Note: Descriptions are shown in the official language in which they were submitted.
CA 02270665 1999-OS-OS
WO 98/21571 PCT/EP97106202 -
_1_
Use of biosensors to diagnose giant diseases
The invention relates to a biosensor suitable for recognising plant diseases)
as well as its use
in the course of a diagnostic process.
Hereinafter, the specific recognition and quantification of plant pathogens
are summarised
under the expression "diagnosis".
Biosensors are measuring instruments whose primary signal is produced by a
biochemical
reaction. The analytical measuring instrument consists of an immobilised
biological material in
close contact with an appropriate transducer arrangement. The transducer
converts the
biochemical signal into a quantifiable electric signal. (Gronow, 1984, Trends
Biochem. Sci. 9,
336-340). The biosensor membrane recognises analytes on a molecular level,
while the
transducer detects the electrochemical, thermal, piezoelectric or optical
changes at its surface.
Sensors may be divided into the following groups according to signal
recognition:
1. Electrochemical sensors
2. Piezoelectric sensors
3. Calorimetric sensors
4. Optical sensors
Electrochemical sensors are described in Wilson) G.S. 1987 in Biosensors:
Fundamentals and
Applications; (Turner, A.P.F., Karube I. & Wilson G.S., Eds.) pp 165-179,
Oxford University
Press, Oxford).
Calorimetric sensors are described in Danielsson, B. & Mosbach) K., 1987, in
Biosensors:
Fundamentals and Applications; (Turner, A.P.F., Karube I. & Wilson G.S., Eds.)
pp 575-595,
Oxford University Press, Oxford).
Piezoelectric sensors are described in t_uong et al. TIBTECH 6, 310-316 (
1988).
Detection is based on optical sensors, for example the measurement of change
in colour,
reflection, refraction index, fluorescence or chemoluminescence. Optical
sensors take
measurements either directly or indirectly; here, either the optical
properties are changed by
means of a reaction between the biological component and the analyte or a dye
is integrated
into the reaction and its depth changes through the reaction between the
biocomponent and
CA 02270665 1999-OS-OS
WO 98I21571 PCTIEP97/06202
_2-
the analyte. Surface plasmon spectroscopy is an example of a direct measuring
method. (Hall)
E. A. H. ( i 986) Enzyme Microb. Technol. 8. 651-658}. In optical
biosensorics, the method of
"internal total reflection spectroscopy" has increased in importance.
(Robinson G.A. ( 1991 )
Biosensors & Bioelectronics 6, 183-191 ).
If a light wave is coupled into a planar waveguide which is surrounded by
media of a lower
refractive index, it is confined by total reflection to the boundaries of the
waveguiding layer. In
the simplest instance, a planar waveguide consists of a three-layer system:
substrate,
waveguiding layer, superstrate (or sample to be investigated), whereby the
waveguiding layer
has the highest refractive index. Additional intermediate layers may further
improve the action
of the planar waveguide.
In chat arrangement, a fraction of the electromagnetic energy enters the media
of lower
refractive index. This portion is described as an evanescent (= decaying)
field. The strength of
the evanescent field is greatly dependent on the thickness of the waveguiding
layer itself, as
well as on the ratio of the refractive indices of the waveguiding layer and
the media
surrounding it. In the case of thin waveguides, i.e. layer thicknesses that
are the same as or
smaller than the wavelength that is to be guided) discrete modes of the guided
light can be
distinguished.
Using an evanescent field for example, it is possible to excite luminescence
in media of
relatively low refractive index, and to do so only in the immediate vicinity
of the waveguiding
region. This principle is called evanescent luminescence excitation.
For analytical purposes, evanescent luminescence excitation is of great
interest, since
excitation is restricted to the immediate vicinity of the waveguiding layer.
The need for early identification of plant pathogens (A. Binder, L. Etienne,
J. Beck, J. Speich &
J. Youd, 1995. Practical value of crop disease diagnostic techniques. In:
Hewitt et al (eds.) A
vital role for fungicides in cereal production, SCI 8 BCPC Proceedings, UK,
237-238) has
increased due to the need for judicious usage of pesticides in plant
protection. Additional
domains are interested in characterising the phytosanitary condition of seeds,
plant material or
the harvested plants. Of the numerous plant pathogens that are important in
the diagnosis of
plant diseases, notable ones are fungi, bacteria, viruses, viroids and
phytoplasma. Which test
method is used depends on the type of pathogen and the plant substrate to be
examined. One
method used originally to examine plant diseases was the visual evaluation of
symptoms.
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97/06202
-3-
Further examinations were normally carried out in the laboratories using
microscopes or by
isolating pathogens on artificial nutrients. Until a short time ago, improved
examination
methods were based on electron microscopy. However, electron microscopy is
very time-
consuming and therefore routine examinations cannot be carried out on a larger
scale. A great
advance was made in the development of serological examination methods based
on
immunological methods (F. M. Dewey & R. A. Priestley (1994): A monoclonal
Antibody-based
for the Detection of the Eyespot Pathogen of Cereals Pseudocercosporella
herpofricoides. In
Modern assays for Plant Pathogenic Fungi CAB international, 9 - 15) and a few
disadvantages
of the above-described methods could thus be eliminated.
Serological methods that are used in crop protection and are based on the
ELISA techniques
are described in an overview by I. Barker (1996) (Serological methods in crop
protection. In
Diagnostics in Crop Protection, BCPC Proceedings, 65, 13 - 22).
Considerable progress has been made in the last 3 years in the development of
testing
methods based on DNA technology (RFPL, PCR) etc.). (J.D. Janse: (1995) New
methods of
diagnosis in plant pathology - perspectives and pitfalls. Bulletin OEPPlEPPO
25, 5.- 7 7).
An alternative analytical process to the ELISA technique, based on the use of
fibre-optic
evanescence field bioaffinity sensors, is described by P. Oroszlan et al.
(Automated Optical
Sensing System for Biochemical Assays: a Challenge for ELISA? Analytical
Methods and
Insfrumentations, Vol. 1, No. 1, 43-51 ).
An overview over the use of these sensors on different bioaffinity systems is
given by
G. Duveneck in Proceedings SPIE) volume 2631, pp 14 - 28 (1996). The potential
improvement in detection limits of bioaffinity sensors, based on the
excitation of luminescence
in the evanescent field of a waveguide through the use of thin-film metal
oxide waveguides as
transducers, is described in WO 33197 and in WO 33198.
Disadvantages of the above-mentioned processes are normally: high costs, since
inter alia the
platforms cannot be regenerated, too long analysis times due to complicated
sample
preparation work, purification steps for working up the plant extracts and too
few samples
being processed) since normally only one pathogen is examined at a time.
There is thus a need to develop a process which allows several samples of
plant material to be
examined for one or more plant pathogens in a parallel manner, i.e.
simultaneously or directly
after one another, without additional purification steps) and in addition
enables the plant
CA 02270665 1999-OS-OS
WO 98I21571 PCTIEP97I06202
-4-
pathogens to be analysed and quantified early, in a highly sensitive manner,
without time-
consuming purification steps for the plant extract, and with a high number of
samples.
Within the context of the present invention) it has now surprisingly been
found that biosensors
may be used in plant diagnostics for the early recognition of plant diseases,
whereby the plant
material to be examined can be used directly in the form of plant extracts
without prior
processing, in the course of the diagnostic process according to the
invention. The use of
biosensors in plant diagnostics results in the fact that from now on plant
extracts can be
examined with high sensitivity, more quickly, cheaper, in a fully automated
manner and with a
higher number of samples than was possible with prior-known processes. These
biosensors
may be employed both in the laboratory and directly in the field, and can be
regenerated.
According to the use of the expression in this application) biosensors are
measuring
instruments whose primary signal is produced by a reaction with biological or
biochemical
analyte molecules. According to the definition used here, biosensors consist
of chemical or
biochemical recognition elements, immobilised on a so-called transducer which)
as a
consequence of the reaction with the biological analytical molecules, creates
a change in state
which can be converted into a quantifiable electronic signal. The transducer
is generally a solid
material. In the following) the expressions "transducer" and "sensor platform"
are used
synonymously. The chemical or biochemical recognition elements recognise
analyte molecules
on a molecular level; contact of the recognition elements with the transducer
enables for
example an electrochemical, piezoelectric, calorimetric or optical effect to
take place as a
consequence of the reaction with the analytes, and this effect can be
subsequently converted
into an electronic signal. Depending on the principle of the signal being
produced, the following
groups of sensors may be classified without restricting their general
application and without
regarding this as a complete list:
1. Electrochemical sensors
2. Piezoelectric sensors
3. Calorimetric sensors
4. Optical sensors
In principle, all of the above-mentioned biosensors, for example
electrochemical sensors,
piezoelectric sensors) calorimetric sensors or optical sensors, are suitable
for the usage
according to the invention in the course of the process according to the
invention. Especially
suitable, and therefore preferred in the context of this invention is the use
of biosensors with
sensor platforms, since these enable several sample solutions to be analysed
with high
CA 02270665 1999-OS-OS
WO 98I21571 PCTIEP97I06202
-5-
sensitivity. Washing or purification steps between the individual measurements
can be omitted,
so that a high number of samples may pass through per unit of time. This is of
great
significance especially for routine examinations or for evaluations in the
course of genetic
engineering.
It has also surprisingly been found that, in a simple manner, a sensor
platform may be
produced on the basis of at least two separate regions on a common substrate,
which is
suitable for the parallel detection of the same or different analytes to
diagnose one or more
plant diseases.
Apart from examining several sample solutions simultaneously, one sample
solution can also
be examined for several analytes contained therein, simultaneously or in
succession, on one
sensor platform. This is particularly advantageous for examination of plant
extracts, which can
be carried out in a particularly rapid and economical manner.
A further advantage of the use of the sensor platform is that the individual
separate regions
may be addressed selectively either chemically or fluidically.
Preference is given to a sensor platform on the surface of which one or more
specific binding
partners are immobilised as chemical or biochemical recognition elements for
one or more)
same or different plant pathogens to be evaluated. .
Especially preferred in the context of the invention are optical biosensors
with a sensor
platform, which are produced on the basis of one, preferably at least two
planar, separate,
inorganic) dielectric, waveguiding regions on a common substrate, and are
suitable for the
parallel evanescent excitation and detection of luminescence of the same or
different analytes
in order to diagnose plant diseases. These separate waveguiding regions may
each contain
one or more grating couplers.
If several sample solutions are analysed at the same time, the separate
waveguiding regions
prevent any cross-talking of luminescence signals from different samples. With
this process,
high selectivity and a low error rate are attained.
Through the separation of waveguiding regions, it is also possible to further
increase selectivity
and sensitivity with the well-directed usage of fight sources of different
wave lengths.
CA 02270665 1999-OS-OS
WO 98/21571 PCT/EP97/06202
-6-
A further advantage of the use of the sensor platform in an optical biosensor
for diagnosing
plant diseases is that the individual separate waveguiding regions may be
selectively
addressed not only chemically or fluidically, but also optically.
Preference is given in the context of the present invention to the use of a
sensor platform to
diagnose plant diseases, which consists of planar, physically or optically
separate waveguiding
regions, in which only one or few modes are guided. They are notable for
especially high
sensitivity with the smallest possible construction. Normally, this
sensitivity is not obtained by
multimodal waveguides of planar construction.
Coupling-in of the excitation light may take place for example using lenses)
prisms, gratings or
directly into the end face of the waveguiding layer.
Coupling-in and, where appropriate, coupling-out using gratings is normally
simpler and more
efficient than with lenses or prisms, so that the intensity of the coupled-in
light wave is similarly
greater, which, in conjunction with low degree of attenuation of the guided
lightwave,
contributes towards very high sensitivity of this arrangement.
Sensitivity may be further augmented by using as strong an evanescent field as
possible. This
offers the possibility of determining even the smallest amounts of luminescent
material on the
surface of the waveguiding layer.
One object of the present invention thus relates to a sensor platform as a
component of a
biosensor) which is especially suitable for diagnosing plant diseases. The
said biosensor)
which is similarly a constituent of the present invention) essentially
comprises a measuring
instrument which contains as a component the sensor platform according to the
invention. The
said sensor platform may be modified and normally contains immobilised) plant
pathogen-
specific, biochemical recognition elements which are in close contact with a
suitable transducer
arrangement. The said biochemical recognition elements are structures which
are specific for
the plant pathogens to be evaluated and therefore enable individual detection
of these plant
pathogens to be made within the course of the diagnosis process according to
the invention.
The said plant pathogens are preferably those selected from the group of
fungi, bacteria,
viruses, viroids and phytopiasms, but especially fungi, selected from the sub-
divisions of
Mastigomycotina, Zycomycotina, Ascomycotina, Basidiomycofina or
Deuteromycofina; bacteria
selected from the group Agrobacterium, Spiroplasma, Clavibacter, Erwinia,
Pseudomonas,
Xanthomonas or Xylella; as well as viruses selected from the group carla
virus, ciostero virus,
CA 02270665 1999-OS-OS
WO 98/21571 PCTlEP97106202
cucumo virus, luteo virus, nepo virus, potex virus, poty virus or tobamo virus
or from the group
phytoplasmosis.
Especially preferred in the course of this invention are sensor platforms
which bear chemical or
biochemical recognition elements that are specific for phytopathogenic fungi
selected from the
group of the genera Aphanomyces, Pythium, Phytophthora, Plasmopara, Bremia,
Pseudoperonospora or Peronospora; Podosphaera, Sphaerotheca, Erysiphe,
Uncinula)
Nectria, Giberella (Fusarium), Glomerella, Claviceps, Sclerotinia)
Cochliobolus, Leptosphaeria
(Septoria)) Pyrenophora, Venturia, Guignardia Uromyces, Puccinia, Hemileia,
Ustilago) Tilletia,
as well as Typhula.
A further object of the invention relates to a sensor platform for diagnosing
plant diseases,
which consists of one or more) but especially two separate regions on a common
substrate.
Also included in the invention is a sensor platform whose signal activation is
based on the
transduction principle of electrochemical, piezoelectric, calorimetric or
optical transduction
mechanism.
A sensor platform whose signal activation is based on an optical transduction
mechanism is
preferred.
In a particular embodiment of the present invention, there is a sensor
platform whose signal
activation is based on the change in resonance conditions to produce a surface
plasmon
resonance by means of interaction between one or more, identical or different
plant pathogens
to be evaluated with one or more specific binding partners as chemical or
biochemical
recognition elements, which are immobilised on the sensor platform.
Especially preferred is a sensor platform whose signal activation is based on
interaction
between one or more, identical or different plant pathogens to be evaluated
with one or more
specific binding partners as chemical or biochemical recognition elements in
the evanescent
field of a waveguide.
Particularly preferred is a sensor platform whose signal activation is based
on the effective
refractive index in the evanescent field of a wave guided in an optical
waveguide through
interaction of one or more) identical or different plant pathogens to be
evaluated with one or
CA 02270665 1999-OS-OS
WO 98I21571 PCTIEP97/06202
-8-
more specific binding partners as chemical or biochemical recognition
elements) which are
immobilised on the sensor platform.
A further object of the invention relates to a sensor platform whose signal
activation is based
on the change in the coupling angle of a grating coupler through interaction
between one or
more, identical or different plant pathogens to be evaluated with one or more
specific binding
partners as chemical or biochemical recognition elements, which are
immobilised on the
sensor platform.
Preference is given to a sensor platform whose signal activation is based on
the change in a
luminescence signal through interaction between one or more, identical or
different plant
pathogens to be evaluated with one or more specific binding partners as
chemical or
biochemical recognition elements, which are immobilised on the sensor
platform.
Especially preferred is a sensor platform based on a planar, dielectric
optical waveguide, but
especially a sensor platform based on a planar) dielectric optical waveguide,
with which
luminescence may be evanescently excited and detected.
Most particularly preferred in the context of this invention is a sensor
platform based on at
least two planar, separate) inorganic dielectric waveguiding regions on a
common substrate.
A specific embodiment of the present invention relates to a sensor platform
for the diagnosis of
plant diseases, which consists of a continuous transparent substrate and a
transparent, planar,
inorganic, dielectric waveguiding layer, which is characterised in that
a) the transparent) inorganic, dielectric waveguiding layer is subdivided at
least in the
measuring region into at least 2 waveguiding regions) such that the effective
refractive
index in the regions in which the wave is guided is greater than in the
surrounding
regions, or such that the subdivision of the waveguiding layer is formed by a
material
on the surface that absorbs the coupled-in light;
b) the waveguiding regions are each provided with or have a common coupling-in
grating,
so that the direction of propagation of the wave vector is maintained after
coupling-in,
and
c) where appropriate, the waveguiding regions are each provided with or have a
common
coupling-out grating.
CA 02270665 1999-OS-OS
WO 98/21571 PCT/EP97/06202
_g_
Similarly included in the present invention is a biosensor for the diagnosis
of plant diseases,
which contains a sensor platform according to the invention and an appropriate
transducer
arrangement.
in addition) the invention relates to processes for diagnosing plant diseases
in plant material
and also in soil or air samples) using the biosensor according to the
invention or the sensor
platform according to the invention.
A further object of the invention relates to the use of the sensor platform
according to the
invention or the biosensor according to the invention in analytical processes
for the diagnosis
of plant diseases.
The present invention relates primarily to a sensor platform as a component of
a biosensor,
which is especially suitable for the diagnosis of plant diseases. The sensor
platform according
to the invention may consist of both one region and two separate regions.
In the present invention, the purpose of the separate waveguiding regions is
to provide one
sensor platform for the simultaneous detection of evanescently excited
luminescence of one or
more analytes.
The terms measuring section and measuring region are used synonymousty in the
context of
the present invention.
The separate waveguiding regions may have any geometric form. This effectively
depends on
the structure of the whole apparatus in which the sensor platform is
installed. Examples of
geometric forms are lines, strips) rectangles, circles, ellipses, cross-
hatches, rhombi,
honeycombs or irregular mosaics. The divisions between the individual
waveguiding regions
essentially run in a straight line. At the ends, they may taper for example,
and they may be
broader or narrower overall than the measuring region.
The waveguiding regions are preferably arranged in the form of separate
strips, rectangles)
circles, ellipses, cross-hatches.
The waveguiding regions are most preferably arranged in the form of parallel
strips. The
waveguiding regions are most preferably in the form of parallel strips less
than 5 mm apart.
CA 02270665 1999-OS-OS
WO 98/21571 PCT/EP97/06202
- 10 -
A furfher preferred embodiment is obtained if the waveguiding regions are
arranged in the
form of parallel strips which are joined at one or both ends, whereby the
direction of
propagation of the wave vector does not change after the coupling-in.
In a further advantageous embodiment, the strips are joined together at one
end, while the
other end is open) whereby the direction of propagation of the wave vector
does not change
after the coupling-in.
Figures 1 a to 1 d and 2a to 2d illustrate a few further possible
arrangements. The reference
numerals show:
the waveguiding layer which has been applied to a substrate;
2 the divisions which are either formed by an absorbing material on the
surface of the
waveguiding layer, or by a reduction in the effective refractive index in the
plane of the layer,
which is achieved most simply by means of an air gap in place of the
waveguiding layer;
3, 3' the coupling-in and coupling-out gratings.
In figure 1 a, the waveguiding regions (= measuring regions) are broken up by
dividing regions.
These dividing regions do not come into contact with the coupling element.
In the case of figure 1 b, coupling-in and coupling-out gratings are jointly
available to all
measuring regions. There is no contact with the dividing regions.
In figure 1 c, the dividing regions extend beyond the coupling element.
Coupling-in is however
unaffected by these in the waveguiding regions.
Figure 1 d contains two grating couplers and otherwise corresponds to figure 1
c.
Figures 2a to 2d show an arrangement in which the gratings couplers are not
continuous, but
an individual grating is assigned to each waveguiding region.
The physically or optically separate waveguiding regions may be produced using
known
processes. There are two possible basic processes. For example, a) the layers
may be
constructed from the start with physical separation in an vapour deposition
method using
masks, or b) a continuous layer is produced and this is subsequently
structured using
appropriate methods. One example of process a) is the vapour deposition of the
inorganic
waveguiding material, whereby a suitably constructed mask covers up part of
the sensor
platform. Such masks are known from the production of integrated circuits.
Here, the masks
CA 02270665 1999-OS-OS
WO 98/21571 PCTIEP97/06202
- 11 -
should be in direct contact with the sensor platform. Positive and negative
masks may be
used.
It is also possible to apply a suspension of the inorganic waveguiding
material to the sensor
platform by means of a suitably constructed mask, and to produce the
waveguiding layer by
the sol-gel technique.
In this way, separate waveguiding regions are produced, whereby the division
is created most
simply by an air gap. However) this gap may also subsequently be filled with
different material
having a lower refractive index than that of the waveguiding layer. If
division into several
waveguiding regions is effected in this way, the difference in the effective
refractive indices
between the waveguiding region and the adjacent material is preferably more
than 0.2, most
preferably more than 0.6 units.
One example of process b} is the vapour deposition of an inorganic waveguiding
material to
form a continuous layer, which is subsequently subdivided into individual
waveguiding regions
by means of mechanical scoring, treatment with laser material) lithographic
processes or
plasma processes.
Vapour deposition normally takes place under vacuum conditions. Plasma
deposition is
similarly possible.
Special mention should be made of treatment with pulsed excimer and solid
state lasers or
continuous gas lasers. in the case of pulsed high-energy lasers, structuring
may be effected
over a large area through a mask. With continuously operating lasers, normally
the focused
beam is guided over the waveguiding layer to be structured) or the waveguiding
layer moves
relative to the beam.
The lithographic processes may be etching techniques, as employed in the
production of
printed circuit boards or microelectronic components. These processes allow an
extraordinarily
large number of geometric patterns to be produced and a fineness of structures
ranging from
micrometers to sub-micrometers.
What is important for all ablative operations is that the waveguiding layer is
completely or
partially removed, but the sensor platform is not completely divided.
Any intermediate layers that are optionally present may similarly be
completely or partially
removed.
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97106202
- 12 -
In a modified variant b) of the process, a continuous layer of an inorganic
waveguiding material
is applied first of all, and in a second step, using an absorbing material
which interrupts the
waveguiding, a structure is applied to this layer so that the waveguiding
regions are divided by
absorbing and thus non waveguiding regions.
The absorbent materials concerned may be inorganic materials such as metals
with a high
optical absorption coefficient, e.g. gold, silver) chromium, nickel or organic
compounds, e.g.
dyed and pigmented polymers. These materials may be applied to the waveguiding
layer as
continuous layers, or in the case of metals) in the form of aqueous colloidal
solutions. Various
methods may be chosen for this.
Deposition processes for structuring, which are carried out under vacuum
conditions, have
already been mentioned above.
Colloidal materials in water or organic solvents, for example gold in water,
may similarly be
employed for the structuring of waveguiding regions.
The deposition of colloidal gold onto surfaces by spontaneous assembly has
been described
for example by R. Griffith et al., Science 1995, 267, 1629-1632. Here, for
example, physically
or fluidically separate laminar part streams of a colloidal gold solution can
be allowed to flow
over the waveguiding layer, whereby the gold particles are deposited e.g. in
the form of strips.
The surface is dried) and separate, waveguiding regions according to the
invention are
obtained. The deposited gold colloids must have a minimum size of 10 to 15 nm
for the
desired absorption to occur. It is preferred if they are 15 to 35 nm in
diameter.
Colloidal gold may also be deposited by stamping the surface. Stamping of
dissolved organic
materials is described by Whitesides as so-called 'microcontact printing' and
has been used for
structuring gold surfaces with liquid alkanethiols (J.L. Wilbur et al., Adv.
Mater. 1994, 6, 600-
604; Y. Xia and G.M. Whitesides, J. Am. Chem. Soc. 1995, 117, 3274-3275). For
example)
colloidal gold solution can be drawn up into an elastomeric stamp having the
desired
structuring pattern, and the structuring pattern can be transferred to the
waveguiding surface
by applying the stamp.
CA 02270665 1999-OS-OS
WO 98121571 PCT/EP97/06202
- 13 -
Processes which operate with organic solvents or water are very flexible and
quick to use.
They enable waveguide structuring to take place directly before carrying out a
luminescence
assay.
Where appropriate, the surface of the waveguiding layer has to be modified
prior to colloidal
deposition of for example gold, so that good adhesion results between the
colloid particles and
the modified surface. Adhesion may be achieved by means of hydrophobic
interaction, van der
Waals forces, dipole-dipole interaction, simple electrostatic interaction or
covalent binding. The
interaction may be produced by functionalisation of the colloids and/or the
surface of the
waveguiding layer.
An appropriate method of modifying the surface and achieving adhesion is for
example
silanisation, as described in Advances in Colloid and Interface Science 6, L.
Boksanyi, O.
Liardon and E. Kovats, ( 1976} 95-137. Such silanisation is also used to
improve the adhesion
of recognition elements in affinity sensing. Mercapto-terminated silane, for
example
(mercaptomethyl)dimethylethoxysilane, is especially suitable for the adhesion
of gold by
creating a covalent sulphur-gold bond.
Another modification of process b) is that, in a second step, the same
inorganic material is
applied in the form of a structure to the continuous layer of an inorganic
waveguiding material)
so that an increase in the effective refractive index is achieved by
increasing the layer
thickness, and thus the propagation of lightwave mode is concentrated in the
resultant
measuring regions. Such 'slab waveguides' and processes for the production
thereof are
described by H.P.Zappe in 'Introduction to Semiconductor Integrated Optics',
Artech House
Inc., 1995.
The width of the strip of waveguiding layers is preferably 5 micrometers to 5
millimetres, most
preferably 50 micrometers to 1 millimetre.
If the width of the waveguiding regions is reduced too greatly, the available
sensor region is
also reduced. The strip width and required sensor region are conveniently
matched to one
another.
The size and width of the individual waveguiding regions may be varied within
a wide range
and basically depend on the purpose of use and the structure of the system as
a whole.
CA 02270665 1999-OS-OS
WO 98I21571 PCTIEP97/06202
- 14 -
The individual waveguiding regions) when formed as strips, preferably have a
length of 0.5 to
50 mm, most preferably i to 20 mm and most preferably 2 to 10 mm.
The number of strips on the sensor platform is preferably 2 to 100Q, most
preferably 2 to 100.
The individual waveguiding regions may be arranged for example as strips on
the substrate in
two or more groups, each respectively having at least two strips, thus forming
a multiple
detection region.
The great practical advantage of multiple detection regions of this
construction is that) between
successive multianalyte measurements, the sensor platform doss not have to be
cleaned or
replaced, but only displaced relative to the excitation unit, fluidics unit
and detection unit.
A further advantage is that such multiple detection regions are economically
more favourable
to produce. A very substantial advantage is that the very time-consuming and
cost-intensive
division into individual sensor platforms may be dispensed with.
Each multiple detection region preferably consists of 2 to 50, most preferably
2 to 20 separate
waveguiding regions.
There are preferably 2 to 100, most preferably 5 to 50 multiple detection
regions on the sensor
platform.
Figures 3a and 3b show a possible arrangement of a sensor platform with
several multiple
detection regions, in which the substrate has the shape of a disc and may be
produced by
press moulding in a similar way to current compact discs. The overall
arrangement may consist
of a disc-shaped sensor platform with several multiple detection regions and a
fluidics disc,
which contains the fluidics supply lines and the actual cell spaces. The two
parts are joined,
e.g. adhered, and form one unit.
The cell spaces in the form of wells may however also be preformed on the disc-
shaped
sensor platform. An embodiment of this type is then covered by a planar lid.
Reference numerals 1 to 3 have the significances indicated above, 4 indicates
an entire
multiple detection region, 5 signifies the substrate and 6 illustrates a
central cut-out portion
which can hold an axle, so that the individual multiple detection regions 4
can be rotated under
excitation and detection optics. 7 and T signify inlet and outlet apertures
for the solutions
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97/06202
required in the course of the assay) which are normally brought into contact,
by means of a
throughflow cell having at least two openings, with the recognition elements
that are
immobilised on the waveguiding regions.
The multiple detection regions may also be arranged on concentric circles. The
spacing
between the individual multiple detection regions may for example be such
that) rotation
through an angle between 5 and 20 degrees brings a new multiple detection
region under the
excitation and detection optics.
Figures 4a and b show an analogous construction of the sensor platform on a
disc, with the
difference that, in comparison with figure 3, the individual multiple
detection regions 4 are
arranged radially instead of tangentially, which leads to improved utilisation
of the surface
area.
A further arrangement is illustrated in figures 5a and 5b. The individual
multiple detection
regions 4 are arranged in the form of a rectangular cross-hatch pattern.
However) the multiple
detection regions may also be arranged as individual images in a film strip.
This film strip may be present as a planar element or may be rolled up.
The individual multiple detection regions may be transported under excitation
and detection
optics in a manner analogous to a film.
The preferences indicated for the separate waveguiding regions also apply to
the multiple
detection regions.
A sensor platform within the context of this invention is a self-supporting
element which may
be shaped as a strip, a plate, a round disc or any other geometric form. It is
basically~planar.
The chosen geometric form is uncritical per se and may depend on the structure
of the
apparatus as a whole in which the sensor platform is installed. However, it
may also be used
as a independent element, physically separate from a source of excitation
light and from the
optoelectronic detection system. Arrangements that allow substantial
miniaturisation are
preferred.
Miniaturised systems are known for example from environmental analytics. These
miniaturised
systems are user-friendly and may also be used directly in the field.
CA 02270665 1999-OS-OS
WO 98/21571 PCT/EP97106202
- 1s -
The substrate may be for example glass of all kinds ar quartz. Glass is
preferably used, as this
has the lowest possible optical refractive index and the lowest possible
degree of intrinsic
luminescence, and it allows the simplest possible optical machining to be
carried out, such as
etching, grinding and polishing. The substrate is preferably transparent, at
least at the
excitation and emission wavelengths. The microscopic roughness of the
substrate should be
as low as possible.
Transparent thermoplastic plastics may also be used as substrates, as are
described for
example in EP-A-0 533 074.
The substrates may be covered with a thin layer, which has a refractive index
lower than or
equal to the substrate and is no thicker than 0.01 mm. This layer may serve to
prevent the
interference of fluorescence excitation in the substrate and also to avoid
superticial roughness
of the substrate, and it may consist of a thermoplastic, a thermally
crosslinkable or a
structurally crosslinked plastics or also of inorganic materials such as Si02.
Where an intermediate layer is present, whose refractive index is lower than
that of the
waveguiding layer and whose layer thickness considerably exceeds the
penetration depth of
the evanescent field (i.e. in general » 100 nm}, transparency of only this
intermediate layer at
excitation and emission wavelength is sufficient, if the excitation light
beams in from the upper
side of the sensor platform. In this case, the substrate may also be
absorbent.
Especially preferred substrates are glass, quartz or a transparent
thermoplastic plastics. Glass
is preferred in particular.
Especially preferred substrates of transparent thermoplastic are
polycarbonate, polyimide or
palymethyl methacrylate.
It is preferable for the refractive index for all waveguiding layers to be the
same, that is, all
waveguiding layers preferably consist of the same material.
The refractive index of the waveguiding layers must be greater than that of
the substrate and
any optional intermediate layers used. The planar, transparent, waveguiding
layer preferably
consists of a material with a refractive index greater than 2.
The materials in question may be far example inorganic materials, especially
inorganic metal
oxides such as Ti02, ZnO, Nb20s, Ta20~, Hf02, or ZrOz.
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97/06202
- 17 -
Ta20s and TiOz are preferred.
The thickness of the waveguiding layers is preferably 40 to 1000 nm, more
preferably 40 to
300 nm, most preferably 40 to 160 nm.
In a preferred embodiment) the thickness of the waveguiding layers is the
same.
The modulation depth of the gratings is preferably 3 to 60 nm, most preferably
3 to 30 nm.
The ratio of modulation depth to the thickness of the layers is preferably
equal to or less than
0.5 and most preferably equal to or less than 0.2
The gratings for coupling in the excitation light or for coupling out the
backcoupled
luminescence light are formed as optical diffraction gratings, preferably as
relief gratings. The
relief structure may have various forms. Suitable forms are for example
sinusoidal, rectangular
or saw-toothed structures. Processes for producing such gratings are known.
Photolithographic or holographic processes and etching techniques are
primarily used to
produce them, as described for example in Chemical, Biochemical and
Environmental Fiber
Sensors V. Proc. SPI E, Vol 2068, 313-325) 1994. For organic substrates,
moulding or
stamping processes may also be employed.
The grating structure may be produced on the substrate and afterwards
transferred to the
waveguiding layer in which the grating structure is then reproduced, or the
grating is produced
in the waveguiding layer itself.
The grating period may be 200 to 1000 nm, whereby the grating advantageously
has only one
periodicity, i.e. it is monodiffractive. The grating period selected is
preferably one that allows
the excitation light to be coupled in the first diffraction order.
The modulation depths of the gratings are preferably of the same magnitude.
The gratings preferably have a bar to space ratio of 0.5 - 2. By bar to space
ratio is understood
for example the ratio of the width of the bars to the width of the spaces in
the case of a
rectangular grating.
CA 02270665 1999-OS-OS
WO 98/2157I PCTlEP97106202
- 18 -
The gratings may serve both to couple excitation light into the individual
waveguiding layers
and to couple out luminescence light backcoupled into the waveguiding layers.
In order to examine different luminescent samples, it may be expedient for all
or part of the
coupling-in or coupling-out gratings to have different grating constants.
In a preferred embodiment, the grating constants for afl gratings are the
same.
If some of the gratings are used for coupting in and some for coupling out the
light) then the
grating constant of the coupling-in gratings) is preferably different from the
grating constant of
the coupling-out gratings}.
The grating distance is preferably B < 3~X,~e, whereby X"e indicates the
length at which the
initial intensity to of the guided beam has fallen to Ide.
One preferred group of embodiments of the sensor platform is characterised in
that
the transparent, planar, inorganic dielectric waveguiding regions on the
sensor platform are
divided from each other at least along the measuring section by a jump in
refractive index of at
least 0.6, and each region has one or two separate grating couplers or all
regions together
have one or two common grating couplers) whereby the transparent) pfarrar,
inorganic
dielectric waveguiding regions have a thickness of 40 to 160 nm, the
modulation depth of the
gratings is 3 to 60 nm and the ratio of modulation depth to thickness is equal
to or less than
0.5.
The jump in refractive index of 0.6 or more is most simply achieved whereby
the waveguiding
layer is divided completely and contains an air gap or, during measurement,
optionally
contains water.
The waveguiding regions preferably guide only 1 to 3 modes) and they are most
preferably
monomodal waveguides.
A further subject of the invention is a modified sensor platform for the
diagnosis of plant
diseases) which is characterised in that one or more specific binding partners
are immobilised
on the surface of the waveguiding regions as chemical or biochemical
recognition elements for
one or more, identical or different analytes.
CA 02270665 1999-OS-OS
WO 98I21571 PCTIEP97/06202
- 19 -
In the course of this invention, a modified sensor platform is preferred, on
the surface of which
binding partners are immobilised as chemical or biochemical recognition
elements, which are
specific for the plant pathogens or properties of pathogens (e.g. fungicide
resistance,
virulence) to be determined and thus enable selective recognition of said
pathogens to take
place.
The biochemical recognition elements are in particular binding partners which
are specific for
indicator substances which are characteristic of the plant pathogens to be
determined.
Specific binding partners which may function as chemical or biochemical
recognition elements
may be in particular antibodies, antigens, binding proteins A, binding
proteins G, receptors,
ligands, oiigonucleotides, single strand RNA) single strand DNA, avidin,
biotin, enzymes,
enzyme substrates, enzyme cofactors, enzyme inhibitors, lectins or
carbohydrates.
The plant pathogens which may be detected in the course of the diagnostic
process according
to the invention may be all plant pathogens from which said specific
recognition elements can
be isolated, but especially pathogens selected from the group comprising
fungi, bacteria)
viruses, viroids and phytoplasms.
Fungal pathogens may be taken from the classification of fungi according to
Ainsworth
(1971,Dictionary of the fungi, 6. ed. Comm. Mycol. Inst. Kew:} and Ainsworth,
Sparrow,
Sussman (1973, The fungi. Vol. IV A, IV B, Academic Press- New York, San
Francisco,
London).
Preferred target organisms among the fungal organisms are to be found within
the division
Myxomycota or Eumycota) and relate in particular to fungal pathogens from the
subdivisions of
Mastigomycotina, Zycomycotina, Ascomycofina, Basidiomycoi'ina or
Deuteromycotina;
Especially preferred are the fungal pathogens of the subdivision
Mastigomycotina, selected
from the group of the genus Aphanomyces, Pythium, Phyrophthora, Plasmopara,
Bremia,
Pseudoperonospora or Peronospora.
Furthermore, those that are especially preferred are fungal pathogens of the
subdivision
Acomycotina selected from the group of the genera Podosphaera, Sphaerotheca,
Erysiphe,
Uncinula, Nectria, Gibereila (Fusarium), Giomerelia, Ciaviceps, Sclerotinia,
Cochiiobolus,
Leptosphaeria (Septoria), Pyrenophora, Venfuria, Guignardia.
CA 02270665 1999-OS-OS
WO 98l21571 PCT/EP97106202
- 20 -
Furthermore, those that are especially preferred are fungal pathogens of the
subdivision
Basidiomycotina selected from the group of the genera Uromyces, Puccinia,
Hemileia,
Ustilago, Tilletia, Typhula.
Furthermore, those that are especially preferred are fungal pathogens of the
subdivision
Deuteromycotina selected from the group of the genera Rhizoctonia, Sclerotium,
Verticillium,
Botrytis, Pseudocercosporella, Pyricularia, Penicillium, Aspergillus,
Rynchosporium,
Cladosporium, Alternaria, Cercospora, Fusarium, Phoma, Ascochyta,
Colletotrichum.
Especially preferred are fungal pathogens selected from the group of the genus
of
Plasmodiophora, Spongospora, Polymyxa.
Especially preferred target organisms in the course of this invention are
Septoria nodorum or
Septona tritici.
Within the bacteria group, the genera Agrobacterium, Spiroplasma, Clavibacter,
Erwinia,
Pseudomonas, Xanthomonas or Xylella are especially notable. These contain a
number of
plant pathogens.
Plant-pathogenic viruses are to be found in particular within the groups carla
virus) clostero
virus, cucumber mosaic virus, luteo virus, nepo virus, potex virus) poty virus
or tobacco mosaic
VIfUS.
Preferred representatives of the phytoplasmoses which may be mentioned are for
example
representatives of proliferation disease and rubber wood disease of the apple.
Suitable chemical or biochemical recognition elements which are immobilised on
the surface of
the sensor platform according to the invention are in particular binding
partners which are
specific for indicator substances that are characteristic for the plant
pathogens to be
determined.
Specific binding partners which may function as chemical or biochemical
recognition elements
may be in particular antibodies, antigens, binding proteins A, binding
proteins G, receptors,
ligands, oligonucleotides, single strand RNA, single strand DNA) avidin,
biotin, enzymes,
enzyme substrates) enzyme cofactors, enzyme inhibitors, lectins or
carbohydrates.
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97/06202
- 21 -
Especially preferred as specific binding partners in the context of this
invention are DNA
sequences from the Internal Transcribed Spacer (ITS) of the ribosomal RNA gene
region,
which are specific for various species and strains of Septoria,
Pseudocercosporella, Fusarium
and Mycosphorella and are described in WO 95I29260.
Especially preferred as specific binding partners in the context of this
invention are also plant
pathogen-specific antibodies or antigens) but especially antigens) which may
be obtained from
the fungal pathogens Septoria nodorum or Septoria tritici, as well as
antibodies which may be
produced to act against these antigens. The antibodies which may be produced
are those in
the process described in EP 0472498 A1.
The said antibodies may be monoclonal or polyclonal antibodies, as selected,
which can be
produced by processes known per se, as are described e.g. in Ivan Roit)
Jonathan Brostoff,
David, K. Maie) Lehrbuch der Immunologie, Georg Thieme Verlag, Stuttgart,
1991, 335f. or in
R.T.V. Fox, 1993: Principles of Diagnostic Techniques in Plant Pathology, CAB,
UK, pp 129-
152.
Preferred indicator substances which are characteristic of certain plant
pathogens may be
selected from the group of receptors, ligands, proteins, antigens)
oligonucleotides, strands of
RNA or DNA, circular RNA, enzymes, enzyme substrates, enzyme cofactors,
inhibitors or
lectins.
Preferred indicator substances which are characteristic of certain plant
pathogens may be
selected from the group of cellulases, chitinases, PR proteins (pathogenesis
related proteins)
cutinases, amylases) pectinases, fatty acids or quinones.
Various specific binding partners can be applied to the surface of a
waveguiding region, the
physical separation thereof within each waveguiding region being unimportant.
They can for
example be present thereon in the form of a random mixture. This is
advantageous when
analytes having different emission wavelengths are to be determined
simultaneously by way of
a coupling-out grating.
The specific binding partners on the surface of each waveguiding region are
preferably
physically separate from one another.
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97/06202
- 22 -
The specific binding partners may be immobilised at various sites on the
waveguiding regions,
for example by photochemical crosslinking) as described in WO 94I27 137.
Another method
comprises the dropwise application of the specific binding partners that are
to be immobilised)
using a multiple-pipette head. This can also be effected using a modified
inkjet printing head
with piezoelectric actuators. This has the advantage that the method can be
carried out rapidly
and that very small amounts can be used. This is a precondition for the
production of thin
strips or other finely structured geometric patterns.
Another preferred method for the physically separate immobilisation of the
specific binding
partners on the waveguiding regions that is very simple to carry out is based
on the use of a
flow cell, it being possible for the separation to be effected in the flow
cell, either mechanically
in the form of dividing bars or fluidically in the case of laminar flow. In
that method, the
geometric arrangement of the part streams supplying the binding partners
corresponds
substantially to the arrangement of the waveguiding regions on the sensor
platform. This
method of immobilisation using a flow cell is advantageous especially when the
specific
binding partners are to be embedded in an environment that is stable only in
the fluid medium,
as is the case for example with lipid-membrane-bound receptors.
In particular) it is possible in this way to deposit specific binding partners
that are covalently
banded to gold colloids, in the same manner as described above for the
production of non-
waveguiding regions. In order to obtain waveguiding in the immobilisation
regions, it is
necessary to use gold colloids of very small diameters of less than 10 nm and
especially of
less than 5 nm.
A further method that is likewise simple to carry out is based on stamping the
surface with the
specific binding partners) or with specific binding partners bonded to metals,
in a manner
analogous to that described above for the production of non-waveguiding
regions.
A preferred metal is gold.
Preferred physically separate patterns are strips, rectangles, circles)
ellipses or cross-hatches
patte ms.
Preference is given especially to a modified sensor platform which is
characterised in that only
one specific binding partner is arranged on the surface of each waveguiding
region.
CA 02270665 1999-OS-OS
WO 98/2I571 PCT/EP97/06202
- 23 -
Another preferred embodiment of the modified sensor platform is obtained if an
adhesion-
promoting layer is located between the waveguiding regions and the immobilised
specific
binding partners.
The thickness of the adhesion-promoting layer is preferably equal to or less
than 50 nm,
especially less than 20 nm.
It is possible, furthermore, for adhesion-promoting layers to be applied
selectively only in the
waveguiding regions or to be passivated in the non-waveguiding regions, for
example by
means of photochemical activation or using wet-chemical methods) such as a
multiple-pipette
head, inkjet printers, flow cells with mechanical or fluidic separation of the
streams, deposition
of colloids or stamping of the surface. The methods have already been
described above for
the direct immobilisation of the specific recognition elements on an
optionally chemically
modified or functionalised surface.
The selective immobilisation of the specific recognition elements exclusively
on the
waveguiding regions, either directly or by way of adhesion-promoting layers,
can, when using a
sample cell that covers both the waveguiding and the non-waveguiding regions,
lead to an
increase in the sensitivity of the detection method, since the non-specific
binding of the
analytes in the regions not used for signal generation is reduced.
The preferences described hereinbefore for the sensor platform apply likewise
to the modified
sensor platform.
The modified sensor platform is preferably fully or partially regenerable and
can be used
several times. Under suitable conditions, for example at low pH, at elevated
temperature, using
organic solvents, or using so-called chaotropic reagents {salts), the affinity
complexes can be
selectively dissociated without substantially impairing the binding ability of
the immobilised
recognition elements. The precise conditions are greatly dependent upon the
individual affinity
system.
A specific form of luminescence detection in an assay consists in the
immobilisation of the
luminescent substances that are used for detection of the analyte directly on
the surface of the
waveguiding regions. These substances may be, for example, a plurality of
luminophores
bound to a protein which can thus be excited to luminescence on the surtace of
the
waveguiding regions. If partners having affinity for the proteins are passed
over that
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97/0G202
- 24 -
immobilised layer, the luminescence can be altered thereby and the quantity of
partners
having affinity can thus be determined. In particular) it is also possible for
both partners of an
affinity complex to be labelled with fuminophores, in order for example to
carry out
determinations of concentration on the basis of the energy transfer between
the two, for
example in the form of luminescence extinction.
Another preferred embodiment of immobilisation for chemical or biochemical
affinity assays
consists in the immobilisation on the surface of the sensor platform of one or
more specific
binding partners as chemical or biochemical recognition elements for the
analytes themselves
or for one of the binding partners. The assays may consist of one or more
stages in the course
of which, in successive steps) one or more solutions containing specific
binding partners for
the recognition elements immobilised on the surface of the sensor platform can
be passed
over the surface of the sensor platform) the analytes being bound in one of
the part steps. The
analytes are detected by the binding of luminescently labelled participants in
the affinity assay.
The luminescence-labelled substances may be any one or more of the binding
partners of the
affinity assay, or an analogue of the analytes provided with a luminophore.
The only
precondition is that the presence of the analytes should lead selectively to a
luminescence
signal or selectively to a change in the luminescence signals.
In order to~increase the chemically active sensor surface, it is also possible
to immobilise the
chemical or biochemical recognition elements on micro particles, so-called
"beads", which in
turn can be fixed to the surface of the sensor platform by suitable methods.
Prerequisites for
the use of beads, which can consist of different materials, such as plastics,
are that, firstly the
interaction with the analyte takes place to a significant extent within the
evanescent field of the
waveguide, and secondly that the waveguiding properties are not significantly
impaired.
In principle, the recognition elements can be immobilised, for example, by
hydrophobic
adsorption or covalent bonding directly on the waveguiding regions or after
chemical
modification of the surface) for example by siianisation or the application of
a polymer layer. In
addition, in order to facilitate the immobilisation of the recognition
elements directly on the
waveguide, a thin intermediate layer, for example consisting of SiOz, can be
applied as
adhesion-promoting layer. The silanisation of glass and metal surfaces has
been described
comprehensively in Literature, for example in Advances in Colloid and
Interface Science 6,
L.Boksanyi, O.Liardon and E. Kovats, (1976) 95-137. Specific possible methods
of carrying out
the immobilisation have already been described hereinbefore.
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97/06202
- 25 -
Suitable recognition elements are, for example) antibodies for antigens,
binding proteins such
as protein A and G for immunoglobulins, biological and chemical receptors for
ligands,
chelators for "histidine-tag components") for example histidine-labelled
proteins, oligo-
nucleotides and single strands of RNA or DNA for their complementary strands,
avidin for
biotin) enzymes for enzyme substrates, enzyme cofactors or inhibitors, or
lectins for
carbohydrates. Which of the relevant affinity partners is immobilised on the
surface of the
sensor platform depends on the architecture of the assay. The recognition
elements may be
natural or may be produced or synthesised by means of genetic engineering or
biotechnology.
The expression antibodies includes both polyclonal and monoclonal antibodies,
and fragments
thereof.
The expressions 'recognition element' and 'specific binding partner' are used
synonymously.
The assays themselves may be either one-step complexing processes, for example
competitive assays, or mufti-step processes) for example sandwich assays.
In the simplest example of a competitive assay, the sample) which comprises
the analyte in
unknown concentration and a known amount of a compound that is identical apart
from being
luminescence-labelled, is brought into contact with the surface of the sensor
platform) where
the luminescence-labelled and unlabelled molecules compete for the binding
sites on their
immobilised recognition elements. In this assay configuration, a maximum
luminescence signal
is obtained when the sample contains no analyte. As the concentration of the
substance to be
detected increases, the observable luminescence signals decrease.
In a competitive immunoassay) the recognition element immobilised on the
surface of the
sensor platform does not have to be the antibody, but may alternatively be the
antigen. It is
generally a matter of choice in chemical or biochemical affinity assays which
of the partners is
immobilised. This is one of the principal advantages of assays based on
luminescence over
methods such as surface plasmon resonance or interferometry, which rely on a
change in the
adsorbed mass in the evanescent field of the waveguiding region.
Furthermore) the competition in the case of competitive assays need not be
limited to binding
sites on the surface of the sensor platform. For example, a known amount of an
antigen can
be immobilised on the surface of the sensor platform and then brought into
contact with the
sample which comprises as anaiyte an unknown amount, which is to be detected,
of the same
CA 02270665 1999-OS-OS
WO 98/21571 PCT/EP97106202
- 26 -
antigen and also luminescence-labelled antibodies. In this case, the
competition to bind the
antibodies takes place between antigens immobilised on the surface and
antigens in solution.
A preferred embodiment is described in application examples B. Septoria
nodorum or tritici
spores are bound by the polyclonal antibodies to Septoria nodorum or Septoria
tritici
immobilised on the sensor plate. Then, the sample is brought into contact with
the surface
which comprises as analyte an unknown amount, to be detected, of the same
antigen of
Septoria nodorum spores or Septoria tritici spores, as well as luminescence-
labelled antibodies
to Septoria nodorum or Septoria tritici. In this case, there is competition
between Septoria
nodorum spores or Septoria tritici spores immobilised on the surface and in
solution for binding
of the Septoria nodorum antibodies or Septoria tritici antibodies.
The simplest example of a mufti-step assay is a sandwich immunoassay in which
a primary
antibody is immobilised on the surface of the sensor platform. The binding of
the antigen to be
detected and of the luminescence-labelled secondary antibody used for the
detection to a
second epitope of the antigen can be effected either by contact with, in
succession, the
solution containing the antigen and a second solution containing the
luminescence-labelled
antibody, or after previously bringing the two solutions together so that
finally the part-complex
consisting of antigen and luminescence-labelled antibody is bound.
An especially preferred embodiment is a multi-step sandwich immunoassay, in
which the
primary antibody and the luminescence-labelled antibody are antibodies which
are directed
against Septoria nodorum antigens or against Septoria tritici antigens, and
the antigen to be
examined is Septoria nodorum antigen or Septoria tritici antigen.
Affinity assays may also comprise further additional binding steps. For
example, in the case of
sandwich immunoassays, in a first step protein A can be immobilised on the
surface of the
sensor platform. The protein specifically binds immunoglobulins to its so-
called F~ portion and
these then serve as primary antibodies in a subsequent sandwich assay which
can be carried
out as described.
There are many other forms of affinity assay, for example using the known
avidin-biotin affinity
system.
CA 02270665 1999-OS-OS
WO 98I21571 PCTIEP97/06202
_ 27 _
Examples of forms of affinity are to be found in J. H. Rittenburg,
Fundamentals of Immuno-
assay; in Development and Application of Immunoassay for Food Analysis, J. H.
Rittenburg
(Ed.), Elsevier, Essex 1990, or in P. Tijssen, Practice and Theory of Enzyme
Immunoassays,
R. H. Burdon, P. H. van Knippenberg (Eds), Elsevier) Amsterdam 1985; US Patent
Number
4,868,105.
A further subject of the invention is a method for the parallel determination
or one or more
luminescences using a sensor platform or modified sensor ptatform for the
diagnosis or plant
diseases) which method comprises bringing one or more Liquid samples into
contact with one
or more waveguidtng regions on the sensor platform) coupling excitation tight
into the
waveguiding regions, causing it to pass through the waveguiding regions, thus
exciting in
parallel in the evanescent field the luminescent substances in the samples or
the luminescent
substances immobilised on the waveguiding regions and, using optoelectronic
components)
measuring the luminescences produced thereby.
The preferences described heretnbefore for the sensor platform and the
modified sensor
platform apply also to the method of diagnosing plant diseases.
Only substantially parallel light is suitable for luminescence excitation.
Substantially parallel is
understood within the context of this invention to mean a divergence of less
than 5°. This
means that the light may be slightly divergent or slightly convergent. The use
of coherent light
for the luminescence excitation is preferred, especially laser light having a
wavelength of 300
to 1100 nm, especially 450 to 850 nm, most particularly 480 to 700 nm.
Examples of lasers that may be used are dye lasers, gas lasers, solid state
lasers and
semiconductor lasers. If necessary, the emission wavelength can also be
doubled by means of
non-linear crystal optics. Using optical elements, the beam can also be
focused further,
polarised or attenuated by means of grey filters. Especially suitable lasers
are argoNion lasers
and heliumlneon lasers which emit at wavelengths of between 457 nm and 5i4 nm
and
between 543 nm and 633 nm respectively. Very especially suitable are diode
lasers or
frequency-doubled diode lasers of semiconductor material that emit at a
fundamental
wavelength of between 630 nm and 1100 nm, since, owing to their small
dimensions and low
power consumption, they allow substantial miniaturisation of the sensor system
as a whole.
CA 02270665 1999-OS-OS
WO 98/21571 PCTIEP97/06202
- 28 -
By "sample" is understood within the context of the present invention the
entire solution to be
analysed, which may contain a substance to be detected - the analyte. The
detection may be
effected in a one-step or multiple-step assay, during the course of which the
surface of the
sensor platform is brought into contact with one or more solutions. At least
one of the solutions
used contains a luminescent substance which can be detected according to the
invention.
If a luminescent substance has already been adsorbed onto the waveguiding
region) the
sample may also be free of luminescent constituents. The sample may contain
further
constituents, such as pH buffers, salts, acids, bases, surfactants, viscosity-
influencing
additives or dyes. In particular) a physiological saline solution can be used
as solvent. If the
luminescent portion is itself liquid, the addition of a solvent can be
omitted. In that case, the
content of luminescent substance in the sample may be up to 100%.
The sample may also be a biological medium, such as solutions of extracts from
natural or
synthetic media, such as soils or parts of plants) liquors from biological
processes or plant
extracts. Soil extracts are especially important for the diagnosis of soil-
borne incidents.
The sample may be used either undiluted or with added solvent.
Suitable solvents are water, aqueous buffer solutions and protein solutions
and organic
solvents. Suitable organic solvents are alcohols, ketones, esters and
aliphatic hydrocarbons.
Preference is given to the use of water, aqueous buffers or a mixture of water
with a miscible
organic solvent.
However, the sample may also comprise constituents that are not soluble in the
solvent, such
as plant cell constituents) pigment particles) dispersants and natural and
synthetic oligomers or
polymers. The sample is then in the form of an optically opaque dispersion or
emulsion.
Functionaiised luminescent dyes having a luminescence of a wavelength in the
range of 330
nm to 1000 nm may be used as luminescent compounds, for example rhodamines,
fluorescein
derivatives, NN382 (CQ5H4gN3013S5Na3), coumarin derivatives, distyryl
biphenyls, stilbene
derivatives, phthaiocyanines, naphthalocyanines) polypyridylJruthenium
complexes such as
tris(2,2'-bipyridyl)ruthenium chloride, tris(1,10-phenanthroline)ruthenium
chloride, tris(4,7-
diphenyl-1,10-phenanthroline)ruthenium chloride and
polypyridyl/phenazinelruthenium
complexes, platinum/porphyrin complexes such as octaethyl-platinum-porphyrin,
long-lived
europium and terbium complexes or cyanine dyes. Dyes having absorption and
emission
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97/06202
- 29 -
wavelengths in the range of about 670 nm are not suitable for analyses in
plant extracts which
contain chlorophyll.
Very especially suitable are dyes, such as fluorescein derivatives , which
contain functional
groups by means of which they can be covalently bonded, for example
fluorescein isothio-
cyanate.
Also very suitable are the functional fluorescent dyes that are commercially
available from the
company LICor, Lincoln) NE, USA, for example NN382 (C45H48N3~13S5Na3), which
are
described for example in K. Behrmann, E. Birckner) E. Fanghaenei, J. Prakt.
Chem. 326, 7 034
(19$4).
The preferred luminescence is fluorescence.
The use of different fluorescent dyes that can all be excited by light of the
same wavelength,
but have different emission wavelengths, may be advantageous, especially when
using
coupling-out gratings.
The luminescent dyes used may also be chemically bonded to polymers or to one
of the
binding partners in biochemical affinity systems, for example antibodies or
antibody fragments,
antigens, proteins, peptides) receptors or their ligands, hormones or hormone
receptors, oligo-
nucleotides, DNA and RNA strands, DNA or RNA analogues, binding proteins, such
as protein
A and G, avidin or biotin) enzymes, enzyme cofactors or inhibitors, lectins or
carbohydrates.
The use of the last-mentioned covalent luminescence labelling is preferred for
reversible or
irreversible (bio)chemical affinity assays. It is also possible to use
luminescence-labelled
steroids) lipids and chelators. In the case especially of hybridisation assays
with DNA strands
or oligonucleotides, intercalating luminescent dyes are also especially
suitable, especially
when - like various ruthenium complexes - they exhibit enhanced luminescence
when
intercalated. When these luminescence-labelled compounds are brought into
contact with their
affinity partners immobilised on the surface of the sensor platform, their
binding can be readily
quantitatively determined using the measured luminescence intensity. Equally)
it is possible to
effect a quantitative determination of the analytes by measuring the change in
luminescence
when the sample interacts with the luminophores, for example in the form of
luminescence
extinction by oxygen or luminescence enhancement resulting from conformation
changes in
proteins.
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97106202
- 30 -
In the method according to the invention, the samples can be both brought into
contact with
the waveguiding regions when stationary, and passed over them continuously, it
being
possible for the circulation to be open or closed.
A further important form of application of the method is based on the one hand
on limiting the
generation of signals - in the case of backcoupling, this applies also to
signal detection - to the
evanescent field of the waveguide, and on the other hand on the reversibility
of the affinity
complex formation as an equilibrium process. Using suitable flow rates in a
throughflow
system, the binding or desorption, i.e. dissociation, of bound, luminescence-
labelled affinity
partners in the evanescent field can be followed in real time. The method is
therefore suitable
for kinetic studies for determining different association or dissociation
constants or for
displacement assays.
The evanescently excited luminescence can be detected by known methods. Those
suitable
are photodiodes, photocells, photomultipliers) CCD cameras and detector
arrays, such as CCD
rows and CCD arrays. The luminescence can be projected onto the latter by
means of optical
elements) such as mirrors, prisms, lenses, Fresnel lenses and graded-index
lenses, it being
possible for the elements to be arranged individually or in the form of
arrays. In order to select
the emission wavelength, known elements, such as filters, prisms,
monochromators, dichroic
mirrors and diffraction gratings can be used.
The use of detector arrays arranged in the immediate vicinity of the sensor
platform is
advantageously, especially when a relatively large number of physically
separate specific
binding partners is present. Optical elements for separating excitation and
luminescence light,
such as holographic or interference filters, are advantageously arranged
between the sensor
platform and the detector array.
One embodiment of the method consists in detecting the isotropicaliy radiated,
evanescently
excited luminescence.
In another embodiment of the method, the evanescently excited luminescence
backcoupled
into the waveguiding region is detected at an edge of the sensor platform or
via a coupling-out
grating. The intensity of the backcoupled luminescence is surprisingly high)
with the result that
very good sensitivity can likewise be achieved using this procedure.
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97/06202
- 31 -
In another form of the method, both the evanescently excited, isotropically
radiated
luminescence and the Luminescence backcoupled into the waveguide are detected
independently of one another but simultaneously. Owing to the different
selectivity of these
two luminescence detection methods, this selectivity being a function of the
distance between
the luminophores and the waveguiding region, this embodiment can be used to
obtain
additional information relating to the physical distribution of the
luminophores. This also makes
it possible to distinguish between photochemical bleaching of the luminophores
and
dissociation of the affinity complexes carrying the luminophores.
Another advantage of the method is that, in addition to the detection of
luminescence, the
absorption of the excitation light radiated in can be determined
simultaneously. Compared with
multimodal waveguides of fibre optic or planar construction, in this case a
substantially better
signal/noise ratio is achieved. Luminescence extinction effects can be
detected with great
sensitivity by means of the simultaneous measurement of luminescence and
absorption.
The method can be carried out by radiating in the excitation light in
continuous wave (cw)
operation, i.e. the excitation is effected with light of an intensity that is
constant over time.
However, the method can also be carried out by radiating in the excitation
tight in the form of a
timed pulse having a pulse length of, for example) from one picosecond to 100
seconds and
detecting the luminescence in a time-resolved manner - in the case of short
pulse lengths - or
at intervals from seconds to minutes. This method is especially advantageous
if for example
the rate of formation of a bond is to be followed analytically or the
reduction in a luminescence
signal resulting from photochemical bleaching is to be prevented using short
exposure times.
Furthermore, the use of suitably short pulse lengths and suitable time
resolution of the
detection make it possible to discriminate between scattered light, Roman
emission and short-
lived luminescence of any undesired luminescent constituents of the sample and
of the sensor
material that may be present, and the luminescence of the labelling molecule,
which ~in this
case is as long-lived as possible, since the emission of the analyte is
detected only once the
short-lived radiation has decayed. In addition, time-resolved luminescence
detection after
pulsed excitation, and likewise, modulated excitation and detection, allows
investigation of the
influence of the binding of the analyte on molecular luminescence decay
behaviour. The
molecular luminescence decay time can be used) alongside specific analyte
recognition by the
immobilised recognition elements and physical limitation of the generation of
signals to the
evanescent field of the waveguide, as a further selectivity criterion.
CA 02270665 1999-OS-OS
WO 98/21571 PCT/EP97/06202
- 32 -
The method can also be carried out by radiating in the excitation fight in an
intensity modulated
manner, at one or more frequencies, and detecting the resulting phase shift
and modulation of
the luminescence of the sample.
Parallel coupling of excitation light into a plurality of waveguiding regions
can be carried out in
several ways:
a) a plurality of laser light sources are used;
b) the beam from a laser light source is broadened using known suitable
optical
components) so that it covers a plurality of waveguiding regions and coupling-
in
gratings;
c) the beam from a laser light source is split using diffractive or
holographically optical
elements into a plurality of individual beams which are then coupled into the
waveguiding regions via the gratings, or
d) an array of solid state lasers is used.
An advantageous procedure is also obtained by using a controllable deflecting
mirror which
can be used for coupling into or out of the waveguiding regions with a time
delay. Alternatively,
the sensor platform can be suitably displaced.
Another preferred method consists in exciting the luminescences with various
laser light
sources of identical or different wavelengths.
Preference is given especially to the use of a single row of diode lasers
(laser array) for the
excitation of the luminescences. These components have the special advantage
that they are
very compact and economical to produce, and the individual laser diodes can be
individually
controlled.
The preferences described for the sensor platform also apply in the case of
the fluorescence
detection method.
Figure fi is a schematic representation of a possible overall construction.
Reference numerals
1 and 3 are as defined hereinbefore and other reference numerals are as
follows:
8 sensor platform
9 filters
seal
t 1 throughflow cell
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97/06202
- 33 -
12 sample space
13 excitation optics
14 detection optics/electronics
The excitation light, for example from a diode laser 13, is coupled via a
first grating 3 into a
waveguiding region 1 of the sensor platform 8. On the underside of the sensor
platform 8 and
pressed tightly against the sensor platform is a throughflow cell 11. The
solutions required for
the assay are flushed through the space 12 in the throughflow cell 11, which
may have one or
more inlet openings and one or more outlet openings. The fluorescence of a
binding partner is
detected at the detector 14 onto which the fluorescence iighi backcoupied
evanescently into
the waveguiding region is coupled out via a second grating 3. The filters 9
serve to filter out
scattered light.
The method is preferably used for analysing samples such as surface water,
soil or plant
extracts, and liquors from biological or synthetic processes.
The present invention also relates to the use of the sensor platform or
modified sensor
platform according to the invention for the quantitative determination of
biochemical
substances in affinity sensing, in the diagnosis of plant diseases.
Since signal generation and detection are limited to the chemical or
biochemical recognition
surface on the waveguide, and disturbance signals from the medium are
discriminated, the
binding of substances to the immobilised recognition elements can be followed
in real time.
The use of the method according to the invention in affinity screening or in
displacement
assays, especially in the diagnosis of plant diseases, by means of the direct
determination of
association and dissociation rates in a throughflow system at suitable flow
rates, is therefore
possible also.
The present invention also includes
a) the use of the sensor platform according to the invention or modified
sensor platform
according to the invention in processes for the diagnosis of plant diseases.
b) the use of the sensor platform according to the invention or modified
sensor platform
according to the invention in analytical processes for the diagnosis of plant
diseases,
preferably for the qualitative or quantitative determination of biochemical
substances in affinity
sensing.
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97/06202
- 34 -
c) the use of the sensor platform according to the invention or modified
sensor platform
according to the invention in an assay.
The assays in question may be assays with a one-step complexing process or a
mufti-step
process.
Preference is given to the use of the sensor platform according to the
invention or modified
sensor platform according to the invention in sandwich assays) most preferably
sandwich
immuno-assays.
Particularly preferred is the use of the sensor platform according to the
invention or modified
sensor platform according to the invention in an assay in which a primary
antibody is
immobilised on the surface of the sensor platform, and binding of the antigen
to be detected
and of the luminescence-labelled secondary antibody used for the detection to
a second
epitope of the antigen can be effected by contact with, in succession, the
solution containing
the antigen and a second solution containing the luminescence-labelled
antibody.
Preference is given to the use of the sensor platform according to the
invention or modified
sensor platform according to the invention in a sandwich immuno-assay in which
a primary
antibody is immobilised on the surface of the sensor platform, and binding of
the antigen to be
detected and of the luminescence-labelled secondary antibody used for the
detection to a
second epitope of the antigen is effected by previously bringing the two
solutions together so
that finally the part-complex consisting of antigen and luminescence-labelled
antibody is
bound.
Preference is given to the use of the sensor platform according to the
invention or modified
sensor platform according to the invention in a competitive assay.
Particular preference is given to the use of the sensor platform according to
the invention or
modified sensor platform according to the invention in a competitive immuno-
assay.
Particular preference is given to the use of the sensor platform according to
the invention or
modified sensor platform according to the invention in a competitive assay in
which
competition is restricted to the binding sites on the surface of the sensor
platform.
Preference is given to the use of the sensor platform or modified sensor
platform in a
competitive assay in which competition takes place between antigens that are
immobilised on
the surface of the sensor platform and those in solution for binding of the
antibodies in
solution.
Particularly preferred is the use of the sensor platform according to the
invention or modified
sensor platform according to the invention in a competitive assay in which a
known amount of
an antigen is immobilised on the surface of the sensor platform and then
brought into contact
CA 02270665 1999-OS-OS
WO 98/21571 PCTIEP97/06202
- 35 -
with the sample which comprises as analyte an unknown amount, which is to be
detected, of
the same antigen and also luminescence-labelled antibodies.
Preference is given to the use of the sensor platform according to the
invention or modified
sensor platform according to the invention in an assay in which Septoria
nodorum or Septoria
tritici antigens are bound by the antibodies to Septoria nodorum or the
antibodies to Septoria
tritici, which are immobilised on the sensor plate, and subsequently the
sample is brought into
contact with the surface which comprises as analyte an unknown amount to be
detected of the
same antigen of Septoria nodorum spores or Septoria tritici) as well as
luminescence-labelled
antibodies to Septoria nodorum or Septoria tritici.
d) the use of the sensor platform according to the invention or modified
sensor platform
according to the invention for the quantitative determination of antibodies or
antigens, proteins,
receptors or ligands) chelators or "histidine-tag components",
oligonucleotides, DNA or RNA
strands) circular RNA, DNA or RNA analogues, enzymes, enzyme substrates,
enzyme
cofactors or inhibitors, lectins and carbohydrates, most preferably for the
quantitative
determination of antibodies or antigens.
e) the use of the sensor platform according to the invention or modified
sensor platform
according to the invention for the selective quantitative determination of
luminescent
components in optically opaque liquids) the optically opaque liquids being
biological liquids
such as samples from environmental analysis, for example surface water,
dissolved earth
extracts or dissolved plant extracts.
f} the use of the sensor platform according to the invention or modified
sensor platform
according to the invention for the detection of plant pathogens, whereby the
above-mentioned
definitions and preferences apply to plant pathogens.
g) the use of the sensor platform according to the invention or modified
sensor platform
according to the invention for the detection of indicator substances which are
characteristic of
certain plant pathogens, whereby the above-mentioned definitions and
preferences apply to
indicator substances.
h) the use of the biosensor according to the invention in processes for
diagnosing plant
diseases.
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97/06202
- 36 -
I) the use of the biosensor according to the invention in analytical processes
for
diagnosing plant diseases, preferably for the qualitative or quantitative
determination of
biochemical substances in affinity sensing.
j) the use of the biosensor according to the invention in an assay.
The assays in question may be assays with a one-step complexing process or a
mufti-step
process.
Preference is given t4 the use of the biosensor according to the invention in
sandwich assays,
most preferably sandwich immuno-assays.
Particularly preferred is the use of the biosensor according to the invention
in an assay in
which a primary antibody is immobilised on the surface of the sensor platform,
and binding of
the antigen to be detected and of the luminescence-labelled secondary antibody
used for the
detection to a second epitope of the antigen can be effected by contact with,
in succession,
the solution containing the antigen and a second solution containing the
luminescence-labelled
antibody.
Preference is given to the use of the biosensor according to the invention in
a sandwich
immuno-assay in which a primary antibody is immobilised on the surface of the
sensor
platform, and binding of the antigen to be detected and of the luminescence-
labelled
secondary antibody used for the detection to a second epitope of the antigen
is effected by
previously bringing the two solutions together so that finally the part-
complex consisting of
antigen and luminescence-labelled antibody is bound.
Preference is given to the use of the biosensor according to the invention in
a competitive
assay.
Particular preference is given to the use of the biosensor according to the
invention in a
competitive immuno-assay.
Particular preference is given to the use of the biosensor according to the
invention in a
competitive assay in which competition is restricted to the binding sites on
the surface of the
sensor platform.
Preference is given to the use of the biosensor according to the invention in
a competitive
assay in which competition takes place between antigens that are immobilised
on the surface
of the sensor platform and those in solution for binding of the antibodies in
solution.
Particularly preferred is the use of the biosensor according to the invention
in a competitive
assay in which a known amount of an antigen is immobilised on the surface of
the sensor
platform and then brought into contact with the sample which comprises as
anaiyte an
unknown amount, which is to be detected, of the same antigen and also
luminescence-labelled
antibodies.
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97106202
- 37 -
Preference is given to the use of the biosensor according to the invention in
an assay in which
Septoria nodorum or Septoria tritici antigens are bound by the antibodies to
Septoria nodorum
or the antibodies to Septoria tritici, which are immobilised on the sensor
plate) and
subsequently the sample is brought into contact with the surface which
comprises as analyte
an unknown amount, to be detected, of the same antigen of Septoria nodorum
spores or
Septoria tritici antigens, as well as luminescence-labelled antibodies to
Septoria nodorum or
Septoria tritici.
k) the use of the biosensor according to the invention for the quantitative
determination of
antibodies or antigens, proteins) receptors or ligands, chelators or
"histidine-tag components",
oligonucieotides, DNA or RNA strands, circular RNA, DNA or RNA analogues,
enzymes,
enzyme substrates, enzyme cofactors or inhibitors) lectins and carbohydrates,
most preferably
for the quantitative determination of antibodies or antigens.
() the use of the biosensor according to the invention for the selective
quantitative
determination of luminescent components in optically opaque liquids, the
optically opaque
liquid being biological liquids such as samples from environmental analysis,
for example
surface water, dissolved earth extracts or dissolved plant extracts.
m) the use of the biosensor according to the invention for the detection of
plant
pathogens, whereby the above-mentioned definitions and preferences apply to
plant
pathogens.
n) the use of the biosensor according to the invention for the detection of
indicator
substances which are characteristic of certain plant pathogens, whereby the
above-mentioned
definitions and preferences apply to indicator substances.
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97/06202 '
- 38 -
The following examples illustrate the invention.
In all the following examples, the unit M of concentration denotes moll, RT is
room
temperature, PAb-Septoria denotes polyctonal antibodies to Septoria.
Examples A: Production of various sensor platforms
Example A1. Production using masks in vapour deposition.
A polycarbonate (PC) substrate is coated with Ti02 by means of vapour
deposition (process:
sputtering, deposition rate: 0.5 Als, thickness: 150 nm). Between the target
and the substrate)
in the immediate vicinity of the substrate, a mask is introduced. This is
produced from
aluminium, in which 6 strips 30 mm in length and 0.6 mm in width have been
cut. The resulting
6 waveguiding regions (measuring regions) have a trapezoidal profile with a
uniform thickness
of 150 nm in the central region, which is 600 pm in width, and a layer
thickness that decreases
at the sides in the form of a gradient (shadowing). Coupled-in laser light is
confined in the
waveguiding region, since the effective refractive index is highest in the
central region owing to
the greatest layer thickness in that region.
Example 2. Production by subsequent division
The operation is carried out using an ArF excimer laser at 193 nm. The
rectangular laser beam
is concentrated using a cylindrical lens to a beam profile 200 p.m wide and 20
mm long
focused on the sensor platform. The sensor platform has a continuous 100 nm
thick layer of
TazOs. At an energy density above 1 J/cm2 the entire layer is ablated with ~a
single laser pulse
( 10 ns).
Example A3. Production by subsequent division
The operation is carried out using an Ar-ion laser at 488 nm. The round laser
beam is
concentrated using a microscope lens (40x) to a diameter of 4 um focused on
the waveguiding
layer. The sensor platform has a continuous 100 nm thick layer of Ta20s and is
located on a
motor-controlled positioning element (Newport PM500). Under continuous laser
irradiation, the
platform is driven perpendicular to the beam at 100 mm/s. At an output of 700
mW, the entire
waveguiding layer is ablated at the focus, with the result that two separate
waveguiding
regions are formed.
Example A4. Production by the application of a structured absorbing cover
layer by the
vacuum method
CA 02270665 1999-OS-OS
WO 98I21571 PCTIEP97/06202
- 39 -
parallel strips of a layer system of chromium/gold are vapour-deposited on the
(continuous)
metal oxide waveguide consisting of Ta20s (vapour-deposition installation:
Balzers BAK 400);
first 5 nm of Cr at 0.2 nm/s) then 45 nm of Au at 0.5 nmls. The coupled-in
models were
interrupted at the absorbing layers.
Example A5. Production by the application of a structured absorbing cover
layer by the
aqueous method
The surface of a metal oxide waveguide consisting of Ta20s is silanised with
(mercapto-
methyl)dimethylethoxysilane in the gas phase at 180°C. With the aid of
a fine pipette) colloid
solution A (GoldSol supplied by Aurion, average colloid diameter = 28.9 nm,
concentration:
As~o = 1, aqueous solution) is applied to the modified surface in the form of
droplets or strips
and incubated for 1 hour. After the incubation, the surface is washed with
water. Guided modal
light is absorbed at the incubated sites. Downstream of the incubated sites,
modal light is no
longer present. The same applies in the case of protein A-covered Au colloid
solution B
(P-9785 supplied by Sigma) average diameter = 18.4 nm, Aszo = 5.5, in 50%
glycerol, 0.15 M
NaCI, 10 mM sodium phosphate, pH 7.4, 0.02% PEG 20, 0.02% sodium azide). The
absorbing
patterns on the waveguide surface are still intact even after flushing several
times with water
and with ethanol, which demonstrates the stability of the structures produced.
By the manual application of rows of microdrops (1 pl) of colloid solution A,
continuous light-
absorbing strips can be produced.
Example A6. Production by the application of a structured absorbing cover
layer by the
aqueous method
The surface of a metal oxide waveguide consisting of Ti02 is silanised with
(mercaptomethyl)-
dimethylethoxysilane in the gas phase at 40°C. Then a portion of the
waveguide surface in
front of and including the second coupling-out grating is incubated far 3
hours with colloid
solution B (P-9785 supplied by Sigma, average diameter = 18.4 nm, As2o = 5.5)
in 50%
glycerol) 0.15 M NaCI, 10 mM sodium phosphate, pH 7.4, 0.02% PEG 20, 0.02%
sodium
azide). The wave propagation at the incubated sites is interrupted completely.
The surface of
the incubated site is examined using an atomic force microscope and the
presence of colloids
and the density of the gold particles anchored to the surface) that is
necessary for the
observed light absorption, are determined. The average separation of the
particles is in the
region of approx. 100 nm.
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97/06202
- 40 -
Example A7. Production by fhe application of a structured absorbing cover
layer by the
aqueous method
The surface of a metal oxide waveguide consisting of Ta20s is silanised with
(mercapto-
methyl)dimethylethoxysiiane (in the gas phase at 180°C}. The waveguide
chip is connected to
a throughflow cell having parallel, fluidically separate laminar part streams
which allow up to
five different streams of fluid to be passed in parallel adjacent to one
another over the length
of the waveguide surface via separate) individually addressable flow openings
(1-5). The
intention. is to produce three waveguiding strips separated by two thinner
strips of deposited
Au colloids. The throughfiow cell is charged at inlets 1, 3 and 5 with buffer
(phosphate-
buffered sodium chloride solution, pH 7.0) and at inlets 2 and 4 with Au
colloid solution. A
colloid solution, the surface of which is blocked with bovine serum albumin
(BSA Gold Tracer
supplied by Aurion, average colloid diameter = 25 nm, ODsao = 2.0), is used.
The flow rates
selected (per channel) are: 0.167 ml/min for the buffer streams 1, 3 and 5,
and 0.05 ml/min for
the two colloid streams 2 and 4. This results in a width of approx. 1 mm for
the colloid stream
and approx. 3 mm for the buffer stream. The ratio of colloid stream width to
buffer stream
width can generally be freely selected via the ratio of the streams. The
streams are applied for
20 mins. (corresponding to an amount of colloid of 1 ml per channel). After 20
minutes
incubation, the waveguide chip is removed, washed with water and dried with a
stream of
nitrogen. Guided modal light is completely absorbed by the colloid-immobilised
strips and
results in three separate light-guiding modes of approx. 3 mm in width.
Application Examples B
Example B1: Detection of a wheat funous antioen (Sepforia nodorum or Seotoria
triticrl usin4
a sensor alatform havin4 a sinote waveouiding re4ion coverino the whole
platform
B1.1 Optical svstem
The fight source used is a laser diode at ~, = 785 nm (Oz-Optics). With the
assistance of an
imaging system, it is adjusted to a beam spot with a diameter in the sensor
plane of 0.4 mm
vertical to the lines of !he coupling grating and 2.5 mm parallel to the
grating tines.
Adjustment of the coupling-in angle and positioning of the beam spot in
respect of the grating
edge is carried out by means of mechanical adjustment uni#s.
The laser power on the sensor platform can be selected within the range P = 0
... 3 mW. For
the experiments described in the following to characterise the grating, P =
1.2 mW was used,
CA 02270665 1999-OS-OS
WO 98I21571 PCTlEP97106202
- 41 -
for the fluorescence measurements p = 0.3 mW. By having rotatable polarising
elements,
linearly polarised light with TE- or TM-orientation can be coupled in as
desired.
A throughflow cell is arranged on the upper side of the sensor platform. It is
sealed against the
sensor with O-rings, and the sample space of this cell is ca. 8 p.l. Various
solutions can be
introduced into the cell using injection pumps and switch valves.
Excitation and detection are effected from the underside of the sensor
platform.
Several measuring channels are available for detection. For the embodiment of
an assay
described in the following, the fluorescence which is excited in the
evanescent field, but is
reflected isotropically into the half space on the other side underneath the
sensor platform, is
recorded. This takes place in a measuring system as described in WO 95/33197.
To avoid spectral cross-talking in the detection of excitation light and
emission light,
interference filters are used in the excitation and emission light paths, in
the emission path with
a band pass 780 nm (30 nm band width, Omega Optical), in the emission path
with a band
pass 830 nm (40 nm band pass, Omega Optical). The fluorescence signals are
recorded by a
Single Photon Counting unit (Hamamatsu H8240-02-B1, with Photomultiplier
R2949). The
outgoing signals thereof can be transmitted to a conventional impulse counter
(Hewlett
Packard 53131A). Si-diodes (UDT PIN 10 D) with a measuring amplifier (UDU 101
C)
connected in series may be used as reference detectors.
B 1.2 Sensor platform
The substrate used is polycarbonate, which is micro-structured in the
following way with two
gratings for coupling-in and coupling-out:
Coupling-in grating with period A~ _ (3T0 t 2 nm), depth t~ = 12.5 nm to 17.5
nm,
Coupling-out grating with period AZ = (580 t 3 nm), depth t2 - 12.5 nm to 17.5
nm,
both gratings with approximately sin4 -shaped profile.
The gratings on the sensor platform are arranged with the following geometric
sizes:
grating distance A = 4 mm, grating width (vertically to lines) B, = B2 = 2 mm,
grating height
(parallel to lines) 4 mm, for dimensions of the sensor platform of 12 x 20
mm2.
CA 02270665 1999-05-05
WO 98I21571 PCT/EP97l06202 '
- 42 -
In order to suppress the polycarbonate intrinsic fluorescence) an intermediate
layer of Si02
with a refractive number n = 1.46 and a thickness of tb~,e, _ ( 100 ~ 10) nm
is applied to this
substrate) and afterwards the high-refractive waveguiding layer of Ti02 with
the refractive
number nf;;m = 2.32 at ~, = 780 nm and the layer thickness them = (i 80 t 5)
nm.
By means of the excitation light, for this grating-waveguide combination, the
modes in the
order of m = 0 can be excited in the waveguide: for TEo coupling-in is
effected at an angle of
o = (-6.3 t 1.1 )°, alternatively for TMo at an angle of o = (-20.4 t
3.3)°.
The fluorescence beam is coupled out with TE-polarisation at an angle range of
o = 29° ... 38°
the excitation light at angles o > 39°. A spectral range of ~, ca. 800
nm ... 830 nm
corresponds to this angle range of fluorescence beam. For TM-polarisation, the
coupling-out
angle is o = 15° ... 23° for the fluorescence and o > 24°
for the excitation beam.
B 1.3. Solutions emaloved
1 ) Buffer A:
8.8 g NaCI, 330m1 phosphate buffer pH7, 50m1 methanol, 0.2 g sodium azide, 1 g
BSA, 5 g
Tween 20 ~ ad 1 I H20.
2) Buffer B:
8.8 g NaCI, 330m1 phosphate buffer pH7, ~Oml methanol) 0.2 g sodium azide ~ ad
11 H20
3) Regeneration buffer:
4i 6.3 ml solution A, 463.7 ml HCI 0.1 M, 120m1 isopropanol ~ pH 1.9
4) Solution A: glycine 0.1 M, NaCI 0.1 M
5) Standards (Septoria nodorum or Septorla tritic~):
S 1 10 million sporeslmi extract from wheat leaves
S2 3 million sporeslml extract from wheat leaves
S3 1 million spores/ml extract from wheat leaves
S4 0.3 million sporeslml extract from wheat leaves
S5 0.1 million spores/ml extract from wheat leaves
CA 02270665 1999-OS-OS
WO 98!2157I PCTlEP97106202
- 43 -
S6 0.03 million spores/ml extract from wheat leaves
6) Buffer C:
40mM Tris, 30mM HCI, 150mM NaCI, 0.1 % BSA, 0.02% sodium azide ~ ad 1 I H20.
set at
pH 7.7
B 1.4 Preparation of the sensor platform
The sensor platforms are silanised with (mercaptomethyl}dimethylethoxysilane
(ABCR GmbH
& Co., Karisruhe) in gas phase (6 hours, 40°C, 0.2 mbar}. After
silanisation, the sensor
platforms are incubated for 2 hours at room temperature with PAb-Septoria
nodorum or PAb-
Septoria tritici (0.3 mg/ml buffer B), washed with H20 and then incubated for
1 hour at room
temperature with Septoria nodorum spores or Sepforia tritici spores (10
million sporeslml buffer
B). The sensor platform is again washed with H20, blown dry with nitrogen and
stored at -80°C
until measured.
Prior to the first measurement, the sensor platforms are incubated (20 miss;
0.5 mllmin) with
buffer A in a throughflow cell in order to neutralise any free adsorption
sites that may possibly
be present on the surface.
B 1.5 Tracer synthesis
300N1 of NN382 (C45H48N3013S5Na3, LiCor, Lincoln, lVE, USA, 1 mglml H20) are
added to
700u1 of PAb-Septoria (0.86mgJml) in C032'IHC03' buffer (pH 9.2). The reaction
mixture is
agitated for 2 hours at room temperature. Afterwards, the mixture is added to
a PD-10 column
(Pharmacia Biotech, Uppsala, Sweden), which was previously equilibrated with
buffer B. The
labelled antibody is eluted with the same buffer. By means of UV/VIS
spectrometry, the
concentration of the NN382-PAb-Septoria is set at 1x10'6 M, the solution is
aliquoted.and
stored at -80°C until measuring. The measuring concentration is
respectively 2.5x10-8 M
NN382-PAb-Septoria in buffer A.
B 1.6 Preparation of extract from wheat leaves
The plant material is placed in a plastic bag and weighed. Then buffer C is
added (1 ml per g
plant material). The plant material in the plastic bag is then extracted using
a macerator
(Homex 6, Bioreba, Reinach).
B 1.7 Measuring method
CA 02270665 1999-OS-OS
WO 98I21571 PCTIEP97/06202
- 44 -
The measuring method consists of the following individual steps
- 5 minutes flushing with buffer A (0.5 mllmin.); recording of background
signal
- 5 minutes supplying the sample (10 ui standard in 1.8 m! tracer, 0.25
mllmin)
- 2 minutes flushing with buffer A (0.5 ml/rnin.)
- 2 minutes supplying regeneration solution (0.5 ml/min.}
- 1 minute flushing with buffer A (0.5 rnl/min.)
The specific signal is calculated from the difference in signal levels at t =
12 mins and t =
mins.
B 1.8 Results
The present assay is a competitive process, forming a sandwich complex
consisting of the
immobilised complex of PAb-Septoria nodorum or tritici and Septoria nodorum or
iritici antigen)
as well as the NN382-PAb-Septoria bound from the sample. Here, competition for
the NN382-
PAb-Septoria tracer takes place between the immobilised antigen and that found
in the
sample. A maximum fluorescence signal is produced at the lowest number of
spores in the
sample (S6}.
specific signal with background signal signal noises
S6
40000 impulses per 2000 impulses per 100 impulses per second
second second
Example B2: Parallel detection of two wheat funous antioens (Seotoria nodorum
and Septoria
trfticrl with different reco4nition elements immobilised on 2 physically
separate wave uiding
regions
B 2.1 Optical sensor platform with two wave uq idin4 re4ions, obtained
according to
example A6
The sensor platform (metal oxide waveguide comprising Ti02 with a surface of
12 mm x 20 mm
with identical parameters to those of example B 1.2) is silanised in gas phase
with (mercapto-
methyl)dimethylethoxysilane (ABCR GmbH & Co., Karlsruhe} (6 hours,
40°C, 0.2 mbar). Using
an added fluid cell, in order to apply an absorbing cover layer in the region
in which the
waveguide is to be interrupted, the surface of the sensor platform is brought
into contact with
colloid solution B (P-9785 from Sigma, average diameter = 18.4 nm, A520 ~ 5.5,
in 50%
glycerol) 0.15 M NaCI) 10 mM sodium phosphate, pH 7.4, 0.02% PEG 20) 0.02%
sodium
CA 02270665 1999-OS-OS
WO 98/21571 PCT/EP97106202
- 45 -
azide). The area of contact with the colloid solution comprises a rectangle of
the dimensions
0.5 mm x 10 mm, which extends beyond the coupling-in and coupling-out grating,
and is
localised in a central position of the grating height, so that 1.75 mm of the
grating remains
unchanged at the top and bottom. In the incubated region, the waveguide is
completely
interrupted.
B 2.2 immobilisation process
After flushing with water, the structured sensor platform is brought into
contact with a fluid cell,
which includes 2 separate flow channels at a distance of 0.5 mm. The
dimensions of the two
flow channels, with a depth of 0.2 mm, are respectively 1.5 mm height
(parallel to the grating
lines of the sensor platform) and 3.5 mm width {vertical to the grating lines
of the sensor
platform). The flow channels are arranged in relation to the sensor platform
in such a way that
the region of interruption of the waveguide lies between the two channels and
at least the
coupling-out grating of the sensor platform lies outside of the flow channel.
!n the region of
channel 1) the sensor platform is incubated for 2 hours at room temperature
with PAb-Septoria
nodorum, and in the region of channel 2 with PAb-Septoria tritici (each with
0.3 mglml in
buffer B). Afterwards, the two channels are washed with H20 and subsequently
incubated for
1 hour at room temperature - channel 1 with Sepforia nodorum spores and
channel 2 with
Septoria tritici spores (respectively 10 million spores/ml buffer B). The
sensor platform is
subsequently washed with H20, dried by blowing nitrogen through and stored at -
80°C until
measuring.
B 2.3 Optical Structure
The light source used is a laser diode at ~, = 785 nm {Oz-Optics). With the
assistance of an
imaging system, it is adjusted to a beam spot with a diameter in the sensor
plane of 0.4 mm
vertical to the lines of the coupling grating and 4 mm parallel to the grating
lines, so that the
whole height of the grating is illuminated.
Adjustment of the coupling-in angle and positioning of the beam spot in
respect of the grating
edge is carried out by means of mechanical adjustment units.
The laser power on the sensor platform can be selected within the range P = 0
... 3 mW. By
having rotatable polarising elements, linearly polarised light with TE- or TM-
orientation can be
coupled in as desired.
CA 02270665 1999-OS-OS
WO 98/2i571 PCTIEP97/06202
- 46 -
A throughflow cell with 2 channels is arranged on the upper side of the sensor
platform in such
a way that the separate channels respectively enclose the similarly separate
waveguiding
regions on which the different recognition elements have been immobilised. The
sample
volume of each channel is ca. 3 p.l. Various solutions can be introduced into
the cell using
injection pumps and switch valves.
Excitation and detection are effected from below the sensor platform.
Several measuring channels are available for detection. For the example which
is described
here, the fluorescence which is excited in the evanescent field in the
separate waveguiding
regions, but is reflected isotropically into the half space on the other side
below the sensor
platform, is recorded. This takes place in a variation of the measuring system
as described in
WO 95I33197 for the simultaneous recording of signals from 2 adjacent
waveguiding regions.
To this end, the fluorescence light from the two separate sensor regions,
which is reflected into
the half space below the sensor platform, is collected by a glass fibre
optics. The inlet cross-
section of the glass fibre optics are designed so that cross-talking of the
signals from the two
sensor regions is avoided and at the same time maximum fluorescence is
recorded. If
required) the coupling-in efficiency into the glass fibres may be further
increased through a
combination with appropriate lenses.
The optical structure otherwise corresponds to the system described in example
B 1.1, but
now designed for 2 separate optical channels to detect fluorescence.
To avoid spectral cross-talking in the detection of excitation light and
emission light,
interference filters are used in the excitation and emission light paths) in
the emission path with
a band pass 780 nm (30 nm band width, Omega Optical), in the emission path
with a band
pass 830 nm (40 nm band pass, Omega Optical). The fluorescence signals from
the two
sensor regions are respectively recorded by a Single Photon Counting unit
(Hamamatsu
H6240-02-B1, with Photomultiplier R2949). The outgoing signals thereof can be
transmitted to
a conventional impulse counter (Hewlett Packard 53131A). Si-diodes (UDT PIN 10
D) with a
measuring amplifier (UDU 101 C) connected in series may be used as reference
detectors.
B 2.4 Solutions emofoyed:
1 ) Buffer A:
8.8 g NaCI, 330m1 phosphate buffer pH 7, 50m1 methanol, 0.2 g sodium azide, 1
g BSA, 5 g
Tween 20 ad 1 I H20.
CA 02270665 1999-OS-OS
WO 98I21571 PCT/EP97/06202
- 47 -
2) Buffer B:
8.B g NaCI, 330m1 phosphate buffer pH 7, 50m1 methanol, 0.2 g sodium azide ~
ad 1 l H20
3) Regeneration buffer:
416.3 ml solution A, 463.7 ml HCI 0.1 M, 120m1 isopropanol ~ pH 1.9
4) Solution A: glycine 0.1 M, NaCI 0.1 M
5) Standards (Septoria nodorum or Septoria tritic~):
S1 10 million sporeslml extract from wheat leaves
S2 3 million sporeslml extract from wheat leaves
S3 1 million spores/ml extract from wheat leaves
S4 0.3 million sporesiml extract from wheat leaves
S5 0.1 million spores/ml extract from wheat leaves
S6 0.03 million sporesiml extract from wheat leaves
6) Buffer C:
40mM Tris, 30mM HCI, 150mM NaCI, 0.1 % BSA, 0.02% sodium azide ~ ad 1 I H20,
set at
pH 7.7
B 2.5 Tracer s~mthesis
300N1 of NN382 (C45H48N3013S5Na3, LiCor, Lincoln, NE, USA, 1 mglml H20) are
added to
700N1 of PAb-Septoria (0.86mg/ml} in C032-IHC03- buffer (pH 9.2}. The reaction
mixture is
agitated for 2 hours at room temperature. Afterwards, the mixture is added to
a PD-~10 column
(Pharmacia Biotech, Uppsala, Sweden), which was previously equilibrated with
buffer B. The
labelled antibody is eluted with the same buffer. By means of UV/VIS
spectrometry, the
concentration of the NN382-PAb-Septoria is set at 1 x10'6 M, the solution is
aliquoted and
stored at -80°C until measuring. The measuring concentration is
respectively 2.5x10'9 M
NN382-PAb-Septoria in buffer A.
B 2.6 Preparation of extract from wheat leaves
CA 02270665 1999-OS-OS
WO 98I21571 PCTIEP97106202 '
- 48 -
The plant material is placed in a plastic bag and weighed. Then buffer C is
added ( 1 ml per g
plant material). The plant material in the plastic bag is then extracted using
a macerator
{Homex 6, Bioreba, Reinach).
B 2.7 Measuring method
Prior to the first measurement, the two separate regions of the sensor
platform are incubated
{20 mins; 0.5 mllmin) with buffer A in a throughflow cell in order to
neutralise any free
adsorption sites that may possibly be present on the surface.
The measuring method with the simultaneous supply of two different analytes to
the two
physically separate sensor regions by means of the sample cell consisting of 2
flow channels
comprises the following individual steps:
- 5 minutes flushing with buffer A (0.5 mllmin.) through both channels and
recording of the
background signal
- 5 minutes supplying the sample: - 10 ~I Septoria nodorum standard in 1.8 ml
NN382-PAb-
Septoria rrodorum (2.5 x 10-9 M, 0.25 mllmin) through
channel i
- 10 ul Septoria tritici standard in 1.8 ml NN382-PAb-
Septoria tritici (2.5 x 10'8 M, 0.25 mUmin) through
channel 2
- 2 minutes flushing with buffer A (0.5 mllmin.) through both channels and
recording of the
fluorescence signal
- 2 minutes supplying regeneration solution (0.5 ml/min.) through both
channels
- 1 minute flushing with buffer A (0.5 mllmin.) through both channels
The specific signal is calculated from the difference in signal levels at t =
12 mins and t =
mins.
Example B3: Alternative detection of two wheat fun4us antioens (Sentoria
nodorum and
Sentoria triticr) with different recognition elements immobilised on 2
ohvsically separate regions
B 3.i immobilisation process
After flushing with water, the sensor platform is brought into contact with a
fluid cell, which
includes 2 separate flow channels at a distance of 0.5 mm. The dimensions of
the two flow
CA 02270665 1999-OS-OS
WO 98I21571 PCTIEP97/06202
- 49 -
channels, with a depth of 0.2 mm, are respectively 1.5 mm height (parallel to
the grating lines
of the sensor platform) and 3.5 mm width (vertical to the grating lines of the
sensor platform).
The flow channels are arranged in relation to the sensor platform in such a
way that the flow
channels are symmetrical to the coupling gratings of the sensor platform and
at least the
coupling-out grating of the sensor platform lies outside of the flow channels.
In the region of
channel 1 ( the sensor platform is incubated for 2 hours at room temperature
with PAb-Sepforia
nodorum, and in the region of channel 2 with PAb-Septoria bitici (each with
0.3 mg/ml in
buffer B). Afterwards, the iwo channels are washed with N20 and subsequently
incubated for
1 hour at room temperature - channel 7 with Septoria nodorum spores and
channel 2 with
Septoria tritici spores (respectively 10 million spores/ml buffer B). The
sensor platform is
subsequently washed with H20, dried by blowing nitrogen through and stored at -
80°C until
measuring.
B 3.2 Optical structure
The light source used is a laser diode at ~. = 785 nm (Oz-Optics), With the
assistance of an
imaging system, it is adjusted io a beam spot with a diameter in the sensor
plane of 0.4 mm
vertical to the lines of the coupling grating and 1.5 mm parallel to the
grating lines, so that the
height of the two flow channels of the sample cell pressed onto the sensor
platform can each
be completely illuminated.
Adjustment of the coupling-in angle and .positioning of the beam spot in
respect of the grating
edge is carried out by means of mechanical adjustment units.
The laser power on the sensor platform can be selected within the range P = 0
... 3 mW. By
having rotatable polarising elements, linearly polarised light with TE- or TM-
orientation can be
coupled in as desired.
A throughflow cell with 2 channels is arranged on the upper side of the sensor
platform in such
a way that the separate channels respectively enclose the similarly separate
regions on which
the different recognition elements have been immobilised. The sample volume of
each channel
is ca. 3 pl. Various solutions can be introduced into the cell using injection
pumps and switch
valves.
Excitation and detection are effected from below the sensor platform.
CA 02270665 1999-OS-OS
WO 98I21571 PCTIEP97l06202
- 50 -
Several measuring channels are available for detection. For the example which
is described
here) the fluorescence which is excited in the evanescent field in the
separate waveguiding
regions, but is reflected isotropically into the half space on the other side
below the sensor
platform, is recorded. This takes place in a measuring system as described in
example B 1.1.
By having an additionally mounted) computer-controlled translation unit with
translation parallel
to the grating lines, the point at which the excitation light meets the
coupling-in grating and this
excites fluorescence in the separate sensor regions can be varied. In this
way, it is possible to
excite and detect alternating fluorescence signals (at time intervals of ca. 8
seconds) from the
separate sensor regions.
B 3.3 Solutions employed:
1 ) Buffer A:
8.8 g NaCI, 330m1 phosphate buffer pH 7, 50m1 methanol, 0.2 g sodium azide, 1
g BSA, 5 g
Tween 20 ad 1 I H20.
2) Buffer B:
8.8 g NaCI, 330m1 phosphate buffer pH 7, 50m1 methanol, 0.2 g sodium azide ~
ad 1 I H20
3) Regeneration buffer:
416.3 ml solution A, 463.7 ml HCI 0.1 M, 120 ml isopropanol ~ pH 1.9
4) Solution A: giycine 0.1 M, NaCI 0.1 M
5) Standards (Sepforia nodorum or Sepforia friticr):
S1 10 million sporeslml extract from wheat leaves
S2 3 million spores/ml extract from wheat leaves
S3 1 million sporeslmi extract from wheat leaves
S4 0.3 million spores/ml extract from wheat leaves
S5 0.1 million spores/ml extract from wheat leaves
S6 0.03 million sporeslml extract from wheat leaves
6} Buffer C:
CA 02270665 1999-OS-OS
WO 98/21571 PCT/EP97/06202 _
- 51 -
40mM Tris, 30mM HCI) 150mM NaCI, 0.1 % BSA, 0.02% sodium azide ~ ad 1 I H20,
set of
pH 7.7
B 3.4 Tracer synthesis
300N1 of NN382 (C45H48N3013S5Na3, LiCor, Lincoln, NE, USA, 1 mgiml H20) are
added to
700p1 of PAb-Septoria (0.86mglml) in C032-IHC03- buffer (pH 9.2). The reaction
mixture is
agitated for 2 hours at room temperature. Afterwards) the mixture is added to
a PD-10 column
{Pharmacia Biotech, Uppsala, Sweden), which was previously equilibrated with
buffer B. The
labelled antibody is eluted with the same buffer. By means of UV/VIS
spectrometry, the
concentration of the NN382-PAb-Septoria is set at 1 x10'6 M, the solution is
aliquoted and
stored at -80°C until measuring. The measuring concentration is
respectively 2.5x10-9 M
NN382-PAb-Septoria in buffer A.
B 3.5 Preparation of extract from wheat leaves
The plant material is placed in a plastic bag and weighed. Then buffer C is
added {1 ml per g
plant material). The plant material in the plastic bag is then extracted using
a macerator
(Homex 6, Bioreba, Reinach).
B 3.6 Measuring method
Prior to the first measurement, the two separate regions of the sensor
platform are incubated
(20 mins; 0.5 ml/min) with buffer A in a throughflow cell in order to
neutralise any free
adsorption sites that may possibly be present on the surface.
The measuring method with the simultaneous supply of two different analytes to
the two
physically separate sensor regions by means of the sample cell consisting of 2
flow channels
comprises the following individual steps: ,
minutes flushing with buffer A (0.5 mllmin.); through both channels and
recording of the
background signal
- 5 minutes supplying the sample: - 10 pl Septoria nodorum standard in 1.8 ml
NN382-PAb-
Septoria nodorum {2.5 x 10-9 M, 0.25 mllmin) through
channel 1
- 10 ~I Septoria tritici standard in 1.8 ml NN382-PAb-
Septoria tritici (2.5 x 10-° M, 0.25 ml/min) through
channel 2
CA 02270665 1999-OS-OS
WO 98I215?1 PCT/EP97106202
- 52 -
- 2 minutes flushing with buffer A {0.5 mllmin.) through both channels and
recording of the
fluorescence signal
- 2 minutes supplying regeneration solution {0.5 ml/min.) through both
channels
- 1 minute flushing with buffer A (0.5 mllmin.) through both channels
The signals from the two separate sensor regions are recorded alternately
during the whole
assay.
The specific signal is calculated from the difference in signal levels at t =
12 mins and t =
mins.
