CA2245013C

CA2245013C - Analytical measurement method and its use

Info

Publication number: CA2245013C
Application number: CA002245013A
Authority: CA
Inventors: Heinz Eipel; Harald Keller; Burkhard Kroger; Sabine Philipp
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 1997-09-25
Filing date: 1998-09-24
Publication date: 2007-11-13
Anticipated expiration: 2018-09-24
Also published as: EP0905515A3; EP0905515B1; CA2245013A1; ATE284536T1; JPH11160314A; DE19742246A1; EP0905515A2; DE59812354D1

Abstract

Disclosed is an analytical measurement method for use in diagnosis, in research looking for active substances, in combinatorial chemistry, in crop protection, in toxicology or in environmental protection. This method makes 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 hte number of measurement points applied per cm2 of the support is greater than or equal to 10. In practise, 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.

Description

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.

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.

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 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.

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 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 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 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).

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.

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.

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.

Claims

1. 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.

2. An analytical measurement method as claimed in claim 1, wherein steps (a) and (b) in the method are carried out in any sequence one or more times, where appropriate excess reagents being removed by washing (c) between the steps in the method or before the measurement (d).

3. An analytical measurement method as claimed in claim 1 or 2, wherein the measurement (d) takes place more than once between the steps in the method or once after the application of the reagents and completion of the time(s) for the reaction or between the steps in the method and after the application of the reagents and completion of the time(s) for the reaction.

4. An analytical measurement method as claimed in any one of claims 1 to 3, wherein the measurement is carried out in an atmosphere which is virtually saturated with water vapor.

5. An analytical measurement method as claimed in any one of claims 1 to 4, wherein the measurement is carried out while cooling the support.

6. An analytical measurement method as claimed in any one of claims 1 to 5, wherein the measurement zones are covered with a hydrophobic layer after application of the reagents.

7. An analytical measurement method as claimed in any one of claims 1 to 6, wherein said method is carried out in diagnosis, in research looking for active substances, in combinatorial chemistry, in crop protection, in toxicology or in environmental protection.