U.S. patent application number 12/197762 was filed with the patent office on 2008-12-18 for chemical assays.
Invention is credited to Kevin Andrew Auton, Paul Thomas Ryan, Sergei M. Schurov, David John Wigley.
Application Number | 20080311665 12/197762 |
Document ID | / |
Family ID | 9903785 |
Filed Date | 2008-12-18 |
United States Patent
Application |
20080311665 |
Kind Code |
A1 |
Ryan; Paul Thomas ; et
al. |
December 18, 2008 |
Chemical Assays
Abstract
An assay device in which to carry out a fluid-phase chemical
assay, comprising means for supporting a test substrate, a sample
chamber for receiving a fluid sample and at least one fluid control
device for controlling the movement of fluid into and/or out of the
sample chamber, wherein the fluid control device comprises a fluid
outlet chamber in fluid communication with the sample chamber, and
a displaceable flexible diaphragm the displacement of which alters
the volume of the outlet chamber so as to allow and/or restrict
fluid flow between the outlet and sample chambers. The invention
also provides assay apparatus incorporating such a device, an assay
station for use as part of such apparatus, a fluid control unit for
use as part of the assay device and a method of conducting an assay
which may involve the use of such apparatus and devices.
Inventors: |
Ryan; Paul Thomas;
(Cambridgeshire, GB) ; Auton; Kevin Andrew;
(Cambridgeshire, GB) ; Schurov; Sergei M.;
(Cambridgeshire, GB) ; Wigley; David John;
(Cambridgeshire, GB) |
Correspondence
Address: |
ST. ONGE STEWARD JOHNSTON & REENS, LLC
986 BEDFORD STREET
STAMFORD
CT
06905-5619
US
|
Family ID: |
9903785 |
Appl. No.: |
12/197762 |
Filed: |
August 25, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10258551 |
Oct 25, 2002 |
7431884 |
|
|
PCT/GB01/05158 |
Nov 22, 2001 |
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12197762 |
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Current U.S.
Class: |
436/52 |
Current CPC
Class: |
B01L 3/50273 20130101;
B01L 2300/1838 20130101; B01F 11/0071 20130101; B01J 2219/00725
20130101; F16K 99/0001 20130101; G01F 1/7088 20130101; B01J
2219/00527 20130101; F16K 7/12 20130101; F16K 99/0034 20130101;
B01J 2219/00286 20130101; B01J 2219/00612 20130101; B01J 2219/00637
20130101; B01L 9/527 20130101; B01L 2400/0481 20130101; B01L
2400/0638 20130101; G01F 11/086 20130101; B01L 2200/0689 20130101;
B01L 2300/1805 20130101; B01J 2219/00689 20130101; F16K 99/0015
20130101; G01F 1/7086 20130101; B01J 2219/00659 20130101; B01F
13/0059 20130101; B01L 2300/0816 20130101; B01J 2219/0063 20130101;
G01F 1/708 20130101; B01L 2300/0822 20130101; B01L 2400/0487
20130101; B01J 2219/00497 20130101; Y10T 436/117497 20150115; B01J
2219/00585 20130101; B01L 2300/0874 20130101; B01L 3/502738
20130101; F16K 99/0059 20130101; B01J 2219/00353 20130101; B01J
2219/00596 20130101; B01J 2219/0061 20130101; B01L 2300/0887
20130101; B01L 2300/1844 20130101; B01J 2219/00605 20130101; B01J
2219/00722 20130101; F16K 2099/0084 20130101; G01P 13/0006
20130101; B01L 2200/027 20130101; B01J 2219/00621 20130101; B01J
2219/00389 20130101; B01L 2300/1827 20130101 |
Class at
Publication: |
436/52 |
International
Class: |
G01N 35/02 20060101
G01N035/02 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 24, 2000 |
GB |
0028647.6 |
Claims
1. A method of conducting a plurality of fluid-phase chemical
assays, which involves supporting a plurality of test substrates in
a corresponding number of assay devices, and conducting a
fluid-phase chemical assay in each device, simultaneously or
sequentially.
2. A method of conducting a plurality of fluid-phase chemical
assays according to claim 1 further comprising: placing fluid
samples in contact with said test substrates via a plurality of
sample chambers, and controlling the movement of fluid into and/or
out of said plurality of sample chambers using at least one fluid
control chamber per assay device.
3. A method of conducting a plurality of fluid-phase chemical
assays according to claim 2 further comprising: inducing fluid
movement within said plurality of sample chambers via a fluid
agitation device, varying the pressure of the fluids supplied to
said sample chambers, and controlling the volume of said plurality
of said sample chambers via a displaceable diaphragm.
4. A method of conducting a plurality of fluid-phase chemical
assays according to claim 3 further comprising: incorporating two
or more of said fluid agitation devices to reciprocally move fluid
back and forth through the plurality of said sample chambers.
5. A method of conducting a plurality of fluid-phase chemical
assays according to claim 4 further comprising: monitoring fluid
flow via a flow rate monitoring device, and controlling the
temperature in said plurality of sample chambers.
6. A method of conducting a plurality of fluid-phase chemical
assays according to claim 5 further comprising: mixing the fluids
in a fluid mixing device to be directed to said plurality of assay
devices.
7. A method of conducting a plurality of fluid-phase chemical
assays according to claim 1 further comprising: immobilizing at
least one probe species on said plurality of test substrates,
binding said probe species to said target test substrate, reacting
said probe species with said plurality of test substrates, and
detecting said bound pair using conventional techniques such as
fluorescence, chemiluminescence, and colored dyes.
8. A method of conducting a fluid-phase chemical assay which
comprises: supporting a test substrate in an assay device,
controlling the movement of fluid in a sample chamber in contact
with said test substrate, incorporating at least one fluid control
device which comprises a fluid outlet chamber in fluid
communication with said sample chamber, and a displaceable flexible
diaphragm, displacing said diaphragm to alter the volume of said
fluid outlet chamber so as to control fluid flow between said fluid
outlet chamber and said sample chamber.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of co-pending U.S. patent
application Ser. No. 10/258,551 filed Oct. 25, 2002, which is the
US national phase of international application PCT/GB01/05158 filed
Nov. 22, 2001, and claims priority from United Kingdom patent
application 0028647.6 filed on Nov. 24, 2000. All prior
applications are herein incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to methods, apparatus and devices for
use in carrying out chemical (which includes biochemical) assays,
in particular for the detection of biological materials such as
proteins and peptides.
BACKGROUND OF THE INVENTION
[0003] Certain types of chemical, in particular biochemical, assays
involve immobilizing on a test substrate a probe species capable of
binding selectively to a target species. A fluid sample, containing
or suspected to contain the target species, is brought into contact
with the test substrate; target species present in the sample will
then bind to the immobilized probe. After washing the substrate to
remove unbound species, the presence of the target-probe pair can
be detected in several known ways, including via chemical "labels"
(for instance, labels capable of chemiluminescence or fluorescence)
attached to the target species.
[0004] This principle is used in a large number of biochemical
assays, for instance to detect the presence of target nucleotide
sequences or proteins. It involves, however, an often complex
sequence of procedures. A suitably selective probe for the target
species must firstly be identified, usually by means of some form
of screening, and immobilized on a test substrate. A sample fluid
must then be maintained in contact with the substrate for a
sufficient period of time, and under suitable conditions, for
target-probe binding to take place (and to take place to a
detectable degree). During this period, the temperature of the
sample often needs to be cycled between quite precise ranges and
over specific time periods, to enable binding to occur. The test
substrate must then be washed, usually with increasing levels of
stringency to remove not only unbound species but also those which
are bound with an unacceptably low degree of specificity. Finally,
the washed substrate must be analyzed to detect the presence and/or
amount of target-probe pairs.
[0005] These procedures can to an extent be automated, but often
still involve significant manual intervention, for instance to
control the introduction of samples and reagents at appropriate
times and locations. Moreover, apparatus for carrying out the
procedures can be both complex and costly, involving large numbers
of separate fluid control devices (valves and pumps) in order to
introduce what is often a large number of necessary sample and/or
reagent fluids.
[0006] Since it may be desirable to assay a large number of samples
at a time, and/or to test a sample against a large number of probe
species, there is a constant need to enhance the efficiency of such
assays, to reduce the complexity of the apparatus in which they are
carried out, to minimize the amount of manual intervention needed,
to maximize throughput and/or to increase accuracy and consistency
in the results. Furthermore, since the samples being assayed are
often scarce (for instance, DNA-containing samples), and typically
need to be screened for more than one target species, it is always
desirable to minimize the amount of sample needed for an assay,
typically by increasing detection sensitivity.
[0007] It is already known to carry out a chemical assay by
spreading a thin layer of a liquid sample over a flat test
substrate, such as a glass microscope slide, on which an "array" of
several, often hundreds or more, probe species has been
immobilized. This allows the sample to be screened simultaneously
for a corresponding number of target species. Such arrays have, for
instance, been disclosed recently for the detection of proteins in
a biological sample; the test substrate may be referred to as a
"protein array" or "protein biochip" [de Wildt, R M T et al, Nat
Biotechnol, 18 (9), 989-94 (September 2000); Mendoza, G,
BioTechniques, 27(4), 781-788 (1999); Bussow, K et al, Genomic 65,
1-8 (2000)]. It would be desirable to be able to use such
substrates in an at least partly automated assay process, and
preferably to be able to process a plurality of substrates
simultaneously.
SUMMARY OF THE INVENTION
[0008] According to a first aspect of the present invention there
is provided an assay device in which to carry out a fluid-phase
chemical assay, the device comprising (i) means for supporting a
test substrate, (ii) a sample chamber for retaining a fluid sample
in contact with a test substrate which is so supported and (iii) at
least one fluid control device for controlling the movement of
fluid into and/or out of the sample chamber, wherein the fluid
control device comprises a fluid outlet chamber in fluid
communication with the sample chamber, and a displaceable flexible
diaphragm the displacement of which alters the volume of the fluid
outlet chamber so as to cause and/or allow and/or restrict fluid
flow between the fluid outlet chamber and the sample chamber.
[0009] The term "fluid-phase chemical assay" means a diagnostic
test for detecting the presence and/or quantity of a target species
in a fluid sample, by means of a chemical reaction. It is intended
to embrace biochemical assays such as for the detection of a target
nucleotide sequence (DNA, RNA etc.) or protein or peptide. It will
typically involve the use of a probe species immobilized on a test
substrate with which the sample is brought into contact, the probe
species being capable of reacting with the target species, via
which reaction the presence and/or quantity of the target species
may be detected. The reaction suitably involves selective binding
of the probe species to the target species, the bound pair being
detectable using conventional techniques such as fluorescence,
chemiluminescence, colored dyes and the like.
[0010] The means for supporting a test substrate may include means,
such as clamps, spring clips and the like, for securing the
substrate in place in the assay device. It preferably also includes
sealing means, such as an appropriately shaped gasket or an O-ring
seal, for sealing areas of contact between the test substrate and
the rest of the device, in particular to help define, and to
prevent fluid leakage from, the sample chamber.
[0011] The assay device may be capable of supporting two or more
test substrates.
[0012] The sample chamber may be defined at least partly by a test
substrate which, in use, is supported in the device. The sample
chamber should then be an enclosed space, save for fluid inlets
and/or outlets such as those providing fluid communication with the
fluid control device(s). Preferably the sample chamber volume is
small, typically between 50 and 120 .mu.L, such as between 100 and
120 .mu.L. More preferably it can enclose a thin layer of the
sample fluid, for instance between 50 and 100 .mu.m deep, adjacent
the active surface of a test substrate supported in the device. The
"active surface" of the substrate is that part of its surface which
carries one or more probe species; the sample chamber ideally
allows the enclosed fluid to contact the whole of the active
surface. The dimensions of the active surface are typically about
20 mm by between 30 and 65 mm.
[0013] Again, the assay device may include two or more sample
chambers which may, in use, be associated with separate test
substrates or with different regions of a single substrate.
[0014] The fluid control device may comprise a fluid flow control
valve such as for controlling the introduction of fluid to and/or
evacuation of fluid from the sample chamber. It may comprise a
fluid agitation device for inducing fluid movement within the
sample chamber, such as by forcing fluid into and/or out of the
sample chamber. It may comprise a fluid storage device in which a
quantity of fluid may be held prior to its introduction into or
following its evacuation from the sample chamber or another part of
the assay device. (The word "comprise" is used in this
specification to mean either "be" or "include".)
[0015] The fluid control device is preferably itself controllable
using a control fluid, which may be supplied to a region of the
flexible diaphragm such that variations in the control fluid
pressure cause displacement (which term includes distortion) of the
diaphragm. The fluid control device therefore preferably comprises
a control chamber and a control port through which control fluid
may be introduced into the control chamber, the diaphragm being
arranged between the control chamber and the fluid outlet chamber
in such a way that displacement of the diaphragm, caused by
pressure changes in control fluid supplied to the control chamber,
alters the volume of the outlet chamber. A suitable control fluid
is compressed air, although many other pressurized liquids or gases
could be useable to the same effect.
[0016] Instead or in addition, the fluid control device may be at
least partially controlled by varying the pressure of one or more
of the other fluids (e.g., sample or reagent fluids) supplied to
it.
[0017] The flexible diaphragm should be made from, at least at its
surface, a material which is inert with respect to the reagents
which will pass through the assay device in use. It must have
sufficient resilience to function in the required manner under the
fluid pressures likely to be applied to it, i.e., to be
displaceable and/or distortable between the required operating
positions. Suitable diaphragm materials include silicone rubbers of
hardness 40 to 60 Shore A and thickness between 0.3 and 2 mm,
typically 1 mm. These may optionally be faced with low or high
density polyethylene (LDPE or HDPE) or polypropylene, of a film
thickness between 10 and 100 .mu.m.
[0018] Where the fluid control device comprises a valve, it
preferably additionally comprises a fluid inlet chamber, the
flexible diaphragm being displaceable between a first position in
which it restricts or prevents fluid communication between the
fluid inlet and outlet chambers and a second position in which
fluid communication between the inlet and outlet chambers is
allowed.
[0019] The inlet and outlet "chambers" may take the form of fluid
conduits. Communication between them may be directly or via one or
more intermediate chambers and/or conduits. In general in this
specification, the term "fluid communication" embraces both direct
and indirect communication, though preferably direct unless
otherwise specified.
[0020] Where the fluid control device comprises a fluid agitation
device, again the diaphragm is preferably arranged between a
control chamber and the fluid outlet chamber in such a way that
displacement of the diaphragm, caused by pressure changes in
control fluid supplied to the control chamber, alters the volume of
the outlet chamber. In this way, displacement of the diaphragm can
cause fluid to be forced either into or out of the sample chamber,
thus generating fluid movement within the sample chamber. Such
movement is generally desirable to maintain a homogeneous sample
fluid and hence increase accuracy and sensitivity of an assay.
[0021] Preferably, the assay device of the invention incorporates
two such fluid agitation devices, which can be reciprocally
operated to move fluid back and forth through the sample chamber.
In such an arrangement, the two agitation devices preferably
communicate with opposite ends of the sample chamber, or at least
with two spaced apart regions of the sample chamber.
[0022] Where the fluid control device comprises a fluid storage
device, it preferably comprises a fluid inlet port for receiving
fluid (typically a sample fluid) and a fluid storage chamber, in
fluid communication with the inlet port, for holding fluid received
at the inlet port. The diaphragm then preferably functions to
control movement of fluid into and out of the storage chamber,
being displaceable between a first position in which fluid is held
in the storage chamber, and a second position in which fluid is
urged out of the storage chamber and into the outlet chamber.
Control of the diaphragm, to displace it between these first and
second positions, may be effected by means of an associated valve
and/or by the application of a pressure change to another part of
the control device, for example directly to the storage chamber,
more particularly by the supply of control fluid to the diaphragm
to displace it within the storage chamber.
[0023] Communication between the storage and outlet chambers may be
via an intermediate chamber. Moreover, a single port may function
as both inlet and outlet, the relative fluid pressures (i) in the
storage and/or intermediate chambers and (ii) at the inlet/outlet
port determining the direction of fluid flow, and the diaphragm
position either allowing or preventing flow as desired. This
arrangement may effectively comprise a combination of a fluid
storage device and a diaphragm-operated valve to control the
introduction of fluid into it, for example via the intermediate
chamber.
[0024] The storage chamber typically holds a small volume, for
instance between 50 and 200 .mu.L preferably between 100 and 150
.mu.L of fluid. It ideally holds at least enough fluid to fill the
associated sample chamber; a 90 .mu.L sample chamber may for
instance be associated with a 150 .mu.l storage chamber, which can
of course be part filled if appropriate. The storage chamber is
particularly suited for the storage of small quantities of scarce
sample fluids, which may be pre-loaded into the assay device and
stored in close proximity to the sample chamber, for introduction
into the sample chamber at an appropriate point in an assay.
[0025] Where the fluid control device has a fluid inlet port or
inlet conduit, the port or conduit may be of any size and shape
suitable to allow the introduction of fluid for example from a
source elsewhere in the assay device or, in the case of a sample
fluid, conveniently via a needle or pipette. The inlet port or
conduit may for example have an opening to the exterior of the
device, the opening being adapted to receive a pipette or other
fluid introducing means.
[0026] The fluid control device may comprise a fluid loading
device, into which fluids may be loaded and/or evacuated and/or
transferred either from outside the assay device or from other
component(s) within the assay device. Such a fluid loading device
comprises a receptacle, such as a cup- or bowl-shaped receptacle,
to accommodate fluid which may be introduced into it for instance
from an external source. Preferably, the receptacle is directly
accessible from the outside of the assay device, and most
preferably, it is adapted to receive a fluid introducing means such
as a pipette.
[0027] A cup- or bowl-shaped receptacle may conveniently be
provided in the exterior surface of a plate or block forming part
of the assay device, as described above. Its capacity may suitably
be between 10 and 500 .mu.L, preferably between 50 and 100 .mu.L,
depending on its intended use. It will have at least a first outlet
which provides fluid communication with another part of the assay
device, typically a fluid storage device or the sample chamber,
such communication conveniently being via another fluid control
device such as a valve.
[0028] The receptacle preferably also has a second outlet through
which fluid may be evacuated, typically to waste. The locations of
the first and second outlets will depend on their intended
functions; suitably the second is positioned, in use, at a higher
fluid level within the receptacle than the first.
[0029] Preferably at least the first outlet is in direct fluid
communication with a valve for controlling fluid flow into and/or
out of the fluid loading device. More preferably still, the first
outlet is in direct fluid communication with, or constitutes, the
fluid inlet chamber of a valve of the type described above which is
operated via a displaceable flexible diaphragm.
[0030] Thus, in a fluid control device (in particular a valve)
which forms part of an assay device according to the invention, any
fluid inlet chamber or port preferably is or comprises a fluid
loading device of the type described above.
[0031] The assay device of the invention preferably comprises more
than one, typically two or more, for instance two, such fluid
loading devices, which may then be used for loading fluids from
externally and/or for transferring fluids between other components
of the device (e.g., sample chambers, fluid storage devices and
reagent or other fluid sources). Each loading device may be
associated with (i.e., in direct or indirect fluid communication
with) one or more storage devices and/or sample chambers, and/or
with one or more other fluid loading devices so that fluid may be
transferred between the loading devices for example via a commonly
connected storage device. The capacity of each loading device
(i.e., of its fluid receptacle) is ideally larger than that of an
associated sample chamber and/or storage device, by an amount
sufficient to accommodate losses and "dead" volumes and still to
provide sufficient fluid to fill the relevant chamber/device. Its
capacity may for example be between 10 and 100% greater than that
of the associated chamber/device.
[0032] The assay device of the invention preferably comprises more
than one fluid control device of the types described above. It may
for instance comprise both a fluid inlet and a fluid outlet valve,
controlling respectively the introduction of fluid into and
evacuation of fluid from the sample chamber. It may comprise more
than one fluid inlet valve, allowing the introduction of more than
one fluid into the sample chamber. Fluid inlet valves may also be
provided for controlling the introduction of fluid(s) into one or
more fluid storage devices. The assay device may comprise one or
more fluid receiving ports associated with one or more of the inlet
valves. It preferably additionally comprises one or more fluid
agitation devices, preferably at least two. More preferably it
additionally comprises one or more fluid storage devices, in which
fluid may be held in close proximity to the sample chamber. Not all
of the fluid control devices need be in direct fluid communication
with the sample chamber.
[0033] Ideally the assay device comprises at least three, more
preferably at least four or five or six fluid control devices, the
fluid ports and chambers of which are defined within a single unit
which may also serve at least partly to support a test substrate.
The assay device can thus comprise integral fluid control devices,
which may be supplied from externally with appropriate sample,
reagent, wash and control fluids. This allows the device to be
relatively simple and compact in construction. It also facilitates
independent temperature control, flow rate analysis and other
necessary processes for a test substrate supported within the
device.
[0034] The unit in which the fluid control devices are provided may
comprise, for instance, a block or plate made from a suitable
material, such as a metal or plastics material, in which the
necessary fluid chambers, conduits and ports may be machined,
molded or similarly provided. Chambers, conduits and ports may be
provided at the face of such a block or plate and may be at least
partly defined by a sealing layer, such as a gasket, positioned
adjacent that face.
[0035] More preferably still, the assay device of the invention
comprises a single flexible diaphragm common to more than one,
ideally all, of its fluid control devices. In other words, a single
diaphragm is arranged to perform more than one function at
different locations in the assay device, the locations
corresponding to the relevant fluid chambers in the fluid control
devices. Typically a separate means for controlling the device
operation will be needed at each such location; this may comprise a
separate control chamber or port, for bringing a control fluid into
contact with the diaphragm at the relevant location to cause a
local displacement of the diaphragm. All the control ports may, in
use, be supplied with control fluid from a single source,
optionally with a separate valve or other means to control the
supply of fluid to each control port. These features can again
simplify the construction of the assay device.
[0036] The diaphragm may for example be positioned between two
adjacent plates or blocks, each providing certain fluid chambers
and channels to form part of the fluid control devices. In a more
preferred embodiment, the assay device comprises more than two (for
instance three) stacked plates, with a diaphragm positioned between
each pair of adjacent plates, so that different fluid control
devices can be defined by different plate pairs. This increases the
versatility of the system, allowing a wider range of devices to be
provided in a single unit and in a wider range of locations within
that unit.
[0037] One or more of the plates may also serve as a support for a
test substrate.
[0038] In regions of the device where the diaphragm contacts, and
provides a seal around, the edges of a fluid conduit or chamber
defined in an adjacent plate or block, raised surface elements
(e.g., ridges) or other forms of surface profiling may be provided
adjacent or close to the perimeter of the conduit or chamber, so as
to amplify, in the region of the conduit or chamber perimeter, the
force applied to clamp the diaphragm in position adjacent the plate
or block. Such surface profiling may be provided on the surface(s)
of either or both of the plates between which the diaphragm is
clamped (preferably that in which the relevant conduit or chamber
is defined), and/or on the diaphragm itself.
[0039] Ideally the assay device of the invention also incorporates
a fluid distribution assembly, by means of which the necessary
fluid(s) may be introduced into the device from external sources
and subsequently removed from the device. This assembly will
typically include one or more fluid inlet ports, directly or
indirectly connectable to external sources of for instance reagent
and wash fluids, and one or more conduits through which fluid may
pass from the inlet port(s) to the fluid control device(s) of the
assay device. It will also include one or more fluid outlet ports,
directly or indirectly connectable for instance to a waste
reservoir, and one or more conduits through which fluid may pass
from the assay device to the outlet port(s).
[0040] Again, the necessary fluid channels may be drilled,
extruded, machined or molded within a unit such as a plate or block
which is appropriately positioned with respect to the fluid control
device(s) of the assay device. Chambers, conduits and ports may be
provided at the face of such a plate or block and may be at least
partly defined by a sealing layer, such as a gasket, positioned
adjacent that face. Most preferably, the fluid ports and conduits
of the distribution assembly are provided in the same plate or
block in which the fluid control devices, or parts thereof, are
located, or at least in an adjacent plate or block.
[0041] The inlet ports of the distribution assembly may in certain
cases correspond to those of the fluid control devices and may for
example comprise fluid loading devices of the type described above.
One or more of the inlet ports may be for the introduction of a
control fluid such as compressed air.
[0042] The incorporation of such a fluid distribution assembly
allows for a plurality (often a very large number) of assay devices
to be connected to a common set of fluid supply and removal lines,
and to the controls for such fluid lines, and hence to be
simultaneously processed in a single assay apparatus.
[0043] A second aspect of the present invention provides an assay
device in accordance with the first aspect, in combination with a
test substrate on which one or more probe species are immobilized.
The substrate may, for example, be a glass slide. The probe species
may be immobilized on the substrate in any known manner. Preferably
the substrate carries a plurality (for instance, up to about
100,000, typically between about 5,000 and 20,000) of immobilized
probe species, in any suitable arrangement such as in an array.
[0044] According to a third aspect of the present invention, there
is provided a device for monitoring the flow rate of a first fluid,
typically a liquid, the device comprising a primary measuring
chamber through which the first fluid may flow, a fluid inlet port
upstream of the primary measuring chamber, through which a volume
of a second fluid (typically a gas, such as in the form of a
bubble) may be introduced into the first fluid flow, and primary
detection means, associated with the primary measuring chamber, for
detecting the presence of the second fluid in the first fluid as
they pass through the primary measuring chamber. The detection
means may be electrical in operation, detecting changes for example
in conductance or capacitance between electrical contacts
positioned at different locations in the flow path of the first
fluid through the measuring chamber. A printed circuit board may
for instance be provided in, and conveniently form one wall of, the
measuring chamber, to detect the presence or absence of the second
fluid in the measuring chamber--where the first fluid is a liquid
and the second an injected gas bubble, for instance, absence of the
liquid indicates the presence of the gas bubble, and can be
detected using the printed circuit board.
[0045] Alternatively, optical detection means may be used, such as
are described for instance in U.S. Pat. No. 4,210,809.
[0046] The time taken for the second fluid to reach the primary
measuring chamber, from its inlet port, may thus be measured and
used to provide an indication of the flow rate of the first fluid
through the device.
[0047] Preferably, the monitoring device additionally comprises a
secondary measuring chamber, in fluid communication with and
conveniently downstream of the primary one, the secondary chamber
having associated with it a secondary detection means, for
detecting the presence of the second fluid in the first as they
pass through the secondary measuring chamber. A more accurate
indication of the first fluid flow rate may then be obtained by
measuring the time taken for the second fluid to travel between the
two measuring chambers. Fluid communication between the measuring
chambers is preferably by means of an extended, more preferably
labyrinthine, fluid conduit, to increase the distance traveled by
the first and second fluids between the measuring chambers.
[0048] The flow rate monitoring device of this third aspect of the
invention may be used in association with an assay device according
to the first aspect, to measure the rate of flow of one or more
fluids through the assay device. The monitoring device is
preferably incorporated in, more preferably integral with, the
assay device, conveniently downstream of the sample chamber. This
can be achieved, for instance, by providing the measuring
chamber(s), fluid conduit(s) and inlet port(s) of the monitoring
device in a unit containing the fluid control device(s).
[0049] An assay device according to the first aspect of the
invention preferably incorporates means for controlling the
temperature inside the sample chamber; this may be particularly
useful in the case where a biochemical assay involving thermal
cycling is to be carried out in the device. The temperature control
means may include conventional devices such as hot air blowers,
ovens, fans, fluid heating and/or cooling baths, etc. The assay
device may for instance comprise a heat sink, of conventional
construction, which can be cooled for instance by means of a fan
which forces a cooling fluid (such as air) through channels
provided in it, and which can preferably also be heated for
instance electrically. Heat may then flow by conduction between the
heat sink and the rest of the device, at least in the region of the
sample chamber.
[0050] Instead or in addition, the temperature control means may
comprise channels within the device or its surrounding apparatus,
through which a heating/cooling fluid may be caused to flow. This
fluid may be externally heated and/or cooled by any convenient
means, for instance electrical resistance heaters, forced (or
natural) air-cooled heat exchangers or peltier devices. It may be
circulated by convection or, preferably, by means of a pump. Such a
form of temperature control can give improved temperature
uniformity, both across the test substrate and also between assay
devices where several are to be processed together. It may make
possible more rapid temperature changes, in particular if several
external reservoirs of heating/cooling fluids are held at different
desired temperatures to be supplied to the assay device at
appropriate times. In use, several assay devices may be supplied by
a common source or sources of heating/cooling fluid(s).
[0051] For more efficient heating and/or cooling the assay device,
or if appropriate groups of assay devices, is/are preferably
enclosed in a chamber to isolate it/them from neighboring assay
devices and from the surrounding environment.
[0052] According to a fourth aspect of the present invention, there
is provided apparatus for carrying out a fluid-phase chemical
assay, the apparatus comprising an assay device in accordance with
the first aspect of the invention, and/or an assay device/test
substrate combination in accordance with the second aspect, and/or
a flow rate monitoring device in accordance with the third
aspect.
[0053] Such apparatus preferably comprises a plurality of assay
devices in accordance with the invention. It may comprise one or
more assay "stations", each of which can accommodate a plurality of
assay devices. Each station ideally has an associated fluid
distribution assembly, communicating with those of its assay
devices, to enable appropriate fluids to be supplied to the assay
devices and spent fluids to be removed to waste.
[0054] A typical such assay station might be capable of supporting
for instance at least four or six or ten or twelve or sixteen assay
devices. Apparatus according to the fourth aspect of the invention
could include for example at least three or four or five or ten
assay stations. This would allow the simultaneous execution of a
large number of assays, each in a respective assay device, and
would lend itself particularly well to at least partial automation,
for instance under the control of a microprocessor. Ideally the
fluid movement through each assay device and/or assay station could
be independently controlled. Similarly, the operating temperature
could be independently controlled for at least each individual
assay station.
[0055] A fifth aspect of the invention provides a fluid
distribution system for use in the apparatus of the fourth aspect,
the system comprising first and second fluid inlet lines via which
first and second fluids may be drawn from respective sources, first
and second fluid flow control devices, each allowing a variable
fluid flow rate, in the first and second fluid inlet lines
respectively, and control means for controlling individually the
flow rates through the first and second fluid flow control devices.
The system preferably additionally comprises a fluid mixing device,
downstream of the fluid flow control devices, for combining the
first and second fluids emerging from the control devices. The
combined fluid stream emerging from the fluid mixing device may
then be directed to a desired location, preferably to one or more
assay devices or assay stations according to the invention.
[0056] The fluid flow control devices are preferably variable rate
pumps, or alternatively valves providing adjustable flow rates.
[0057] Such a fluid distribution system allows two fluids to be
combined in a desired ratio. Ideally the fluid flow rates through
the first and second fluid flow control devices are continuously
variable between their minimum and maximum values, allowing for
continuous variation of the first:second fluid ratio in the mixture
emerging from the system. This could be of particular use, for
instance, in supplying varying concentrations of reagent or wash
solutions to an assay device (in which case the first fluid might
be a suitable reagent in concentrated form and the second fluid a
diluent such as water).
[0058] The fluid distribution system may include more than two
fluid inlet lines with more than two respective fluid flow control
devices. In this case any desired number of fluid mixing devices
may be included to achieve any desired combination of the fluids
passing through the system.
[0059] The fluid distribution system may form part of apparatus
according to the fourth aspect of the invention, and may be used to
supply one or more sample, reagent or wash fluids to the assay
device(s). Preferably apparatus according to the fourth aspect
includes more than one such distribution system, for introducing
more than one fluid mixture into the assay device(s).
[0060] According to a sixth aspect of the present invention, there
is provided a fluid control unit for use as part of an assay device
according to the first aspect, the unit comprising a fluid control
device as described above, i.e., comprising a fluid outlet chamber
which is connectable to an assay device sample chamber in use, and
a displaceable flexible diaphragm the displacement of which alters
the volume of the fluid outlet chamber so as to cause and/or allow
and/or restrict, in use, fluid flow between the fluid outlet
chamber and the sample chamber. The unit preferably comprises a
plurality of such fluid control devices, which may include (as
above) valve(s), fluid agitation device(s), fluid storage device(s)
and/or fluid loading device(s).
[0061] The unit is preferably constructed from two or more adjacent
plates having a flexible diaphragm positioned between each pair of
adjacent plates, at least some of the fluid chambers and ports of
the fluid control device(s) being defined in those faces of the
plates which are adjacent the diaphragm(s). One of the plates may
also serve as a support for a test substrate, in use. The unit
preferably also comprises means (such as fluid inlet and outlet
ports) for connecting it to external fluid inlet and outlet
conduits (for instance, leading to fluid sources and/or to waste),
to one or more supplies of a control fluid such as compressed air,
and/or to a sample chamber when the unit forms part of a complete
assay device.
[0062] The unit of the sixth aspect of the invention preferably
additionally comprises a flow rate monitoring device in accordance
with the third aspect, and/or temperature control means as
described in connection with the first aspect.
[0063] A seventh aspect of the invention provides an assay station
comprising means for accommodating one or more assay devices,
preferably according to the first aspect of the invention, and a
fluid distribution assembly (for instance as described above) which
is connectable, for example via fluid ports, to external fluid
conduits for the supply of fluids to, and/or their removal from,
assay devices held at the station, the fluid distribution assembly
having one or more fluid conduits to allow fluid communication
between the assay devices and one or more external fluid conduits.
More preferably, the assay station comprises one or more,
preferably a plurality of, fluid control units in accordance with
the sixth aspect of the invention, the fluid distribution assembly
allowing communication between the external fluid conduits and each
of the fluid control units. More preferably still, the assay
station comprises two or more adjacent plates with a flexible
diaphragm positioned between each pair of adjacent plates, wherein
the fluid control devices of the individual units, and at least in
part the fluid conduits of the distribution assembly, are provided
within the two plates.
[0064] The devices, apparatus, assemblies and units of the
invention may include at least partially automated control means,
for instance comprising a computer.
[0065] According to an eighth aspect of the present invention,
there is provided a method of conducting a fluid-phase chemical
assay which involves the operation of an assay device or other
apparatus in accordance with the invention. In particular, the
method involves supporting a test substrate in an assay device in
accordance with the first aspect of the invention, and controlling
the movement of fluid into and/or out of the sample chamber in
contact with the test substrate using at least one fluid control
device which comprises a fluid outlet chamber in fluid
communication with the sample chamber, and a displaceable flexible
diaphragm the displacement of which alters the volume of the fluid
outlet chamber so as to cause and/or allow and/or restrict fluid
flow between the fluid outlet chamber and the sample chamber.
[0066] The method preferably involves locating a plurality of test
substrates in a corresponding number of assay devices (suitably
using apparatus according to the fourth aspect of the invention)
and conducting a fluid-phase chemical assay in each device,
simultaneously or sequentially.
BRIEF DESCRIPTION OF THE DRAWINGS
[0067] In the accompanying illustrative drawings:
[0068] FIG. 1 shows apparatus according to the fourth aspect of the
invention;
[0069] FIG. 2 shows one of the assay stations A to D from the FIG.
1 apparatus;
[0070] FIG. 3 shows the fluid control devices for one of the
cassettes seen in FIG. 2;
[0071] FIGS. 4a and 4b are a stylized "plan" view and a partially
exploded cross section respectively of one of the valves seen in
FIG. 3;
[0072] FIGS. 5a and 5b are a stylized plan view and a cross section
respectively of an alternative valve useable in the FIG. 3
cassette;
[0073] FIG. 6 is a cross section through one of the fluid agitation
devices seen in FIG. 3;
[0074] FIGS. 7a and 7b are a stylized plan view and a cross section
respectively of a combination of fluid control devices such as
those shown in FIGS. 4 to 6;
[0075] FIGS. 8a and 8b are a stylized plan view and a cross section
respectively of an alternative combination of fluid control
devices;
[0076] FIGS. 9a and 9b are a stylized plan view and a perspective
view respectively of a fluid control unit in accordance with the
sixth aspect of the invention, for use in the apparatus of FIGS. 1
and 2;
[0077] FIG. 10 is a section through one of the valves seen in FIG.
9a;
[0078] FIGS. 11a and 11b are a stylized plan view and a cross
section respectively of a sample chamber of one of the cassettes
seen in FIG. 2;
[0079] FIG. 12 is a cross section through an assay device in
accordance with the first aspect of the invention, for use in the
apparatus of FIG. 1;
[0080] FIGS. 13a and 13b are a stylized plan view and a cross
section respectively of part of an assay station for use in the
FIG. 1 apparatus;
[0081] FIG. 14 is a cross section through a flow rate monitoring
device in accordance with the invention, for use in the FIG. 1
apparatus;
[0082] FIG. 15 is a cross section through part of an assay station
of the type shown in FIG. 13, in combination with temperature
control means;
[0083] FIG. 16a is a side view and FIG. 16b a stylized plan view of
a "blanked-off" cassette from the FIG. 13 assay station;
[0084] FIGS. 17a and 17b are a stylized plan view and a cross
section respectively of an alternative type of valve for use in
apparatus or assay devices according to the invention;
[0085] FIG. 18 is a cross section through one embodiment of the
FIG. 17 valve;
[0086] FIG. 19 is a cross section through an alternative embodiment
of the FIG. 17 valve;
[0087] FIG. 20 is a cross section through a fluid loading device
for use in an assay device according to the invention;
[0088] FIG. 21 is a section through part of the FIG. 20 device
during a typical fluid loading operation;
[0089] FIG. 22 shows an arrangement of fluid loading devices and
other fluid control devices useable in an assay device according to
the invention;
[0090] FIGS. 23a, b and c are sections through parts of fluid
control devices in accordance with the invention, illustrating an
alternative construction;
[0091] FIGS. 24a and b are respectively a section through part of a
fluid control device in accordance with the invention and a
stylized plan view of the same;
[0092] FIG. 24c is a section corresponding to that in FIG. 24b
showing the device during operation;
[0093] FIG. 25 is a section corresponding to that in FIG. 24a but
through part of an alternative fluid control device in accordance
with the invention; and
[0094] FIGS. 26a and 26b are a stylized plan view and a cross
section respectively of an alternative type of valve for use in
apparatus or assay devices according to the invention.
[0095] All figures are schematic.
DETAILED DESCRIPTION OF THE INVENTION
[0096] Embodiments of the present invention will now be described,
by way of example only, with reference to the accompanying
drawings.
[0097] FIG. 1 shows schematically apparatus according to the fourth
aspect of the invention, for use in carrying out several
simultaneous chemical assays, in particular using protein arrays.
The apparatus comprises reservoirs 1 of the required reagent fluids
(including buffers, detergents, catalysts, wash solutions and the
like and typically also, for in situ dilution of other reagents,
distilled water and/or other solvents). An appropriate number of
fluid supply lines, here illustrated schematically as a single
conduit 2, carrying fluids from the reservoirs 1 to assay stations
3, each of which houses a number of slide "cassettes" as described
below in connection with FIGS. 2 and 3. The assay stations are here
labeled A to D; in apparatus according to the invention there can
be any desired number of such stations supplied from the same fluid
reservoirs. The apparatus of the invention allows all stations to
be processed simultaneously but also, if necessary,
independently.
[0098] Supply line 4 carries a control fluid such as compressed
air, via pump 5, to each of the assay stations 3. Conduits 6 carry
fluids from the assay stations to a waste reservoir 7.
[0099] FIG. 2 shows, also schematically, one of the assay stations
A to D from the FIG. 1 apparatus. The station supports a suitable
number of, in this case twelve, slide "cassettes" 8. Each cassette
holds one test substrate, typically a microscope slide having an
array of probe materials (for example, antigens and/or antibodies)
immobilized on it. Each cassette also provides, in association with
the assay station, the fluid control devices necessary to conduct
fluids to and from the test substrate it holds; these devices are
described below in connection with FIGS. 3 to 10 and 17 to 25.
[0100] The assay station also includes fluid conduits, valves and
pumps to control the flow of reagent and control fluids into and
out of the slide cassettes. Fluid supply line 4 carries the control
fluid (in this case compressed air) to the slide cassettes, via a
corresponding number of valves 9. Reagent fluids are fed to the
cassettes via fluid supply lines 10 (corresponding to conduit 2 in
FIG. 1), selecting valves 11, pumps 12 and a further mixer 13. Any
desired number of valves 11, pumps 12 and mixers 13 may in practice
be used, according to requirements. A "purge valve" 14 allows the
fluid conduits in the station to be purged to waste (reservoir 7,
as shown in FIG. 1) with a chosen reagent fluid, bypassing the
slide cassettes 8. Fluids pumped through the cassettes drain to a
single outlet conduit 15 (corresponding to 6 in FIG. 1) and thence
to the waste reservoir.
[0101] The FIG. 2 apparatus incorporates a fluid distribution
system in accordance with the fifth aspect of the invention, which
allows the relative concentrations of certain reagent fluids to be
automatically and continuously varied.
[0102] Each valve 11 can in this case be selected to feed one of a
pair of fluids, for instance either that carried by conduit 10a or
that carried by conduit 10b, into a pump 12. The output of two or
more (in this case all three) of the pumps 12 is combined in the
mixing device 13, and the resultant mixture can then be fed to the
slide cassettes. If only one of the pumps 12 is operated, then only
a single fluid is sent to the cassettes. If more than one of the
pumps is operated then a mixture of fluids may be supplied. By
varying the pumping rate for each pump then the overall volumetric
flow rate may be set as well as the mixing ratio(s) between the
fluids feeding the pumps. In this way, either a preset mix ratio
may be achieved or, if the pumps are operated at a controlled,
time-varying rate, the constitution of the fluid mixture can be
varied with time. For instance, two pumps may be operated with a
constant combined pumping rate, but varying their pumping rate
ratio between 1:0 and 0:1. The composition of the resulting fluid
mixture may thereby be varied from 100% of one fluid to 100% of
another, the variation following any desired pattern with time,
whether continuous or step-wise.
[0103] This arrangement is of particular use for instance in
supplying wash solutions of varying concentrations to the test
substrates. A concentrated wash solution may be supplied to one of
the pumps 12 and a diluent such as distilled water to another,
allowing the wash solution to be diluted to any desired level by
altering the two pump rates.
[0104] When there are three pumps, as shown in FIG. 2, two of them
may be used to pump concentrated active ingredients, with variable
blending between the two, whilst the third pumps a diluent (again
typically water) to set the overall concentration of the active
ingredient mixture. In this way the FIG. 1 apparatus need only be
fed from smaller bottles of concentrated active ingredients. The
pumps 12 are preferably of the positive-displacement type such as
piston, peristaltic, gear or diaphragm pumps. To vary their pumping
rate they are preferably driven with an electronic speed controller
that optionally includes a microprocessor to calculate the pumping
speed and control its variation with time.
[0105] An alternative to the variable rate pumps 12 would be the
provision of variable valves in pressurized fluid supply lines. The
fluid flow rates downstream of the valves would depend on the
upstream pressures, the valve openings and the back pressure from
downstream fluid control devices. Flow rate monitors could be
incorporated to allow feedback control over the fluid flows.
[0106] Other sensing devices may be provided downstream of the
mixing device 13, to provide indications of flow rate, pH,
conductivity and/or other desired parameters. Measurements obtained
from such sensors may then be used to adjust fluid flow rates to
obtain a desired mix. Such adjustment may be real-time, using
measurements taken during processing to correct the fluid mixing in
a dynamic way. Alternatively, the measurements may be used during a
separately conducted calibration or characterization process. In
this latter case, the performance of the pumps (or other fluid flow
control devices) is characterized by analyzing the fluid mix
resulting from various predetermined operating rates.
[0107] The sensing devices should also be calibrated; this can be
done semi-automatically using standardized solutions in the reagent
reservoirs, the standards being passed individually (and unaltered)
through the sensors. The sensor outputs may be used to correct
subsequent sensor outputs during an assay, to achieve greater
accuracy. Alternatively, calibration may be performed by setting
the system to create a mix of particular (dynamically measured and
real-time corrected) characteristics. This fluid may be collected
and checked manually, and the process may be repeated for a variety
of mixes and the results used to correct inaccuracies in the mixing
and monitoring systems.
[0108] Fluid connections to the slide cassettes 8, and fluid
conduits within the cassettes, are arranged to offer similar
resistance-to-flow for each cassette. This means that the fluid
flow will divide evenly between the cassettes. Resistance-to-flow
can be matched between the cassettes by matching the length and
aperture of conduits to, through and from the cassettes. Where
matching is not practical for a part of a conduit (for example, in
the case of a manifold), its internal cross section must be made
larger and/or its length shorter to ensure its resistance-to-flow
does not affect the division of fluid flow between the cassettes.
Thus, the operating rates of pumps such as 12 may be used to
control the rate of fluid flow through all of the cassettes.
[0109] Clearly apparatus according to the invention may include
more than one mixing device 13, and a corresponding number of
"sets" of valves and pumps, to allow greater versatility in the
number and ratio of fluids which can be supplied to the slide
cassettes.
[0110] FIG. 3 shows in more detail, although still schematically,
the fluid control devices in one of the FIG. 2 cassettes 8. Each
cassette is an assay device constructed in accordance with the
first aspect of the invention; it contains all the fluid control
devices necessary for carrying out a chemical assay on a test
substrate held within it, in particular devices for storing small
volumes of sample fluids and injecting them into a sample chamber
containing the test substrate, for agitating fluids in the sample
chamber and for removing fluids from it.
[0111] FIG. 3 shows a test substrate 16 (in this case, a microscope
slide carrying a protein array as described above) held in the
cassette. The slide is removable from the cassette and replaceable
so as to carry out further assays using the same cassette. A cover
17 provides an enclosed sample chamber over the active surface of
the substrate 16.
[0112] The fluid control devices, all contained within the cassette
as described below in connection with FIGS. 9, 12 and 13, include a
fluid inlet valve 18 and outlet valve 19 which connect the cassette
to the fluid supply lines 10 of the FIG. 2 apparatus and to the
outlet conduit 15 respectively. They also include sample loading
means 20 and 21, each of which comprises a fluid loading device as
described below in connection with FIG. 20 and a fluid storage
device as described in connection with FIG. 7. These allow storage
of sample fluids and their introduction into the sample chamber.
The cassette also includes agitation devices 22 and 23 which
operate in tandem to move fluids back and forth through the sample
chamber.
[0113] Each of the fluid control devices 18 to 23 is individually
supplied with control fluid (e.g., compressed air) from the supply
line 4 (see FIG. 2). The control fluid supply may be independently
controlled for each of the fluid control devices, using
conventional fluid flow controls (not shown).
[0114] The fluid control devices 18 to 23 are all constructed using
two adjacent plates with a flexible diaphragm sandwiched between
them. The construction of the inlet valve 18, for instance, is
shown schematically in FIG. 4a ("plan" view) and FIG. 4b (cross
section). It is formed in upper and lower plates 24 and 25
respectively, with a flexible, impervious diaphragm (membrane) 26
clamped between them. The upper plate 24 carries a control port 27,
to which a control fluid (typically compressed air, at for example
300 kPa) can be selectively supplied, and a control chamber 28.
Lower plate 25 contains a fluid outlet port 29, leading to the
sample chamber of the cassette, and a fluid inlet conduit 30. Fluid
from the supply lines 10 is supplied to the conduit 30 at a
moderate differential pressure (typically 20 kPa) relative to that
at the outlet port 29. In the absence of pressure, the control port
27 may be vented or, optionally, a negative pressure (relative to
that at outlet port 29) may be applied.
[0115] Pressure at control port 27 forces the diaphragm 26 against
the upper face of plate 25 over the area of the control chamber 28.
The diaphragm thus seals the end of conduit 30, preventing fluid
flow through the device. If port 27 is vented (or a negative
pressure applied to it) then the diaphragm is no longer clamped to
the plate 25 and may move away, aided if necessary by the pressure
of incoming fluid in conduit 30. This incoming fluid may then flow
to the outlet port 29, and thence into the sample chamber of the
cassette. In the case where the valve is "open" when its control
port 27 is vented, the valve constitutes a restriction to fluid
flow, which may be overcome by fluid pressure in the inlet conduit
30. In contrast, an arrangement in which a vacuum is applied to the
control port in order to open the valve can present less of a
restriction to fluid flow.
[0116] The supply of all necessary fluids to the sample chamber may
be controlled using valve(s) of the FIG. 4 type.
[0117] The diaphragm 26 may be made from any of a variety of
materials or even a combination. If the diaphragm material is thin
and/or soft then little pressure is required to force fluid through
the valve (supposing port 27 to be vented). In contrast, if it is
thicker, harder and compressed by the clamping of the plates then a
substantial pressure differential is required between ports 27 and
29 to overcome the natural sealing force provided by the diaphragm.
In this latter case, fluid flow through the valve may be controlled
by varying the pressure of the fluid feed through inlet conduit 30,
the pressure at the control port 27 remaining constant (e.g.,
vented).
[0118] Clearly the diaphragm should be inert with respect to the
fluids passing through the valve. This may be achieved either by
fabricating the diaphragm from a suitably inert material or by
using a laminated structure in which a material chosen for its
mechanical properties is faced by a preferably thin layer of a
different, inert material. Typical diaphragm materials would be
silicone rubber sheet (of hardness 45 Shore "A") faced by a
polypropylene sheet. It is not necessary for the components of the
laminate to be joined mechanically for the valve to function but it
may aid assembly.
[0119] The operating pressure of the valve is dependent also on its
dimensions. A smaller diameter for the control chamber 28 and/or a
thicker diaphragm (with a correspondingly increased clamping force)
would lead to a higher operating pressure. Typical dimensions for
the FIG. 4 valve would be a control chamber diameter of between 3
and 6 mm, preferably between 4 and 5.5 mm, a control chamber depth
of between 0.2 and 2 mm, preferably between 0.5 and 1.5 mm, such as
1 mm, and a diaphragm thickness of between 0.2 and 1.5 mm,
preferably between 0.7 and 1.3 mm, such as 1 mm, for a rubber
diaphragm of hardness 40-60 Shore A (preferably a silicone rubber
of hardness 45 Shore A). Control port pressures in the region of
70-300 kPa, preferably 100-200 kPa, such as 150 kPa, would be
required to operate such a valve.
[0120] To prevent undesirable fluid leakage at the edges of a
fluid-containing conduit or chamber such as the control chamber 28,
outlet port 29 or inlet conduit 30, particularly when the fluid is
at a relatively high pressure, the modification illustrated in FIG.
23 may be utilized. FIG. 23a shows schematically part of a fluid
control device similar to the FIG. 4 valve, in which a flexible
diaphragm 158 is clamped between essentially flat upper and lower
plates 159 and 160 respectively, spanning the open end of a fluid
conduit or chamber 161. The risk of fluid leakage from the chamber
161 depends on the pressure applied to the diaphragm 158
immediately adjacent the chamber edges.
[0121] To reduce this risk, as shown in the exploded sectional view
of FIG. 23b, one of the internal plate surfaces may carry raised
portions such as the ridges 162 adjacent or close to the chamber
perimeter; these serve to concentrate the clamping force applied to
the diaphragm 158 around the chamber edges, as shown in FIG. 23c.
As a result, effective leak-proof sealing can be achieved by
applying a lower clamping force.
[0122] Although FIGS. 23b and c show the provision of raised
surface elements in the lower plate 160, such elements could
instead or in addition be provided in the upper plate 159 and/or in
the diaphragm itself, in the region immediately surrounding the
fluid conduit or chamber. Other forms of surface profiling, which
achieve the same force-concentrating effect as the ridges 162, may
be used.
[0123] The FIG. 23 modification may be used in any part of a fluid
control device according to the invention where sealing of a
flexible diaphragm is required around a fluid-carrying channel or
cavity. In particular, the modification may be used in devices such
as the valves, fluid storage devices, fluid agitation devices and
fluid loading devices described below in connection with FIGS. 5 to
8, 10, 12, 13, 17 to 20, 24 and 25, and/or to enhance sealing
around sample chambers.
[0124] An alternative inlet/outlet valve, useable as valve 18 or 19
in FIG. 3, is shown schematically in FIGS. 5a ("plan" view) and 5b
(cross section). Parts corresponding to those of the FIG. 4 valve
are correspondingly numbered, and similar comments apply as to
their construction and operation.
[0125] In the FIG. 5 valve, fluid inlet/outlet conduits 31 and 32
are formed as blind-ended channels in the lower plate 25; both may
function as either inlet or outlet conduits in use or the valve may
be bi-directional. Control is again affected through control port
27, as described in connection with FIG. 4.
[0126] In a valve such as that of FIG. 4 or 5, it is preferred that
the fluid inlet and outlet ports (the ends of the fluid inlet and
outlet conduits 30 and 29 in FIG. 4) be located as close as
possible to the central longitudinal axis of the control chamber
(28 in FIG. 4), since efficient valve operation, and in particular
effective sealing between the diaphragm and the fluid ports, is
less easily achieved towards the periphery of the relatively large
diameter control chamber. For example, one of the ports may be
positioned on or very close to the central axis of the control
chamber. The other port may also be closer to the central axis than
to the perimeter of the control chamber, or at least as close.
[0127] For example, the valve control chamber may be generally
cylindrical in shape and have a cross sectional diameter of about
5.5 mm. The fluid inlet, outlet and control ports might each
typically have a diameter of between 0.5 and 2.0 mm, such as about
1.0 mm. In more general terms, the cross sectional diameters of the
fluid ports or conduits are typically between 1/20 and 1/5 of that
of the control chamber, and the smallest distance between the
perimeters of the inlet and outlet ports (typically measured along
a diameter of the control chamber) is then preferably between 1/10
and 1/2 times the control port diameter.
[0128] In the FIG. 4 valve, one of the inlet/outlet ports is
positioned coaxially with the control chamber. The central
longitudinal axis of the second port might then be spaced by 2 mm
from that of the control chamber (i.e., the smallest distance
between the perimeters of the first and second ports would be 1
mm).
[0129] A particularly preferred alternative form of the FIG. 4 or 5
valve is constructed as shown in stylized plan view in FIG. 26a and
in cross section in FIG. 26b. Reference numeral 177 represents the
control chamber, 178 the control port and 179 and 180 the fluid
inlet and outlet ports. 181 and 182 are upper and lower plates
respectively, between which a flexible diaphragm 183 is clamped.
All three fluid ports approach the control chamber with their
central longitudinal axes substantially parallel to that of the
control chamber. Here, the central longitudinal axis of each of the
inlet and outlet ports is suitably located within a distance of 1/8
to 1/4 times x from the central longitudinal axis of the control
chamber, where x is the cross sectional diameter of the control
chamber.
[0130] Further alternative valve constructions are shown in FIGS.
17 to 19. These valves, the general construction of which is
illustrated in FIG. 17, are set to be either closed (the FIG. 18
valve) or open (FIG. 19) in the absence of control fluid
pressure.
[0131] Referring to the schematic "plan" view of FIG. 17a and cross
section of FIG. 17b, a valve is constructed between an upper plate
120 and a lower plate 121, with a flexible diaphragm 122 clamped
between them. Provided in the lower plate are a control port 123
and control chamber 124, which allow pressurized control fluid to
displace the diaphragm locally against the opening of a fluid port
125 provided in the upper plate. Fluid may normally flow in either
direction between the fluid port 125 and an annular groove 126 and
channel 127, passing between the upper plate 120 and the diaphragm
122. If however the control port 123 is pressurized, such fluid
flow is prevented.
[0132] Two alternative forms of such a valve are shown in schematic
cross section in FIGS. 18 and 19; their operation depends on the
depth of the upper plate surface in the region 128, around the
opening of the fluid port 125 adjacent the diaphragm. If the
surface region 128 extends fully into the annular groove 126, as in
FIG. 18, it distends the diaphragm 122. In this case the elasticity
of the diaphragm provides a sealing force to close the fluid port
125, and the valve is "normally closed". The sealing force can be
overcome either by excess fluid pressure (in either the fluid port
125 or the channel 127), or by application of a relatively low
pressure at the control port 123.
[0133] A "normally open" valve is shown in FIG. 19. Here, the
surface region 128 extends only partially into the groove 126 and
is therefore clear of the diaphragm. The valve is therefore open
unless a relatively high pressure is applied at the control port
123.
[0134] A "normally closed" valve is generally desirable where it is
necessary to seal against fluid flow in the absence of
energisation. A typical example might be a valve associated with a
fluid storage or loading device, where it is desirable to load the
device remotely from the rest of the assay apparatus.
[0135] A "normally open" valve has a lower resistance to flow at
any given control port pressure and might therefore be preferred in
locations where pressure drop is a potential problem, for instance
where fluids are distributed between several assay devices and
variable pressure drop across the inlet and outlet valves could
cause a variable division of flow between the devices.
[0136] FIG. 6 shows how the fluid agitation devices 22 and 23 in
FIG. 3 may be constructed in a similar fashion to the valves 18 and
19. The FIG. 6 device comprises an upper plate 33, a lower plate 34
and a flexible diaphragm 35 clamped between them. A control chamber
36, typically larger than that of the valve 18, is provided in the
lower surface of the plate 33. Control port 37 is supplied with
control fluid from supply line 4. Fluid inlet/outlet port 38
communicates with the sample chamber of the cassette.
[0137] Using the pressure at port 38 as reference, the application
of negative pressure at control port 37 draws the diaphragm 35 away
from the lower plate 34 so that fluid is drawn into the device,
from the sample chamber, through port 38 to fill the space between
the diaphragm 35 and the lower plate 34. This situation is
illustrated in FIG. 6, the arrows indicating the directions of
fluid flow. Negative differential pressure at control port 37 can
be achieved by applying either a negative gauge pressure to the
control port or a positive gauge pressure at the port 38.
Conversely, a positive pressure at control port 37 ejects fluid in
the device back out through port 38.
[0138] In this way, fluctuations in applied pressure can be used to
move small amounts of fluid into and out of the sample chamber,
thus ensuring continuous fluid movement in the region of the test
substrate. By operating a pair of such devices in tandem through a
sealed volume, back and forth fluid motion can be caused simply by
applying positive gauge pressure to the control ports of the two
devices alternately.
[0139] Generally speaking, gentle rather than vigorous fluid
movement will be desirable throughout the assay device, in
particular within the sample chamber. To achieve this, moderate
pressures (e.g., up to 120 kPa, for instance about 100 kPa) should
ideally be applied to the fluid device control chambers (such as
chamber 36 in the FIG. 6 device), and changes in control fluid
pressure should be effected gradually, for instance by including a
flow restrictor in the control fluid flow. Suitably a period of
between 0.5 and 2.5 seconds, preferably between 1 and 2 seconds,
should be allowed for a device such as a valve to be switched
between states (e.g., between "open" and "closed" or between "on"
and "off").
[0140] The sample volume displaced by the FIG. 6 device is
dependent on the volume and cross sectional area of control chamber
36 and the movement of diaphragm 35. These can be set in two ways.
If the control chamber is relatively deep and the diaphragm
relatively stiff then the degree of diaphragm movement is
determined by the applied differential pressure. This may be an
advantage in some circumstances where it is desired to change the
displaced volume by remote control; varying the applied pressure,
either manually or automatically, can be used to set the displaced
volume. In contrast, if the chamber 36 is relatively shallow and
the diaphragm more flexible then the diaphragm may be displaced by
applied pressure until it substantially contacts the top face of
the chamber. In this case the displaced volume is dependent more on
the dimensions of the control chamber and less on the applied
differential pressure. The advantage of this latter arrangement is
that a predetermined volume of sample fluid, which does not vary
significantly with applied differential pressure, can be displaced.
This could be useful, for example, where the fluid pressure at port
38 is uncertain.
[0141] A device similar in construction to that of FIG. 6 may be
used to store a small quantity of fluid (typically a sample fluid)
prior to its introduction into the sample chamber of the cassette.
Another sealing device (typically a valve) is required to hold the
fluid within the cavity formed between the diaphragm 35 and the
lower plate 34. If this sealing device is opened (which may be
arranged to occur automatically under the action of excess fluid
pressure) then application of pressure at control port 37 will
force stored fluid out of the cavity and into the sample chamber.
Such an arrangement is illustrated in FIG. 7.
[0142] Two or more fluid control devices such as those of FIGS. 4
to 6 and 17 to 20 may be constructed together in a single unit.
FIG. 7 illustrates schematically both in "plan" (FIG. 7a) and in
cross section (FIG. 7b) how this might be achieved. Control port
39, inlet port 40, control chamber 41 and "intermediate" channel 42
together form a valve. Control port 43 and storage chamber 44
together function as a fluid storage device, communicating with the
valve via intermediate channel 42. Fluid can be trapped in the
storage chamber 44 by the action of the valve. Valve sealing may be
effected either by pressure applied at port 39 or by the natural
elasticity of the diaphragm 45.
[0143] To load the FIG. 7 device with for example a sample fluid,
the fluid is injected under pressure through port 40. With
sufficient differential pressure between ports 39 and 40, fluid is
pushed from port 40, between the diaphragm 45 and the lower plate
46, into the intermediate channel 42. From channel 42 it flows into
the storage chamber 44, filling the space between the diaphragm and
the lower plate and distending the diaphragm as it does so.
[0144] Once the storage chamber 44 is filled, the fluid is retained
by the valve (either with applied pressure or by elasticity) until
pressure is applied to port 43 in the upper plate 47. This
pressurizes the fluid to overcome the sealing of the valve (any
pressure at port 39 may be reduced or removed) and the fluid exits,
via port 40, to the sample chamber. The storage chamber may thereby
be either wholly or partially evacuated.
[0145] Another useful combination of fluid control devices is
illustrated in schematic FIGS. 8a ("plan" view) and 8b (cross
section). Here, a valve (control port 48, control chamber 49 and
channels 50 and 51) is combined with a fluid agitation device
(control port 52, control chamber 53 and sample fluid port 54). An
outlet chamber 55 is provided in the lower plate 56, between the
flexible diaphragm 57 and the port 54. In use, the fluid path is
from channel 50, through the valve to channel 51 and through the
agitation device chamber 55 to port 54. Reverse flow is also
possible. In the absence of pressure at valve control port 48,
fluid applied under pressure to channel 50 displaces the diaphragm
57 in the valve control chamber 49 to allow flow through to channel
51. If pressure is applied to control port 48 then this flow is
prevented. Flow through channel 51 fills chamber 55 and the fluid
can then exit through port 54 into the sample chamber. Fluid flow
through the chamber 55 will substantially clear bubbles from it
provided its dimensions are not too large in comparison to the
typical fluid meniscus dimension. Once the chamber 55 is
substantially filled with fluid the valve may be closed by
application of pressure at valve control port 48.
[0146] To ensure fluid movement in the sample chamber, pressure,
and optionally vacuum, can be applied cyclically to the agitation
device control port 52. This makes the diaphragm 57 displace
between the chambers 53 and 55, thereby displacing fluid back and
forth through port 54.
[0147] An advantage of this over the previously described agitation
arrangement (FIG. 6) is that the chamber 55 may be thoroughly
cleared of bubbles prior to agitation. A similar arrangement may be
employed in a fluid storage device such as that seen in FIG. 7, by
the provision of an additional fluid port to allow the introduction
of wash or other fluids into the storage chamber. Once filled with
such fluid(s), the storage device can be purged by operating it as
previously described, and thus washed clean and purged of bubbles
prior to its re-use with fresh fluids.
[0148] A typical assay device (cassette) in accordance with the
first aspect of the present invention would have two of the FIG. 8
device combinations connected to its sample chamber, as seen in
FIG. 3. Preferably, the sample fluid ports 54 of the two devices
would communicate with opposite ends of the sample chamber so that
operation of the agitation devices in opposition would cause fluid
displacement over substantially the whole active area of the test
substrate.
[0149] In an arrangement such as that shown in FIG. 8, the valve
may be used to relieve excess pressure in the sample chamber, such
as might be induced by raising the temperature in the chamber
during a processing operation. This is achieved by applying a
predetermined "threshold" pressure to the valve control chamber 49,
causing the valve to open if the fluid pressure in the sample
chamber exceeds that threshold.
[0150] Generally speaking, for all of the fluid control devices
described above, very high or very low control fluid pressures are
likely to cause undesirably rapid switching between operating
positions (e.g., between closed and open valve positions). This in
turn may lead to sudden fluid movements which again are
undesirable, especially in the sample chamber. Thus, moderate
control fluid pressures are ideally used, and changes in fluid
pressures effected as gradually and smoothly as possible.
[0151] It will be evident from the above that a complete set of
fluid control devices for each cassette can be constructed simply
from two adjacent plates and a flexible diaphragm or membrane
between them Each device type can be characterized by an
arrangement of chambers, conduits and fluid ports provided in the
adjacent plate faces.
[0152] FIG. 9 illustrates a fluid control unit which can form part
of an assay device (e.g., the FIG. 3 cassette) in accordance with
the first aspect of the invention. The unit combines two sample
fluid storage devices 58 and 59, two fluid agitation devices 60 and
61 and two valves 62 and 63 (one for fluid entry into and one for
fluid exit from the cassette). The storage devices 58 and 59 are
shown with their associated valves, as in FIG. 7--note that the
valves in this case are of the "normally closed" type illustrated
in FIG. 18; they have no control port and are opened instead by
excess sample fluid pressure. FIG. 9a is a stylized plan view of
the fluid control unit and FIG. 9b a perspective view.
[0153] All of the fluid control devices are constructed within
upper and lower plates 64 and 65 respectively, between which is
clamped a flexible diaphragm 66. Holes in the upper face of plate
64 provide control ports 67 to 72, as well as reagent fluid ports
73 and 74. Sample fluid ports, which will communicate with the
sample chamber in use, are provided as holes (e.g., 58a and 59a) in
the lower face of plate 65.
[0154] FIG. 10 is a schematic section through the valve labeled 63
in FIG. 9a. Hole 74 is a fluid inlet port, 75 a sample fluid port
intended to communicate with a sample chamber and 72 the control
port. When the valve is activated, fluid is allowed to flow through
port 75, between diaphragm 66 and lower plate 65, through
intermediate channel 76 and through port 74 (which passes through a
hole in the diaphragm). Fluid may flow either from 74 to 75 or vice
versa.
[0155] An important feature of the slide cassette (assay device)
described above is the means to enclose a small volume of fluid in
contact with the active surface of a test substrate. During a
typical chemical assay, liquid needs to be passed over the
substrate surface to wash it and to apply reagents. However a
critical requirement is to be able to leave a small quantity of a
scarce or valuable liquid (such as a biological sample) in contact
with the substrate for extended periods, typically many hours.
Ideally the liquid should be spread in a thin layer, covering as
much as possible of the active surface of the substrate. A larger
area of coverage allows a larger array of probe species to be
included. While the liquid remains in contact with the substrate,
it is essential to prevent its depletion by leakage, evaporation or
absorption. These requirements can be met, according to the present
invention, in a compact assay device of relatively simple
construction.
[0156] A preferred way in which to achieve efficient sealing of the
sample chamber, in for example the cassettes 8 of FIG. 2, is
illustrated schematically in FIG. 11a, which is a stylized plan
view of a test substrate and its support, and 11b, which is a cross
section of the same.
[0157] In the FIG. 11 arrangement, the test substrate 77 is clamped
against a plate or block 78 with a gasket 79 between them.
Typically the test substrate is a thin glass plate such as a
microscope slide. A clamping plate 80 presses on the back of the
slide, which is achieved using any suitable clamping means. The
clamping means preferably includes some mechanical means, such as a
spring, to accommodate small differences or changes in the overall
thickness of the assembly. It is necessary that sufficient clamping
force be applied under all combinations of component tolerance and
thermal expansion/contraction or compression set (e.g., of the
gaskets and diaphragms in the device).
[0158] Aperture 81 in the gasket 79 defines a sample chamber (82)
adjacent the active surface 83 of the test substrate. It is in this
active area of the substrate that an array of probe species will
previously have been placed. Gasket 79 not only seals the substrate
against the plate 78 but also sets the depth of the sample chamber
82. Material for the gasket is chosen for impermeability, softness
to conform to the mating surfaces and incompressibility to maintain
a reliable thickness under clamping pressure. With a typical slide
size of approximately 26 mm by 76 mm, a suitable overlap of the
slide over the gasket at each edge is around 2 mm.
[0159] Though as small as possible a fluid depth is desirable to
minimize the volume required to fill the sample chamber 82,
irregularities in the slide and plates make it expensive (because
of the tighter finishing tolerance required) to maintain a
consistent thickness of less than a few tens of microns.
Consequently, a typical sample chamber depth would be around 70
microns. At these dimensions, suitable materials for the gasket are
low density polyethylene (LDPE), high density polyethylene (HDPE)
and polypropylene (PP). These materials are readily available in
extruded films of controlled thickness; gaskets may be cut from
such films by known techniques such as punch-and-die or laser
cutting or using knife tools.
[0160] For optimum sealing, it is preferable to use the softest
possible material consistent with the operating temperature range.
For instance, for operation between about 5 and 40.degree. C., LDPE
is suitable, whereas at higher temperatures HDPE or PP should be
used. This is because, of the three polymers, LDPE has the lowest
softening temperature and PP the highest. If the gasket is made
from a polymer that softens at a low temperature compared to the
operating temperature of the cassette then the clamping pressure
may make the gasket extrude from between the test substrate 77 and
the supporting plate 78.
[0161] Since polymers with higher softening temperatures are
normally harder at any given temperature there is a potential
sealing problem when working over a wide temperature range. A
polymer suitable for withstanding the maximum operating temperature
may be too hard at the minimum temperature and so not conform to
the mating faces, allowing leakage. A solution to this is to use a
multilayer material, for instance having a core of a harder
polymer, capable of withstanding the higher temperatures, and a
thinner, softer polymer laminated onto its two faces. The softer
material affects the sealing but does not suffer excessive
extrusion because it is such a thin layer. Its viscosity, even at
the maximum operating temperature, does not allow it to extrude
from between the plates even over extended processing periods.
[0162] In use, fluids may be passed to and taken from the sample
chamber 82 through small diameter conduits (not shown) provided in
plate 78. The internal surfaces of these conduits, and the upper
surface of plate 78, are both in contact with sample fluid for
extended periods and so must be made from inert materials. The
surfaces must also be, and remain, flat and smooth both for
efficient sealing and to prevent unwanted binding between the
surfaces and species in the sample fluid. Suitable materials
include stainless steel (preferably grade 316) and polymers such as
polyetheretherketone (PEEK), polyoxymethylene (POM--otherwise known
as acetal), polytetrafluoroethylene (PTFE) or polypropylene
(PP).
[0163] A significant problem with certain polymers is their
absorption of liquid solvents, particularly water. Assay samples
are often formulated in aqueous solution so it is essential to
minimize water absorption into the components enclosing a sample
during potentially extended processing periods. Absorption into the
gasket 79 is not normally a significant problem because little
solvent can be absorbed into the small amount of material involved
and because the area exposed to the sample fluid is very small.
Absorption into plate 78 and into the test substrate itself could
be much more critical. The test substrate normally poses no problem
since glass, which is the typical substrate material, shows minimal
absorption. Absorption into plate 78, however, may prevent use of
polymers. A preferred material for the plate is therefore stainless
steel, which has very low water absorptivity and may be finished to
a high degree of flatness and polish (by chemical polishing,
abrasive polishing or diamond facing for instance).
[0164] The sample chamber construction illustrated in FIG. 11 is
compatible with the fluid control unit of FIG. 9 to create a
complete assay device in accordance with the first aspect of the
invention. Such a device is shown in schematic cross section in
FIG. 12. It consists of three parallel plates, an upper 84, an
intermediate 85 (corresponding to the lower plate 78 in FIG. 11 and
also to the lower plate 65 in FIG. 9) and a lower "fluidic" plate
86 corresponding to the upper plate 64 in FIG. 9. A test substrate
87 is clamped between plates 84 and 85, with a gasket 88 (analogous
to gasket 79 in FIG. 11) which serves to define an enclosed sample
chamber 89. A flexible diaphragm 90 is clamped between plates 85
and 86.
[0165] The fluid controls for the FIG. 12 device are made up of
chambers and channels (as described in connection with FIGS. 4 to
10 and 17 to 20) defined in the adjacent faces of intermediate
plate 85 and fluidic plate 86, together with fluid conduits through
the two plates and the diaphragm 90. Such chambers, channels and
conduits are omitted from FIG. 12, for simplicity.
[0166] In this case, the assay device might include: [0167] i) two
sample storage and injection devices, as illustrated in FIG. 7,
communicating with the sample chamber 89; [0168] ii) two
inlet/outlet valves, as illustrated in FIGS. 4, 5, 10 or 17 to 19,
to control fluid flow from external reservoirs to the sample
chamber or from the sample chamber to waste; and [0169] iii) two
fluid agitation devices, as illustrated in FIG. 6 or FIG. 8, to
move fluid contained in the sample chamber back and forth across
the active surface of the test substrate 87.
[0170] An assay device such as that of FIG. 12 may also incorporate
one or more fluid loading devices of the type shown in schematic
cross section in FIG. 20. This comprises an open cup-shaped recess
129, of approximate volume 50-100 .mu.L, provided in the outer
surface of upper plate 130. The "cup" 129 has first and second
outlets 131 and 132 respectively.
[0171] Outlet 131 leads to a valve which is constituted by a
control chamber 133, a control port 134, an intermediate chamber
135 and a fluid outlet port 136. The valve is operated by the
supply of control fluid to the control chamber 133 which causes
displacement of the diaphragm 137 and thereby controls fluid flow
either into or out of the cup 129. In this case, fluid outlet port
136 leads to a sample chamber.
[0172] The second outlet, 132, from the cup leads to waste (outlet
port 138).
[0173] The FIG. 20 device is provided in a three-plate construction
which includes not only the upper plate 130 but also a pair of
lower plates 139 and 140. A second flexible diaphragm 141 is
located between the two lower plates. Such a construction has the
advantages described below in connection with FIG. 25.
[0174] Fluid may be loaded into the cup 129 either directly or, as
shown in FIG. 21, by insertion of a pipette tip 142 into the first
outlet 131. The mouth of outlet 131 is specially adapted to
accommodate a standard pipette tip.
[0175] The valve associated with the FIG. 20 loading device is
analogous in operation to those described in connection with FIGS.
4, 5, 10 and 17 to 19. To open the valve, a low pressure is applied
to its control chamber 133, This causes a local downwards
displacement of the diaphragm 137, which allows fluid flow either
to or from the cup 129. The application of a higher pressure to the
control chamber 133 seals the diaphragm 137 against the upper plate
130, preventing fluid flow into or out of the cup.
[0176] The FIG. 20 device may be used to load fluids into other
parts (in particular the sample chamber and/or fluid storage
devices) of the assay device, and to evacuate fluids from other
parts such as the sample chamber. It may also be used in the
washing of the sample chamber and other apparatus parts.
[0177] A typical sample loading operation would involve dispensing
sample liquid either directly into the cup 129 or via a pipette as
shown in FIG. 21. If the cup has previously been washed (as
described below), then a small quantity of wash liquid will remain
in it and this ensures that the sample can be delivered to the
valve inlet under a liquid surface so that no bubbles can be
trapped.
[0178] Sample aspiration may then be achieved by opening the
control valve so as to draw the sample liquid through into the
sample chamber by vacuum applied downstream of the valve. This
could be done for instance by operating a fluid storage device (of
the type described in connection with FIG. 7) to suck the liquid
in.
[0179] Just as liquid can be drawn in from the cup 129 by vacuum,
similarly it may be expelled to the cup (or to a pipette tip
inserted into the cup outlet 131) by appropriately applied
pressure.
[0180] A typical washing operation may be achieved using the FIG.
20 device by introducing a wash liquid to the valve (for instance
via the port 136) under slight pressure. Vacuum is then applied to
the control chamber 133, allowing the wash liquid to feed into the
cup 129. Vacuum applied to the waste port 138 removes excess liquid
from the cup, taking contaminants away with it. Any bubbles trapped
in the valve, in particular in its inlet conduit, are also purged
in this process. Once the supply of wash liquid is stopped, fluid
in the cup drains down to approximately the level of the waste
outlet 132.
[0181] FIG. 22 shows schematically how a group of fluid loading
devices, of the type shown in FIG. 20, may be used together in an
assay device according to the invention. Here three loading
devices, 143 to 145, are illustrated. Each has an associated valve
(146 to 148 respectively). Items 149 and 150 are fluid storage
devices, each associated with a sample chamber in which an assay is
to be conducted. Items 151 to 155 are further fluid flow control
valves. Conduit 156 is connected to a source of wash fluid, and
conduit 157 leads to waste.
[0182] All three loading devices are in fluid communication with
storage device 149, and loading device 145 is additionally in fluid
communication with storage device 150. All three loading devices
may be used to deliver fluids to storage device 149 and to evacuate
fluids from it (for instance, previously stored or assayed sample
fluids, or wash liquids). All three loading devices may be washed
with fluid supplied via the storage device 149. In addition, device
145 may deliver fluids to or receive fluids from the storage device
150. Fluids may also be transferred between all three loading
devices via the storage device 149.
[0183] The capacities of the loading device "cups" may be
sufficiently great that they may be only partially filled or
emptied in any given fluid "transaction". Thus, for example, device
145 may be used to dispense aliquots of fluid to both the storage
devices 149 and 150.
[0184] A major advantage of the FIG. 22 arrangement is the
flexibility it offers in terms of fluid movements. It makes
possible not only the storage of small quantities of a number of
different fluid samples, but also the drawing of more than one
fluid sample from each loading device and the supply of fluid to
any given sample chamber or storage device via more than one
loading device. Operation of the loading devices can moreover be
automated, conveniently via operation of other fluid control
devices in the surrounding apparatus, with which the fluid loading
devices are operationally compatible.
[0185] The fluid loading devices also offer the ability to purge
air bubbles trapped in the device, whilst their compatibility with
conventional laboratory pipettes makes them straightforward to
use.
[0186] Ideally, fluid loading device(s) are located in the assay
device of the invention in reasonably close proximity to the sample
chamber. There may however be cases in which one or more fluid
loading devices are provided at a different location, for instance
as part of an assay station at which one or more assay devices are
to be processed.
[0187] Fluid connections are made to the FIG. 12 assay device
through holes (not shown) in the lower face of fluidic plate 86,
for entry and exit of sample and reagent fluids and also of control
fluid for the valves and other devices. These connections may be
made by individual tubes. However, in a system which has several
such assay devices or "cassettes" at an assay station, there will
be many such connections and high cost may result. An alternative
connection method is to combine the fluidic plates 86 of several
cassettes into a single plate. In the lower face of this plate are
fabricated channels to distribute and collect the various fluids to
and from the cassettes. At least some of the fluid control devices
and fluid distribution conduits may therefore be common to more
than one assay device.
[0188] Such an arrangement is shown in stylized "plan" view and in
cross section in FIGS. 13a and 13b respectively, FIG. 13b being a
section along the line B-B in FIG. 13a. Here, fluidic plate 91 is
common to seven assay cassettes 92 (in general, there may be any
desired number of cassette locations in an assay station such as
that of FIG. 13). Each cassette comprises a diaphragm 93, an
intermediate plate 94, a gasket 95 and an upper clamp plate 96. A
test substrate 97 is shown clamped inside the cassette seen in FIG.
13b, in an arrangement similar to that of FIG. 12.
[0189] The lower face of the fluidic plate 91 provides a number of
channels 98 which run beneath all of the cassette locations. The
channels 98 are closed at either end except for fluid connection
ports 99 through the upper face of the plate. Though they may be
fabricated as closed tubes within the body of plate 91 (e.g., by
drilling or extrusion), it is more economic to fabricate them in
the plate face and then close them off with a sealing gasket 100
and gasket plate 101, as shown in FIG. 13b. The gasket plate 101
may be clamped to fluidic plate 91 by any convenient method.
[0190] Holes through the fluidic plate 91 in selected positions
allow communication between the channels 98 and the fluid control
devices in the upper face of the plate and/or the intermediate
plate 94, for each cassette position. Using the fluid ports 99,
liquids and gases may thus be supplied to the cassettes 92 via the
channels 98. Any of the channels 98 may connect either to a single
fluid control device of a cassette or to a particular type of
device across all the cassettes. A preferred set of channel
functions is listed below. [0191] (i) liquid (typically wash and/or
reagent liquid) source to all cassettes [0192] (ii) to (viii)
control fluid source for input valves in cassettes 1 to 7
respectively [0193] (ix) control fluid source for agitation device
A (all cassettes) [0194] (x) control fluid source for sample A
storage/injection device (all cassettes) [0195] (xi) control fluid
source for sample B storage/injection device (all cassettes) [0196]
(xii) control fluid source for agitation device B (all cassettes)
[0197] (xiii)-(xix) control fluid source for output valves in
cassettes 1 to 7 respectively [0198] (xx) liquid outlet from all
cassettes.
[0199] With this combination of fluid control devices it is
possible to blank off individual cassettes so that the assay
station may be processed with fewer than seven test substrates in
place. Otherwise control is common to all cassettes so that they
run synchronously.
[0200] A "sub-assembly" such as that of FIG. 13 includes all of the
fluid control devices necessary for each cassette. The sub-assembly
can form part of an assay station as shown in FIG. 1, the remaining
control elements including the pumps and valves shown in FIG. 2.
Preferably, the FIG. 13 sub-assembly is built as a module that can
be easily and quickly removed from a station of which it forms a
part. Fluid and electrical connections to the sub-assembly can be
made via ganged connectors to facilitate this.
[0201] In fluid control devices of the types described above, and
in assemblies or sub-assemblies incorporating such devices, it may
be necessary for a fluid conduit or chamber provided in one plate
to overlay, at least partly, another conduit or chamber provided in
an adjacent plate. Such a situation is illustrated in FIG. 24a,
which is a section through part of a fluid control device formed
between upper and lower plates 163 and 164 respectively, in which
fluid ports 165 and 166 and their common fluid conduit 167 are
separated by the flexible diaphragm 168 from fluid conduit 169 in
the lower plate. The arrangement is shown in stylized "plan" view
in FIG. 24b, the conduits within the structure being illustrated by
dashed lines. FIG. 24c is a section corresponding to FIG. 24a, but
illustrating how, when the fluid pressure in conduit 169 is higher
than that in conduit 167, displacement of the diaphragm 168 may be
sufficient to allow fluid leakage from conduit 169 along the
interface between the diaphragm 168 and the lower plate 164.
[0202] This problem may be overcome or at least mitigated by
providing an "inner" plate between the two plates in which the
relevant overlapping conduits/chambers are defined. Such an
arrangement is illustrated in FIG. 25, in which overlapping fluid
conduits 170 and 171, provided in upper plate 172 and lower plate
173 respectively, are separated by a plate 174 sandwiched between
two flexible diaphragms 175 and 176. All three plates are made from
a suitable rigid material such as stainless steel, a ceramic
material or a rigid plastics material. The inner plate 174
transmits clamping forces over the whole mating surface of each of
the upper and lower plates, avoiding unsupported regions in the
flexible diaphragms which might otherwise-allow fluid "tunneling"
between the diaphragms and adjacent plates.
[0203] Any of the fluid control devices of the invention, including
those described above and in particular the FIG. 26 valve, may be
constructed using an arrangement of the form shown in FIG. 25.
[0204] A further optional feature of apparatus in accordance with
the invention is a device for monitoring the fluid flow rate
through one or more of the apparatus parts, in particular through
assay devices. This is desirable firstly in order that malfunctions
may be detected and secondly so that pumping rates may be adjusted
to achieve a desired flow rate.
[0205] A flow rate monitoring device in accordance with the third
aspect of the present invention, for use for example in the
apparatus of FIG. 1, is shown in schematic cross section in FIG.
14. It is provided in a fluidic plate 102, which may simply be an
extension of the fluidic plate, such as 91 in FIG. 13, of an assay
"cassette". Two cavities, 103 and 104, are machined into the
fluidic plate. They are closed by clamping a printed circuit board
(PCB) 105 to the upper face of plate 102 with a gasket 106 between.
The cavities communicate with each other via a labyrinth 107 of
fluid conduits provided in the body of plate 102 (this labyrinth
may have any desired geometry). Fluid from an assay cassette may
enter the device at port 108 and exit, typically to waste, at port
109. Port 110 allows injection of a gas, the purpose of which is
described below. The arrows indicate the directions of fluid flow
in use.
[0206] Fluid (typically liquid) filling the cavities 103 and 104
comes into contact with the face of the PCB 105. Its presence can
be detected in either cavity by detecting a change of conductance
or capacitance between conductor traces provided on the lower face
of the PCB; the electronics to do this may be provided either on
the PCB itself or remotely. This provides a digital indication of
the presence or absence of liquid in the cavities.
[0207] To measure flow rate, a small bubble of gas is injected, via
port 110, into the liquid flowing through the FIG. 14 device. The
dimensions of the fluid conduits and cavities in the device are
such that the bubble fills the cross section and propagates along
with the liquid flow. As it passes the PCB sensors associated with
cavities 103 and 104, a controlling computer detects this and
measures the time taken for the bubble to pass through the
labyrinth 107. From this, an approximate liquid flow rate can be
calculated.
[0208] An additional benefit of the FIG. 14 device is that it can
also check for the presence of bubbles during "normal" liquid flow,
for instance as a quality check during purging.
[0209] If the speed of gas injection at port 110 can be held
sufficiently uniform, it may be possible to achieve sufficient
accuracy with a single cavity and PCB sensor, the interval between
gas injection and sensing being the measured parameter.
[0210] Clearly, the PCB must be compatible with the fluids present;
this may be achieved for instance by constructing the PCB from
gold-plated tracks on an epoxy substrate.
[0211] Optical or other alternative detection means may be used
instead of the PCB to detect the presence or absence of liquid in
the cavities.
[0212] The FIG. 14 device may be inserted in any desired liquid
flow. For instance, if the flow rate through each of the cassettes
needs to be measured independently then one device per cassette is
required. Preferably the device is positioned downstream of the
cassette, to prevent the injected gas bubbles affecting assays
being conducted in the cassette.
[0213] The gas injection port could conveniently be common to a
number of cassettes. To prevent liquid flowing along this common
connection, which would affect the gas injection, a valve of the
type shown in FIG. 5 could be inserted into the gas supply line.
This valve could be operated either through its control port 27 or
simply by the pressure of the gas feed overcoming the natural
sealing of the diaphragm 26.
[0214] It will be evident that both the flow rate monitoring device
and any associated valve(s) can be fabricated as features in the
plates 91, 94 and 101 of a cassette (see FIG. 13), making use of
the diaphragms 93 and 100 between them. This minimizes the overall
cost of the cassette as the components of all the fluid control
devices can be fabricated at the same time.
[0215] During a chemical or biochemical assay it is usually
necessary to control the temperature of the test substrate and the
fluids in contact with it. This may involve heating to temperatures
above ambient and/or cooling below ambient, often "cycling" between
different operating temperatures at different times during the
processing.
[0216] Using apparatus in accordance with the present invention,
temperature control may be effected in a variety of known ways such
as by: [0217] i) passing, heated or cooled air over the whole
assembly; [0218] ii) allowing heated or cooled liquid to flow
against or through any convenient part of the assembly, provided
there is good thermal conductivity between that part and the
remainder; [0219] iii) electrical resistance heating of such a
part; and/or [0220] iv) Peltier heating or cooling of such a
part.
[0221] A preferred temperature control means is illustrated in
schematic cross section in FIG. 15, in combination with a cassette
of the type shown in FIG. 13 (like parts are labeled with the same
numerals). In this case, the lowermost plate 111 is an extruded
(for instance, aluminum) heat sink of the type conventionally used
in electronic systems, having a number of cooling "fins" 111a. In
this case the heat sink can also itself be heated, by means of
electrical heaters 112 connected to it. Applying power to these
heaters raises the temperature of the heat sink 111 and, by
conductivity, of the associated cassette. The temperature may be
monitored by any convenient means, such as platinum resistance or
thermocouple sensors. Using an automatic temperature controller,
the temperature of the assembly may be stabilized at any desired
level above ambient.
[0222] An enclosure 113 fits around the whole assembly to minimize
convection and other draughts and hence temperature differences
within the assembly. However the natural cooling rate of the
assembly, in the absence of heater power, is then reduced, which in
turn could slow down its overall operating rate and even be
deleterious to the test process itself. Faster cooling may
therefore be achieved using a flow of air forced through the heat
sink 111 by an electrically driven fan (not shown). Such a fan
could also be under the control of the automatic temperature
controller system so as to be activated automatically when cooling
is required. If cooling below the ambient temperature is required
then air drawn in by the fan must be precooled using any of a
variety of known techniques.
[0223] Apparatus in accordance with the fourth aspect of the
present invention may include any desired number of assay stations,
as shown in FIG. 1, each station having a plurality of cassette
locations. The apparatus described above, in connection with FIGS.
1 to 15 and 17 to 25, allows processing with any combination of
cassettes and assay stations active.
[0224] As a result of common fluid connections, active cassette
positions within a station can be partly interdependent. Each
station, however, can operate entirely independently. A controlling
computer or other sequencer may be used to operate the valves,
pumps, heaters and other devices necessary to execute a
pre-programmed processing sequence at one or more stations.
[0225] The following describes how the apparatus might typically be
operated, by reference to one assay station. Other stations in the
apparatus may be operated, synchronously or asynchronously, in a
similar way.
[0226] Preparation
[0227] Before a processing run may be performed, all washes,
reagents etc. must be in the relevant reservoirs. It is typically a
manual task to check and fill reservoirs. Waste bottles must also
have sufficient remaining capacity. The apparatus may be connected
to "main-line" services such as purified water, gas and waste in
some circumstances. Automatic checking of reservoir levels may be
included by any of a variety of known techniques (e.g., weight
sensor or weight balance).
[0228] Station Configuration and Sample Storage
[0229] If fewer test substrates are to be processed than the
maximum station capacity, unused cassette locations must be made
inactive. Depending on the exact configuration of the fluid control
devices and the station assembly, this may require no further
action. In the preferred arrangement, however, it is necessary to
"blank-off" unused cassette positions to prevent leakage of control
gas or other fluids from exposed ports. This is achieved as shown
schematically in FIG. 16.
[0230] FIG. 16a is a side view and FIG. 16b a stylized plan view of
a "blanked-off" cassette from the apparatus described above. As in
FIG. 15, item 111 is a plate which functions both as support and
also as a heat sink, item 91 is a fluidic plate carrying fluid
control devices and fluid channels and item 100 is the diaphragm
between these two plates. A backing plate 114 and plain sealing
gasket 115 are clamped in place on top of fluidic plate 91, by
means of a spring clamp 116 and toggle clamp 117.
[0231] Ideally, the apparatus is arranged so that even in the
"blanked-off" cassettes, the fluid control devices are periodically
(and preferably automatically) operated, i.e., fluid is passed
through them, to prevent or at least reduce adhesion between the
flexible diaphragms and the adjacent plates.
[0232] For each "active" cassette, a diaphragm 93 and intermediate
plate 94 (see FIG. 13) are used in place of the backing plate 114
and gasket 115, secured using similar spring and toggle clamps.
With these in place, one or more small quantities of sample fluid
or other reagent may be pre-loaded into one or more of the storage
devices of the cassette, ideally via fluid loading devices, using
for instance a pipette or syringe. (The syringe is ideally fitted
with a hollow tip suitable for sealing into the inlet port of the
relevant fluid control device.)
[0233] Fitting Test Substrates
[0234] Once the sample and other necessary fluids have been
pre-loaded, the gasket 95, test substrate 97 and clamp plate 96
(FIGS. 13 and 15) may be fitted to each active cassette. Again
these are conveniently held in place by spring clamps, as described
in connection with FIG. 16. The test substrate carries at least one
"probe" species which will react (preferably selectively) with a
target species contained in or thought to be contained in the
sample under test. A typical test substrate for a biochemical assay
is a glass microscope slide coated with streptavidin, with one or
more (preferably an array of) biotin-tagged probes, such as
nucleotide sequences, antigens or antibodies, immobilized on it.
The use of avidin-biotin binding to immobilize biological reagents
on a substrate is entirely conventional.
[0235] Fitting the Station Sub-Assembly to the Assay Station
[0236] Once a station sub-assembly of the type shown in FIG. 13
(containing a plurality of cassettes) has been prepared with all of
the required test substrates, sample fluids and sealing plates for
unused positions, it is fitted into one of the assay stations 3 of
the FIG. 1 apparatus. In doing so, fluid and electrical connections
are made between the fluid and thermal control devices of the
station and those of the sub-assembly.
Automated Processing
[0237] The apparatus may then be used to carry out an almost fully
automated chemical assay, typically under the control of a
pre-programmed microcomputer or other process control means. This
is set up to operate the pumps, valves, thermal controller and
other requisite devices in a pre-programmed sequence. A typical
such sequence involves: [0238] i) purging the system--in turn, open
each valve between reservoirs and each pump. The pump is run with
the manifold bypass valve 14 (FIG. 2) open.
[0239] Liquid runs to waste, clearing all fluid conduits and
manifolds as it does. [0240] ii) washing the substrates--selected
reservoirs, containing for instance concentrated wash liquid and
distilled water, are connected to the pump(s) by opening the
corresponding valve(s). The pump(s) are run at a rate dependent on
the required fluid mixing ratio and flow rate, the latter depending
on the number of active cassettes in the assay station. Inlet and
outlet valves for selected cassettes are opened (by appropriate
operation of the associated control valve applying pressure or
vacuum or vent to the valve control ports). Outflow from the
cassettes goes to waste. Active cassettes may be washed
independently or together by appropriate control of their inlet and
outlet valves. [0241] iii) applying reagent(s)--as in step (ii),
but using reagent fluid reservoir(s) as the source(s). Reagent
fluids may include buffers, surfactants, electrolytes, catalysts,
reaction initiators and/or terminators, blocking agents, labeled
reagents and the like. [0242] iv) injecting sample--with the
cassette inlet valves closed but their outlet valves open, apply
pressure to the corresponding control ports of the sample storage
devices. Liquid stored in the devices is injected into the sample
chambers of the cassettes.
[0243] Concurrently with the other steps, the temperature of the
cassette assemblies may be set to a predetermined value by the
thermal control system. This may involve heating or cooling, as
previously described.
[0244] During the assay, pressure (and optionally vacuum) is
applied alternately to the agitation devices of each pair of
devices in each active cassette (the inlet and outlet valves being
closed). This moves the liquid in the sample chambers back and
forth.
[0245] Subsequent assay steps may involve washing, heating and/or
cooling, agitating and/or supplying further reagents or samples to
the test substrates and sample fluids, all as described above.
[0246] When the assay is complete, gas or air may be pumped through
the cassettes to remove most of the liquid in the sample chambers.
The test substrates may then be removed from the cassettes and
appropriately imaged to obtain the desired test results.
[0247] In the washing step(s), the apparatus of the invention
allows the wash solution concentration to be altered as desired.
This in turn makes possible several successive washing steps,
typically with increasing degrees of stringency.
[0248] It can be seen from the above that apparatus in accordance
with the present invention can possess several key advantages,
namely: [0249] i) a small volume of reaction or wash fluid can be
enclosed against the test substrate; [0250] ii) multiple test
substrates can be simultaneously and economically processed; [0251]
iii) small quantities of sample fluid can be pre-loaded and
efficiently stored for each substrate under test; [0252] iv)
multiple samples can be stored for each test substrate, allowing
multiple probing with for instance several different antibodies;
[0253] v) operation can be at least partially, preferably fully,
automated; [0254] vi) the sample fluid can be agitated over the
active surface of the test substrate; [0255] vii) multiple wash or
reagent fluids can be introduced into the sample chamber; [0256]
viii) wash and/or reagent fluids can be blended to achieve desired
concentrations or mixes that can be varied continuously with time;
[0257] ix) large numbers of samples can be assayed simultaneously,
with independent control of the processing conditions (for example,
the temperature and fluid movement) for each; [0258] x) the fluid
control devices, such as valves and agitators, are relatively
simple and compact in construction, being incorporated into the
cassettes. This allows the use of large numbers of reagent and
sample fluids without undue size, complexity and cost in the
apparatus as a whole;
* * * * *