U.S. patent application number 09/859677 was filed with the patent office on 2002-01-03 for set-up of measuring instruments for the parallel readout of spr sensors.
Invention is credited to Dickopf, Stefan, Schmidt, Kristina, Vetter, Dirk.
Application Number | 20020001085 09/859677 |
Document ID | / |
Family ID | 7888969 |
Filed Date | 2002-01-03 |
United States Patent
Application |
20020001085 |
Kind Code |
A1 |
Dickopf, Stefan ; et
al. |
January 3, 2002 |
Set-up of measuring instruments for the parallel readout of SPR
sensors
Abstract
The invention relates to a set-up of measuring instruments for
the parallel readout of SPR sensors. The aim of the invention is to
provide a measuring arrangement for the parallel readout of a
plurality of SPR sensors, wherein the readout process should be
terminated within an integration period of less than 30 minutes. To
this end, a wavelength-selective component (5) and an optical
imaging system (L2, L3) are arranged downstream of a light source
(3). Said optical imaging system (L2, L3) is in such a manner that
at a first wavelength it guarantees a parallel illumination of the
light entrance sides of the waveguides (13) which are provided with
SPR-compatible sensor zones, and that the light emerging from the
individual light waveguides (13) may simultaneously be imaged onto
a CCD chip (20) via an optical system (L4) in such a way that the
light emerging from each individual light waveguide (13) is
respectively detectable by several adjacent CCD pixels of the CCD
chip (20), and from these pixel areas a respective light intensity
value is calculable by means of image processing software, and,
after data storage of an intensity value, a set wave length and
coordinate in the waveguide array (10), an adjustment of the
wavelength-selective assembly (5) to a second, arbitrarily
providable, further light wavelength is performable by means of a
computer (30) via a control line (31).
Inventors: |
Dickopf, Stefan;
(Heidelberg, DE) ; Schmidt, Kristina; (Heidelberg,
DE) ; Vetter, Dirk; (Heidelberg, DE) |
Correspondence
Address: |
Gary M. Nath
NATH & ASSOCIATES PLLC
6th Floor
1030 15th Street, N.W.
Washington
DC
20005
US
|
Family ID: |
7888969 |
Appl. No.: |
09/859677 |
Filed: |
May 18, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09859677 |
May 18, 2001 |
|
|
|
PCT/EP99/08977 |
Nov 16, 1999 |
|
|
|
Current U.S.
Class: |
356/445 |
Current CPC
Class: |
G01N 21/253 20130101;
G01N 21/553 20130101 |
Class at
Publication: |
356/445 |
International
Class: |
G01N 021/55 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 20, 1998 |
DE |
198 54 370.0 |
Claims
1. A measurement assembly for reading out in parallel SPR sensors
that in form of a plurality of waveguides (13) form a waveguide
array (10), in which the distances between the individual
waveguides (13) correspond to a regular matrix and each individual
waveguide (13) has an SPR-compatible sensor area (16), which may be
associated with a respective sample, characterized in that a
wavelength-selective assembly (5) and an optical imaging system
(L2, L3) are arranged downstream of a light source (3), said
optical imaging system (L2, L3) being configured so that it ensures
a parallel illumination of the light entrance sides of said
waveguides (13) at a first wavelength, and the light emerging from
the individual light waveguides (13) may simultaneously be imaged
onto a CCD chip (20) via an optical system (L4; L6, L7) in such a
way that the light emerging from each individual light waveguide
(13) is respectively detectable by several adjacent CCD pixels of
the CCD chip (20), and from these pixel areas a respective light
intensity value is calculable by means of image processing
software, and, after data storage of an intensity value, a set wave
length and coordinate in the waveguide array (10), an adjustment of
the wavelength-selective assembly (5) to a second, arbitrarily
providable, further light wavelength is performable by means of a
computer (30) via a control line (31).
2. The measurement assembly as set forth in claim 1, characterized
in that a polarisator (6) is arranged downstream of said
wavelength-selective assembly (5).
3. The measurement assembly as set forth in claim 1, characterized
in that said waveguides (13) are configured comb-shaped and their
matrix is adapted to the well arrangement of a microtiter plate
(60), at least the bottom portion (62) of said wells being
configured optically transparent so that light from said waveguide
array (10) incident in said wells (61) is detected.
4. The measurement assembly as set forth in claim 1 or 3,
characterized in that dispersing means (9) are assigned to the
light exit side of said waveguide array (10).
5. The measurement assembly as set forth in any of the claims 1, 3
or 4, characterized in that masking and/or absorption means are
assigned to the light entrance side of said waveguide array
(10).
6. The measurement assembly as set forth in claim 5, characterized
in that masking means in the form of a perforated mask (8) are
assigned to said light entrance side of said waveguide array
(10).
7. The measurement assembly as set forth in claim 3 or 5,
characterized in that said masking and/or absorption means are
assigned to ensure that the light enters only the bottom portion
(62) of said wells of a microtiter plate (60) in which the
SPR-compatible sensors are arranged.
8. The measurement assembly as set forth in claim 3 or 5,
characterized in that said absorption means assigned to said light
entrance side of said waveguide array (10) take the form of a
microtiter plate whose well sidewalls are fabricated of a
light-absorbing material.
9. The measurement assembly as set forth in claim 5, characterized
in that said absorption means assigned to said light entrance side
of said waveguide array (10) take the form of a light-absorbing
cast separating said waveguides (13) from each other.
10. The measurement assembly as set forth in claim 1, characterized
in that said wavelength-selective assembly (5) is formed by an
incrementally variable monochromator and said optical imaging
system (L2, L3) is followed by a folding mirror (7) ensuring
parallel illumination of said light entrance side of said
waveguides (13) at a suitable angle.
11. The measurement assembly as set forth in claim 1, characterized
in that said wavelength-selective assembly (5) is formed by an
incrementally variable monochromator and said optical imaging
system (L2, L3) ensures parallel perpendicular illumination of said
light entrance side of said waveguide array (10), each optical
waveguide (13) being provided upstream with a lens (L5) ensuring
divergent illumination of said SPR-compatible layers (16) of said
waveguides (13).
12. The measurement assembly as set forth in claim 10,
characterized in that said light exit side end of said waveguide
array (10) is additionally provided directly downstream with a
further folding mirror (71) ensuring imaging of said waveguide
array (10) on a CCD chip (20) inclined to the optical axis.
Description
DESCRIPTION
[0001] The invention relates to a measurement assembly for parallel
readout of surface plasmon resonance (SPR) sensors.
[0002] In the search for new active substances combinatorial
chemical systems hold high promise in finding ligands matching a
receptor molecule. Miniaturizing and automating synthesis and
parallelization thereof is salient to assaying as large a number of
ligands as possible. Due to the small resulting amounts of ligand,
these requirements (miniaturizing, automating and parallelizing)
apply likewise to detecting the ligand receptor binding for which
the high sensitivity of the SPR method can be used in which the
light reflected from a thin gold film is detected. Under a suitable
resonance condition (angle of incidence and wavelength of the light
and thickness of the gold film) the intensity of the reflected
light is reduced. The energy of the light is then transformed into
charge density waves of the electron gas in the gold film, these
charge density waves being termed plasmons.
[0003] There are two methodical approaches to observing the
resonance: either monochromatic light is used in plotting the
intensity of the reflected light as a function of the angle of
incidence or the angle of incidence is maintained constant and the
wavelength of the light is varied. In both cases the resonance
curve is shifted with a change in the refractive index of the
medium provided on the side of the gold film facing away from
incident light.
[0004] This effect is made use of in biochemical analysis. The
receptor or ligand is immobilized on the gold surface. After
addition of the ligand or receptor the resonance condition is
changed on molecular association.
[0005] The simplest assembly for measuring this effect is a glass
prism which is illuminated with light and the angle of incidence of
which is varied (see e.g. "Biospecific interaction analysis using
biosensor technology" Malmqvist, M., Nature 361 (1993)
186-187).
[0006] A more sophisticated method is the parallel detection of
multiple angles in which the gold surface is illuminated with a
slightly divergent beam of monochromatic light (aperture angle
.about.10.degree.) and the reflected light is directed to a
position resolving light detector for obtaining an explicit
assignment between the angle of reflection and the position on the
detector. This construction has the advantage of sensing the angle
range of interest with no moving parts. This is why use is made of
this kind of detection in a few commercial systems as disclosed
e.g. in WO 90/05295 A1 or EP 797 091 A1. One disadvantage of these
assemblies is that only one prepared array of gold sensors
(one-dimensional detection) or but a few arrays of sensors arranged
along a line (using a two-dimensional detection) can be assayed
each time, i.e. this not permitting simultaneous measurement of a
two-dimensional sensor array by this angle detection method. After
having installed the prepared gold film in systems of this kind,
obtaining thermal equilibrium takes, however, some minutes (at
least 15 minutes), i.e. the actual measurement then lasting at
least until equilibrium of the molecular association is attained
which may also take up some minutes. This is why systems of this
kind lack good suitability in detecting the binding of a large
number of different ligands, since the time and expense involved in
measuring and changing the samples is relatively high.
[0007] A parallel approach to analyzing a sample array is SPR
microscopy (SPM) (see: EP 388 874 A2 or M. Zizisperger, W. Knoll,
Prog Colloid Polym Sci. 1998, 109 pages 244-253) in which the gold
surface applied to a prism is coated in various portions with
various samples and the gold surface imaged on a CCD chip at the
SPR angle. During measurement the angle is varied by a mechanical
scanner. However, this method is restricted to small object
diameters.
[0008] A more recent SPR method is disclosed in WO 94/16312A1 in
which detecting the binding of small amounts of substance is
achieved by optical fiber guides partially coated with a gold film.
However, here too, the problem still exists in designing a system
required to assay a plurality of samples in parallel in accordance
with this principle. Such an array of gold-coated fibers is, on the
one hand, expensive and highly sensitive to mechanical stress, and,
on the other, producing the array in parallel as proposed therein
is difficult to achieve technically.
[0009] Optical fiber guides are also employed as it reads from WO
98/32002 A1. To protect them from being damaged physically the
fiber cable is housed in a pipette. To achieve an array it is
proposed to use a series arrangement of such pipettes. However,
miniaturizing such an arrangement is difficult to achieve,
especially for parallel measurement of many different samples.
[0010] The invention is based on the object of defining a
measurement assembly for simultaneous readout of a plurality of SPR
sensors, more particularly exceeding a hundred or a thousand, in
which readout is completed with a measuring time of less than
thirty minutes. To achieve this object, use is made of a specially
configured array in an assembly with imaging methods to permit
simultaneous readout. The array used for this purpose comprises a
plurality of waveguides, it being noted that waveguide in this
context is understood within the scope of the present invention to
be an optical medium in which the light is guided in at least one
dimension and which has at least two parallel interfaces.
[0011] This object is achieved by the characterizing features of
claim 1. Advantageous embodiments are contained in the
sub-claims.
[0012] The invention will now be detailed by way of examples
illustrated diagrammatically in the drawings, in which:
[0013] FIG. 1 is an illustration of one variant of part of a
waveguide array employed,
[0014] FIG. 2 is an illustration of how a single SPR-compatible
sensor element is assigned to the pixels of a CCD array,
[0015] FIG. 3 is an illustration of a first variant of a
measurement assembly in accordance with the present invention,
[0016] FIG. 4 is an illustration of a second variant of a
measurement assembly in accordance with the present invention,
[0017] FIG. 5 is an illustration of a third variant of a
measurement assembly in accordance with the present invention,
[0018] FIGS. 6a and 6b are illustrations of two different optical
beam paths through the SPR-compatible surface area,
[0019] FIG. 7 is a CCD image of a SPR waveguide array,
[0020] FIG. 8 is a plot of the intensity profile of a single
SPR-compatible waveguide,
[0021] FIG. 9 is a plot of how the intensity profile is shifted for
differences in the sample concentration and
[0022] FIG. 10a and b illustrate the difference between
illuminating the waveguide array 10 with divergent light in
accordance with one embodiment as shown in FIG. 4 and with parallel
light as shown in FIGS. 3 and 5.
[0023] Referring now to FIG. 1 there is illustrated how use is made
to advantage within the scope of the present invention of a planar
SPR sensor 1 suitable for being fabricated by known silicon
semiconductor technologies and arranged into a waveguide array.
Part of one such waveguide array is shown in FIG. 1. A wafer of
silicon 11 is provided with a layer of SiO.sub.2 12 serving as an
optical buffer relative to the waveguiding layer 13 and the silicon
base material 11. The waveguide consists of a layer of silicon
oxynitride 13 in a thickness down to approx. 10 .mu.m. The silicon
oxynitride layer 13 is patterned by a dry etching process so that
parallel strips 14 materialize having widths in the range 10 .mu.m
to 2000 .mu.m on a center-spacing in the range 10 .mu.m to 5000
.mu.m. Once the waveguide strips 14 have been patterned, the
complete wafer is protected by a cover (not shown) except for the
areas intended to form the substrate for the SPR-compatible metal
layer 16 in a later process. Subsequently, the exposed locations of
the waveguide left unprotected in the previous step in the process
are metallized in a thickness compatible with the requirements of
the SPR measurement. The remaining cover of the wafer is removed.
Depending on the technology employed for producing the comb-like
recesses 15 in the substrate, these recesses may be produced before
or after cited metallization.
[0024] The technology as described permits accommodating a
plurality of parallel arrangements of waveguide patterning on a
wafer which are then singled by means of etching silicon or sawing
the silicon wafer. Separating each waveguide from the other, at the
end locating the SPR sensor, is achievable by wet chemical etching
the silicon or by a sawing process. Another variant in producing
the waveguides consists of the possibility of producing polymers in
thin films e.g. by centrifuging them onto a substrate. The polymers
(e.g. PMMA, polycarbonate, UV-curing adhesives or siliconated
polymers (cyclotenes or ORMOCERES)) present in dissolved or
non-cured form are centrifuged or poured onto the substrate
material. The refractive index of the substrate material must be
smaller than that of the polymers to be applied, later representing
the waveguide. When using UV-curing polymers, after homogenous
application of the layer, the non-exposed portions are etched away
so that narrow parallel strips of polymer remain on the substrate.
Other polymers may be produced as strips by embossing or other
replication techniques, the material remaining at the locations at
which no light is to be guided needing to be dimensioned
correspondingly thin. After the waveguide strips have been
patterned, here too the complete wafer is protected by a cover
except for the areas intended to carry the SPR-compatible
metallization 16. In the next step these exposed areas are coated
with the SPR-compatible metallization 16, after which the
protective layer covering the remaining areas is removed.
[0025] The variants described permit production of a great many
waveguides in parallel on a wafer. After fabrication of the
waveguides with the SPR sensor surface areas, single strips
consisting of a plurality of parallel waveguides are prepared from
the processed wafer by a separating method, e.g. sawing. This
singling process results in new face areas 17 which are prepared so
that light can be coupled thereinto and thereout of.
[0026] The variants as described permit planar fabrication of a
great many SPR sensors arranged in a row. To achieve an array of
sensors several of these strips are arranged stacked horizontally.
After assembly, such an array can be casted in a portion outside of
the SPR-compatible metallization in a polymer to provide the SPR
waveguide array with additional support. The arrangement and
spacing of the individual SPR-compatible sensors may be made in
accordance with the arrangement and spacing of the wells of a
microtiter plate 60 (see FIG. 3) to be presented. When this is the
case, the SPR waveguide array is brought into contact with the
microtiter plate 60 carrying the samples 61 to be characterized,
for measuring or coating the SPR-compatible layers 16 by
introducing the SPR waveguide array into the microtiter plate 60
sufficiently until the SPR-compatible metallization is totally
wetted by the samples 61. The individual vertical arranged
waveguides can be arranged horizontally on the center-spacing of an
optional microtiter format.
[0027] A waveguide array of the aforementioned kind is made use of
in a first variant as will now be described with reference to FIG.
3.
[0028] Referring now to FIG. 3 there is illustrated how only light
in a bandwidth of approx. 0.5 to 5 nm is transmitted from the white
light of a halogen lamp 3 after having passed through a suitable
beam adapter optical system L1, an IR filter 4 inserted in the
example to protect the optical components and a monochromator 5. As
an alternative, selecting the wavelength may also be implemented
with a filter wheel, requiring a corresponding number of filters
having a similar bandwidth. Thereafter, in the example, the
polarization direction is selected by a polarisator 6 for TM waves
in respect of the SPR-compatible metallization 16 of the employed
optical waveguide 13. An optical beam spreader L2, L3 then ensures
parallel illumination of the complete waveguide array, in this
example, via a folding mirror 7 applied so that the light is
incident in the waveguide 13 at an angle of 70.degree. to
90.degree. relative to the normal of the gold film of the
waveguides, since it is only light in this angle range that is
capable of exciting plasmon resonance. Using a folding mirror in
this case merely serves to make for a more compact configuration of
the measurement assembly. As an alternative, illumination could
also be made directly at this angle. A perforated mask 8 provided
in the example as shown in FIG. 3 which may take one of many
configurations, shades the portions behind which no waveguide is
located to prevent scattered light from gaining access by some
unwanted way to a detector furthermore provided. When some other
technologically more complicated means of applying and locating a
plurality of waveguides arranged in parallel is provided, as
commercially available with a core diameter of approx. 200 .mu.m,
for example by embedding the individual waveguide sections in a
common substrate, likewise in the scope of the present invention,
then use may be made of a non-transparent material or a material
having a non-transparent coating for the substrate material
employed, as a result of which the perforated mask 8 as mentioned
above can be eliminated. The waveguides themselves are located by
their sensitive portion in a liquid which can be changed for
implementing reference measurements in the various solutions to
swap the target molecules or to implement washing. At the end of
the waveguide remote from the sensitive waveguide portion the light
emerges at an angle the same in amount as that with which it was
incident into the other side (see FIG. 6). In the scope of the
invention a dispersing means 9 is furthermore assigned to the
waveguide sections at the light exit end, causing dispersion of the
light emerging from the waveguides. This is achievable, for
example, by means of a dispersing lamella to be applied separately,
a suitable coating, or the like. This strongly divergent light is
imaged directly by the objective lens L4 on a CCD chip 20, the
objective lens L4 being designed to "see" the entirety of the
waveguide array, but only a small proportion of the light emitted
from an optical waveguide permitting detection. For detecting such
small amounts of light, use is made in the example of a cooled
high-sensitivity CCD chip 20, the necessary exposure time of which
may amount to a few seconds.
[0029] It is provided for in the scope of the present invention
that the light emerging from each waveguide is imaged on several
CCD pixels simultaneously to enhance accuracy of detection. Thus,
in the example, imaging is provided for on several camera pixels
since a CCD chip has a great many more pixels than the waveguide
array provided for in this case at the individual waveguides.
Within the scope of the present invention, an image processing
software is employed for assigning the portion of several CCD
pixels to each waveguide. This is indicated diagrammatically in
FIG. 2, e.g. waveguide 2,2 being assigned the pixel portion
{(11,12), (11,13), . . . , (14,13)}, i.e. the portion of the CCD
chip 20 for imaging the waveguide 2,2 comprises 12 pixels. This
assignment is memorized in a computer 30 and remains available for
the complete duration of the subsequent measurement, since the
array is no longer moved further. A fast program algorithm obtains
the sum of these camera pixel portions assigned to each waveguide
so that a discrete intensity value is obtained and memorized for
each waveguide. The wavelength of the light incident from
underneath in the example, is then shifted by approx. 1 nm with the
aid of the monochromator 5 signalled by the computer 30 to obtain
the next intensity value for all waveguides to thus obtain an
intensity spectrum specific to each and every waveguide. To
optimize the measuring time needed for imaging such a spectrum, the
exposure of the CCD camera is instantly restarted, once the data
relative to the last wavelength have gained access to the computer
memory and a new wavelength is set at a monochromator 5 signalled
by a stepper motor. Summing the results in the computer as to the
pixel maps can then be undertaken during the time needed for
exposing the new wavelength, When this time is sufficiently long,
the time needed for computing the summation is neglible. The time
needed for each wavelength is dictated substantially by the
exposure time, which in the example is approx. 5 seconds, 16
minutes thus being needed for a spectrum of 200 nm. Now, to
determine the molecules associating with each SPR sensor of every
individual waveguide, it is not the absolute intensity spectrum of
a single measurement, but the difference in the minima of the
intensity spectra for a measurement in a pure buffer solution as
compared to those of a second measurement in the presence of the
target molecule, i.e. it is this shift in the wavelength that first
permits arriving at an indication as to the molecular association.
Accordingly, sequencing reference and sample measurements would be
possible within 32 minutes for all waveguide sensors
illuminated.
[0030] Referring now to FIG. 4 there is illustrated
diagrammatically another variant of the assembly in accordance with
the invention which reduces the time needed for the measurement.
Since it is the time needed for the exposure which is the
time-limiting factor, the measurement time can be shortened by
increasing the amount of light in the waveguides. For this purpose
a more powerful light source 3, such as e.g. a Xenon arc lamp may
be employed, The same as in the arrangement as shown in FIG. 3, the
light first passes through an IR filter 4, a monochromator 5 and a
polarisator 6, an optical beam spreader L2, L3 then assuring
parallel illumination of the waveguide array 10, upstream of which
in this embodiment spherical or gradient lenses L5 are inserted. To
illuminate all waveguides in the example as shown in FIG. 4,
however, several new positionings of the waveguide array 10,
relative to the lenses L5 provided, are needed. However, assigning
a separate lens to each waveguide is likewise within the scope of
the present invention whereby this lens may also be a component in
the wells 62 of the microtiter plate concerned.
[0031] The parallel light is focussed by the lenses L5 into the
waveguides, coated with gold, for example, such that in the
sensitive portion of the waveguide the diverging rays are incident
at the provided metallization of the waveguide also at the angle
permitting SPR detection. As compared to the exposure arrangement
as shown in FIG. 3 the lenses L5 as provided in the example permit
inputting roughly 100 times the light intensity into the
waveguides. The entrance windows 17 of the waveguides in this
example are positioned at the focal point of the lenses, i.e. at a
distance of a few 100 .mu.m. In this arrangement it is to be
assured that no light bypasses the waveguide, for instance due to
any interspaces or transparencies, into the space for light exit
from the waveguides. Here too, this can be avoided by providing the
means as described above, for example a perforated mask 8. At the
light exit end of the waveguides the light emerges from the optical
waveguide again divergent and is likewise imaged on a CCD chip 20
with the aid of a dispersing means 9 and an objective lens L4.
[0032] The further assignment and analysis of the light intensities
assigned to each optical waveguide is the same as described with
reference to FIG. 3. For each wavelength, the time needed can now
be reduced to approx. 1 second due to the higher light intensity.
When restricting the arrangement to e.g. a spectrum of sixty
wavelengths, the time needed for measuring all spectra in parallel
in the example is one minute, thus making it possible to implement
e.g. kinetic binding assays simultaneously with a large number of
ligands.
[0033] Referring now to FIGS. 10a and 10b the difference between
illuminating the waveguide array 10 with divergent light in
accordance with the embodiment as shown in FIG. 4 and with parallel
light as shown in FIG. 3 will now be demonstrated. These Figs.
illustrate the marginal rays after having passed through the
perforated mask 8 against the background of a line of sensors. In
the divergent case (FIG. 10a) the marginal rays sweep several
measuring ranges so that after emergence the marginal ray can no
longer be assigned to a single measuring range, this being the
reason why when illuminating with divergent light, interfaces 40
are to be included at the margin of the sensor area to ensure total
reflection, whereas when illuminating with parallel light (FIG.
10b) these margin areas can be eliminated. In this arrangement the
light beam is guided solely by total reflection at the sensor area
and the opposite surface. In this case the light beam penetrates
the waveguides at a angle suitable for SPR resonance right from the
start. When then being successful in imaging the entirety of the
parallel emergent light on a CCD detection, it is likewise possible
to increase the intensity and thus to shorten the time needed for
measurement, except that in this case unlike the situation as shown
in FIG. 3, the angle of the incident light is fixedly defined.
[0034] Referring now to FIG. 5 there is illustrated a more
complicated embodiment, working on the principle of total parallel
ray path and achieving additionally improved imaging performance
(less edge shading, lower sensitivity to scattered light). In this
case the light is guided through an optical fiber 50 via an optical
imaging system L1,L1' to the monochromator 5 and through a further
optical fiber 51 to an optical beam spreader L2, L3. This has the
advantage that guiding the illuminating beam can be adjusted
independently of the light being coupled into and out of the
monochromator 5. Illuminating the waveguide array 10 is done the
same as described in FIG. 3 with a spread parallel bundled rays at
the plasmon resonance angle, a perforated mask (not shown in FIG.
5) being employed optionally as shown in FIG. 3. In emerging from
the sensor the light is either parallel displaced (for an even
number of reflections, cf. FIG. 6b) and thus at the angle .alpha.
the same as on entry, or for an uneven number of reflections (FIG.
6a) it emerges at an angle -.alpha.. The length L and width B of
the SPR-compatible waveguide are specially dimensioned in the
example as shown in FIG. 5 so that the number of reflections is
uneven and thus the light emerges exclusively in the direction of a
second folding mirror 71. Unlike the situation as shown in FIG. 3
in which imaging on a CCD camera is achieved with a scattering
layer, in this case the parallelism of the emerging light is made
use of to practically reverse the illuminating light path. Via an
achromatic collimator lens L6 of long focal length (e.g. f=1000 mm)
and large diameter and a suitable objective lens L7 (e.g. f=100 mm)
the surface area of the sensor array is imaged on the CCD chip like
in a telescope. Since the surface of the waveguide array 10 to be
imaged does not stand perpendicular to the optical axis of the
arrangement, using a conventional camera, i.e. with the objective
lens parallel to the CCD chip would result in only a line of the
object being sharply focussed because of the inadequate depth of
focus. This is why on the imaging side the CCD chip 20 needs to be
likewise tilted relative to the optical axis, as indicated in FIG.
5, to ensure a sharp image of the whole sensor array surface area
10. This is achievable by tilting the CCD camera with a goniometer
(not shown) relative to the objective lens.
[0035] Another advantage of telescopic imaging is its low
sensitivity to scattered light, since it is only the light emerging
at the detection angle from the plane of the object that is imaged
on the CCD chip.
[0036] Referring now to FIG. 7 there is illustrated a sharp image
of a sensor array 10 as produced by the optical arrangement as
shown in FIG. 5. Such an image is detected for each wavelength and
in the computer 30 the intensity integral as a function of a sensor
surface area is formed, as described above with reference to FIG.
2. It is in this way that for each and every sensor a drop in the
intensity as materializing in surface plasmon resonance, is
detected in the spectrum as shown in FIG. 8 by way of example for a
single case. The broken line curve in the upper portion of FIG. 8
shows the spectrum with the sensor surface area located in air,
this substantially corresponding to mathematically folding the lamp
spectrum and monochromatic transmission. The solid line corresponds
to the measurement in the presence of a buffer; this spectrum is
superimposed by the surface plasmon resonance. As shown in the
lower portion of FIG. 8 obtaining the pure surface plasmon
resonance spectrum is done by scaling to the spectrum in air.
[0037] To verify that the detected drop in the spectrum is surface
plasmon resonance, the refractive index of the solution is varied
by making the measurement in various sucrose concentrations. It is
evident from the upper portion of FIG. 9 how the drop in the
spectrum is shifted, as expected, with increasing sucrose
concentration. Adapting the measurement data with a Gauss function
furnishes numerical values for the location of the minima of the
spectra, from which, with the known refractive index of the
solution, a calibration curve is obtained as shown in the lower
portion of FIG. 9. For a sufficiently small range of the refractive
index an approximately linear relationship is assumed. In the
present example a shift of 1.4 nm is obtained for a change in the
refractive index of 10.sup.-3 in enabling the minimum to be
determined with an accuracy of approx. 0.3 nm, a typical value for
a resonance curve of approx. 50 nm half-value width. Thus changes
in the refractive index of 2.multidot.10.sup.-4 can be detected.
This demonstrates the sensitivity of the method for parallel
measurement of several hundred or thousand samples within a few
minutes.
List of Reference Numerals
[0038] 1 - planar SPR sensor
[0039] 10 - waveguide array
[0040] 11 - wafer of silicon
[0041] 12 - SiO.sub.2 layer
[0042] 13 - optical waveguide
[0043] 14 - waveguide strips
[0044] 15 - comb-like recesses
[0045] 16 - SPR-compatible layer
[0046] 17 - face areas (entrance windows) of 13
[0047] 20 - CCD chip (camera) 20
[0048] 3 - light source
[0049] 4 - IR filter
[0050] 5 - wavelength-selective assembly
[0051] 6 - polarisator
[0052] 7 - folding mirror
[0053] 71 - second folding mirror
[0054] 8 - perforated mask
[0055] 9 - light dispersing means
[0056] 30 - computer
[0057] 31 - control line
[0058] 40 - reflective interfaces
[0059] 50, 51 - optical fiber
[0060] 60 - microtiter plate
[0061] 61 - sample in a well of a microtiter plate
[0062] 62 - wells
[0063] B - width of SPR-compatible waveguide
[0064] L - length of SPR-compatible waveguide
[0065] .alpha.- angle of light entry/exit
[0066] L1, L1', L2, L3, L4, L6, L7 - optical imaging systems
[0067] L5 - lenses
* * * * *