Note: Descriptions are shown in the official language in which they were submitted.
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ANALYTICAL MEASUREMENT METHOD AND ITS USE
The invention relates to an analytical measurement method.
The invention also relates to the use of the analytical
measurement method in diagnosis, in research looking for active
substances, in combinatorial chemistry, in crop protection, in
toxicology or in environmental protection.
A main task of research looking for active substances in crop
protection or in medicine is to identify novel lead structures
and to develop active substances derived from these structures.
In classical research looking for active substances, the
biological effect of novel compounds has been tested in random
screening on the whole organism, for example the plant or the
microorganism. Employed for this purpose were complex in vitro
and in vivo test methods with which only a few hundred substances
could be tested each year.
In this case the biological testing was the limiting factor with
respect to the synthetic chemistry.
The provision of molecular test systems by molecular and cell
biology has led to a drastic change in the situation. These
molecular test systems, such as receptor binding assays, enzyme
assays or cell-cell interaction assays can, as a rule, readily be
carried out in microtiter plates in reaction volumes of from 5 to
250 l and can easily be automated. This involves use of
microtiter plates with 96, 384, 864 and recently even with 1536
reaction vessels on a single support. Automation and
miniaturization of these test systems permits the sample
throughput to be high. This development makes it possible to test
large numbers of different chemicals for possible use as lead
structure in research looking for active substances.
A modern automated test system allows 100,000 or more chemicals
to be tested for their biological effect each year in mass
screening. Microtiter plate assays are very often used because,
as a rule, they are very robust.
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One disadvantage of the available test systems, for example in
research looking for active substances, in diagnosis, in
environmental protection or crop protection, is that the reagents
required for many test systems, such as enzymes, antibodies,
receptors, fluorescent dyes, radioactively or otherwise labeled
ligands, cytokines, activators, inhibitors or other reagents, are
costly, difficult to prepare andJor not available in a quantity
sufficient for the automated tests. This gives rise to
considerable costs in the automated screening for active
substances. Further miniaturization of the test mixtures is
therefore desirable.
Canadian laid-open patent application no. 2,260,807 describes a solid support
for analytical measurement methods which makes further miniaturization of the
reaction volumes possible. It is possible with these supports to utilize
advantageously the surface tension, which hinders further miniaturization of
the
present microtiter plate technique to ever smaller reaction cavities (=
wells),
because thereby in very small microtiter plate recesses forces such as
adhesion
of the reaction liquid to the surface of the microtiter plates or the
capillary forces
are of increasing importance, and thus make it impossible to fill the reaction
cavities and thus carry out a measurement, to reduce further the reaction
volumes. This application claims an analytical measurement method in which
the various reagents for the test are applied with the aid of a micrometering
system (supplied by Microdrop). The disadvantage of this method is that
although the same reagent is used for all the measurement zones, it has to be
applied in sequential steps to the various measurement zones with the aid of
the
micrometering system.
It is an object of the present invention to develop a simple, rapid and low-
cost
analytical measurement method and to make it available for research looking
for
active substances, diagnosis, environmental protection, crop protection,
toxicology or combinatorial chemistry.
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We have found that this object is achieved by an analytical measurement
method using a solid support which is essentially composed of an inert solid
support material on which hydrophilic measurement zones which may be
provided with a surface loading are separated from one another by at least one
hydrophobic coating in the form of separate zones around the hydrophilic
measurement zones, where the number of measurement points applied per cm2
of the support is greater than or equal to 10, wherein the following steps are
carried out:
a) application one or more times of mutually different reagents singly or in a
mixture to the individual hydrophilic measurement zones on the support,
b) treatment of the support with at least one reagent common to all the
hydrophilic measurement zones so that the reagent is placed
simultaneously on a plurality or all of the hydrophilic measurement zones,
c) optionally washing all the measurement zones together after the
application of the reagents or after the completion of the time for the
reagents to react with one another,
d) measurement, together or singly, of the measurement zones.
Figures 1 and 2 illustrate examples of supports as used in the above method.
The solid support used in the method according to the invention is an inert
solid
support which can consist of a level, planar plate of an exactly similar block
or of
a sheet of any desired shape and size, into which small depressions (see
Figure
2) can, where appropriate, be introduced at the locations of the measurement
zones. Flat supports (see Figure 1) are preferred. Rectangular or square
supports are preferred, and rectangular supports with the size of a standard
microtiter plate (127.5 mm x 85.5 mm) or integral multiples of microtiter
plates
which can be larger or smaller than, for example, the Terasaki plates (81 mm x
56 mm, 60 measurement points) are particularly preferred. The preferred size
of
the supports according to the invention has the advantage that all the
peripherals of the automated microtiter plate technique can be used without
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conversion. Another preferred embodiment of the support comprises supports in
the form of commercial slides for microscopy or of integral multiples thereof,
because they make low-cost evaluation easy, for example using a microscope
or a scanning microscope and an advantageously automated evaluation system.
The supports may consist, for example, of material such as glass, ceramic,
quartz, metal, stone, wood, plastic, rubber, silicon, germanium or porcelain.
The
materials can be used in pure form, as mixtures, alloys or blends or in
various
layers or after coating with, for example, a plastic or a paint for producing
the
supports according to the invention. Transparent supports made of quartz,
glass, plastic, germanium or silicon, which are suitable for all visual tests
such
as microscopic, camera-assisted and laser-assisted tests, are preferably
produced.
Suitable transparent plastics are all amorphous plastic materials
which in a single-phase or multiphase manner with identical
refractive index as polymers of acrylonitrile/butadiene/styrene
or multiphase manner with different refractive index, in which
the domains of the plastic components form zones which are
smaller than the wavelength of the light, such as the block
copolymers of polystyrene and butadiene (polystyrene/butadiene
blends).
Particularly suitable transparent plastics which may be mentioned
in this connection are polystyrene, styrene/acrylonitrile,
polypropylene, polycarbonate, PVC (= polyvinyl chloride),
poly(methyl methacrylate), polyesters, silicones,
polyethylene/acrylate, polylactide or cellulose acetate,
cellulose propionate, cellulose butyrate or mixtures thereof.
Silicon or germanium supports are particularly suitable for
applications in which detection or induction of the reaction
using near infrared light is necessary.
It is also possible to use supports in the form of a conveyor
belt which, when the assays are automated, can move past the
charging, incubation or detection stations.
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Hydrophilic measurement zones on the support (5) mean areas on
the support on which or in which the measurement is carried out
after application of the reaction liquid and thus of the reagents
or reactants (see number 1 in Figures 1 and 2). They thus
correspond to the wells in conventional microtiter plates and are
referred to hereinafter as "measurement zones or measurement
points".
The hydrophilic measurement zones on the support are
advantageously surrounded by a hydrophobic zone (see number 2 in
Figures 1 and 2). This hydrophobic zone can be composed of at
least one hydrophobic coating which covers the support completely
or only partly with discontinuities.
The measurement zones, and the hydrophobic zones which separate
them from one another (see number 2 in Figures 1 and 2), can be
applied, for example, by microlithography, photoetching,
microprinting or a micropunch technique or can be sprayed on
using a mask technique. Photochemical processes which can be used
to make the surfaces of the plates or rolls specifically
hydrophobic at particular points and hydrophilic at other points
are known from the techniques for producing printing plates. It
is possible with this technique to produce, for example, an array
of several thousand regularly arranged hydrophilic measurement
zones (see number 1 in Figures 1 and 2), surrounded by
hydrophobic margins (see number 2 in Figures 1 and 2), in a
simple manner on a support (5), eg. a glass or metal plate. This
may entail firstly one or more hydrophobic coatings being
arranged on the support, and subsequently the measurement zones
being applied to the required points or, conversely, firstly the
hydrophilic measurement zones and then the hydrophobic zones, or
both simultaneously, being arranged. It is also possible to apply
a plurality of hydrophilic measurement zones to the same point.
In the case of hydrophobic support materials it is sensible to
apply hydrophilic measurement points to the support.
The measurement zones can have any desired shape, for example
dots, circles, polygons or crosses, and circular measurement
zones are preferred.
The hydrophilic measurement points may advantageously have, to
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improve immobilization of reagents, further reactive groups which
make noncovalent or covalent linkage of reagents possible.
Preferred reagents are those for which covalent linkage is
possible, for example all coupling reagents from peptide or
nucleic acid chemistry, such as those having aldehyde, epoxide,
isothiocyanate, carbodiimide, hydroxyl, sulfhydryl, amino and/or
carboxyl groups. Linkage via biotin/avidin may also be utilized
advantageously.
It is advantageous to use, in order to maximize the density of
measurement points with reactive groups in the hydrophilic
measurement points, polymers which are able to provide a
multiplicity of binding sites. Examples which may be mentioned
are polymers having acidic or basic groups, such as polyimines,
proteins, polylysine, nucleic acids or (meth)acrylic acid
copolymers. The reagents can be linked to these polymers directly
and/or via bifurictional coupling reagents.
The hydrophobic coating or coatings may be applied coherently to
the support or else be provided with discontinuities of any
design. They may also be in the form of separate zones around the
measurement zones, with hydrophobic rings separating the
hydrophilic measurement zones from one another being preferred.
It is possible in principle to apply any desired number of
measurement points to a support, but the number of measurement
points per cm2 is preferably greater than or equal to 10,
particularly preferably greater than or equal to 15 and very
particularly preferably greater than or equal to 20. Supports
having a number of measurement points per cm2 greater than or
equal to 30 are most preferred. Moreover the reaction volumes
applied are from a few nl up to some l, with volumes of less
than 5 l being preferred, and of less than or equal to 1 l
being particularly preferred.
The measurement points can be applied in any desired arrays to
the support, and square or rectangular arrays are preferred.
Methods suitable for applying sample material and reagents are
all those able to meter amounts of liquid from a few nl to a few
l, such as techniques used in ink jet printers (see
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DE-A 40 24 544) or in flow cytometry or in cell sorters (Horan,
P.K., Wheeless, L.L., Quantitative Single Analysis and Sorting,
Science 198 (1977) 149 - 157). Drop formation can in this case
take place by piezoelectric drop formation (Ultrasound),
piezoelectric drop ejection or ejection by evaporation (ink jet
technique). It is possible to use systems with permanent drop
production or systems which produce drops on demand.
These techniques can be used to place individual droplets in an
accurately metered and targeted manner on the individual
hydrophilic measurement points of the multianalysis surface of
the support by, for example, moving the support under one or more
nozzles, which are arranged in parallel, in accordance with the
rhythm of the metered liquid and in accordance with the preset
array. It is also possible likewise to move the metering device,
for example consisting of at least one nozzle, over the support
in accordance with the rhythm of the metered liquid and in
accordance with the preset array.
It is possible and advantageous with these methods which apply
individual drops for reagents which differ from one another to be
applied one or more times, singly or in a mixture, to the
individual hydrophilic measurement points on the support (step a
in the method).
It emerges, contrary to expectations, that electrostatic charges
and airflow between the nozzles and the supports have no effect
on accurate placing of the reagents on the support. On the
contrary: these inaccuracies, related to the technique, in the
placing of the reagents are corrected by an automatic refocussing
via hydrophilic/hydrophobic interactions between the
advantageously aqueous or polar reagent liquids and the
hydrophobic and hydrophilic zones on the support. Aqueous reagent
liquids are preferred.
It has additionally been found that reagents which are applied,
for example, to the measurement points by immersion or washing
over the complete support surface are also automatically
demarcated from one another and focussed on the measurement
zones. Further advantageous methods for applying the reagents
together are application using a knife or using a sponge or
another absorbent material. It is also possible and advantageous
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for the supports to be sprayed with the reagents. It is possible
with this method advantageously to apply at least one common
reagent one or more times simultaneously to several or all of the
hydrophilic measurement points on the support (step b in the
method).
Time-consuming sequential, and thus costly, application of the reagents, as
described in Canadian laid-open application no. 2,260,807, is thus no longer
necessary.
It is also possible for the washing steps which are, where
appropriate, necessary between the individual steps (a) and/or
(b) in the method or before the measurement (d) to be carried out
advantageously by immersion, washing over or wiping over with an
absorbent material. These washing steps for all the measurement
zones can take place after the application of the reagents or
after completion of the time for the reagents to react together.
Washing steps can also be carried out, when reagents which are
mutually different are applied several times to the individual
hydrophilic measurement zones on the support, between the
individual applications, or else a joint washing step can be
carried out at the end of the treatments.
If one or more reactants have been linked to the hydrophilic
measurement zones on the support, and if common reagents are
subsequently applied by the abovementioned methods, it is in fact
possible for the reaction to be carried out, for example, in an
immersion bath without the need to separate the individual
measurement points physically from one another.
Even if small amounts of free reactants are present in the
liquid, this would result in only very slight cross-contamination
as long as care is taken_that the ratio between the actual
incubation time and the duration of the joint contact of all the
reactants and all the measurement points is favorable.
Steps (a) and (b) in the analytical measurement method according
to the invention can be carried out in any sequence one or more
times with removal where appropriate of excess reagents by
washing (c) and/or wiping off between the steps in the method or
before the measurement(s) (d).
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The mutually different reagents may be applied singly or in a
mixture to the individual hydrophilic measurement points
(= measurement zones) on the support.
As described above, it is possible with this measurement method
for different reagents and/or single cells to be placed on the
predetermined sites (= measurement points) on the surface of the
support and to be reacted. It is advantageous that, with the
small volumes in the range from a few nanoliters to a few
microliters, mixing of the reactants by diffusion takes place
very quickly so that no special mechanical mixing device is
necessary. It is also possible, before the addition of liquid
droplets for carrying out the actual analysis, for certain
ligands, eg. proteins or nucleic acids, to be present on the
support in adsorbed or chemically bound form before metering in
the measurement samples and the reagents.
Further advantages of the measurement method according to the
invention are the saving of substances such as chemicals to be
tested, enzymes, cells or other reactants, of time through a
further increase in parallel reaction mixtures, which are
automated where appropriate, of space and staff requirements, due
to further miniaturization of the reaction mixtures.
The reagent droplets placed on the supports can also be applied
in the form of gel droplets which subsequently solidify where
appropriate and thus reduce evaporation of the reaction liquid.
Evaporation of the reaction liquid (see number 3 in Figures 1 and
2) can also be reduced by coating with a hydrophobic liquid (see
number 4 in Figures 1 and 2), in which case the hydrophobic
coating or coatings act like an anchor. Low-viscosity oils such
as silicone oils are preferably used for the coating.
Evaporation can also be reduced by incubating the supports in an
atmosphere which is virtually saturated with water vapor.
Reduction in evaporation is likewise possible by cooling the
supports.
Evaporation can be reduced by using single elements of those
mentioned or combinations thereof.
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It is also possible in the analytical measurement method
according to the invention, depending on the objective and the
reactants used, perfectly to tolerate interim drying of the
individual reaction mixtures on the hydrophilic measurement
points.
The analytical measurement method according to the invention is
suitable in principle for all analytical methods now carried out
in microtiter plates, such as colorimetric, fluorimetric or
densitometric methods. It is possible in these cases to use and
measure light scattering, turbidity, wavelength-dependant light
absorption, fluorescence, luminescence, Raman scattering, ATR
(= attenuated total reflection), radioactivity, isotope labeling,
10 pH shifts or ion shifts, advantageously alone or in combination,
to mention only a few of the possible measured quantities here.
Analytical methods which can be carried out in the measurement
method according to the invention and which may be mentioned here
are the binding of antibodies to antigens, the interaction
between receptors and ligands, the specific cleavage of substrate
molecules by enzymes, the polymerase chain reaction (PCR),
agglutination tests or the interaction between different or
identical cell types such as enzyme assays, titration assays such
as virus titration assays, erythrocyte or platelet aggregation
assays, agglutination assays with latex beads, ELISA
(= Enzyme-linked immunoaorbent Issay) or RIA
(= Radioimmunogssay).
It is possible in the analytical measurement method according to
the invention for the reaction to be measured several times
between the steps, or part steps (= multiple repetition of a
step), in the method, or once after the application of the
reagents and completion of the time for the reaction, or between
the steps in the method and after the application of the reagents
and completion of the time for the reaction.
The analytical measurement method according to the invention can
be employed, for example, in diagnosis, in research looking for
active substances, in combinatorial chemistry, in crop
protection, in toxicology, in environmental protection, for
example for cytotoxicological tests, in medicine or in
biochemistry.
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The analytical measurement method according to the invention is
particularly suitable for mass screening.
All modern image-acquiring and image-analyzing systems are
suitable for the measurement method according to the invention.
The following examples serve to illustrate the invention further
without restricting it in any way.
Example 1
Production of a support from a glass slide for use in the method
according to the invention
Firstly, the glass slide was cleaned with a 20% strength aqueous
solution of an acidic cleaner (Reacalc(D, supplied by Chemotec
GmbH) in an ultrasonic immersion bath for 10 minutes. The glass
slide was subsequently rinsed with water and then with absolute
ethanol and dried at about 23 C.
A micropunch was used to apply the hydrophobic coating in the
form of hydrophobic rings (see Figure 1 and 2) on the hydrophilic
support (5). The hydrophobic layer was applied using a 1%
strength hexadecyltrimethoxysilane solution in isopropanol/H20
(9:1). The punch was dipped in the silane solution and then
briefly, for about 5 sec, pressed on the support, and then the
support was dried at 1000C for 15 minutes. Two types of punches
were used to apply 12 and 25, respectively, measurement points
per square centimeter.
