 |
Description  |
|
|
This invention relates to devices for use in chemical (especially
biochemical or clinical) test procedures, to processes for their
manufacture, and to the use of the devices.
The devices are, in certain embodiments, intended for use in procedures for
detecting and measuring inorganic ions in liquid aqueous samples, and in
other examples for specific binding assay procedures.
Previously, micro-scale procedures have often been carried out using
so-called microtitre wells, conventionally of about 0.5 ml working
capacity, amongst a variety of other liquid containers for the assay
reaction liquids. Other devices and arrangements for handling micro-scale
assay materials are described in Specifications Nos. EP 0 31 993, GB 1 571
872, GB 1 584 129 and GB 1 414 479, for example.
In particular, the prior art contains numerous disclosures of analytical
devices for handling and metering small volumes of test samples.
GB 2 090 659 (Instrumentation Laboratory, Inc.) describes test strips
constructed with a self-filling metering channel and a lip or inlet on
which a sample of more than about 10 microlitres of for example whole
blood can be placed, so that (for example) 10 microlitres is taken up by
capillary action to react with a reagent carried on a fibrous pad above a
filter layer beneath a transparent window. The result can be viewed by the
unaided eye, e.g. as a colour reaction.
GB 2 036 075 (H E Mennier), GB 1 104 774 (J P Gallagher), EP 0 057 110, 0
034 049, 0 010 456 (Kodak), all describe some other aspect of the uses of
capillary channel or chamber dimensions for handling biological or test
fluids.
The prior art also includes many disclosures of chemically-sensitive or
selective electrodes: see "Ion-Selective Electrodes in Analytical
Chemistry" (ed. H. Freiser, Plenum Press 1978).
According to the invention to be described here, capillary fill cell
devices which can be conveniently manufactured, are provided to facilitate
electrically-monitored tests, for example conductivity measurements,
specific ion analysis, enzymatic reactions, and specific binding assays,
using very small liquid samples.
According to the invention we provide a specifically-reactive
sample-collecting and testing device possessing a cavity or cavities each
having a dimension small enough to enable sample liquid to be drawn into
the cavity by capillary action, wherein said cavity includes an electrode
structure for making measurements of one or more electrically measurable
characteristics of the sample, and wherein a surface of a wall of the
cavity optionally also carries a coating of a material appropriate to the
test to be carried out in the device.
According to the present invention there is also provided a method of
manufacturing specifically-reactive sample-collecting and testing devices,
comprising the steps of (a) (usually) forming a coating on the surface of
a sheet material which is to provide part of a multiplicity of the
devices, (b) forming an additional structure which together with said
coated sheet material provides for each device of the multiplicity of
devices a cavity of capillary dimension for collecting and retaining by
capillarity a volume of sample liquid in contact with the reactive
coating, (c) forming an electrode structure within each cavity, and (d)
separating the sheet material into portions each providing one or a
plurality of the sample-collecting and testing devices.
Usually it is found convenient, as in the case of examples described below,
first to form conductive layers on the surface of a carrier which will
later constitute a wall of a capillary cell, then to add any layers of
ancillary materials, and lastly to assemble the cell or cells.
The coatings can be reagents such as buffers which are to be released into
liquid sample material taken up into the device, or they can be
specifically-reactive materials such as proteinaceous binding agents or
enzymes: they can be confluent or continuous or divided into a pattern,
e.g. of discrete portions, e.g. as a 2-dimensional array of patches.
Another useful example of a coating is an ion-selective membrane coated on
to an electrode previously formed or coated on a wall to form part of the
capillary cell cavity. Where such patches are formed they can be made by
first forming a continuous coating and then removing or inactivating
portions of it to leave the desired pattern, e.g. the array of discrete
portions. The coating can be a releasable reagent coating, e.g. a coating
of releasable antigen or antibody, or derivative thereof, or else for
example an immobilised specific binding material such as a covalently
bound antigen or antibody or derivative thereof to form an immunosorbent,
with specificity appropriate to a desired assay. The additional structure
which completes the capillary cell(s) can for example be a further sheet
material bonded to the first sheet material by suitable bonding adhesive,
and spaced therefrom by a capillary space, e.g. less than about 1 mm, to
allow uptake of sample liquid between the sheets by capillarity,
preferably in a defined reproducible volume. The separation of units can
be achieved for example by scribing and breaking or by sawing or cutting
the sheet materials, e.g. of glass, siliceous or plastics material, and in
the examples described below is carried out so as to leave an external
loading surface or aperture to which sample liquid can be loaded or
applied and from which it can be drawn onto the cavity of the device. The
external loading surface has preferably a capacity to contain or hold at
least enough liquid (e.g. in the form of a drop of material spread out
over the surface) to load the cavity fully.
In examples of the devices such as the one illustrated in FIG. 1, the sheet
carrying the electrodes extends out beyond the end of the capillary cell
as completed by the additional structure, e.g. the second sheet, so as to
provide an anchoring-point for electrical connections to an external
circuit.
According to the invention we also provide specifically reactive
sample-collecting and testing devices made by the processes described
herein.
The fabrication of the electrode structures in the capillary cell devices
of the invention can be carried out in any of a number of ways.
For example, a surface of a glass, silica or plastics substrate which is to
form a wall of the capillary cell can be provided in per se known manner
with a conductive coating of tin oxide SnO.sub.2, especially one that has
been partly etched away to leave an electrode pattern. Such a pattern of
plural electrodes can be used for example for conductivity or general
impedance measurements. Such conductive electrodes can be overlain by any
of the reactive or ligand-binding coatings mentioned elsewhere in this
specification.
Alternatively, a surface of a glass, silica, crystal, ceramic or plastics
surface which is to form a wall of the capillary cell can be provided with
one or a plurality of conductive coatings each of which forms an
electrochemical half-cell, e.g. a coating forming a silver-silver chloride
electrode, optionally provided with an ion-selective or other
chemically-selective membrane overlayer.
An example of an electrode structure to be included in a capillary cell
according to an embodiment of the invention is a composite electrode
structure comprising a non-conducting matrix (e.g. a body of polymer such
as polyvinyl chloride), wherein the matrix incorporates conductive
particles. The conductive particles are such as to allow the provision of
a conductive connection from the particle-containing composite to an
external electrical circuit. In an example to be described below, the
conductive particles provide the composite electrode with a conductivity
corresponding to resistivity of a few milliohm-cm. This value is by no
means critical: resistivities several orders of magnitude higher can be
used, but the resistance of the electrode material should generally be
small in comparison with that of the external circuit (often many
megohms).
Such an electrode structure can be formed on a substrate (forming a wall of
the capillary cell) which is also made of non-conducting polymeric
material.
Besides polyvinylchloride, other useful nonconducting solid matrices or
materials from which the substrate can be formed are for example
polyurethane, polystyrene, polyvinylacetate, epoxy resin (especially as a
matrix for the conductive particles), and methacrylate plastics, as well
as inorganic matrices e.g. such as glass. In what follows, mention of
polyvinylchloride (pvc) implies also the possible use of any such
substitute matrix material. In the structures provided by the invention,
the conductive particles can be for example graphite, silver, platinum,
gold, or copper. If desired, the matrix in which the particles are
incorporated can be an organic matrix derived by solidification from the
paint vehicle used as the base of conductive paints known and available
for use in thick-film microcircuit manufacture, e.g. for the purpose of
painting or screen-printing conductive tracks on flat insulating
substrates such as ceramic substrates. Suitable particle sizes for the
conductive particulates can be chosen from a wide range, e.g. from (of the
order of) 10-20 to (of the order of) 200 microns. Suitable levels of
incorporation of the particles into the composite can include for example
those of the order of 10%-80%, e.g. 50 %, by volume (based on the volume
of the composition), or, dependent on the nature of the particles,
whatever level is needed to reach a desired degree of electrical
conductivity. One example of a electrode structure comprising silver-pvc
has 3:1 silver particles: pvc particles by weight, with 50 micron (maximum
particle size) silver particles.
One suitable form of electrode comprises a conductor and a non-conductive
membrane matrix material, (e.g. glass or organic polymer), in direct
contact with the conductor, wherein the conductor comprises a matrix
incorporating conductive particles, as described above, the organic
membrane material comprises a sensitising component such as an ionophore,
for sensitising the electrode to a particular analyte to be investigated,
and the organic membrane material is securely bonded to the matrix of the
conductive composite. For example, the membrane material and the matrix
can, conveniently, both be of a similar polymer and can be fused or bonded
together in any convenient manner, for example solvent-welding.
When pvc is used as matrix material for conductive particles, then it is
preferred (for the pvc of the zone that incorporates the conductive
particles) to use pvc incorporating either no plasticiser or only very
small quantities of plasticiser. (This we describe as "pure" pvc).
(Preferably, of the order of 10% by weight of plasticiser or less can be
present in this zone. Preferably there is less, e.g. <5%, e.g. <1%).
In the case of the membrane material, examples of suitable contents for the
ionophore or other sensitising component are for example within the range
5-10% by weight of the whole membrane composition. The
ionophore-containing zone can be for example 0.1-0.5 mm thick. The pvc of
the zone that incorporates the ionophore or other sensitising component
should usually incorporate conventional relatively large amounts of
plasticiser, as used in previous ionophore-pvc membranes, e.g. the
ionophore-containing layer can for example be composed largely of a
pvc:plasticiser composition in proportions for example in the range 1:1 to
1:2 by weight. Examples of plasticisers useful in connexion with
ion-selective polymer membranes, especially pvc membranes, include dioctyl
phenyl phosphonate, diethyl adipate, dioctyl sebacate, trioctyl phosphate,
and o-nitrophenyl phenyl ether. Examples of useful ionophores include
calcium diisooctyl phenyl phosphate (for calcium-sensitive electrodes)
valinomycin (for potassium-sensitive electrodes), tridodecylamine (for
hydrogen-ion-sensitive electrodes), particles of silver chloride, bromide,
or iodide (for corresponding halide-sensitive electrodes), particles of
silver sulphide (for sulphide-sensitive electrodes), mixtures of particles
of silver and copper sulphides (for electrodes sensitive to copper as well
as to sulphide), and more generally a finely divided particulate form of
any of the materials previously used in crystalline form for making
single-crystal electrodes, can be incorporated into the polymer or other
non-conducting matrix of the electrode membrane, in sufficient amount to
put the particles in electrically effective contact to enable the
electrode to respond to the presence of the corresponding constituent of
the surrounding solution to be tested or measured.
Capillary cell devices according to the invention can also in certain
examples have features as follows:
(a) If desired, at least one of the walls surrounding the cavity can be
transparent to light, e.g. to visible and/or ultraviolet light, with
optically regular, generally smooth surfaces, so as to enable
photoelectrical measurements and/or optical analysis in situ of the
products of the sample collection and reaction with the specific binding
capacity, as well as the electrical measurements enabled by the
electrode(s).
(b) In some examples, the cavity of the device can be a thin planar cavity
between two opposite walls forming a cell, and preferably cemented or made
into an integral unit. In some cases, for example, such an embodiment can
include a bonded structure of plates similar to the structure of an
unfilled liquid crystal display device as obtained as an intermediate
stage in manufacture of liquid crystal displays.
According to an aspect of the invention there is provided a (preferably
disposable) (possibly translucent or transparent) capillary cell, which
can be made by the methods described herein, for carrying out specific
binding assays, comprising a pair of opposite plates spaced less than
about 1 mm apart, and sealed together to form an apertured
liquid-retentive cell able to take up and retain by capillary action a
(preferably defined) volume of (usually aqueous) liquid, and carrying on
at least one of its internal surfaces a coating of reagents such as
enzymes, dye molecules, antibody, antigen or buffer salts, as appropriate
to the test to be carried out, and also including electrode(s) or an
electrode structure for making measurements of one or more electrically
measurable characteristics of the sample. "Defined volume" means a volume
that is determined substantially by the shape and configuration of the
cell itself and not appreciably by the volume of sample if applied in
excess.
Cells of the type described above can be assembled from glass or plastics
sheeting, and where plastics sheeting is used, it can be in the form of
precision mouldings, e.g. provided with spacers such as ridges to achieve
controlled spacing of the component walls of the capillary cell cavities.
The cell can have an outer surface portion or lip to which a quantity of
sample sufficient to fill the cell can be applied and from which it can
easily be made to run into the capillary cell by capillary action. Such a
lip can easily be formed by an extension of one of the plates, outwardly
beyond the cell aperture, for a distance sufficient to give a surface area
large enough for convenient sample loading. An alternative form of inlet
is one formed by an aperture in one wall of the capillary cell, e.g. a
hole that exposes an area of an opposite wall of the cell on to which a
sample can be loaded. The lip or aperture can be provided with a porous
filter such as a porous plug or e.g. a filter paper or dialysis membrane,
to allow any desired degree of filtration or dialysis to be applied to
that part of the sample which is drawn into the capillary cell.
Preferably the sealing of the cell can be achieved by using a line of epoxy
resin, leaving an aperture, e.g. extending the resin along two opposite
sides of a rectangular capillary cell, to give a filling aperture and a
further aperture left to allow the exit of air from the capillary cell as
it fills up. Suitably, the resin can comprise solid particles to ensure a
desired spacing for the plates as they bed down on the resin. Particles
such as substantially monodisperse ballotini (fine glass particles) of
diameter about 100 micron or otherwise corresponding to the chosen
capillary gap, or short glass fibre lengths of for example 8 micron
diameter and 50-100 micron long (e.g. made by mortar-grinding of long
glass fibre and exclusion of long residual fibres by sieving), are
suitable to regulate small spacings of the order of the diameter of the
ballotini or the fibres. Generally, spacings in the range 5 to 500 microns
can be chosen, by way of non-limitative example. Fibres are preferred for
very narrow gaps, as they are more easily obtainable in diameters less
than about 50 micron than are monodisperse ballotini: ballotini are
preferred for the wider gaps.
The material or one of the materials which forms a coating on a wall of the
capillary cell cavity can for example be an enzyme, an antigen or
antibody. Among suitable examples are urease, glucose oxidase,
concanavalin A or antiglobulin antibody. Especially in the case of enzyme
coatings, the enzyme can be either immobilised or coated in releasable
form. Such proteins can be immobilised to the glass or silica or plastics
surface in any of the ways otherwise practised for such immobilisation.
For example, it can be useful simply to coat and dry the binding agent and
sucrose on to the carrier surface, either simultaneously or successively.
Covalent and other immobilisation can be achieved where desired in any of
the ways mentioned in EP specification 0 014 530 (Unilever), and
references cited therein, especially in the case of plastics materials,
and in any of the ways mentioned in "Immobilised Enzymes for Industrial
Reactors" (ed. Messing, Academic Press, 1975; especially Filbert, chap 3),
or in for example U.S. Pat. No. 3,652,761 or GB 1 530 997, for a wide
variety of carrier materials including siliceous materials such as glass
and silica.
Especially in the example cases of potentiometric or amperometric
electrodes mentioned below, a material forming one or more coatings on the
surface of the capillary cell can be an ionic salt, e.g. a buffering salt,
for example a thin coating of salt in admixture with a glaze-forming inert
substance such as sucrose or other nonionic humectant.
Especially in the case of amperometric electrodes, the electrode structures
can be coated with enzymes and a mediator which facilitates electron
transport from an enzymatic reaction to the conducting part of the
electrode structure, e.g. ferrocene which can facilitate transport of
electrons from glucose oxidase to an electrode.
Especially in the case of impedance-measuring devices, the area between two
electrodes on one wall of the cell can be coated with a specific binding
agent which can bind conducting particles such as gold sol particles as
used in certain immunoassays.
Embodiments of the invention are illustrated for example by the
accompanying FIGS. 1-9 and associated description.
FIG. 1 shows in diagrammatic plan a capillary cell device containing
electrodes, according to an embodiment of the invention.
FIG. 2 schematically shows a diagrammatic section through another
disposable capillary cell device.
FIG. 3 shows a diagrammatic plan of the cell device of FIG. 2, and includes
a line I--I to show the line of section of FIG. 2.
FIG. 4 shows in diagrammatic fragmentary plan an intermediate stage in the
manufacture of a plurality of devices as of FIGS. 2-3.
FIGS. 5-8 show in schematic cross-section the electrode arrangements
provided in further embodiments of the invention.
FIG. 1 shows a form of capillary cell device according to the invention.
The cell device shown in FIG. 1 comprises an upper plate 1, spaced from a
lower plate 2 by bonding tracks 3. A plurality of capillary cavities,
three in number, is formed by four bonding tracks 3 which both space the
plates and define the lateral boundaries of the cavities. The device of
FIG. 1 has an interdigitating electrode structure composed of electrodes
10 and 11 each composed of part of an overall tin oxide (SnO.sub.2)
conductive coating which has been etched away to form the pattern shown.
The conductive tin oxide layer is prepared and etched in a manner which is
itself well known in the semiconductor and liquid crystal industry and in
itself forms no part of this invention.
If desired, one or more coating layers (not shown in FIG. 1) can be formed
either on top of the tin oxide coating electrode structure and/or on the
inner face of the opposite plate 2.
Electrodes 10 and 11 continue out beyond the capillary cell on to surface 6
of plate 2, and terminate in connexion points 12 and 13 for connexion to
other electrical circuitry and devices. (If desired, electrodes 10 and 11
may alternatively be continued out to connexion points at another part of
the cell, passing out under bonding layer 3 to suitable connexion points
elsewhere than shown in the drawing).
One pair of electrodes 10 and 11 is provided for each constituent cell of
the device. Only one pair is shown in full and fully referenced in the
drawing. In this device platforms 6 are not used for sample loading, but
plate 2 also extends out beyond plate 1 at the other side of the device to
provide a sample loading platform, divided by extensions 14 of the two
centre tracks of the bonding tracks 3 to divide the platform into three
sample loading areas 15. The three pairs of connectors 12 and 13 are
arranged to be capable of mating with corresponding wires 16 of an
edge-connector device 17 into which the whole of the capillary cell device
can fit by the edge of plate 2 that bears the connexion points 12 and 13.
Such a capillary cell device is useful inter alia for making electrical
conductivity measurements, which in certain cases can also involve other
conductive materials, e.g. conductive particles such as gold sol particles
coated with specific binding agents.
FIGS. 2-3 show in schematic form the capillary cell features of a device
according to an embodiment of the invention except for omission from the
drawing of the electrode structure(s). The device is of a size to be
handled easily, e.g. about 3 cm.times.1.5 cm. The device comprises upper
(e.g. plastics, glass, pvc or silica) plate 1 and lower (e.g. similar)
plate 2 (about 1 mm thick) fixed together in parallel opposed and spaced
relation, less than 1 mm apart, by bonding tracks 3 of suitable (e.g.
epoxy) adhesive to form a capillary cell cavity 4, open at both ends,
which communicates with the outside through a first discontinuity in the
bonding 3 arranged to form a cell aperture at side 5 of plate 1. Another
discontinuity is present at the other end of bonding 3, to leave another
aperture, to allow exit of air when a sample liquid is loaded into the
cell. Plate 2 is larger than plate 1 and has a portion 6 extending away
from the aperture. Portion 6 of plate 2 acts as a platform or threshold or
lip onto which a drop of sample liquid can be applied, so that this liquid
can be made to fill the capillary cell cavity 4 by capillary flow. Cavity
4 attracts and contains a definite and adequately reproducible volume of
liquid when loaded in this way.
Immobilised to the inner surface of the capillary cell is a layer 7 of
material relevant to the test procedure in which the capillary cell is to
be used. In the example shown in the drawings the layer 7 is a patch of
material carried on plate 2, or in an alternative arrangement carried on
plate 1. For the purpose of a test based on enzyme activity, e.g. urea
measurement by conversion to ammonium ions by means of urease, it can be
for example an area of immobilised enzyme, e.g. urease enzyme. Otherwise
the layer can be e.g. an immobilised antibody, relevant to an immunoassay.
There can be more than one such layer, e.g. a layer on plate 1 as well as
plate 2, or a superimposition and/or side-by-side plurality of layers on
either plate. Although not shown in FIGS. 2-3, layer 7 or other layer(s)
lining the internal surface(s) of the capillary cell includes an
electrically conductive layer or layers as described in connextion with
FIG. 1, and conductive external connections are provided (not shown in
FIGS. 2-3) by means of conductive tracks or connectors from the interior
of the cell to the exterior of the cell, if desired, passing between
bonding layer 3 and the surface of the plates. These can be made in a
manner known per se and used in the conventional surface fabrication of
conductive tracks as often employed in the manufacture semiconductors and
liquid crystal displays.
The section shown as FIG. 2 presents plates 1 and 2 spaced apart because
the line of section does not extend through the bonding tracks 3.
The fabrication of a plurality of cells such as that of FIGS. 2-3 is
illustrated by FIG. 4, a fragmentary plan diagram showing an intermediate
stage in the manufacture of such cells. A large plate 8 of glass or other
material to make plates 2 is cleaned and coated in any appropriate way
with patches of material 7 of any of the kinds described above and
patterned electrode layers as well as tracks of bondable adhesive 3. A
second plate, not shown, is then adpressed to plate 8, optionally after
forming on it bonding tracks corresponding to track 3, and optionally
after forming patches or tracks of any other desired material, and the
adhesive is cured. Then the assembly is broken or cut along lines shown as
dotted lines 9 in FIG. 4, and corresponding lines in the upper plate (not
necessarily in registration with lines 9, though). The result is to give
cells like the cells shown in FIGS. 2-3.
The arrangements of FIG. 4 have been described in relation to the use of
sheet glass as a substrate method: especially but not exclusively where
plastics sheet is used, it can be convenient to use other than plain sheet
material, e.g. spacer ridges, inlet apertures and filter arrangements as
described elsewhere herein can be incorporated as part of such sheets
before the capillary cells are assembled.
The capillary cell device of FIGS. 2-3, among other devices made according
to this invention, can if desired be provided with any convenient form of
handling-piece or holder and for this purpose may be provided with any
convenient form of fixed or releasable connexion arrangement to engage
with such a holder where this is not formed in one piece with the cell
device.
In general, thin coating layers of biochemical reagents can be present;
they can be either immobilised (i.e. non-releasable) or releasable
coatings, e.g. formed by air-drying protein-sucrose mixtures in thin films
on the plates. These can be selected and combined according to the
particular test chemistry to be performed in the device. The range of
chemical or binding reactions that can form part of the tests to be
carried out includes electrochemical, enzymatic, binding and quenching
reactions of any kind, but it is emphasised that some of the tests may not
require such reactions to take place in the capillary cells at all.
The formation of reactive immobilised protein layers (e.g. enzymes or
antibodies) on the inner surfaces of the cells can be achieved for example
as follows.
A sheet of (e.g. soda) glass for example about 1 mm thick, and large enough
to contain a 2-dimensional array of cell areas, with a plurality of
several cell units in each direction, is cleaned by any suitable method,
e.g. by detergent and ultrasonic treatment and if need be by solvent
vapour degreasing in known manner, or by successive hot (80.degree. C.)
treatments with ammonia hydrogen peroxide and hydrochloric acid/hydrogen
peroxide, water-rinsing and airdrying, e.g. at 115.degree. C. for 30
minutes. A pattern of patches of a desired protein or other coating is
then applied by the following or equivalent technique. Covalent coupling
of antigen or antibody or other protein is achieved by first reacting the
glass with a silane-based coupling compound in known manner (e.g. with a
terminal amino-alkyl trimethoxysilane, or another reagent substantially as
e.g. the 3-aminopropyl compound another reagent substantially as described
in U.S. Pat. No. 3,652,761, suitably about 2% v/v in acetone), then
reacting the amino terminals immobilised on to the glass with (e.g. 2% pH
7)glutaraldehyde, removing excess reagents and exposing the activated
glass with immobilised aldehyde groups to reaction with the protein in
solution (e.g. 1 mg/ml antibody immunoglobulin), according to component
techniques well known in themselves. For example, treatment at about pH
9.5 for 2 hours at 37.degree. C. has been found suitable here. A suitable
final active protein loading rate on the glass surface can be for example
about 0.5 microgram/cm.sup.2. This is thought to constitute a continuous
or near-continuous layer The dosage or density or specific activity of the
immobilised layer is determined by the sensitivity requirements of the
particular assay chemistry, which in itself forms no part of this
invention. Excess reagents can be removed for example washing in strong
buffer (0.1M acetate, 0.5M NaCl, pH4-5), then neutral buffer washing, (pH
7-7.4), followed by pH 9-10 washing and neutralisation.
If it is afterwards desired to etch or to inactivate any part of the
protein (e.g. enzyme coating), the following technique can be used. The
coated sheet can then be placed in a confined atmosphere substantially
free of air draughts, e.g. it can be brought close to another flat inert
surface to reduce the air gap on the coated side to about 1 mm or less.
The sheet is then illuminated with an ultraviolet patterned image (using
preferably light of as narrow as practicable a waveband around c. 280 nm)
in a pattern corresponding to areas from which the coating is to be etched
away or inactivated, e.g. a grid pattern, to leave a pattern of surviving
active protein patches. Illumination can for example be carried out using
a GE 7-watt mercury lamp spaced a few centimetres from the plate, for a
period of about 5-20 minutes. The illumination pattern can be produced by
masking close to the plate, or by a real imaging system. The ultraviolet
etching used here is thought to rely on the same principle as the u.v.
etching process described by J A Panitz, I Giaver, in Surface Science, 97
(1980) pp 25-42, to which reference is made.
Then a uv-curable epoxy adhesive is printed on to the patch-coated glass
plate in a desired pattern for forming a connexion with an upper spaced
plate. The epoxy adhesive is applied by a silk-screen technique which is
conventional in itself, and in itself forms no part of this invention.
The epoxy resin can have a small content of short-length glass fibre, about
20 micron in diameter and about 100-200 micron long, (made for example by
grinding long glass fibre in a mortar and sieving to remove residual long
fibres). A preferred alternative to the glass-fibre pieces is a content of
ballotini in the epoxy resin, used as follows. In order to produce a gap
of for example 100 micron, correspondingly-sized ballotini are
incorporated in the epoxy: a layer of epoxy a little thicker than the
desired spacing between the plates, e.g. 10% thicker, e.g. about 110
micron for a desired spacing of 100 micron, can be laid down by
screen-printing, and the addition plate pressed gently into position to
spread the epoxy slightly.
If desired, a mirror-image of the first pattern of epoxy adhesive can be
applied as a pattern to a second similar sheet of glass, either coated
patchwise with the same or a different protein or other coating material,
or otherwise uncoated, and the two sheets then brought together, subjected
to vacuum or deoxygenation if needed for curing, and cured by ultraviolet
illumination. The ultraviolet is applied as an image with a pattern that
avoids the patches of coated protein or other material which are to be
retained in active form.
After adhesive curing, the two plates can be scribed and broken down into
individual cell units in any convenient known manner as used in stages in
the manufacture of liquid crystal devices, and in particular by the
methods referred to in Specifications Nos. CH-627 559, and 629 002,
concerning fabrication of liquid crystal display devices. Corresponding
steps in the methods of those specifications and of this invention can be
performed by similar methods, mutatis mutandis.
A convenient form of cell obtainable by this process comprises two
substantially parallel opposed layers of glass, air-spaced by about 5-500
micron, which, together with an incomplete frame of bonding material
located between them, (having at least one opening for the inward passage
of liquid and possibly also the outward passage of air), form a capillary
cell able to take up a defined volume of aqueous liquid. One of the glass
layers can extend out beyond the opening of the cell to enable a drop of
liquid to be placed on its surface and pass either wholly or partly into
the cell. Especially in versions of the devices which are made of plastics
material, an aperture can be made or left in one of the walls of the cell
to allow sample loading, preferably with a filter device as described
above.
FIGS. 5-9 show in diagrammatic cross-sectional scheme five further
electrode-containing capillary cell devices according to embodiments of
the invention. In each Figure of the group 51 & 52 represent opposite
walls of the capillary cell, and the other structure apart from the
electrodes is omitted for clarity. In each the capillary gap can
conveniently be of the order of 0.1-1 mm.
In FIG. 5, a pair of spaced-apart electrodes 55 and 56 are shown as layers
fixed to the surface of wall 52. Electrode 55 is an ion-selective (e.g.
potassium ion-selective) electrode and 56 is an ion-insensitive electrode,
e.g. an electrode coated with a thin layer of polystyrene, to work
effectively as a reference electrode. Suitable modes of construction of
the electrodes are described below.
In FIG. 6, there is shown an electrode-containing capillary cell device to
measure potassium ion concentrations, comprising two spaced-apart
| | |
