Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02260807 1999-O1-14
1
SOLID SUPPORTS FOR ANALYTICAL MEASURING PROCESSES,
A PROCESS FOR THEIR PREPARATION, AND THEIR USE
The invention relates to solid supports for analytical measure-
ment~methods which are 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, where the number of measure-
ment points applied per cm2 of the support is greater than or
equal to 10. The invention furthermore relates to a process for
producing the supports, and to the use of the supports in diag-
nostic methods, 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 biologi-
cal effect of novel compounds has been tested in random screening
on the whole organism, for example the plant or the microorgan-
ism. 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 mo-
lecular test systems, such as receptor binding assays, enzyme as-
says or cell-cell interaction assays, can, as a rule, readily be
carried out in microtiter plates in reaction volumes of from 50
to 250 ~l and can easily be automated. Automation and miniaturiza-
tion 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 re-
search 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 entail low costs, are very reliable and have
little susceptibility to faults.
0050/47114 CA 02260807 1999-O1-14
2
In order to be able fully to exploit the efficiency of these test
systems, novel solid-phase syntheses have been and are still be-
ing developed in combinatorial chemistry.
Combinatorial chemistry makes it possible to synthesize a wide
variety of different chemical compounds, called substance li-
braries. This is particularly true when combinatorial chemistry
makes use of automated solid-phase synthesis (see, for example,
review articles I. [sic] Med. Chem. 37 (1994) 1233 and 1385).
Solid-phase synthesis has the advantage that a large number of
compounds can be synthesized, and that by-products and excess
reactants can easily be removed, so that elaborate purification
of the products is unnecessary.
The large number of synthesized compounds in combinatorial chem-
istry means that the efficiency of modern automated test systems
can be fully exploited with regard to chemical diversity. Since,
however, in contrast to classical active substance synthesis, the
chemicals to be investigated are not available in any desired
amount on synthesis by means of combinatorial chemistry, only a
restricted number of test systems can be examined because of the
amounts of chemicals required in the test systems.
Another disadvantage of present test systems, for example in re-
search looking for active substances, in diagnostic methods, in
environmental protection or crop protection, is that the reagents
required for many test systems, such as enzymes, antibodies, re-
ceptors, fluorescent dyes, radioactively or otherwise labeled li-
gands, cytokines, activators, inhibitors or other reagents, are
costly, difficult to prepare and/or not available in a quantity
sufficient for the automated tests.
DE-A 44 35 727 describes an approach for reducing the reagents
required for a test.
The disadvantage of this process is that the support for the mea-
surements must first be produced in an elaborate multistage pro-
cess.
Another disadvantage is that the reactions which can be carried
out with this support material are confined to reactions linked
to solid phases, such as reactant binding between antibodies, an-
tigens, haptens or nucleic acids. It is not possible with this
method to carry out reactions in solution.
0050/47114 CA 02260807 1999-O1-14
3
It is an object of the present invention to develop a novel ana-
lytical measurement method which can be carried out without the
stated disadvantages and provide it for research looking for ac-
tive substances, diagnostic methods, environmental protection,
crop protection, toxicology or combinatorial chemistry.
We have found that this object is achieved by using the solid
support described at the outset for the measurement method.
It has been found that the surface tension which hinders further
miniaturization of the present microtiter plate technique to ever
smaller reaction cavities (= wells), because thereby forces such
as adhesion of the reaction liquid to the surface of the micro-
titer plates or the capillary forces are of increasing impor-
tance, and thus make it impossible to fill the reaction cavities
and thus carry out a measurement, in very small microtiter plate
wells, can be utilized advantageously for the supports according
to the invention.
Hydrophilic measurement zones on the support 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 reactants (see
number 2 in Figures 1, 3 and 4). They thus correspond to the
wells in microtiter plates and are referred to hereinafter as
"measurement zones or measurement points".
The hydrophilic measurement zones on the support are advanta-
geously surrounded by a hydrophobic zone (see number 1 in Figures
1 to 4). This hydrophobic zone can be composed of at least one
hydrophobic coating which covers the support completely or only
partly with discontinuities. These discontinuities (see number 5
in Figures 1 to 4) are advantageously hydrophilic.
Figures 1 to 4 serve to illustrate the supports according to the
invention by way of example.
The measurement zones, and the hydrophobic zones which separate
them from one another (see number 1 in Figures 1 to 4), can be
applied, for example, by microlithography, photoetching, micro-
printing or a micropunch technique or can be sprayed on using a
mask technique. Photochemical processes which can be used to make
the surfaces of plates or rolls specifically hydrophobic at par-
ticular 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, a grid of several thou-
sand regularly arranged hydrophilic measurement zones (see number
2 in Figures 1, 3 and 4), surrounded by hydrophobic margins (see
005047114 CA 02260807 1999-O1-14
4
number 1 in Figures 1 to 4), in a simple manner on a support, eg.
on a glass or metal plate. This may entail firstly one or more
hydrophobic coatings being applied to the support, and subse-
quently the measurement zones being applied to the required
points or, conversely, initially the hydrophilic measurement
zones and then the hydrophobic zones, or both simultaneously, be-
ing applied. It is also possible to apply a plurality of hydro-
philic measurement zones to the same point.
Figure 2 depicts by way of example a support according to the in-
vention having the size of a microtiter plate.
The measurement zones can have any desired shape, with circular
measurement zones being preferred.
The hydrophobic coating or coatings may be applied coherently to
the support or else be provided with discontinuities of any de-
sign. They may also be in the form of separate zones around the
measurement zones, with hydrophobic rings separating the hydro-
philic measurement zones from one another being preferred.
The hydrophobic coating or coatings are intended to prevent the
measurement zones spreading into one another and thus to make ac-
curate measurement of individual reaction mixtures possible.
It is possible in principle to apply any desired number of mea-
surement points onto a support, but the number of measurement
points per cm2 is preferably greater than or equal to 10, particu-
larly preferably greater than or equal to 15 and very particu-
larly preferably greater than or equal to 20. Moreover, the reac-
tion volumes applied are from a few nl up to some wl, with volumes
of less than 5 ~1 being preferred, and of less than or equal to
1 ~.1 being particularly preferred.
The measurement points can be applied in any desired grids to the
support, and square or rectangular grids are preferred.
The inert solid support may consist of a level, planar plate of a
block of the same type or of a sheet of any desired shape and
size, which may have small depressions (see Figure 4) at the mea-
surement zone points, with flat supports (see Figure 3) being
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, for example the Terasaki plates
(81 mm x 56 mm, 60 measurement points), are particularly pre-
ferred. The preferred size of the supports according to the in-
0050/47114 CA 02260807 1999-O1-14
vention has the advantage that all the peripherals of automated
microtiter plate technology can be used without alteration.
The support may consist, for example, of materials such as glass,
5 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 coat-
ing 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 [lacuna] in a single-phase or multiphase manner with iden-
tical refractive index as polymers of acrylonitrile/butadiene/
styrene or in a multiphase manner with different refractive in-
dex, in which the domains of the plastic components form zones
which are smaller than the wavelength of 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, poly-
propylene, 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 in-
duction of the reaction using near infrared light is necessary..
The support according to the invention may also be designed in
the form of a conveyor belt which, when the assays are automated,
can move past the charging, incubation or detection stations.
One process for producing the supports according to the invention
starts, for example, from ceramic, quartz or glass plates. The
support is for this purpose expediently first cleaned with a
cleaner, for example an alcohol, an alkaline cleaner or an acidic
cleaner such as Reacalc~ (which contains, according to the sup-
plier Chemotec GmbH, phosphoric acid and surfactants). The clean-
ing can advantageously be improved by carrying it out in an
ultrasonic bath. After cleaning, the support is dried, immedi-
ately or after rinsing with water and/or alcohol or with an alco-
hol/water mixture. The hydrophobic coating of the support takes
place, for example, with a 1 ~ strength hexadecyltrimethoxysilane
solution in a solvent such as isopropanol/HZO (9:1) using a punch
0050/47114 CA 02260807 1999-O1-14
6
technique. The punch is briefly pressed on the glass slide to
apply the 1 % strength hexadecyltrimethoxysilane solution. The
support is subsequently dried. The glass support is advantageous-
ly dried at elevated temperatures, ie. above 80°C. The support is
preferably rinsed once again after drying to remove excess hexa-
decyltrimethoxysilane, for example with an alcohol/water mixture
such as isopropanol/H20 (9:1).
This punch technique may be used to apply an additional surface
loading in the region of the hydrophilic measurement points. This
surface loading can be generated, for example, by applying pro-
teins, acidic or basic polymers such as polylysine or acidic or
basic molecules.
Methods suitable for applying sample material and reagents are
all those able to meter amounts of liquid from a few nl to a few
~1, such as techniques used in ink jet printers (see DE-A
40 24 544) or in flow cytometry, 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 hydro-
philic 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
grid. 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 accor-
dance with the preset grid.
It is possible with these techniques if necessary to place dif-
ferent reagents and/or individual cells on the predetermined
sites (measurement points) on the support surface and bring about
reaction thereof. It is advantageous that, with the small volumes
preferred according to the invention, in the range from a few
nanoliters to a few microliters, mixing of the reactants by dif-
fusion takes place very quickly so that no special mechanical
mixing device is necessary. It is also possible, before the addi-
tion of liquid droplets for carrying out the actual analysis, for
certain ligands, eg. proteins or nucleic acids, to be present on
0050/47114 CA 02260807 1999-O1-14
7
the support in adsorbed or chemically bound form before metering
in the measurement samples and the reagents.
Further advantages of the supports 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 appropri-
ate, of space and staff requirements, due to further miniaturiza-
tion of the reaction mixtures and thus finally also of money.
The droplets placed on the supports may also be applied in the
form of gel droplets which subsequently solidify where appropri-
ate and thus reduce evaporation of the reaction liquid.
Evaporation of the reaction liquid (see number 3 in Figures 3 and
4) can also be reduced by coating with a hydrophobic liquid (see
number 4 in Figures 3 and 4), in which case the hydrophobic coat-
ing or coatings act like an anchor (Figure 3 and 4).
I,ow-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 sup-
ports.
Evaporation can be reduced by using single elements of those men-
tioned or combinations thereof.
The supports according to the invention are 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-dependent light absorption, fluorescence,
luminescence, raman scattering, ATR (= Attenuated Total Reflec-
tion), radioactivity, isotope labeling, 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 on the supports ac-
cording to the invention and which may be mentioned here are the
binding of antibodies to antigens, the interaction between recep-
tors 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
0050/47114 CA 02260807 1999-O1-14
8
assays, erythrocyte or platelet aggregation assays, agglutination
assays with latex beads, ELISA (= Enzyme-1_inked immuno~orbent
essay) or RIA (_ _Radioimmuno_assay).
The supports according to the invention can be employed, for ex-
ample, in diagnostic methods, in research looking for active sub-
stances, in combinatorial chemistry, in crop protection, in tox-
icology, in environmental protection, for example for cytotoxico-
logical tests, in medicine or in biochemistry.
The supports according to the invention are particularly suitable
for mass screening.
The supports according to the invention are particularly suitable
for all modern image-acquiring and image-analyzing systems.
The following examples serve to illustrate the invention further
without restricting it in any way.
Example 1
Production of a support according to the invention from a glass
slide
Firstly, the glass slide was cleaned with a 20 % strength aqueous
solution of an acidic cleaner (Reacalc~ 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 to 4) on the hydrophilic
support. 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 100°C for 15 minutes. Excess silane solution was removed
from the support by immersing the support in isopropanol/H20 (9:1)
for about 1 minute. Two types of punches were used to apply 12
and 25, respectively, measurement points per square centimeter.
0050/47114 CA 02260807 1999-O1-14
9
Example 2
Protease inhibitor assay with the supports according to the in-
s vention
A protease inhibitor assay was carried out using a support pro-
duced by the process described in Example 1.
96 samples each comprising 100 nl of a solution of casein labeled
with fluorescein isothiocyanate (20 ~g/ml) in 10 mM tris/HC1 buff-
er (pH 8.5) were applied, in a chamber with a relative humidity
exceeding 95 ~ using a micrometering system supplied by Micro-
drop, Norderstedt, to a slide produced as described above. The
reaction droplets were arranged in 8 rows and 12 columns in a
2 x 2 mm grid in accordance with the hydrophobic zones (= barrier
layers) applied by the punch. The width of the hydrophobic rings
was 0.4 mm in each case.
Subsequently, 1 nl of each of various protease inhibitors in a
concentration of 1 mM in 10 mM tris/HC1 buffer (pH 8.5) was added
to the reaction samples using the Microdrop apparatus. Addition
took place precisely into the fluorescent-labeled casein solution
which had previously been applied. One nanoliter of 10 mM tris/
HC1 buffer (pH 8.5) was used as control.
Finally, 10 nl of the protease trypsin in a concentration of
10 mg/ml in tris/HC1 buffer (pH 8.5) were added to the reaction.
The reaction droplets were subsequently covered either with min-
eral oil, silicone oil or liquid paraffin, which was applied us-
ing the microdrop metering system, to reduce evaporation.
After incubation for 30 minutes, the assay was measured. This was
done using an inverted fluorescence microscope for excitation,
from the underside of the slide, with linearly polarized light in
the range from 450 to 485 nm and for detection of fluorescence in
the range from 515 to 530 nm. A coolable CCD camera in front of
which there was a polarization filter which could be rotated by a
motor was used for detection.
The anisotropy of polarization of the casein molecules was deter-
mined using the following equations:
- 0050/47114 CA 02260807 1999-O1-14
Iperpendicular - Iparallel
A = (I)
Iperpendicular + 2 X Iparallel
5 Iperpendicular - Iparallel
A = III)
Iperpendicular '~ Iparallel
where:
A is the anisotropy
P is the polarization
Iparallel is the measured intensity of the fluorescent light on
polarization parallel to the polarization of the ex-
citing light and
Iperpendicular is the measured intensity of the fluorescent light
with crossed polarization filters.
The anisotropy is a measure of the rotational diffusion coeffi-
cient of molecules and can be employed to estimate the hydrody-
namic sizes of molecules (G. Weber, Biochemie, Vol. 51, 1952,
145-155).
On cleavage of the protein labeled with fluorescein isothiocya-
nate by the protease, a polarization in the range from 50 to
75 x 10-3 was measured. On inhibition of the protease trypsin, the
measured polarization was greater than 150 x 10-3.
For parallel analysis of all 96 reaction mixtures, the complete
measurement field with the 96 points was covered at one time with
a lens of a stereo magnifier and was analyzed using an image-pro-
cessing program.
Example 3
Protease inhibitor assay with 1536 parallel measurement points
A trypsin inhibitor assay was carried out as described in Exam-
ple 2 with 1536 parallel measurement points on the size of a
microtiter plate (see Figure 2).
