Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Device and method for carrying out quantitative
fluorescence-marked affinity tests
The invention relates to a device for carrying out, in
particular, quantitative fluorescence-marked affinity
tests by means of evanescent field excitation. This may
involve a wide variety of known biochemical assays of
general receptor/ligand systems, such as
antibody/antigen, lectin/carbohydrate, DNA or
RNA/complementary nucleic acid, DNA or RNA/protein,
hormone receptor, enzyme/enzyme cofactors, [sic]
protein or protein A/immunoglobin [sic] or
avidin/biotin. Preferably however, antibody/antigen
systems are evaluated.
Fluorescence immunotests, or fluorescence
immunosensors, use an antibody/antigen system and have
long been used widely. They are used primarily to
quantify an unknown amount of a particular chemical or
biochemical substance in a liquid sample matrix. In
this context, antibodies are bound selectively to the
substance to be determined. The substance to be
determined is referred to by the person skilled in the
art as an antigen. In fluorescence immunotests, the
analyte-specific antibodies are marked with a marking
substance which is optically excited at a particular
substance-specific wavelength ~,eX and the fluorescent
light with a different wavelength, which is generally
longer, is used with a suitable detector with
evaluation of the fluorescent light intensity. The use
of evanescent field excitation when implementing such
fluorescence immunotests, or respectively the
fluorescence immunosensors, already belongs to the
prior art. For example, a variety of solutions have
already been described in WO 94/27137, by R.A. Badlay,.
R.A.L. Drake, I.A. Shanks, F.R.S., A.M. Smith and
P.R. Stephenson in "Optical biosensors for
immunoassays: fluorescence capillary-fill device",
Phil. Trans. R. soc. Lund. B 316, 143 to 160 (1987) and
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D. Christensen, S. Dyer, D. Flowers and J. Herron,
"Analysis of Exitation [sic] and Collection Geometries
for Planar Waveguide Immunosensors", Proc. SPIE-Int.
Soc. Opt. Eng. Vol. 1986, Fiber Optic Sensors in
Medical Diagnostics, 2 to 8 (1993). However, the known
solutions generally have the disadvantage that they
require relatively great outlay in order for the light
needed to generate the fluorescence to be coupled into
an optical fiber or for the fluorescent light to be
extracted, which form an essential part of hitherto
customarily used devices.
Further, US 3,939,350 describes a solution in which
fluorescence immunoassays are carried out by means of
evanescent field excitation.
In this case, light from a light source is directed at.
an.angle through a prism onto an interface, so that _
total reflection takes place and the fluorescence
caused in a sample can be measured with a detector. The
entire sample volume is in this case accommodated in a
sealed closed space, so that on account of the
relatively large sample volume only diffusion
controlled end-point detection can be carried out and
this is susceptible to error.
WO 90/05295 describes an optical biosensor system in
which [lacuna] an elaborate optical system excitation
light can be directed onto sensitive regions of a
likewise elaborate channel system, through which the
sample volume is fed by means of controlled valves and
pumps, and the fluorescent light emerging through
windows from the sensitive regions can be re-directed
onto a detector with a view to intensity measurement.
Besides the aforementioned disadvantageous complex and
elaborate structure, this system requires, before and
after a test is carried out, cleaning both of the pumps
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and of the entire channel system in order to preclude
the possibility of subsequent measurement errors.
WO 90/06503 describes a sensor in which excitation
light is directed at a suitable angle through an
optically transparent substrate onto an interface to
form an optically transparent buffer layer, over which
an extra waveguide layer, to which the analytes to be
determined are in turn bound, is applied.
The refractive index of the buffer layer is in this
case lower than that of the substrate and of the
waveguide. If a suitable choice is made for the angle
of the excitation light, total reflection takes place
at the substrate/buffer boundary layer [sic] and, by
means of the resulting evanescent field, the excitation
light is coupled into the waveguide lying over the
buffer layer. The light coupled into the waveguide is
guided by means of total reflection in the waveguide,
and the resulting evanescent field is correspondingly
employed for fluorescence excitation.
The sample may be accommodated in one or more cavities,
the only restr_~ction on the corresponding dimensioning
of such a cavity being that its size permits the sample
to be transported into the cavities by means of
capillary force. After the sample has been taken in by
the cavities, no further flow or movement of the sample
takes place.
WO 89/09408 A1 discloses a similar solution, which once
more uses the light source for the excitation light and
the detector for the fluorescent light on the same
side. The sample to be detected is accommodated in a
cavity between a waveguide and a cover plate. here
[sic) again no further flow or movement of the sample
takes place after the sample has been taken up.
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A feature of preferred embodiments of the invention is
therefore to provide the possibility of carrying out
quantitative fluorescence-marked affinity tests, with a
variety of known biochemical assays, with a very simply
constructed device.
In accordance with an embodiment of the present invention thee
is provided device for carrying out quantitative fluorescence-
marked affinity tests by means of evanescent field excitation,
having at least one light source which emits almost
monochromatic light and directs light beams, having a
wavelength which causes fluorescence of a marking substance
bound to a chemical or biochemical partner of a general
receptor/ligand system, at an angle a to be defined by a
predeterminable penetration depth d for the evanescent field
onto an interface of an optically transparent baseplate made
of a material whose refractive index n1 is greater than the
refractive index n2 of the material above the interface,
characterized in that the light is directed through the
baseplate onto the interface of a reception region which is in
the form of a cuvette and has a thickness of between 0.001 and
0.5 mm, a sample container for receiving the sample is
arranged in such a way that a link is formed via a first
possible connection, which is at least partially an opening
between the reception region in the form of a cuvette and the
sample container and a second possible connection is linked to
the reception region in the form of a cuvette, the receitpion
region being covered on the opposite side from the baseplate
with a cover plate, and a detector for picking up the
fluorescent light being arranged on the same side of the
baseplate as the light source.
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In accordance with another embodiment of the present invention
there is provided method for carrying out fluorescence immuno
tests by means of evanescent field excitation, characterized
in that a sample volume is taken from a sample container
through a reception region in the form of a cuvette by
suction, pressure or capillary forces and the marked chemical
or biochemical components which are to be determined according
to the biochemical assay are bound to the corresponding
complementary chemical or biochemical components which are
fixed on the surface in the reception region, and the
fluorescent light is measured with a detector by evanescent
field excitation by means of at least one light source.
With the device designed in accordance with the invention, it
is possible to carry out fluorescence immunotests in a variety
of procedures (assays) and further quantitative fluorescence-
marked affinity tests. This provides the opportunity, on the
one hand, for carrying out competitive assays and sandwich
assays as well as other known assay forms may further be used.
The procedure adopted when working with the device according
to the invention is similar to that already known in the prior
art. In this context, a fluorophore is used as marking
substance and analyte-specific antibodies are marked using it.
The bound fluorophore
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[lacuna] excited with evanescent field excitation and
the fluorescent intensity which this has caused makes
it possible to quantify the marked antibodies, and it
thus becomes possible to quantify the analyte as well.
In the device according to the invention, light from a
light source is directed at an angle a onto the
interface between two media with different refractive
indices. In this context, a light source is selected
which emits almost monochromatic light having a
wavelength which is suitable for exciting the marking
substance, in this case the fluorophore. Suitable light
sources for this include, in particular, laser diodes
since they have a suitable beam profile and sufficient
optical power, together with a small overall size and
low energy consumption.
Other light sources which emit monochromatic light may,
however, also be employed.
In this case, care should then be taken that all the
optically transparent objects are each transparent to
the wavelengths which are used.
The angle a at which the emitted light is delivered to
the interface determines, besides the refractive index
of the material arranged in the optical path in front
of the interface and the material which follows,
together with the wavelength of the light, the
penetration depth d for the evanescent field. In this
case, the refractive index n1 of the material which is
arranged in the optical path in front of the interface
must permit total reflection at the interface, and
should therefore be greater than the refractive index n2
of the other material arranged thereafter, at the
wavelengths of the light sources used. The angle a is
preferably chosen so as to satisfy: sin(a) > n2/nl. If
this prerequisite is met, all the light is reflected at
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the interface and total reflection is therefore
achieved. When this condition is met, however, a
relatively small part of the light penetrates through
the interface into the material which is arranged in
the optical path after the interface, and an evanescent
field is created. The penetration depth d is defined as
corresponding to the distance from the interface at
which the intensity of the evanescent field has fallen
to the value 1/e, a being equal to the natural
logarithm base. The penetration depth d can be
calculated using the following equation:
d - a. * 1
2~c n; * sine (a) - nz
With a wavelength 7~ - 700 nm, a refractive index n1 -
1.51 and a refractive index n2 - 1.34, if the angle of
incidence of the light is a - 65° then a penetration
depth d of about 400 nm is obtained, and a penetration
depth d of about 173 nm is obtained if the angle of
incidence a - 80°. The consequence of this is that,
using the evanescent field, it is only possible to
optically excite those marking substances which are in
the immediate vicinity of the interface. The result of
this, as regards carrying out fluorescence immunotests,
is that exclusively the marking substances of the
antibodies or antigens which are bound on the surface
of the interface are excited. The fluorescent intensity
of the light emitted by these fluorophores is therefore
directly proportional to the concentration of the
marked antibodies bound to the surface and, depending
on the biochemical assay used, proportional or
inversely proportional to the antigen concentration.
The device designed in accordance with the invention
now uses at least one light source which emits almost
monochromatic light and directs it onto a baseplate
which is transparent to this light at an angle a which
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predetermines the penetration depth d for the
evanescent field. The refractive index n1 of the
baseplate should be greater than 1.33. On the other
side of the baseplate, a reception region designed in
the form of a cuvette is arranged between a cover
plate. Between the baseplate and the reception region
designed in the form of a cuvette, the aforementioned
interface is formed and the evanescent'field can act
with the predetermined penetration depth d within the
reception region in the form of a cuvette on marked
chemical or biochemical partners of a receptor/ligand
system which are bound to the surface, and excite the
fluorophores used as marking substance.
The fluorescence caused by this is measured with the
corresponding intensity by a detector. The detector is
in this case arranged on the same side of the baseplate
as the light source.
The detector used may in this case be an individual
photosensitive detector, or a one-dimensional or two-
dimensional arrangement of a plurality of photo-
sensitive detectors.
Although, when laser diodes are used, the delivered
light is almost monochromatic, a weak broad-spectrum
fluorescence background is observed in the laser light.
For this reason, a narrow-band excitation filter, which
preferably has a spectral bandwidth < 10 nm and only
transmits light in this narrowly limited wavelength
range matched to the light source, is preferably
arranged between the light source and the baseplate.
Advantageously, a relatively broadband second filter
(emission filter) may also be arranged in front of the
detector. This filter prevents light from the light
source which is scattered in the material of the
baseplate and on reflection at the interface from
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reaching the detector and vitiating the measurement
result. Favorable options for the two filters are the
interference filters or edge filters, or a combination
of the two.
It is particularly advantageous to direct polarized
light onto the sample to be determined. To this end, a
polarizer may be arranged in the optical path of the
light following on from the light source.
l0
In the case of polarized excitation and detection, it
is favorable to make use of the fact that the
fluorophores of the marked antibodies, which are firmly
bound to the surface, are restricted in their mobility.
Consequently, their fluorescent light is oriented in
exactly the same polarization plane as the excitation
light, and can be detected.
Conversely, the fluorescent light from the fluorophores
of the chemical or biochemical substance (antibody),
which are not bound to the surface and can consequently
move freely, will be oriented in another polarization
plane. By using polarized light for the excitation, it
is possible to suppress the fluorescent light from the
fluorophores bound to the antibodies or antigens, which
although lying within the range of the evanescent field
are not bound to the surface. The advantage with
polarized excitation and detection is apparent if the
dimensions of an antibody, about 10 nm, are considered
in relation to the penetration depth of the evanescent
field, up to about 500 nm. Polarization during
excitation and detection, a polarizer also being
arranged in front of the detector for this purpose, can
improve the ratio of the useful signal to the
background signal.
The device designed in accordance with the invention
has some essential advantages over those hitherto used.
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It has a very simple structure which places little
demands on the optical components to be used and, in
particular for coupling the light in to excite the
fluorophores and/or for extracting the fluorescent
light, does not involve any special requirements.
Furthermore, a wide variety of biochemical assays can
be readily carried out and the essential components can
be produced economically, so that even a single use, at
least of some parts, is entirely possible. Furthermore,
the requisite separation of the chemical components
from the sample volume, in relation to the components
bound to the surface, is guaranteed.
This is also achieved in that the baseplate and the
cover plate, as well as the spacers arranged in
between, are made of simple and inexpensively available
materials which can be processed using standard
technologies. Thus, plastics may be used for the
baseplate and cover plate, and a biocompatible adhesive
film, which is designed to adhere on both sides, may be
used in an advantageous form for the spacer.
The spacer has a thickness of from 0.001 to 10 mm,
preferably between 0.001 and 0.5 mm and particularly
preferably 50 ~,m, and by means of a recess forms the
reception region for the sample.
The essential basis of the invention is that a def fined
sample volume is taken through the reception region in
the form of a cuvette and subjected there to evanescent
field excitation, as described above. The sample volume
may in this case be taken through the reception region
in the form of a cuvette by suction, pressure [lacuna]
capillary forces.
For the binding of a chemical or biochemical component
to its complementary chemical or biochemical component
fixed on a surface, two physical transport effects must
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be taken into account in a flowing system, namely
convection and diffusion. In this case, the thickness
of a diffusive boundary layer (dependent on the flow
rate and viscosity) has an essential effect on the
binding in terms of quality and rate. If the diffusive
physical transport predominates with a relatively thick
diffusive boundary layer, rebinding of already bound
chemical or biochemical components may take place and
vitiate the measurement result. This can be
counteracted with the invention by keeping the free
cross section of the reception region in the form of a
cuvette relatively small, in particular as regards the
height. If the flow rate is maintained, this has the
cooperative effect that the diffusive boundary layer is
negligibly small in the actual detection region. The
measurement can thereby be taken substantially faster
with satisfactory or, under certain circumstances, even
higher accuracy. In many cases, it is even possible to
do away with end-point detection, that is to say the
entire sample volume does not necessarily have to be
taken into account.
In an advantageous embodiment, at least one possible
connection, which is at least partly an opening, is
provided in a cover plate, and a sample container can
be fitted or arranged in it, in this case, the opening
is arranged in the cover plate in such a way that a
link can be made between the sample container and the
reception region. In addition, a second opening is
present as a further such possible connection, and is
likewise joined to the reception region in the form of
a cuvette.
The second opening may likewise be provided in the
cover plate. An external pump may be connected to this
second opening, or it may have its own pump fitted in
it. If it has its own pump, this will preferably
consist of a cylindrical hollow body which can be
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fitted tightly into the second opening. An absorbent
material, for example a nonwoven material or paper, is
arranged at the bottom of the cylindrical hollow body.
The cylindrical hollow body may be closed off with a
stopper, a cap or a film. The pumping process is
activated by removing the stopper or cap or by making a
hole in the film.
The invention may be refined in that a sleeve, which
can be closed off at the bottom by a membrane, can be
fitted into the aforementioned sample container. In
this case, antigens, preferably, may be fixed on the
membrane and the requisite marked analyte-specific
antibodies needed for carrying out the test may be
located on the inner wall of the sleeve.
The invention will be described in more detail by way
of example below.
Fig. 1 shows the measurement principle of a
competitive assay;
Fig. 2 shows the measurement principle of a
fluorescence measurement with evanescent field
excitation of surface-bound marked antibodies;
Fig. 3 shows the measurement principle when performing
a sandwich assay;
Fig. 4 shows the principle when performing an improved
competitive assay;
Fig. 5 shows a part of the device according to the
invention for a continuous flow arrangement;
Fig. 6 shows a schematic representation of an
embodiment of a device designed in accordance
with the invention having a light source;
Fig. 7 shows a schematic representation of a second
embodiment having two light sources which are
used;
Fig. 8 shows a schematic representation of a further
embodiment having one light source;
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Fig. 9 shows a refined example according to Fig. 6;
Fig. 10 shows the arrangement of a sample container and
a pump in relation to the device according to
the invention;
Fig. 11 shows a development of the device according to
Fig. 10, with an additionally insertable
sleeve, and
Fig. 12 shows a further embodiment of a baseplate with
a spacer.
Fig. 1 schematically discloses the measurement
principle for carrying out a competitive assay. A known
amount of fluorophore-marked analyte-specific antibody
Ak is mixed with an unknown amount of antigen Ag to be
quantified, the amount of antibody Ak having to be
greater than the amount of antigen Ag. The marked
analyte-specific antibodies Ak bind to the antigens Ag
and, since the amount of marked analyte-specific
antibodies Ak is greater than that of the antigens Ag,
some marked analyte-specific antibodies Ak remain
unbound. The resulting mixture of unbound, or free,
marked analyte-specific antibodies Ak is brought into
contact with a surface (O) on which the antigen Ag has
been fixed. For greater clarity, this phase of the
assay is separated by the dashed line in the
representation. The still free marked analyte-specific
antibodies AK bind to the fixed antigens Ag, while
those already bound remain in the solution above the
surface. The amount of analyte-specific antibodies Ak
which are bound to the surface-fixed antigen Ag is
inversely proportional to the antigen concentration
which is to be investigated.
The amount of marked analyte-specific antibodies Ak
bound to the surface can be quantified by evanescent
field excitation and measurement of the fluorescent
intensity.
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This measurement principle is disclosed by Figure 2. In
this case, light having a wavelength with which the
fluorophore as marking substance can be excited is
directed at an angle a parallel against the surface
which acts as an interface 20 and on which the fixed
antigens are bound, through a medium having the
refractive index n1 which will subsequently be referred
to as the baseplate 1 in the description of Figures 5
to 9. The medium having the refractive index n1 is here
arranged under the interface 20.
At this surface which serves as an interface 20, total
reflection takes place and an evanescent field is
formed above this surface, and causes the excitation of
the fluorophores.
This being the case, Figure 2 also represents the
intensity I with the curve 21 of the evanescent field
as a function of the distance X from the surface acting
as the interface 20, it being clear to see that the
intensity 21 falls exponentially with increasing
distance.
Figure 3 schematically discloses the procedure of
another assay format, a so-called sandwich assay, the
phases being once more separated by the dashed line
which is drawn. A prerequisite in this case is that
there are a pair of analyte-specific antibodies Ak
which can both simultaneously bind to the antigen Ag,
without these analyte-specific antibodies Ak hindering
one another. In this case, one of the two analyte-
specific antibodies is marked with a fluorophore. The
other of the analyte-specific antibodies Ak is fixed on
a surface. After the reaction and surface binding of
the second antibody, the fluorophores are then excited
with exposure to light of a specific wavelength, and a
fluorescence signal is obtained which, in this form of
the immunotest, is directly proportional to the antigen
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concentration. This has the advantage that just a
relatively small antigen concentration produces a
measurable signal, and even small changes in the
antigen concentration can be sensitively registered.
In comparison with this, the competitive assay
described above (cf. Fig. 1) has the disadvantage that
even small antigen concentrations produce a large
signal, and small changes can accordingly be measured
only with difficulty.
It is generally known that, in sandwich assays, the
detection threshold is lower and the sensitivity is
higher than with competitive assays. Sandwich assays
have the disadvantage with respect to this that there
must be a second analyte-specific antibody Ak, and this
is only possible if the molar mass of the antigen Ag is
substantially in excess of 200 daltons. Antigens with a
low molecular weight, for example environmental
pollutants, cannot consequently be detected, or can
only be detected with difficulty.
It follows from this that competitive assays are
universally usable, but have lower sensitivity,
whereas, although a sandwich assay is better in these
regards, it nevertheless cannot be used in all cases.
Figure 4 describes the principle of a novel assay, for
which more detailed explanations, in particular
regarding the device to be used, will be given in the
description of Figure 11.
The basis of this novel assay is once more a
competitive assay. The mixture of free and bound marked
analyte-specific antibodies Ak is fed through a
membrane 22, to the surface of which the corresponding
antigen Ag is fixed. By way of example, a
nitrocellulose membrane may be used as the membrane
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material. Exclusively the free marked analyte-specific
antibodies Ak bind to the antigens Ag fixed there,
while those already bound pass through the membrane 22.
The liquid following on from the membrane 22 is brought
into contact with a surface O on which a protein P is
fixed. This protein is capable, independently of the
specificity of the marked analyte-specific antibody Ak
which is used, of recognizing and binding the latter.
The protein P used may, for example, be protein A or an
anti-antibody. The fluorescent intensity signal
obtained in this assay behaves in accordance with the
sandwich assay, and the sensitivity is consequently
greater and the possible detection threshold is
correspondingly low.
Figure 5 represents the outline structure of a part of
the device according to the invention. The three parts
illustrated there, the baseplate 1, the spacer 4 and
the cover plate 3, may be connected to. one another
before the fluorescence immunotest is carried out, or
may already form a completed unit and be equivalent in
their structure to a continuous-flow cell and a
measuring cuvette.
In this case, the baseplate 1 is made of a high-index
transparent material, for example glass or a plastic,
such as a polymer (PMMA or PC) having a refractive
index n1 > 1.33. The thickness of the baseplate may lie
in a range from 0.01 to 10 mm, preferably between 0.5
and 1 mm.
The spacer 4 is preferably a thin sheet, which is
provided on both sides with an adhesive film, or else a
thin adhesive film and thus bondable to the baseplate
1, on the one hand, and to the cover plate 3, on the
other hand. The total thickness of the spacer,
including the adhesive which is used, should lie in a
range preferably between 0.001 and 0.5 mm, and quite
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particularly preferably at a thickness of 50 Vim. A
hole, which creates a reception region 2 in the form of
a cuvette, is made in the spacer 4.
Figure 5 furthermore shows the cover plate 3 in which
through-openings 11' and 11 are formed, in the present
example as bores. Their function will be returned to in
more detail below. The openings 11' and 11 are in this
case arranged in such a way that they at least partly
overlap the region of the reception region 2 of the
spacer 4. The spacer 4 may preferably be made of a
biocompatible adhesive film, which is preferably
provided on both sides with a peel-off protection layer
and is already commercially available.
Figure 6 shows the outline structure of a device
according to the invention. In this case', light from a
laser diode 7 is directed through a polarizer 18,
through an excitation filter 19, which is of narrow
optical band design, onto the reception region 2 formed
in the spacer 4 through the baseplate 1. By total
reflection at the interface 20 between the baseplate 1
and the reception region 2, evanescent field excitation
takes place in the reception region 2 and causes
fluorescence of the fluorophores used as marking
substance. The fluorescent light passes from there, via
a lens 16, through a broadband filter 8, by which the
scattered light from the laser diode 7 is kept away
from the detector 5. The fluorescent light passes
through the downstream polarizer 6 with the aid of the
lens 16' onto the detector 5, in front of which a
diaphragm 17 is placed. With the detector 5, the
fluorescent intensity is picked up and, accordingly,
the fluorescence immunotest is carried out and
corresponding quantitative determination is made
possible.
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In the example shown in Figure 6, the light from the
laser diode 7, as described above, is advantageously
directed via the polarizer 18 and an excitation filter
19 through an optically transparent body 25 and the
baseplate 1 onto the interface 20. Of course, it is
also possible for the light to be directed, with
omission of the polarizer 18 and excitation filter 19,
directly onto an end face 24 of the transparent body
25. The transparent body 25 consists of a material
having a refractive index higher than that of the
material which is arranged above the interface 20. The
transparent body 25 preferably has the same refractive
index as the baseplate 1.
Preferably, a body which, for example, is designed as a
flattened glass prism or plastic prism, is used as the
transparent body 25, in which case a variety of
plastics having the said refractive index and other
favorable optical properties are to be used.
The transparent body 25 may on the one hand be joined
to the baseplate 1 using an adhesive, it being
preferable to use an adhesive with the same optical
properties. A further possibility consists, in order to
obtain optimum optical contact between the high-index
optically transparent body 25 and the baseplate 1, to
introduce a very thin film 26 (matching fluid) between
the two of them, the refractive index of the fluid
being the same as the refractive index of the baseplate
1 and the transparent body 25 in the most favorable
case. The adhesive or film form [sic] an optical layer
26 for mediating the optical contact between the body
25 and the baseplate 1.
The use of the transparent body 25 has the effect that
the majority of the light from the laser diode 7 can
reach total reflection at the interface 20 between the
baseplate 1 and the reception region 2.
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It is in this case particularly favorable to configure
and orientate the end face 24 or 24' in such a way that
the light from the laser diode 7 impinges orthogonally
on this end face 24 or 24', and maximum light yields
can thus be coupled in in order to achieve total
reflection at the interface 20.
In the simplest case, the baseplate 1 and the
transparent body 25 may be formed as a common
component, so that the join described above, or the use
of the very thin film 26 between the transparent body
25 and the baseplate 1, may be omitted.
In this example, use is advantageously also made of a
collimator (lens) 16, 16' with which the fluorescent
light can be directed concentrated onto the detector 5.
In the example shown, the collimator consists of two
separate lenses 16 and 16' which are arranged opposite
and between which the filter 8 and the polarizer 6 may
be located. It is of course also possible for a one-
piece lens to be used as the collimator 16.
The distance 9 between the transparent body 25, or the
baseplate 1 in the case when it is of one-piece design
according to Figure 8, and the lens 16 should be in the
region of between 0 to about 1000 mm.
The example, represented in Figure 7, of a device
designed in accordance with the invention corresponds
essentially to the example which was described above
and is shown in Figure 6. In this case, there are
additionally only a second light source 7', a filter
19' and a polarizer 18'. The light source 7' delivers
light having a wavelength which differs from the first
light source 7. In this example as well, polarized
light is preferably used. The device shown in Figure 7
can advantageously be used when different marking
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substances, which can be excited at different
wavelengths, are used. Examples of this are the
fluorophores Cy5 and Cy7. In this case, in order to
excite the Cy5 fluorophore, a laser diode having light
with a wavelength of between 635 and 655 nm is used,
and a laser diode which delivers light having a
wavelength of between 730 to 780 nm is used for the Cy7
fluorophore.
In this embodiment, the way in which the measurement is
taken is by using either alternately switched diodes 7,
7' or, for example, correspondingly synchronized
choppers, so as to ensure that, at any time, only light
from one light source 7 or 7' can reach the sample to
excite it, and therefore that no spurious results
occur.
However, since it is in this case necessary for two
different fluorescence signals to be transmitted by the
same filter, it is no longer possible to use a
broadband filter 8. Two filters 8, 8', which
selectively block the wavelengths of the exciting light
sources 7, 7' should therefore be arranged one after
the other. To this end, for example, notch filters may
be used. A further possibility consists in bringing the
corresponding filter mechanically into the optical path
between the lenses 16 and 16', or removing it
therefrom, correspondingly synchronized with the laser
diode choppers or the switching on and off. A further
possibility consists in leaving the light sources 7, 7'
continually switched on and alternately bringing the
corresponding filters 8, 8' mechanically into the
optical path between the lenses 16, 16' or removing
them therefrom.
With this arrangement, on the one hand, it is possible
to obtain a reference signal which permits internal
calibration of the measurement signal. For the
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reference measurement, a reference antibody which is
not targeted against an antigen from the sample is
used. The reference antibody is quantified beforehand
and made discriminatable from the analyte-specific
antibody Ak to be determined by using a different
marking substance. The quantity of reference antibodies
actually bound to the surface can be determined using a
second light source 7', which causes light of a
fluorescence [sic] of different marking substance, a
l0 second scattered-light filter 8' and the detector 5.
Using this determination, it is possible to account for
the losses of marked analyte-specific antibodies Ak or
antigens Ag not bound to the surface.
Besides obtaining a reference signal, it is also
possible to carry out two immunotests which are run
independently of one another, the discrimination being
carried out with the aid of the different fluorophores.
Advantageously, a one-dimensional or two-dimensional
arrangement of photosensitive detectors may be used as
the detector 5. By means of this, it is possible for a
plurality of analytes to be detected in a parallel if,
depending on the biochemical assay, either different
antigens (in the case of a competitive assay) or
different analyte-specific antibodies (in the case of a
sandwich assay) are fixed in the reception region 2 at
different positions, and differently marked analyte-
specific antibodies are contained in the sample
container 10 in accordance with the amount of different
antigens or antibodies. The differently marked analyte-
specific antibodies are bound, corresponding to the
biochemical assay, at different positions in the
reception region 2 and, by focusing the fluorescent
light using the collimator 16 onto the one-dimensional
or two-dimensional arrangement of a plurality of
photosensitive detectors, the fluorescent light is
detected with spatial resolution. When only one marking
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substance is used, for example Cy5, this permits
independent parallel quantification of a plurality of
analytes from a sample. The detector 5 represented in
the figures is then formed by a corresponding
arrangement of a plurality of photosensitive detectors.
The illustrative embodiment shown in Figure 8
corresponds essentially to the example explained above
in the description of Figure 6. In this case, however,
the structure has been somewhat simplified in that the
baseplate 1 undertakes the tasks of the transparent
body 25 and is accordingly of larger design.
The baseplate 1 shown in this example has a rectangular
cross section with which, although input coupling
losses have to be accepted, the manufacturing cost is
reduced. The baseplate 1 may, of course, also be
designed as is the case for the transparent body 25
shown in Figure 6, and the end faces 24 and 24' are
inclined in such a way that the light from the laser
diode 7 can be orthogonally incident.
Figure 9 represents a further illustrative embodiment
which provides a refinement of the invention. Although
the majority of the structure represented has been
adopted from the illustrative embodiment shown in
Figure 6, it is also possible for the other embodiments
to be supplemented accordingly.
In this example, use is made of a diaphragm 17 which
can be moved relative to the detector 5 and the
baseplate 1.
The aperture dimensions of the diaphragm 17 may be
tailored to the application, so that circular, oval or
slit-shaped holes may be used for the diaphragm 17. The
size of the free diaphragm cross section may also form
a further selection criterion.
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For constant free cross section of the diaphragm 17, it
may be favorable to configure the diaphragm 17, or the
detector 5, in such a way that their mutual separation
can be altered. A further possibility consists in
providing a rotatable body in which there are a
plurality of different diaphragms 17, which differ by
size and/or position, so that different regions of the
reception region 2 can be imaged and can be acquired
with spatial resolution by the detector 5.
Since, when the diaphragm 17 is used, only a fraction
of the fluorescent light is focused onto the detector
5, a displaceable diaphragm 17 is represented in Figure
9.
Preferably, the diaphragm 17 is arranged in the focal
plane 28 of the imaging lens system made up of the
half-lenses 16 and 16~ and arranged in front of the
detector 5 in the optical path of the fluorescent
light.
By displacing the diaphragm 17, it is possible
sequentially to scan the surface of the reception
region 2.
When the biochemical assays which have been carried out
are ones in which different or identical chemical or
biochemical components are fixed at different positions
3 0 in the reception region 2 , and the sample container 10
which is represented contains complementary marked
chemical or biochemical substances in accordance with
the number of fixed components respectively found
there, the binding of the marked complementary
substances can be picked up with spatial resolution.
This is done by moving the diaphragm 17 parallel and/or
orthogonal to the focal plane, the full area of the
reception region 2 being comprehensively scanned.
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Figure 10 represents the way in which a sample
container 10 is arranged with respect to the opening 11'
in the cover plate 3 and a link can thus be made
between the sample container 10 through the opening 11'
to the reception region 2. In this case, the sample
container 10 forms the receptacle in which the known
amount of antibodies Ak fluorophore-marked with the
marking substance axe mixed in with the sample to be
l0 determined. The sample container 10 should
advantageously always be filled with the same amount,
in order to make it possible to obtain reproducible
results. It should in this case favorably always be
filled to maximum capacity. In all forms of assays
which can be carried out, the specific antibody Ak can
in each case be located on the surface of the sample
container 10 and, through contact with the liquid
sample, detached from the surface and passed into the
sample. One simple method which is already known
20 consists in applying lyophilized antibodies to the
surface of the sample container 10. This makes it
possible to store the test fox a relatively long time
before the immunotest is actually carried out. The
reception region 2 defines the area on the baseplate 1
on which, depending on the assay format, the
respectively corresponding chemical or biochemical
substances are fixed.
Figure 10 furthermore represents a preferred
30 cylindrical hollow body 12 which accommodates a plunger
13 or another suitable cover (stopper, cap, film), the
two of which act together as a pump. If the plunger 13
is moved out from the cylindrical hollow body 12, a
vacuum is created which sucks the sample material from
the sample container 10 through the reception region 2
in the direction of the cylindrical hollow body 12.
Through capillary forces in the reception region 2 and
through a liquid-absorbing material, on the bottom of
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the cylindrical hollow body 12, the flow is sustained
until the entire sample volume has been taken through
the reception region 2. The cylindrical hollow body 12
is mounted, or has a hole in the bottom, so that there
is a link with the reception region 2. This can be
achieved by the second opening 11 as a possible
connection in the cover plate 3. If no cover plate 3 is
used, the possible connection may also be designed in a
different way.
It is, however, also possible to connect a separate
external pump to the opening 11.
After the sample has been applied, (with the sample
container 10), it is necessary to wait a corresponding
length of time so that the desired binding between the
antigens Ag and the marked antibodies Ak can take place
fully. Following this, the pump 12, 13 is activated,
and a wait is made until all of the liquid has been
pumped through the reception region 2. After excitation
with the light source 7, or the light sources 7 and
it is then possible to determine the antigen
concentration, in which case the structure according to
the invention as has been represented in Figures 6 and
7 should be employed.
The structure, as has been represented and described
above, can be used for a wide variety of biochemical
assays.
In competitive assays, analyte-specific antibodies AK
marked with a fluorophore are contained unfixed in the
sample container 10, and the corresponding antigen Ag
is fixed in the reception region 2 on the baseplate 1,
which is made of a high-index glass or a polymer or
other suitable plastic.
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The sample container 10 is then filled with the sample
and the analyte binds to the marked antibody Ak. After
the reaction, the pump 12, 13 is activated and the
marked antibodies which are still free bind to the
fixed antigen Ag in the reception region 2 on the
baseplate 1. The corresponding amount of marked
analyte-specific antibodies Ak can be quantified by
measuring the fluorescent intensity, as has been
described above.
A further biochemical assay can be carried out as
follows . There is a membrane (not shown) , on which the
antigen Ag is fixed, on the bottom of the sample
container 10, and lyophilized analyte-specific
antibodies Ak marked with a fluorophore are contained
on the walls of the sample container 10. On the surface
of the baseplate 1, which is made of a material
described above, an anti-antibody or protein A which is
targeted against the analyte-specific antibody Ak is,
for example, fixed. The sample container 10 is then
filled with the sample and the antibodies Ak bind to
the analytes. After the reaction, the pump [sic] 12, 13
is [sic] activated and the antibodies Ak which are
still free bind to the antigens Ag on the membrane. The
analyte-bound antibodies are bound on the surface of
the reception region 2, and the corresponding amount
can then, as described above, be quantified by using
evanescent field excitation.
An important point with all the biochemical assays is
for there to be a relatively large sample volume in the
sample container 10, all of which is pumped past the
small measurement area formed by the reception region
2. Since the height of the reception region 2 is
comparatively small, it may be assumed that the
corresponding antibodies Ak (free in a competitive
assay and bound in a sandwich assay) can reliably reach
the surface through the processes of convection and
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diffusion, and this is actually the case over a large
range of flow rates. It is in this way possible to
achieve, on the one hand, concentration of the
antibodies Ak at the surface and, on the other hand,
stable operating reliability so that the process is
virtually independent of the through-flow rate.
Figure 11 gives a further possible illustrative
embodiment of the device, with which biochemical assays
can be carried out, as was described above in outline
in the description of Figure 4.
In this case, a sleeve 15 which can be pressed into the
sample container 10 is additionally used. The sleeve 15
is closed up at the bottom with a membrane 23. On the
surface of the inside of the sleeve 15, the lyophilized
analyte-specific antibodies and, where appropriate, the
reference antibody are contained, and the corresponding
antigen is fixed on the surface of the membrane. The
assembly can be stored in this condition for a
relatively long time. In order to carry out this test
according to Figures 4 and 9, the device represented in
Figure 7 is preferably used, the different antibodies
Ak, or the reference antibody, being marked with
different fluorophores. The quantification is then
carried out in the manner described above.
In the example shown in Figure 11, it is again possible
for a cylindrical hollow body 12 with fitted plunger 13
to be used, as was described above with reference to
the example shown in Figure 8.
Figure 12 shows a further refinement of the device
according to the invention, in which links 27 and 27 ~ ,
through which the chemical or biochemical substances to
be determined can be introduced into the reception
region 2 and taken out again, as was described above
with reference to the other examples, are formed on
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both sides of the reception region 2. The spacer 4
shown in Figure 12 can again be formed in simple
fashion by punching, it being possible for everything
to be carried out in one punching procedure. In
addition, the opening 12 of the cylindrical hollow body
12, which is integrally incorporated in the cover plate
3 arid on whose bottom there is again a liquid-absorbing
material, or the opening 11 in which there is a liquid-
absorbing material, is provided with a film cover 33
which, in the initial condition, hermetically seals the
opening 11, or the opening of the cylindrical hollow
body 12, and the flow of the sample through the
reception region 2 can be initiated simply by breaking
the cover sheet 33.
