Note: Descriptions are shown in the official language in which they were submitted.
WO 95124632 w' ~ PC'fIGB95100507
- 1 -
The present invention relates to spectrophotometric
techniques using particular device
s.
In EP-A-0171148, devices are disclosed for use i
n
chemical (especially biochemical or clinical) test
procedures, particularly for use in specific binding
assay procedures. The devices typically possess a
cavity into which samp.Le liquid is drawn. One surface
of the cavity is a sura_ace of a transparent solid plate
which acts as a light transmissive waveguide
for us
i
;
e
n
specific binding assays, this surface carries an
immobilised reagent appropriate to the assay to be
carried out in the device. The plate has an edge which
is substantially optically smooth and transverse to the
plane of the plate and in use in an assay procedure the
device can transmit, far example, fluorescence from an
adsorbed fluorescent material along the plate acting as
a waveguide, transfer of useful light across the
boundary of the plate occurring by the evanescent wave
located very close to the interface.
We have now found that devices similar to those
in EP-A-0171148 may be used as a cuvette in
spectrophotometric assays. In the normal mode of use of
the devices as described in EP-A-0171148 there is only a
thin film of sample (typically of the order of 0.1 mm)
present in the device; passing radiation through a path
length of this dimension is not suitable for reliable
spectrophotometric measurement. We have now found,
however, that totally internally reflecting radiation
within a modified form of the device allows the
radiation to pass through the sample many times and
therefore enables a suitable path length (for example,
of up to 1 cm) to be built up. Such a
ath le
th i
p
ng
s
~'JO 1512~G32 ,~, ~ ~ ~~ ~~ ~ ~ PC'TIGB95I00507
- 2 -
then suitable for reliable spectrophotometric
measurement.
Devices for analysing substances and which emgloy
multiple internal reflection are already known. In US
5273633 (Tiansong Wang) a'device for capillary zone
electrophoresis is disclosed which comprises a long
transparent capillary tube with a reflecting surface
surrounding the outer surface of the capillary, the
reflecting surface having an incident and exit window
for the electromagnetic radiation. Similarly, in
Analytical Chem., vol 55, no. 6, 1983, pp 951-955 (Wei
Lei et al) a long capillary cell, in which in use
multiple internal reflection of electromagnetic
radiation occurs, is employed for the colorimetric
determination of phosphorus. US-3431424 (Henry W.
Allen) discloses a complex optical fluid sampling device
which in use also empoys multiply reflected radiation.
These prior art devices are all based on a circular
geometry and this presents certain disadvantages. -As
specifically acknowledged in US-5273633 the fact that
radial reflection occurs places constraints cn the size
and orientation of the incident window and also, since
the exiting radiation will be distributed around the
entire circumference of the capillary, on the preferred
shape of the exit window and the detection apparatus.
Furthermore, these prior art devices are relatively
large Iof the order of 50-100 cm) and this makes them
impractical for use in routine analytical work. The use
according to the present invention of a device as
described above (the modified form of the device
disclosed in EP-A-0171148) overcomes these
disadvantages.
Thus, according to one aspect of the present
invention, we provide the use of a sample-collecting
device as a cuvette in spectrophotometric assays, the
device possessing a cavity or cavities each having a
WO 95/24632 PCTlCB95100507
- 3 -
dimension small enough to enable sample liquid to be
drawn into the cavity by capillary action, wherein a
surface of the cavity is a surface of a transparent
solid plate having a substantially rectangular cross-
section and forming a wall of the cavity, the opposite
' surface of the cavity being an additional structure
having a substantially rectangular cross-section and
forming a wall of the cavity, and wherein a portion of
said plate carries a reflective coating and a portion of
said additional structure is reflective.
According to a further aspect of the present
invention there is provided a method of
spectrophotometric assay of a species in a liquid sample
using a device as defined above which comprises
(a) filling said device with the sample liquid;
(bl irradiating said device with light from a suitable
light source such that the radiation is totally
internally reflected within said device; and
(c) monitoring the radiation emerging from said device
by conventional methods in order to determine
whether and/or the extent to which the species is
present in the sample.
Preferably the additional structure of the device
is also a transparent plate a portion of which carries a
reflective coating. However, this is not a necessary
requirement and it i.s suitable merely to use a structure
part of which is fabricated of an appropriate reflective
material; in such an embodiment the material concerned
is preferably inert with regard to the sample liquid to
be contained within the cavity.
The reflective coating carried on the or each
transparent plate is preferably carried on the external
surface i.e. the surface remote from the cavity.
Fiowever, the coating on the or each transparent plate
may instead be carried on the internal surface i.e. the
W095I2:1632 ~ ~ PCTIGB95IUOSQ7
- 4 -
surface opposite the external surface (being that which
forms part of the capillary cavity wall). Alternatively,
where two transparent plates are present, one may carry
the coating on the external surface whereas the other
may carry the coating on the internal surface.
The reflective portions of the transparent plate
and of the additional structure facilitate total
internal reflection of radiation within the device in
use in order to build up a suitable path length of the
radiation through the sample.
The choice of where the reflective coating is
carried on the or each transparent plate is made in the
light of this result to be achieved.
A suitable coating for the transparent plates) is
a metal coating as conventionally used in the
preparation of coated glass, e.g. an aluminium coating.
The devices for use according to the present
invention are conveniently manufactured, enable
convenient sample collection by virtue of their
capillarity and are especially suitable for handling and
accurately metering small liquid samples, the volume of
the cavity containing the sample being of the order of
0.1 ml in typical embodiments.
The devices for use according to the present
invention may be constructed by similar methods to those
described in detail for the devices of EP-A-0171148, but
with the additional step of applying a reflective
coating to the appropriate surface of the transparent
plate(s).
Thus, typically a process for manufacturing sample-
collecting devices as described hereinbefore would
comprise the steps of
(a) applying a coating of reflective material to a
portion of a surface of a transparent sheet
material which is to provide part of a '
multiplicity of the devices;
WO 95l24G32 ~ ~ ~ ~ ~ ~ ~ p(.°fIGB9~fOQ507
- 5 -
(b) either prior to or subsequent to step (a),
attaching to said sheet material in parallel,
spaced re7.ation thereto an additional
structure which together with said 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, a
portion of the additional structure being
reflectivE.; and
(c) separating the assembled laminate into
portions each providing one or a plurality of
the sample-collecting devices.
Where the additional structure is a transparent
sheet material, a portion of which carries a coating of
reflective material, an additional step would comprise
the application of this coating.
The orientation of the attachment in (b) will-
determine on which surfaces (external or internal) of
the cavity the coatings) of reflective material is(are)
carried.
The transparent plates) of the device for use
according to the present invention may be fabricated
from a variety of materials Which. can be transparent to
infrared, visible and/or ultraviolet light depending on
the spectrophotometric method to be carried out using
the device. Suitable materials are, for example, glass
e.g. sheet soda glass about 1 mm thick, silicas and
plastics sheet material e.g. acrylic plastics material
as well as material's conventionally used in the
fabrication of spectroscopic cells such as quartz, NaCl
and CaF2. Where part of the additional structure is
simply fabricated from a reflective material, this may,
for example, be a <:hemically inert reflective metal such
as gold or platinum.
CA 02182080 2006-05-24
- 6 -
A number of modifications of the devices as
disclosed in detail in EP-A-0171148 are applicable to the
device for use according to the present invention
including for example the use of a barrier for selective
takeup of sample liquid into the cavity, the use of a
collection surface of the device onto which a drop of
sample liquid can be placed and the provision of a fixed
or releasable holder for the device. The present
invention extends to the use of devices as described
above incorporating such features.
The sample-collecting device as defined above would
be used in conventional spectrophotometric detection
methods and would find particular utility in the
detection and determination of chemical moieties in
biological samples. For example the devices are able to
draw up a very small sample of blood e.g. from a finger
spot and thus analysis of such a sample for routine
moieties including haemoglobin, glucose and urea is
envisaged.
In certain test methods ancillary reagents may be
needed and these can be dosed separately, or they can be
carried in dry releasable form on a part of the device
to be contacted in use by sample liquid e.g. a surface
of the capillary cavity or a surface of a filter, if
present. Such techniques are illustrated in
EP-A-0171148.
In accordance with another aspect of the present
invention there is provided a method of spectrophotometric
assay of a species in a liquid sample using a sample-
collecting device, said device comprising a cavity or
cavities each having a dimension small enough to have a
sample liquid drawn into the cavity by capillary action,
wherein a surface of the cavity is a surface of a
CA 02182080 2006-05-24
- 6a -
transparent solid plate having a substantially rectangular
cross-section and forming a wall of the cavity, the
opposite surface of the cavity is an additional structure
having a substantially rectangular cross-section and
forming a wall of the cavity, and wherein a portion of
said plate carries a reflective coating and a portion of
said additional structure is reflective, said method
comprising: (a) filling said device with the sample
liquid; (b) irradiating said device with light radiation
from a suitable light source such that the radiation is
totally internally reflected within said device; and
(c) monitoring the radiation emerging from said device in
order to determine whether the species is present in the
sample.
In accordance with yet another aspect of the present
invention there is provided a sample-collecting 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 a surface of the
cavity is a surface of a transparent solid plate having a
substantially rectangular cross-section and forming a wall
of the cavity, the opposite surface of the cavity being an
additional structure having a substantially rectangular
cross-section and forming a wall of the cavity, and
wherein a portion of said plate carries a reflective
coating and a portion of said additional structure is
reflective, said device having a length of between about
20 mm and about 60 mm, a width of between about 5 mm and
about 20 mm and a gap between the transparent plate and
the additional structure of about 0.1 mm to about 1 mm.
Figure 1 shows a diagrammatic section through a
device according to one embodiment of the invention.
CA 02182080 2006-05-24
- 6b -
Figure 2 shows a diagrammatic plan of the device of
Figure 1, and includes a line I-I to show the line of
section of Figure 1.
The device illustrated in Figures 1 and 2 comprises
a transparent plate 1 and a transparent plate 2 fixed
together in parallel opposed and spaced relation,
preferably less than 1 mm apart, by bonding tracks 3 of
suitable (e. g. epoxy) adhesive to form a capillary cell
WO 95124632 ~ ~ ~ ~ ~ ~ ~ PCTlGB95100507
cavity 4, open at both ends, which communicates with the
outside though a first discontinuity in the bonding 3
arranged to fornl 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 has a portion 6 extending away from the aperture which
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. On a portion of each external surface of both
plate 1 and plate 2 are respectively carried reflective
coatings 7 and 8.
In the manufacture of such a device the coating of
the outside surfaces of the plates is carried out by
conventional methods e.g. by the use of a vacuum and
evaporation of a suitable metal such as silver or
aluminium. The sealing is preferably achieved by screen
printing onto one plate lines of epoxy resin which
suitably comprise solid particles to ensure the desired
spacing (e. g. substantially monodisperse ballotini).
The two sheets are then brought together, subjected to
vacuum and the adhesive cured by ultraviolet
illumination. The plates are then scribed and broken
into individual cell units.
Devices for use according to the present invention
are preferably fabricated with a gap of between 0.1 mm
and 0.5 mm, more preferably about 0.2 mm. The
transparent plates) preferably have a thickness of
between 0.5 and 1.5 mm, more preferably about 1.0 mm.
The devices are also preferably fabricated so that in
use in spectrophotometric assays the path length of the
radiation through the sample is between 0.1 and 2.0 cm,
more preferably about 1.0 cm. The devices are
WO 95124632 ~ ~ Q ~ ~ ~ ~ PCT/GB!)5100507
_ g _
preferably of a length between 20 mm and ~0 mm, more
preferably about 40 mm, and have a width of between 5 mm
and 20 mm, more preferably about 10 mm.
Thus, according to a further aspect of the present
invention we provide a sample-collecting device as
defined above in which the device has a length of
between about 20 mm and 60 mm, a width of between about
5 mm and 20 mm and a gap between the transparent plate
and the additional structure of about 0.1 mm to 1 mm.
Figure 3 schematically illustrates the use of a
device according to the present invention in
spectrophotometric measurement, the incoming radiation
being internally reflected in the device and exiting
after a suitable path length has been built up.
The path length through the sample would be
controlled by the length of the device, the thickness of
the plates, the positioning of the reflective metal
layers on the surfaces of the device and the angle of
incidence of the radiation. Placing one or both of the
coatings on the internal surface of the plates) as
opposed to the external surface would also affect the
overall path length, since it would alter the skip
distance for each bounce of radiation within the device.
For example, for a device as illustrated in Figure
3 where the width of each transparent plate is 1.1 mm
and the angle of incidence of the radiation is 20°, each
bounce of the light would result in a skip distance of
approximately 1.75 mm (4.8 x tan 20°) down the device
with the light path through the sample being
approximately 0.42 mm. Thus a device of this type which
is at least 35 mm long would have a path length through
the sample of at least 0.8 cm. In practice the device
would be slightly longer, for example in the region of
mm, to allow for ease of handling. Assuming that the
35 internal width of the device is 10 mm (between the '
bonding tracks) then the sample volume would be
WO 45/24632 ~ PCTJGB45100507
_ g _
approximately 80 ~1.
As an alternative configuration it is possible to
allow for a sample and reference beam within the device,
" as illustrated schematically in Figure 4. Ideally one
light source which is split into two and chopped would
be used to illuminate both sides of the device. The
device would be formed with two separate cavities, one
cavity containing the sample of interest and through
which the sample beam travels, the other cavity
containing the reference solution (essentially the
'solvent' of the sample solution) through which the
reference beam passes. As an alternative, two distinct
and separate devices could be used akin to a
conventional double beam spectrophotometric technique.
In either case, the use of a single detector is
beneficial to avoid the problems of matching more than
one detector.
