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BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to sensors for use in biological, biochemical and
chemical testing and in particular to immunosensors used to monitor the
interaction of antibodies with their corresponding antigens.
2. Description of the Related Art
When antibodies are immobilized on a surface, the properties of the surface
change when a solution containing a corresponding antigen is brought into
contact with the surface to thus allow the antigen to bind with the
antibody. In particular, the change in the optical properties of the
surface can be monitored with suitable apparatus.
The phenomenon of surface plasmon resonance (SPR) can be used to detect
minute changes in the refractive index of the surface as the reaction
between the antigen and the antibody proceeds. Surface plasmon resonance
is the oscillation of the plasma of free electrons which exists at a metal
boundary. These oscillations are affected by the refractive index of the
material adjacent the metal surface and it is this that forms the basis of
the sensor mechanism. Surface plasmon resonance may be achieved by using
the evanescent wave which is generated when a p-polarized light beam is
totally internally reflected at the boundary of a medium, e.g. glass,
which has a high dielectric constant. A paper describing the technique has
been published under the title "Surface plasmon resonance for gas
detection and biosensing" by Lieberg, Nylander and Lundstrom in Sensors
and Actuators, Vol. 4, page 299. Illustrated in FIG. 1 of the accompanying
drawings is a diagram of the eqipment described in this paper. A beam 1 of
light is applied from a laser source (not shown) onto an internal surface
2 of a glass body 3. A detector (not shown) monitors the internally
reflected beam 4. Applied to the external surface 2 of glass body 3 is a
thin film 5 of metal, for example gold or silver, and applied to the film
5 is a further thin film 6 of organic material containing antibodies. A
sample 7 containing antigen is brought into contact with the antibody film
6 to thus cause a reaction between the antigen and the antibody. If
binding occurs, the refractive index of the layer 6 will change owing to
the size of the antibody molecules and this change can be detected and
measured using the surface plasmon resonance technique, as will now be
explained.
Surface plasmon resonance can be experimentally observed, in the
arrangement of FIG. 1, by varying the angle of the incident beam 1 and
monitoring the intensity of the internally reflected beam 4. At a certain
angle of incidence the parallel component of the light momentum will match
with the dispersion for surface plasmons at the opposite surface 8 of the
metal film. Provided that the thickness of metal film 5 is chosen
correctly, there will be an electromagnetic coupling between the
glass/metal interface at surface 2 and the metal/antibody interface at
surface 8 as a result of surface plasmon resonance, and thus an
attenuation in the reflected beam 4 at that particular angle of incidence.
Thus, as the angle of incidence of beam 1 is varied, surface plasmon
resonance is observed as a sharp dip in the intensity of the internally
reflected beam 4 at a particular angle of incidence. The angle of
incidence at which resonance occurs is affected by the refractive index of
the material against the metal film 5--i.e. the antibody layer 6--and the
angle of incidence corresponding to resonance is thus a direct measure of
the state of the reaction between the antibody and their antigen.
Increased sensitivity can be obtained by choosing an angle of incidence
half way down the reflectance dip curve, where the response is
substantially linear, at the beginning of the antibody/antigen reaction,
and then maintaining that angle of incidence fixed and observing changes
in the intensity of the reflected beam 4 with time.
Known systems of the type described with reference to FIG. 1 utilize a
prism as the glass body 3. A diagram showing this arrangement is given in
FIG. 2 which is simply an experimental set up intended to demonstrate
surface plasmon resonance. The prism is shown under reference 8 and has
applied to its undersurface a thin film 5 of metal. Light 1 from a laser
source (not shown) is incident on the prism where it is refracted at point
9 before entering the prism. The internally reflected beam 4 is likewise
refracted (at point 10) upon exiting from the prism.
One problem with the known SPR systems is the slowness of operation
relative to changes in the refractive index of the antibody layer. Another
problem, particularly related to the use of the prism shown in FIG. 2, is
that, as the angle of incidence is changed, either by moving the source,
or rotating the prism, or both, the point on surface 2 at which the
incoming beam is incident moves. Because of inevitable variations in the
metal film 5 and the coating 6 of antibody, the angle of incidence which
results in resonance changes as this movement occurs, which in turn
introduces a further variable factor into the measurement and thus makes
comparisons between the initial, unbound, state and the bound state of the
antibody layer 6 less accurate.
SUMMARY OF THE INVENTION
In the present invention, the speed of response is improved by providing
that the incoming beam of radiation which is internally reflected at the
glass/metal interface takes the form of a fan-shaped beam of
electromagnetic radiation, usually in the visible or near-visible region.
In this way, the progress of the resonant condition, as the reaction
between the sample and the antibody layer proceeds, can be monitored. In
one example, this can be achieved by taking a "solid" input beam from a
source of electromagnetic radiation, and bringing it (the beam) to a focus
at the point of incidence of the beam on the glass/metal interface. The
input beam thus becomes equivalent to several beams incident upon the
glass/metal interface over a range of angles. The equipment can be chosen
so that the range of angles spans the angle of dip corresponding to
surface plasmon resonance together with a range of angles thereabout. The
corresponding internally reflected beam is likewise effectively several
beams and may be monitored by a large area detector, or by an array of
angularly spaced detectors positioned to collect the several emergent
beams. Thus the detectors can encode the information from the whole of the
dip within milliseconds. In this way, the progress of the resonant
condition, as the reaction between the sample and the antibody layer
proceeds, can be monitored.
The use of a fan-shaped beam highlights the problems of the prism (see
above) and, in order to avoid these, it is provided that the surface of
the transparent, usually glass, body onto which the incoming light is
incident is a curved, preferably circular, surface and is arranged, with
respect to the input beam of electromagnetic radiation, such that the beam
enters orthogonally to the tangent to the surface at the point of entry.
Preferably likewise, that surface from which the internally reflected beam
emerges is a curved, preferably circular, surface.
In a first embodiment of the invention, the transparent body takes the form
of a glass hemispherical body whose flat surface is covered with a thin
metal film and a sensitive overlayer in the manner described above. The
source of input electromagnetic radiation, for example a light source, is
arranged so that the input beam enters the hemispherical body orthogonally
to the tangent at the point of incidence, and thus the beam passes through
unrefracted and is incident at the center of the circular flat surface.
The point of incidence on the flat surface is thus the same for all parts
of the fan-shaped beam.
Shapes other than hemispherical can be used; for example semicylindrical,
which gives a line incidence, rather than a point, or truncated
hemispherical or hemicylindrical in which the top is cut off--i.e. to form
a body having two flat, probably parallel, surfaces with arcuate sides
joining the surfaces.
The fan-shaped beam may be constrained to be substantially planar by being
projected through a slit lying in a plane passing through the point of
incidence and oriented vertically to that of the glass/metal interface.
Alternatively, the expression "fan-shaped" may refer to a shape of a
section of the input beam, and the beam itself may extend in other
planes--for example wedge-shaped (giving a line of incidence), or conical
shaped.
Although the layer applied to the metal film is described herein as an
antibody layer for use in immunoassays, it will be seen that any sensitive
layer whose refractive index changes upon an event occurring can be used
to thus provide a sensitive detector having a wide variety of applications
in the fields of biology, biochemistry and chemistry. As an example, the
sensitive layer could be a DNA or RNA probe which would, during the test,
bind with its complement in solution as represented by the sample to be
tested.
The metal film material is commonly silver or gold, usually applied by
evaporation. The film needs to be as uniform as possible in order to cater
for minute movement in the point of incidence of the incoming beam. It is
assumed that a structured metal film will give the best resonance and
there are various ways in which the glass body can be pretreated to
improve the performance of the metal film and in particular to control the
natural tendency of such films to form discontinuous islands:
1. Immersion in molten metal nitrates and other molten salts. This has the
effect of introducing ions into the surface in a manner which can be
structured and which can act as foci for island formation.
2. Ion bombardment of cold or hot glass to introduce nucleating sites. The
removal of the more mobile ions has been demonstrated to reduce the
thickness at which the evaporated film becomes continuous.
3. Electroless plating or electroplating over lightly evaporated films (0
to 100 angstroms thick). Electroless plated films survive to a greater
thickness than evaporated films and could form more stable nuclei for
subsequent coating.
4. Evaporating on to electroless plated films. The electroless plated films
have a stronger tendency to an island structure and to bigger islands with
greater spacing than evaporating films. This could be of advantage in
tuning to light of a prescribed wavelength.
Coating performance can also be improved by:
1. Controlling the glass surface temperature during coating. Using a higher
temperature substrate increases the islands' size and the spacing between
them and conversely.
2. Evaporating in the presence of a magnetic or electrostatic field or
electron emission device to control the ion content of the vapor stream.
The state of charge of the substrate is know to affect the island
structure.
3. Controlling the angle of incidence of the evaporated vapor stream
relative to the glass surface. The mobility of the evaporated atoms and
hence their ability to form bigger islands is greater when the momentum of
the atoms relative to the glass surface is increased.
BRIEF DESCRIPTION OF THE DRAWINGS
In order that the invention may be better understood, some embodiments
thereof will now be described by way of example only and with reference to
the accompanying drawings in which:
FIGS. 1 and 2 are diagrams of known experimental arrangements for
demonstrating the surface plasmon resonance effect;
FIG. 3 shows, in schematic outline, a cross-sectional view of a sensor in
accordance with one example of the invention;
FIG. 4 is a diagrammatic side view of another example of a sensor according
to the present invention; and
FIGS. 5(a) and 5(b) illustrate the performance of which an arrangement in
accordance with the invention is capable.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Referring now to FIG. 3, a collimated beam 13 of electromagnetic radiation
of width 2 r from a source, which is not shown but may conveniently
comprise a laser diode collimator pen such as that manufactured under the
model number TXCK 1200 by Telefunken Electronic, is incident upon a
hemi-cylindrical focussing lens 14 of focal length f.sub.1, which causes
the light to converge to a point 15 on an interface 27 between an
optically transmissive component, generally shown at 28, and a reflective
layer 19 in the form of a metallic coating. The optical component is, in
this example, made up of a glass support plate or slide 16 having a first
surface (upon which the reflective layer is coated,) and a hemispherical
lens 11 having a second, curved, spherical surface with its center of
curvature located at the point 15. A suitable index matching fluid is
provided, as shown at 29, between the facing surfaces of plate 16 and lens
11 and the arrangement is such that all light rays in the convergent beam
which emerges from lens 14 travel radially of the optically transmissive
component 28 and thus undergo no refraction and are focussed centrally on
the point 15. A slit 30 constrains the convergent beam to a substantial
planar fan shape, so that only a small area of reflective layer 19 is
illuminated to reduce any effects due to non-uniformity of the metal
coating.
The light internally reflected from point 15 travels as a divergent,
planar, fan-shaped beam back out of the component 28, through a third
surface thereof contiguous to the second surface as shown in FIG. 3, and
is incident upon a focussing lens 31 which transmits such light as a light
beam 32 which is substantially parallel-sided, or at least of reduced
divergence compared to the fan-shaped beam of light emergent from
component 28. Beam 32 is arranged to be incident upon a detector 18, for
example an array of photo-sensitive detectors, and in particular an
angular array, and it will be appreciated that the main purpose of lens 31
is to reduce stray reflections in the array 18 ensuring that beam 32 is
normal to its surface. If, however, the stray reflections are not of
significance or if the array 18 can be conveniently placed close to the
exit surface of component 28 (possibly even attached to or deposited on
that surface) lens 31 is not required.
The array of detectors is arranged to generate electrical signals
indicative of the variation of intensity of light with position across the
beam 32; the SPR effect dictating that strong absorption will occur at a
particular angle as determined by material in the fluid to which the
reflective layer 19 is exposed. These electrical signals are sampled and
digitized and fed to a suitable analyzing arrangement which may include a
microprocessor or larger computer.
It can be desirable, in the interests of minimizing the disturbing effects
of extraneous light without having to resort to the expense and
inconvenience of shrouding the entire arrangement, or at least the
components 5 and 28, to arrange that a characteristic modulation is
impressed upon the light and that the detectors and/or the processing
circuits are "tuned" to respond preferentially to such modulation.
A second embodiment of the invention will now be described by reference to
FIG. 4. Referring to FIG. 4, the apparatus comprises a hemispherical body
11 made of transparent material such as glass or quartz housed within a
casing 12. A source (not shown) of electromagnetic radiation produces a
collimated input beam 13 of electromagnetic radiation. The frequency of
the radiation must be such as to result in the generation of surface
plasmon waves and in practice will be within or near the visible region.
Suitable sources include a helium neon laser or an infrared diode laser,
but an ordinary light source, with suitable filters and collimators, could
be used.
A lens 14 is used to bring the parallel input beam 13 to a focus at a point
15 spaced just above the center of the circular flat surface of the
hemispherical body 11. The point 15 lies on the surface first surface of a
slide 16 made of transparent material such as glass whose refractive index
is equal or close to that of the hemispherical body 11. The arrangement is
such that the point 15 lies at the approximate center of curvature of the
curved spherical, surface second surface of the hemispherical body.
Radiation which is internally reflected at point 15 passes, through a third
surface contiguous to said second surface, out of the hemispherical body
in the form of a divergent beam 17 and passes into a radiation sensitive
detector 18 which gives an electrical output signal for analysis by
external circuitry (not shown) in the manner described above. The detector
may, for example, be a diode array, or a charge couple device or similar
imaging device.
In a practical realization of the apparatus, the metal film layer, shown
under reference 19, is applied to the surface of the aforementioned slide
16. The point 15 to which the input beam is focussed thus lies on the
interface between the metal film and the slide 16. Applied to the surface
of the metal film is a sensitive layer 20 whose refractive index changes
as the test progresses. The sensitive layer may, for example, be an
antibody layer. The sensitive layer 20 is restricted to a relatively small
active zone about the point 15 and within a central hole provided in a
circular disc 21 of absorbent material. Overlying disc 21 are two further
discs 22, 23 of non-absorbent material. A central aperture in upper disc
23 defines a well 25 into which a sample to be tested is placed. A central
aperture 24 in disc 22 is of a size to cause liquid in well 25 to travel
by capillary action into the active zone above layer 20. The thickness of
disc 21 is such as to define a depth for the active zone such as to
promote radially outward movement of the sample liquid emerging from
aperture 24 by capillary action. The absorbent disc 21 absorbs sample
which has flowed past the active zone.
The whole unit comprising slide 16, disc 21 and discs 22 and 23 is
disposable so that a fresh unit, including sensitive layer 20 can be used
for each test. The slide 16 is placed upon the flat surface of the
hemispherical body 11, preferably after applying to the flat surface a
thin layer of optical oil or grease to ensure good optical coupling
between the hemispherical body and the slide. Optionally, the
hemispherical body itself may be disposable, provided it can be produced
cheaply enough, in which case there would be no need to include a separate
slide 16, and the metal film 19 can be applied direct to the hemispherical
body.
In order to use the apparatus, a sample to be tested, and containing an
antigen capable of binding with the antibody molecules in layer 20, is
placed in the well 25 and passes through aperture 24 by capillary action.
Emerging from aperture 24, the liquid sample commences to flow radially
outwards in all directions towards the absorbent disc 21, passing as it
does so the antibody layer 20. The sample adjacent the layer 20 is thus
being constantly replenished during the course of the test, which ensures
maximum sensitivity.
As the sample flows past the layer 20 any antigen within the sample capable
of binding with the antibody in layer 20 will do so, thus altering the
refractive index of layer 20 as the reaction proceeds. This change in
refractive index is continuously monitored during the test by directing at
the point 15 the focussed light beam 13. Provided that conditions are
correct--in particular the angle of incidence at the point 15 is
correct--the application of beam 13 will result in the generation of a
plasmon wave, thus extracting energy from the input beam and causing an
attenuation in the intensity of the output beam 17 at a particular angle
of incidence. The input beam is arranged such that the mid-angle of the
range of angles of the input beam is approximately halfway down the
reflectance dip, as described above, and the test is carried out at a
constant angle of incidence, monitoring the intensity of the reflected
beam above and below this mid point level. This gives a linear and highly
sensitive output.
The initial reflectance dip which is chosen for setting up the angle of
incidence should be the dip which results when some neutral or buffer
solution is passed through the cell, or when the sample under test is
passed through the cell but before any reaction thereof has taken place.
In connection with the latter method, which is currently preferred, it is
to be noted that, as sample begins to flow past the active zone adjacent
layer 20 the refractive index does not start to change immediately due to
the antibody/antigen reaction. There is thus sufficient time to take an
initial reading with the unreacted sample flowing past, which reading can
be utilized, using feedback circuitry to rapidly adjust the angle of
incidence to an appropriate value halfway down the reflectance dip, so
that the rest of the test can be performed at this fixed angle.
In an embodiment of the invention, the hemispherical body 11 is replaced by
a semicylindrical body. In this case FIGS. 3 and 4 can be regarded as
sections through a suitable apparatus, with the semicylindrical body 11
extending above and below the paper. The use of a semicylindrical body
gives the possibility of a line area of resonance instead of the single
point 15, and hence a linear active zone. The aperture 24 becomes a slit,
and the well 25 becomes elongate. The light source is operable to generate
a "sheet" output beam which may be focused by a cylindrical lens of the
type shown in FIG. 3 by reference numeral 31 onto a line extending through
point 15. The detector 18 is likewise linear in extent and is preferably
composed of separate detectors or detector arrays, each arranged to
monitor a specific section along the length of the line 15.
The semicylindrical lens 11 has the advantage that it can be used to
perform several tests simultaneously on a single sample. To this end, the
layer 20 takes the form of a series of distinct sensitive areas, each
comprising a different antibody, with each separate area being monitored
by its own detector 18. A single sample introduced into well 25 will flow
through the slot 24 into the active area and will react simultaneously
with the various sensitive areas, giving individual output readings which
can be monitored at detectors 18.
Although the hemispherical/semicylindrical body 11 is shown as having a
complete 180.degree. curvature, in fact it will be noted that only that
part near the flat surface is used and therefore a substantial portion of
the body 11 can be cut away to form a truncated hemispherical or
semicylindrical body, as indicated, by way of example, by the dotted line
26 in FIG. 4.
As will be appreciated from the foregoing, the invention enables a whole,
or at least a significant part of, the spread of angles of interest to be
investigated at once; the speed of investigation being limited only by the
response characteristics of the detectors in the array 18 and of the
associated sampling and computing circuits. This enables initial
transients and other shifts which may occur during the analysis to be
monitored and allowed for and also permits rapid calibratory checks to be
made. Furthermore it has been found that, if each analysis, or assay, is
started at a fixed value of reflectivity (as determined by a suitable
output from the computing circuits) then the absolute refractive index of
the fluid sample, which may well vary between samples, is unimportant.
Importantly, the invention enables the desired reflectivity characteristic
to be determined on a time scale so short that it is less than the time
taken for the chemical bonding, necessary to SPR, to be achieved between
the relevant constituent of the fluid sample and the reflective layer.
A further advantage of the invention is that it permits calibratory scans
to be conducted with fluids of known SPR characteristics to generate
compensating data which can be held in the computing circuits, and
automatically applied as corrections if desired during clinical analysis.
This compensating data can be used, for example, to allow for variations
in reflectivity over the point 15, a phenomenon which can occur
particularly if the reflective layer is produced by evaporation.
FIG. 5 shows a representation of a video signal derived from the detector
18 in the arrangement of FIGS. 3 and 4, as displayed on an oscilloscope
screen. The SPR resonance can be clearly seen.
The detector is electronically scanned, typically at approximately 200
times per second, to allow the movement of the resonance to be viewed in
"real-time" as biochemicals are bound to the surface of the metal coated
plate 16. The reflectivity curve in FIG. 5a has been modulated by the
approximately Gaussian profile of the beam from the laser diode source.
This profile can be removed by appropriate signal processing as shown in
FIG. 5b, which was produced by subtraction of the fixed backround due to
ambient light and division by the signal without any resonance.
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