant, which may be of
ionic, molecular, or macromolecular nature may be immobilized on the
reaction layer by strong physical forces or by being bonded in some
manner, such as covalent chemical coupling, to the surface of the
microporous, liquophilic reaction membrane layer. As employed herein, the
term "surface" or "surface area" refers not only to the gross surface(s)
of the structure but also, in those cases where a microporous structure
such as a membrane is under consideration, to the surfaces of the
micropores, i.e., the interior surfaces of the structure which are
contacted by fluid during use. As distinguished from "surface area" or
"surface", the exposed planar or gross area of the material is herein
referred to as the "reaction layer area", "microporous reaction layer
area", "reaction layer membrane area", or the like.
Wettability or liquophilicity is a requisite of the materials used for the
microporous reaction layer of the present invention. It is particularly
preferred that such materials be capable of being spontaneously wetted.
Some of the materials which are suitable or preferred for use as the
reaction layer in the present invention are intrinsically hydrophilic or
water-wettable. Others may be modified to render them hydrophilic.
BIODYNE.RTM. is an N66 polymide, microporous membrane commercially
available from Pall Corporation which is inherently water-wettable by
virtue of its method of manufacture (see U.S. Pat. No. 4,340,479).
Polyvinylidene fluoride membranes are not inherently water-wettable but can
be rendered such by an appropriate surface treatment. Microporous,
polyvinylidene fluoride membranes which have been treated to render them
hydrophilic are commercially available. As discussed above, wettability or
liquophilicity is a function of the critical surface energy of the solid
structure and the surface tension of the liquid. Wettability may also be
expressed in terms of intrusion pressure which may be defined as the
applied pressure required for liquid to penetrate into the pores of the
membrane. Although a function of the properties of the liquid used, such
as surface tension, materials which are particularly preferred for the
reaction layer of the composite membrane have intrusion pressures of or
close to zero.
Materials which are preferred for the reaction layer also have large
surface areas. This feature permits a greater amount or higher
concentration of reactant to be immobilized in the reaction layer.
Accordingly, higher sensitivities may be achieved using the test plate of
the present invention.
Polyamides preferred for use in the present invention include nylons of the
type described in
U.S. Pat. No. 4,340,479, which is incorporated herein by reference. As
noted above, a membrane material of this description which is particularly
useful for the present invention is a microporous, hydrophilic nylon
membrane commerically available from Pall Corporation under the trademark
BIODYNE.RTM..
Another preferred membrane useful as the reaction layer is
IMMUNODYNETM.TM., available from Pall Corporation. IMMUNODYNETM.TM. is a
modified CARBOXYDYNE.RTM. membrane, also available from Pall Corporation.
CARBOXYDYNE.RTM. is a hydrophilic, microporous, skinless nylon 66 membrane
with controlled surface properties formed by the cocasting process
described in U.S. Pat. No. 4,707,266, as discussed below, specifically by
cocasting nylon 66 and a polymer containing an abundance of carboxyl
groups to form a membrane having controlled surface properties
characterized by carboxyl functional groups at its surfaces.
IMMUNODYNETM.TM. membranes may be prepared from CARBOXYDYNE.RTM. membranes
by treating them with trichloro-s-triazine in the manner described in U.S.
Pat. No. 4,693,985, discussed below.
Also included among the preferred polyamide membranes for the present
invention are hydrophilic, microporous, skinless polyamide membranes with
controlled surface properties of the type described in (1) U.S. patent
application Ser. No. 850,061, filed Apr. 7, 1986, now U.S. Pat. No.
4,707,266, which is a continuation application of U.S. patent application
Ser. No. 459,956, filed Jan. 21, 1983, now abandoned, which in turn is a
continuation-in-part application of U.S. patent application Ser. No.
346,118, filed Feb. 5, 1982, now abandoned and in (2) U.S. patent
application Ser. No. 848,911, filed Apr. 7, 1986, now U.S. Pat. No.
4,702,840, which is a continuation application of U.S. patent application
Ser. No. 460,019, filed Jan. 21, 1983, now abandoned which is a
continuation-in-part application of U.S. patent application Ser. No.
346,119, filed Feb. 5, 1982, now abandoned.
All of the aforementioned U.S. patent applications are specifically
incorporated herein by reference. These hydrophilic, microporous,
substantially alcohol-insoluble polyamide membranes with controlled
surface properties are formed by cocasting an alcohol-insoluble polyamide
resin with a water-soluble, membrane-surface-modifying polymer having
functional polar groups. Like the preferred hydrophilic, microporous nylon
membranes which do not have controlled surface-modified polar groups
present, the polyamide membranes of the present invention having
controlled surface properties are also skinless; that is, they are
characterized by through pores extending from surface-to-surface which are
of substantially uniform size and shape. If desired, however, materials
having tapere through pores, i.e., pores which are larger at one surface
of the sheet, narrowing as they approach the opposite surface of the
sheet, may be used.
The surface-modifying polymers used to prepare the polyamide membranes with
controlled surface properties, useful in the present invention, comprise
polymers which contain substantial proportions of chemically functional
groups, such as hydroxyl, carboxyl, amine, and imine groups. As a result,
the membranes include, at their surfaces, high concentrations of
functional groups such as hydroxyl, carboxyl, imine, or a combination of
any of the above groups which do not react with one another. These
polyamide membranes having controlled surface properties have higher
concentrations of carboxyl or imine groups at their surfaces than the
preferred microporous, hydrophilic, skinless polyamide membranes described
above which do not have controlled surface properties, i.e., those which
are formed from the preferred polyamide resin but are not cocast with
surface-modifying polymer.
The reaction layer may be treated by any method known to one of skill in
the art to deposit and/or bind reagents thereto. As indicated above, the
reagent may be of an ionic, molecular, or macromolecular nature. When used
as a diagnostic tool to provide a visible change, the reagent may be one
or a combination of substances which is initially colorless and which,
upon reaction with a suitable material, provides an optically measurable
response. Other possible variations include the use of suitable labels,
such as the formation between the deposited reagent and the material for
which testing is being conducted of a complex or compound which is
appropriately labeled by any known technique, such as enzymatic/substrate
labels or the like.
Although treatment of the reaction layer with a suitable reagent(s) may be
performed at the time at which diagnostic tests are to be performed,
including addition of the test reagent(s) both immediately preceding and
following introduction of the sample containing the analyte to the
well(s), the present invention is expected to have greatest applicaion to,
and a preferred embodiment includes, a composite membrane in which the
reaction layer has been pretreated with at least one test reagent.
Typically, pretreatment is conducted after the composite membrane has been
sealed to the wells but before the device is shipped to a user. If the
reagent(s) is not heat sensitive, the membrane may be treated before
assembling the composite membrane.
A useful method of binding reagents of a molecular nature, especially
macromolecules, and particularly those of a biological nature, is
disclosed in U.S. Pat. No. 4,693,985, specifically incorporated herein by
reference. This patent describes a method for immobilizing a wide range of
biologically active substances as acceptor molecules on active membranes.
The acceptor-bound membranes described in the application are capable of
immobilizing and binding a wide variety of biologically-active compounds,
specifically ligands, to the acceptor molecules. Using such reaction
layers or membranes permits the testing of bodily fluids, such as blood,
serum, plasma, urine, saliva, and the like, and testing for particular
substances by chemical assays or immunoassays, such as those where a
specific label is employed, such as one indicating enzyme activity or an
electromagnetic energy absorbing and/or emitting label, such as a
fluoroescent label. The macromolecules used as reagents and bound to the
reaction layer or which are assayed for using the device of the present
invention generally include materials of a biological nature and are
frequently proteinaceous in nature. The reagent or acceptor molecule bound
directly to the reaction layer or the ligand being tested for include such
substances as immunoglobulins or antibodies, either polyclonal or
monoclonal, antigenic substances, apoproteins, receptors, glycoproteins,
lectins, carbohydrates, hormones, enzymes, carrier proteins, heparin,
coagulation factors, enzyme substrates, inhibitors, cofactors, nucleic
acids, etcetera.
Placed below, preferably in intimate contact with or, most preferably,
adhered, bonded, or otherwise secured to the reaction layer, particularly
at the periphery of each well, is a porous sealing layer which serves
several purposes. Strongly preferred for use as a sealing layer is a very
porous liquophobic structure. The term "liquophobic" as used herein is
effectively the obverse of the term "liquophilic", that is, a liquophobic
material has a critical surface energy lower than the surface tension of
the applied liquid and is not readily or spontaneously wetted by the
applied liquid(s). Liquophobic materials are characterized, then, by a
high contact angle between a drop of liquid placed on the surface and the
surface. Such a high contact angle indicates poor wetting. The porous
sealing layer assists in bonding the reaction layer to the barrier layer.
It also makes it possible to form a liquophobic seal at the periphery of
the well, thereby eliminating cross-talk by diffusion or wicking. The use
of such material in the sealing layer also provides the composite membrane
with the ability to withstand or maintain a pressure differential. For
example, it prevents, without the application of vacuum or high pressures,
penetration of liquid through the composite membrane and the concomitant
loss of liquid from the well by dripping. Thus, at specified pressure
conditions, generally close to or at atmospheric pressure, liquid is
retained within a well. However, when reduced pressure is applied to the
downstream side of the composite membrane or super atmospheric pressure is
applied to the upstream side of the membrane, liquid readily drains from
the well.
Materials suitable for use as the sealing layer, in addition to being
liquophobic, preferably are hydrophobic and are also significantly more
porous than the reaction layer. Such material may be present in sheet or
fiber form, either woven or unwoven. Suitable materials include
polyamides, linear polyesters, such as esters of ethylene glycol and
terephthalic acid, polyolefins, such as polypropylene, polyethylene,
polymethylpentene, and polyisobutylene, as well as copolymers formed by
copolymerizing the monomers used to form the aforementioned homopolymers,
such as ethylene-propylene copolymers. Mixtures or blends of such polymers
can also be used. The polyolefins are preferred and polypropylene is
particularly preferred.
The third or downstream layer constitutes a barrier layer which,
preferably, is in intimate contact with or, most preferably, is adhered,
bonded, or otherwise secured to the sealing layer, particularly at the
periphery of each well. It is this layer, provided with at least one, and
preferably only one, aperture located substantially at the center of the
well, which permits a drop of liquid to pass from the well, under
application of positive or negative gauge pressure, i.e., super
atmospheric pressure applied upstream of the composite membrane or reduced
pressure applied downstream of the membrane, and to drop from the plate
without radial migration to another well. The apertures can also serve as
restriction means, controlling the flow rate of liquid through the
composite membrane. Thus, the barrier layer, because of the nature of the
material used therein, i.e., a highly liquophobic, preferably hydrophobic,
material, enhances isolation between wells and forces the liquid passing
through the composite membrane to form small drops at the hole in the
barrier layer. These drops, rather than spreading radially when passing
through a porous or microporous liquophilic material (such as the material
of the reaction layer used alone or in conjunction with the sealing layer)
and forming a small contact angle with the surface of such material, tend,
when passing through the barrier layer, to form a large contact angle with
the surface of liquophobic material. Thus, even if the device is raised at
one end so that the bottom surface is not completely horizontal, liquid
drops passing through each well tend to drop from the device rather than
rolling to one side and potentially contaminating fluid in an adjacent
well.
The size and shape of the apertures formed in the barrier layer depend on a
number of variables including the porosity of the barrier layer, the
number of apertures in the barrier layer, the porosity and number of
apertures formed in the other layers (discussed below), the number of
wells, the flow rate sought, ease of manufacturing, etc. However, when a
single aperture is used in the barrier layer of each well, it may be
formed in a variety of shapes. A suitable size for the aperture is about
1/32 to about 1/4 inch. An aperture or hole simply formed in the shape of
an "X" in the barrier layer, i.e., without a corresponding aperture in the
sealing layer or reaction layer, provides an operable system.
The material used to form the barrier layer should have a greater
liquophobicity than either the reaction layer or the sealing layer and may
be either microporous or nonporous. Another way of expressing the
suitability of a material as the barrier layer relates to the wetting
resistance characteristics of the material. A suitable material should be
capable of resisting a liquid intrusion pressure greater than to the
height of the column of liquid above the barrier layer, i.e., the height
of the liquid placed in the well. Suitable materials include polyolefins,
such as polypropylene, polyhalogenated polyolefins, particularly
perfluorinated polyolefins, such as polytetrafluoroethylene, and
polyvinylidene difluoride, as well as sulfones. Polytetrafluoroethylene is
most preferred.
In addition to the aperture formed in the liquophobic barrier layer, the
reaction layer may also be provided with at least one, and preferably no
more than one, aperture. When a single aperture is provided in the
reaction layer, the diameter of the aperture may be in the range of about
3 to about 100 microns. When present as a plurality of apertures, each
aperture may have a diameter of about 3 to about 100 microns. The choice
of using a membrane either with or without an aperture in the reaction
layer is determined by such factors as the source and type of vacuum or
pressure apparatus employed to force a liquid through the composite
membrane, the number of wells present in the device, the overall porosity
of the composite membrane, the porosity or bubble point characteristics of
the reaction layer, and the dimensions of the aperture(s) in the barrier
layer. All of these factors affect the rate of fluid flow through the
membrane at a given pressure drop.
The provision of one or more apertures in the reaction layer of the
composite membrane in each well assures total voiding of liquid through
the composite membrane when positive or negative gauge pressures are
employed. When the reaction layer is provided with an aperture, a
continuous, rather than an intermittent, application of positive or
reduced pressure is necessary since the vacuum or positive pressure will
be continuously bleeding away. However, depending on the factors listed
above, when a reaction layer having no apertures passing therethrough is
provided in the composite membrane, a continuous application of positive
or reduced pressure, preferably the latter or a single or intermittent
application of positive or negative gauge pressures using, for instance, a
syringe and check valve combination may be employed. These latter
alternatives are generally preferred.
In some instances when manufacturing the composite membrane of the present
invention, it is desirable to initially provide each of the layers with
the appropriate number of apertures. Thus, a needle(s) may be used to
simultaneously form the apertures in each layer in alignment with each
other. However, with the porous sealing layer, the aperture will usually
not be much larger than the existing pores and when a fibrous material is
used, the fibers will generally move back to the position they occupied
before insertion of the needle. In this embodiment, the diameters of the
apertures in the reaction and barrier layers are the same. This embodiment
provides for ease of manufacturing in that the apertures may be formed
after the composite membrane is assembled and the layers are secured to
one another.
Typically, reduced pressure or vacuum-assisted fluid flow is accomplished
with the present invention by means of a vacuum manifold which is provided
with a means for connection to a vacuum source, such as a projecting tube
which can be inserted into vacuum tubing. Such manifolds also include a
means to form an air-tight seal between the well(s)-containing diagnostic
plate device and the manifold, such as a pliable gasket or the like. Such
manifolds commonly include a vertical wall portion, the internal surface
of which configurationally conforms to the outer surface of a vertical
wall portion of the plate device such that mating of the two wall surfaces
occurs. Other designs, however, may be employed, such as the diagnostic
plate device being provided with an internal wall surface which mates with
an external wall surface of the manifold. Alternative structures in which
mating surfaces of the manifold and diagnostic plate device are provided
and in which an air-tight seal may be established may also be employed
with the present invention.
In use, a first test or reagent liquid is normally placed in each of the
wells of the diagnostic plate device, and the device is then inserted into
the vacuum manifold such that the gasket or sealing means contacts mating
surfaces in both the diagnostic plate device and the manifold.
Communication between a source of vacuum and the manifold is then
established. As the pressure in the manifold decreases, the diagnostic
plate device is drawn against the gasket, improving the seal. As this
occurs, liquid is drawn through the wells into the vacuum line and to a
waste trap. Solutions of samples to be tested or reagents are then
introduced into each of the wells.
To provide a means for isolating and retaining fluids passing through the
diagnostic plate device of the present invention for those situations in
which it is desirable to conduct further tests on the liquid, a second
modified form of the diagnostic plate device may be used within the vacuum
manifold. The modified form of the plate is positioned intermediate the
vacuum manifold and the diagnostic plate device in which test samples are
analyzed. The modified form of the device is one in which the wells have
solid impermeable bottoms, that is, the wells do not have open bottoms
covered with the composite membrane of the present invention but rather
the membrane is replaced with the same material from which the walls of
the wells are made or some similar material. In most cases, the modified
plate is of unitary construction in which the side walls and bottom are
formed integrally in a single step. The sealing means is still maintained
between the diagnostic plate device of the present invention and the
vacuum manifold when the device is placed into operation.
In some situations, it may be desirable to use the diagnostic plate device
of the present invention in situations where a vacuum line connected to a
centralized source of vacuum or a vacuum pump is not available. An
embodiment of the present invention, nevertheless, permits analysis in
such instances. The manifold employed includes a modified outlet means for
connecting to a vacuum source. Specifically, the outlet includes a one-way
valve of a type generally known to the valve art which permits fluids to
be withdrawn from the manifold but does not permit fluids to enter the
manifold. In addition, the connection means permits a syringe tip to be
inserted into the manifold so that a large syringe may be used as the
vacuum source. Such a means for connection to a syringe tip may be a Luer
locking device or the like. When a syringe is used as the vacuum source,
this will normally be able to displace a much smaller volume of air than
will a continuously operating vacuum pump. Accordingly, the vacuum
manifold and diagnostic plate device adapted to be used with the manifold
will be of a smaller size with fewer than 96 wells which is a typical
number for such multiple well diagnostic plate devices.
As with the larger diagnostic plate device described above, the periphery
of the plate may have any suitable configuration as long as adequate
mating and sealing surfaces are provided between the manifold and the
diagnostic plate device. To sufficiently reduce the volume of the manifold
when the device is to be used with a syringe as the source of vacuum, the
periphery of the plate is preferred to have a circular configuration, such
as that shown in FIG. 1, the wells also preferably being arranged in a
circular configuration. With such a configuration, various means may be
provided for the attachment of the plate to the manifold including that
described above in which the well-containing plate is merely pressed into
the manifold with a sealing means separating two mating surfaces.
Alternatively, in addition to sealing means, threading or bayonet mounts
may be provided on corresponding mating surfaces of the plate and
manifold. However, a preferred structure is one that is disposable in
which the diagnostic plate is formed integrally with the manifold.
| | |
